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WO2015142875A1 - Compositions and methods using modified salmonella - Google Patents

Compositions and methods using modified salmonella Download PDF

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Publication number
WO2015142875A1
WO2015142875A1 PCT/US2015/021002 US2015021002W WO2015142875A1 WO 2015142875 A1 WO2015142875 A1 WO 2015142875A1 US 2015021002 W US2015021002 W US 2015021002W WO 2015142875 A1 WO2015142875 A1 WO 2015142875A1
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gene
salmonella
bacterium
bacterial
infection
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French (fr)
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Igor E. BRODSKY
Meghan A. WYNOSKY-DOLFI
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The Trustees Of The University Of Pennsylvania
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Microbial infection triggers assembly of inflammasome complexes that promote caspase-1 -dependent antimicrobial responses.
  • Inflammasome assembly is mediated by members of the Nucleotide binding domain-Leucine-Rich Repeat (NLR) protein family that respond to cytosolic bacterial products or disruption of cellular processes.
  • NLR Nucleotide binding domain-Leucine-Rich Repeat
  • Flagellin injected into host cells by invading Salmonella induces inflammasome activation through Nucleotide binding domain Leucine Rich Repeat Family Card Domain Containing 4 (NLRC4), while Nucleotide binding domain Leucine Rich Repeat Family Pyrin Domain Containing 3 (NLRP3) is required for inflammasome activation in response to multiple stimuli, including microbial infection, tissue damage, and metabolic dysregulation, through mechanisms that remain poorly understood.
  • NLRC4 Nucleotide binding domain Leucine Rich Repeat Family Card Domain Containing 4
  • NLRP3 Nucleotide binding domain Le
  • Salmonella avoids NLRC4 inflammasome activation by downregulating flagellin expression. Macrophages exhibit delayed NLRP3 inflammasome activation following Salmonella infection, suggesting that Salmonella may evade or prevent the rapid activation of the NLRP3 inflammasome.
  • Pattern recognition receptors that detect and respond to evolutionarily conserved microbial structures such as lipopolysaccharide (LPS) or peptidoglycan, as well as pathogen-specific virulence activities, are critical for host immune defense 71 ' 102 .
  • microbial pathogens inject virulence factors into the cytosol of infected cells to disrupt or modulate critical host physiological processes 19 .
  • contamination of the target cell cytosol by microbial components triggers cytosolic PRRs of the Nucleotide binding domain-Leucine-Rich Repeat (NLR) family 52 . Diverse NLRs respond to a variety of endogenous and exogenous signals associated with infection, tissue stress or damage.
  • NLR Nucleotide binding domain-Leucine-Rich Repeat
  • NL C4 responds to microbial products such as bacterial flagellin or structurally- related specialized secretion system components that are injected into the cytosol of infected cells during infection by bacterial pathogens including Pseudomonas, Legionella, and Salmonella spp. 72 ' 75 ' 97 NLRs recruit pro-caspase-1 to multiprotein complexes termed inflammasomes, where pro-caspase-1 is processed and activated, leading to cleavage and secretion of caspase- 1 -dependent cytokines 66 ' 67 , as well as pyroptosis, a caspase-1 -dependent pro-inflammatory cell death 5 .
  • microbial products such as bacterial flagellin or structurally- related specialized secretion system components that are injected into the cytosol of infected cells during infection by bacterial pathogens including Pseudomonas, Legionella, and Salmonella spp. 72 ' 75 ' 97 NLRs recruit
  • Inflammasome activation and subsequent production of caspase-1 -dependent cytokines is important for both innate and adaptive antimicrobial responses 63 , as IL-1 family cytokines released upon inflammasome activation promote neutrophil migration to infected tissues and drive T H 17 and T H 1 responses against mucosal pathogens 16 ' 40 . How pathogens evade inflammasome activation, and whether persistent bacterial pathogens evade or suppress inflammasome activation in order to establish or maintain persistence remains poorly understood.
  • Salmonella enterica species cause a range of disease from severe gastroenteritis to persistent systemic infection 4 .
  • Salmonella enterica serovar Typhimurium ⁇ Stm invades host cells by means of a type III secretion system (T3SS) encoded on Salmonella pathogenicity island I (SPI-1) 18 ' 57 .
  • Salmonella subsequently replicates within a Salmonella-containing vacuole (SCV) that is established and maintained by the activity of a second T3SS, encoded on a second pathogenicity island, SPI-2 17 ' 35 .
  • T3SS type III secretion system
  • Intestinal inflammation during Stm infection is triggered by NLRC4-dependent responses to Stm flagellin, accompanied by caspase- 1- dependent cytokine secretion and pyroptosis 29 .
  • Activity of a SPI-1 effector protein, SopE also contributes to SPI-1 -dependent inflammasome activation in intestinal epithelial cells 79 .
  • flagellin expression is downregulated at systemic sites 20 ' 21 , and enforced flagellin expression enhances NLRC4 activation and bacterial clearance, indicating that inflammasome activation in response to bacterial flagellin is detrimental for Stm replication during systemic infection 73 ' 95 .
  • NLRP3 responds to a wide variety of structurally unrelated molecules and activities, including extracellular ATP, bacterial pore-forming proteins, bacterial nucleic acids, crystals, and unsaturated fatty acids 39 ' 45 ' 64 ' 68 107 . While ATP, crystals, and the Yersinia T3SS all induce rapid formation of an NLRP3 inflammasome that leads to caspase-1 activation within 1-2 hours 8 ' 64 ' 68 , Stm induces delayed activation of a non-canonical NLRP3 inflammasome 12-16 hours post-infection 9 . This non-canonical NLRP3 inflammasome is independent of the activities of the SPI-1 T3SS and instead is regulated by caspase-11 and TLR4-dependent production of type I interferon 10 ' 34 ' 86 .
  • NLRs detect exogenous and endogenous molecules that serve as indicators of infection or tissue stress. Together with the NAIP proteins, NLRC4 detects the cytosolic presence of bacterial flagellin and inner rod proteins of bacterial T3SSs, leading to rapid inflammasome activation and pyroptosis 48 113 . Downregulation of flagellin expression or inactivating the genes encoding flagellin enables pathogens to evaded NLRC4 detection 14 ' 20 ' 73 . Pathogens also utilize active mechanisms of inflammasome suppression, either by downregulating NLRC4, or by targeting other host pathways, such as autophagy, that regulate inflammasome
  • the pathogenic Yersinia species possess several distinct effector-mediated mechanisms for inflammasome modulation 8 ' 54 .
  • the activation of the AIM2 inflammasome in response to Mycobacteria is blocked by the ESX system of virulent mycobacteria 91 .
  • NLRP3 inflammasome responds to a wide variety of structurally and chemically unrelated signals, and, along with NLRC4, contributes to host defense against Stm infection 9 .
  • NLRP3 induces delayed inflammasome activation in response to Stm independently of the SPI-1 and SPI-2 T3SSs via a non-canonical pathway involving a TLR4-TRIF-IFN signaling pathway that requires caspase-11 10 ' 34 ' 86 .
  • an attenuated Salmonella e.g., Salmonella enterica Typhimurium bacterium is provided that is deficient in the gene encoding one or both of the bacterial Citric Acid Cycle (TCA) enzymes, aconitase and isocitrate dehydrogenase.
  • TCA Citric Acid Cycle
  • an antibiotic resistance gene is inserted in the bacterium in place of the naturally occurring, deleted gene.
  • a heterologous gene is inserted in the bacterial chromosome.
  • composition comprising attenuated Salmonella, e.g., Salmonella
  • Salmonella enterica Typhimurium bacterium deficient in the gene encoding one of the bacterial TCA enzymes selected from aconitase and isocitrate dehydrogenase and a pharmaceutically acceptable carrier in another aspect, a composition is provided comprising attenuated Salmonella, e.g., Salmonella enterica Typhimurium bacterium deficient in the gene encoding one of the bacterial TCA enzymes selected from aconitase and isocitrate dehydrogenase and further containing a heterologous gene.
  • a method for inducing the innate immune response of an animal against infection by a Salmonella strain e.g., Salmonella enterica Typhimurium
  • a method of inducing the innate immune response of an animal against a microbial infection or cancer comprises administering to an animal in need thereof a composition containing an attenuated Salmonella strain, e.g., Salmonella enterica
  • the heterologous gene is from another infectious microorganism or from a cancer cell or tumor cell.
  • a method of generating a Salmonella vaccine comprises deleting a naturally occurring gene encoding a TCA enzyme selected from aconitase or isocitrate dehydrogenase from the chromosome of a wildtype or attenuated Salmonella strain, e.g., Salmonella enterica Typhimurium bacterium.
  • the method employs bacteriophage transduction or homologous recombination to effect the deletion or replacement.
  • a method of generating a composition that induces the innate immune response against a target gene comprises deleting a naturally occurring gene encoding a TCA enzyme selected from aconitase or isocitrate dehydrogenase from the chromosome of a wildtype or attenuated Salmonella strain, e.g., Salmonella enterica Typhimurium bacterium and inserting into the bacterial genome a heterologous target gene from another infectious microorganism or from a cancer or tumor cell.
  • the method employs bacteriophage transduction or homologous recombination to effect the deletion or insertion.
  • FIG. 1 A are data generated from in vitro screens for bacterial mutants that fail to inhibit inflammasome activation.
  • fliCfljB Tnl0d::tet library was grown in 96-well format under SPI-1 inducing conditions and screened for ability to induce LDH release in immortalized Nlrc4 ' ' ' macrophages.
  • FIG. 1 A shows two bar graphs providing data representative of a plate (left graph) from indicated screen (right graph).
  • LPS+ATP is a positive control
  • fliCfljB parent strain is a negative control. All experiments other than initial screen were performed in triplicate and are representative of at least 3 independent experiments. * /> ⁇ 0.05.
  • FIG. IB provides data generated from infecting B6, Nlrc4 ' and Casp Casp ll '1' bone marrow derived macrophages (BMDMs) in triplicate with initial bacterial mutants that exhibited elevated lactate dehydrogenase (LDH) release in primary screen. Supernatants were harvested 4 hours post- infection, and assayed for LDH release as descried in FIG. 1A. All experiments other than initial screen were performed in triplicate and are representative of at least 3 independent experiments. * /> ⁇ 0.05.
  • FIG. 1C is a bar graph showing IL- ⁇ released from B6, N/rc "A and Caspl '1' Caspl l '1'
  • BMDMs was measured by ELISA 4 hours post-infection. All experiments other than initial screen were performed in triplicate and are representative of at least 3 independent experiments. * /> ⁇ 0.05.
  • FIG. ID shows bars graph data generated from measuring IL-6 released from infected cells 4 hours post-infection as a control cytokine. All experiments other than initial screen were performed in triplicate and are representative of at least 3 independent experiments. * /X0.05.
  • FIG. 2A is a schematic of S. Typhimurium TCA cycle.
  • FIG. 2B shows that deletion of Salmonella citrate or isocitrate TCA cycle enzymes induces NL P3 inflammasome activation.
  • FIG. 2B. is a graph showing data from the infection of Casp 1 ' ' ' Casp 11 ' ' ' BMDMs with Stm carrying targeted deletions in indicated TCA cycle genes. The cells were lysed, bacteria diluted, and intracellular bacterial CFUs determined at 1, 8 and 24 hours post-infection. All data are representative of at least three independently performed experiments. * /> ⁇ 0.05.
  • FIG. 2C is a bar graph showing that targeted mutations in specific core TCA cycle genes lead to NLRP3- and caspase-l/caspase-11 -dependent cell death.
  • LPS+ATP was used as a positive control for cell death
  • fliCfljB used as a negative control (parent strain). All data are representative of at least three independently performed experiments.
  • FIG. 2D is a Western blot for active caspase-1 (plO) that was performed on lysates from B6, Nlrp3 ' , and Nlrc4 ' BMDMs 3 hours post-infection with indicated bacterial mutant strains, ⁇ -actin was blotted for as a loading control. Molecular weight markers in kD are indicated at left. All data are representative of at least three independently performed experiments. UI - Uninfected, B6 - C57BL/6. * / 0.05.
  • FIG. 2E are bar graphs showing supernatants from B6, Nlrc4 ' , Nlrp3 ' and Caspl ' ' ' Caspll '1' BMDMs infected with TCA mutants for 4 hours and analyzed for IL- ⁇ measured by ELISA. All data are representative of at least three independently performed experiments. B6 - C57BL/6. * /? ⁇ 0.05.
  • FIG. 2F are bar graphs showing that IL-6 released from infected cells was measured as a control cytokine. All data are representative of at least three independently performed experiments. B6 - C57BL/6. * / 0.05.
  • FIG. 3 A provides data showing that deletion of bacterial TCA genes activates the canonical NL P3 inflammasome.
  • FIG. 3A is a bar graph showing data generated from infecting B6, Caspl '1' , Caspll '1' and Caspl ' ' ' CaspH ' ' ' BMDMs with bacterial TCA cycle mutants. Cell death was measured 4 hours post-infection by LDH release assay. LPS+ATP was used as a positive control and fliCfljB (parent strain for indicated mutants) was used as a negative control. All experiments were performed three independent times in triplicate, and data from representative experiments are shown. * /? ⁇ 0.05.
  • FIG. 3B is a bar graph showing data from supernatants from infected cells analyzed 4 hours post- infection by ELISA for IL-lp. Experiments and data were performed and shown as described for FIG. 3A.
  • FIG. 3C is a bar graph showing data from supernatants from infected cells analyzed 4 hours post-infection by ELISA for IL-6. Experiments and data were performed and shown as described for FIG. 3A.
  • FIG. 4A provides data showing that NLRP3 inflammasome activation in response to Salmonella TCA cycle mutants requires the bacterial SPI-1 T3SS.
  • FIG. 4A is a bar graph showing that supernatants of Nlrc4 ⁇ / ⁇ BMDMs were analyzed 4 hours post-infection with indicated bacterial strains for LDH release. All experiments were performed three independent times in triplicate and representative data are shown. * p ⁇ 0.05, ** / ⁇ .01.
  • FIG. 4B is a bar graph showing that supematants of Nlrc4 ⁇ ' BMDMs were analyzed 4 hours post-infection with indicated bacterial strains for IL- ⁇ release. All experiments were performed three independent times in triplicate and representative data are shown. * p ⁇ 0.05, ** /K0.01.
  • FIG. 4C is a bar graph showing that supematants of N/rc "A BMDMs were analyzed 4 hours post-infection with indicated bacterial strains for IL-12 release. All experiments were performed three independent times in triplicate and representative data are shown. * /? ⁇ 0.05, ** /K0.01.
  • FIG. 4D is a bar graph showing data generated when N/rc "A BMDMs were infected with indicated parental bacterial strains or isogenic sseC mutants and assayed 4, 8 and 20 hours post-infection for cytotoxicity by LDH release. All experiments were performed three independent times in triplicate and representative data are shown.
  • FIG. 4E is a bar graph providing data generated by infecting Nlrc4 ⁇ / ⁇ BMDMs with parental or sopEBE2 mutant bacterial strains and assaying supematants 4 hours post- infection for cell death by LDH release by ELISA. All experiments were performed three independent times in triplicate and representative data are shown. * /? ⁇ 0.05, ** /? ⁇ 0.01.
  • FIG. 4F is a bar graph providing data generated by infecting Nlrc4 ⁇ / ⁇ BMDMs with parental or sopEBE2 mutant bacterial strains and assaying supematants 4 hours post-infection for IL- ⁇ release by ELISA. All experiments were performed three independent times in triplicate and representative data are shown. * /? ⁇ 0.05, ** /? ⁇ 0.01.
  • FIG. 4G is a bar graph providing data generated by infecting Nlrc4 ⁇ / ⁇ BMDMs with parental or sopEBE2 mutant bacterial strains and assaying supematants 4 hours post-infection for IL-12 release by ELISA. All experiments were performed three independent times in triplicate and representative data are shown. * / 0.05, ** / ⁇ .01.
  • FIG. 5A provides data that Salmonella TCA cycle mutants trigger NLRP3
  • FIG. 5 A shows a photograph and bar graph resulting from production of mitochondrial superoxide in Sim-infected Nlrc4 ⁇ / ⁇ BMDMs assayed four hours post-infection with aconitase, i.e., acnB strain.
  • aconitase i.e., acnB strain.
  • fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 ⁇ . * p ⁇ 0.05.
  • FIG. 5B shows a photograph and bar graph resulting from production of mitochondrial superoxide in Sim-infected Nlrc4 ' BMDMs assayed four hours post- infection with aconitase, i.e., icdA strain.
  • aconitase i.e., icdA strain.
  • fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 ⁇ . * /? ⁇ 0.05.
  • FIG. 5C is a bar graph showing B6 and MCAT BMDMs infected with indicated bacterial strains, for which cell death was assayed 4 hours post-infection.
  • fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 ⁇ . * /? ⁇ 0.05.
  • FIG. 5D is a bar graph showing IL- ⁇ in supernatants of B6 and MCAT BMDMs infected as described in FIG. 5C and measured by IA A.
  • fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 ⁇ . * /? ⁇ 0.05.
  • FIG. 5E is a bar graph showing IL-12 in supernatants of B6 and MCAT BMDMs infected as described in FIG. 5C and measured by I A.
  • fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 ⁇ . * /? ⁇ 0.05.
  • FIG. 5F is a bar graph showing data generated when Nlrc4 ⁇ / ⁇ BMDMs were pretreated with either vehicle control or ⁇ MitoQ 3 hours prior to infection. Cell death was assayed 4 hours post- infection by measuring release of LDH in cell supernatants. fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 ⁇ . * / 0.05.
  • FIG. 5G is a bar graph showing data when supernatants of samples treated with vehicle control or MitoQ as described in FIG. 5F were assayed for IL- ⁇ .
  • FIG. 5H is a bar graph showing data when supernatants of samples treated with vehicle control or MitoQ as described in FIG. 5F were assayed for IL-12.
  • fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 ⁇ . * p ⁇ 0.05.
  • FIG. 6A is a schematic with bar graphs for each component, showing that NLRP3 inflammasome activation by Salmonella aconitase and isocitrate dehydrogenase mutants correlates with excess bacterial citrate production and requires citrate synthase.
  • FIG. 6B is a bar graph showing data from the analysis of bacterial metabolites from bacteria grown under SPI-I inducing conditions using GC-MS analysis.
  • Nlrc4 ' BMDMs were infected with indicated bacterial mutants. Supernatants were assayed 4 hours post-infection for levels of LDH release.
  • fliCfljB is the parent strain background for all indicated bacterial mutants. Experiments were performed with four independently grown bacterial cultures for each strain. * p ⁇ 0.05.
  • FIG. 6C is is a bar graph showing data from the analysis of bacterial metabolites from bacteria grown under SPI-I inducing conditions using GC-MS analysis.
  • Nlrc4 ' BMDMs were infected with indicated bacterial mutants. Supernatants were assayed 4 hours post-infection for levels of IL- ⁇ .
  • fliCfljB is the parent strain background for all indicated bacterial mutants. Experiments were performed with four independently grown bacterial cultures for each strain. * /? ⁇ 0.05.
  • FIG. 6D is a bar graph showing data from the analysis of bacterial metabolites from bacteria grown under SPI-I inducing conditions using GC-MS analysis.
  • Nlrc4 ' BMDMs were infected with indicated bacterial mutants.
  • Supernatants were assayed 4 hours post-infection for levels of IL- 12.
  • fliCfljB is the parent strain background for all indicated bacterial mutants. Experiments were performed with four independently grown bacterial cultures for each strain. * /? ⁇ 0.05.
  • FIG. 7A is a graph showing deletion of bacterial TCA cycle aconitase leads to a decrease in bacterial virulence.
  • the data is generated from infecting C57BL/6 mice i.p. with 5x10 2 CFU. Spleens were harvested on days 3 and 5, and CFUs were enumerated by plating dilutions of tissue homogenates on selective plates. Dashed lines indicate the limit of detection. Mann-Whitney U test and unpaired two-tailed Student's t test were used to determine statistical significance for mouse CFU and cytokine data, respectively. Bars represent geometric means for CFU, and arithmetic mean for cytokine data. * /? ⁇ 0.05, ** p ⁇ 0.0l , * ** / 0.001. Data are representative of two independently performed experiments.
  • FIG. 7B is a graph showing the data from the experiment of FIG. 7A, but livers were harvested on days 3 and 5, and CFUs were enumerated by plating dilutions of tissue homogenates on selective plates. Data are representative of two independently performed experiments. Symbols are the same as described in FIG. 7A.
  • FIG. 7C is a graph showing data generated from infecting C57BL/6 and Caspl '1' Caspl l '1' mice with 2x10 7 CFU orally. Seven (7) days post-infection spleens were harvested for bacterial enumeration. Mice that succumbed to infection prior are designated with an 'X'. Data are representative of pooled data from two to three independent experiments, otherwise symbols and data are as described in FIG. 7A.
  • FIG. 7D is a graph showing data generated from the experiment of FIG. 7C, except that 7 days post-infection serum was harvested for IL-18 ELISAs. Symbols are described in FIGs. 7A and 7C. Data are representative of pooled data from two to three independent experiments.
  • FIG. 7E is a graph showing data generated from infecting C57BL/6 and Nlrp3 ' mice with 2x10 7 CFU orally. Seven (7) days post- infection spleens were harvested for bacterial enumeration. Symbols are described in FIGs. 7A and 7C. Data are representative of pooled data from two to three independent experiments.
  • FIG. 7F is a graph showing data generate from the experiment of FIG. 7E, except that 7 days post-infection serum was harvested serum for IL-18 ELISAs. Symbols are described in FIGs. 7A and 7C. Data are representative of pooled data from two to three independent experiments.
  • FIG. 8 A shows that deletion of bacterial TCA cycle aconitase leads to a defect in bacterial persistence.
  • 129S6/SvE mice were infected with lxl O 3 CFU of bacteria i.p., and mesenteric lymph nodes (MLNs were harvested on days 21 and 60, and CFUs enumerated by plating dilutions of tissue homogenates.
  • dashed lines indicate limit of detection.
  • Mann- Whitney U test and unpaired two-tailed Student's t test were used to determine statistical significance for mouse CFU and cytokine data, respectively. Bars represent geometric means for CFU, and arithmetic mean for cytokine data. * /? ⁇ 0.05, ** / O.01. Data are pooled from two independently performed experiments, except competitive index and survival curve studies, which indicate individual experiments.
  • FIG. 8B provides data from the experiment of FIG. 8A, except that spleens were harvested on days 21 and 60, and CFUs enumerated by plating dilutions of tissue
  • FIG. 8C provides data from the experiment of FIG. 8A, except that livers were harvested on days 21 and 60, and CFUs enumerated by plating dilutions of tissue
  • FIG. 8D provides data from an experiment in which 129S6/SvE mice were co-infected with lxl O 3 CFU of each indicated bacterial strain i.p., and MLNs, spleens (left) and livers (right) were harvested on day 21 and competitive index (CI) was determined by plating tissue homogenates on different selective plates.
  • FIG. 8E provides data from an experiment in whichl29S6/SvE mice were infected i.p. with lxl O 4 CFU of fliClfljB or isogenic acnB mutant bacterial strains and survival of the mice was monitored over 21 days.
  • FIG. 8F provides data from an experiment in whichl29S6/SvE mice were infected with lxlO 4 CFU fliCfljB or isogenic acnB mutant bacteria, and 7 days post-infection CFU per gram of spleens from mice was determined by plating tissue homogenates.
  • FIG. 8G provides data from the experiment of FIG. 8F except that 7 days post- infection CFU per gram of livers from mice was determined by plating tissue homogenates.
  • FIG. 8H provides data from the experiment of FIG. 8F except that serum levels of IL- 18 from mice were assayed at day 7 post-infection.
  • FIG. 81 provides data from the experiment of FIG. 8F except that serum levels of IL-6 from mice were assayed at day 7 post-infection.
  • the inventors determined that Salmonella lacking certain TCA enzymes trigger NLRP3 inflammasome activation in infected macrophages, leading to elevated inflammatory responses and reduced virulence.
  • Stm genes that modulate NLRP3 inflammasome activation during infection were identified.
  • Four of the genes identified in the examples below ⁇ acnB, bcfB, rcsD, and melB) were previously found in a genome-wide screen for Salmonella persistence genes 56 , consistent with the possibility that modulating inflammasome activation might promote long-term systemic infection.
  • bcfB encodes a fimbrial chaperone
  • rcsD encodes a member of a two-component system previously found to contribute to persistent infection and regulate resistance to antimicrobial peptide responses 23 ' 26
  • melB encodes a symporter of melibiose and monovalent cations.
  • acnB encodes the enzyme aconitase, which mediates conversion of citrate to isocitrate as a key step in the TCA cycle.
  • several other TCA cycle enzymes contribute to persistent infection by Salmonella, as well as a number of other bacterial pathogens ' ' ' .
  • Stm mutants deficient in aconitase or isocitrate dehydrogenase, but not other TCA cycle enzymes induce rapid canonical NLRP3 inflammasome activation in BMDMs.
  • the inventors have identified a link between mitochondrial Reactive Oxygen Species (ROS) and NLRP3 inflammasome activation in response to alteration of Salmonella citrate and isocitrate metabolism.
  • ROS mitochondrial Reactive Oxygen Species
  • NLRP3 inflammasome activation in response to alteration of Salmonella citrate and isocitrate metabolism.
  • Stm can limit adaptive immune responses through a variety of mechanisms including the induction of host nitric oxide as well as production of bacterial L-asparaginase 51 ' 90 .
  • inflammasome activation leads to release of IL- ⁇ and IL-18, which contribute to T H 1 and T H 17
  • Salmonella as used herein refers to any strain of Salmonella, including any strain of Salmonella enterica, including Salmonella enterica serovar Typhimurium.
  • Salmonella enterica serovar Typhimurium The serovars of S. enterica that may be used as the attenuated bacterium of the live compositions described in accordance with various embodiments herein include, without
  • Salmonella enterica serovar Typhimurium (“ ⁇ .typhimurium "), Salmonella montevideo, Salmonella enterica serovar Typhi (“S. typhi”), Salmonella enterica serovar, Paratyphi B (“S. paratyphi 13"), Salmonella enterica serovar Paratyphi C (“S. paratyphi C”), Salmonella enterica serovar Hadar (“S. hadar”), Salmonella enterica serovar Enteriditis (“S. enter iditis "), Salmonella enterica serovar Kentucky (“S. Kentucky”), Salmonella enterica serovar In/antis (“S. infantis”), Salmonella enterica serovar Pullorurn (“S.
  • Salmonella enterica serovar Gallinarum (“S. gallinarum”
  • Salmonella enterica serovar Muenchen (“S. muenchen”
  • Salmonella enterica serovar Anaturn (“S. anatum”
  • Salmonella enterica serovar Dublin (“S. dublin”)
  • Salmonella enterica serovar Derby (“S. derby”)
  • Salmonella enterica serovar minnesota (S. minnesota), among other known strains.
  • Stm refers to Salmonella enterica serovar Typhimurium.
  • fliCfljB refers to Salmonella flagellins.
  • AcnB refers to aconitase or aconitate hydratase 2 (citrate hydro-lyase 2), which is a TCA enzyme found in Salmonella.
  • the nucleotide sequence of this enzyme is found at the chromosome locus of wildtype Salmonella strain SL1344 at tag 0159.
  • the nucleic acid sequence of the gene acn and the amino acid sequence of the acnB enzyme in Salmonella enterica Subsp. enterica serovar Typhimurium is found in the KEGG database at the Entry No. SL1344 0159 SEQ ID NOs: 1 and 2 respectively.
  • Other publically available database entries for this gene and enzyme are found for the same or other Salmonella strains in the NCBI database, e.g., under GI378698137 or
  • IcdA refers to isocitrate dehydrogenase, which is a TCA enzyme found in Salmonella.
  • the nucleotide sequence of the gene icdA and the amino acid sequence of the icdA enzyme in Salmonella enterica Subsp. enterica serovar Typhimurium is found in the KEGG database at the Entry No. SL1344 1176 SEQ ID NOs: 3 and 4, respectively.
  • NL P3 refers to Nucleotide binding domain Leucine Rich Repeat Family Pyrin
  • NLRC4 refers to Nucleotide binding domain Leucine Rich Repeat Family Card Domain Containing 4;
  • NRAMPl refers to Natural Resistance Associated Macrophage Protein 1 ;
  • AceA refers to isocitrate lyase
  • Caspl refers to caspase-1 ;
  • Caspl 1 refers to caspase-11 ;
  • SPI-1 refers to Salmonella Pathogenicity Island 1 ;
  • SPI-2 refers to Salmonella Pathogenicity Island 2;
  • BMDM refers to bone marrow derived macrophages
  • MLN refers to mesenteric lymph nodes.
  • nucleic acid sequence when used in singular or plural form, generally refers to any nucleic acid sequence, polyribonucleotide or
  • polydeoxribonucleotide which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or include single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • polynucleotide specifically includes cDNAs.
  • the term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases.
  • polynucleotide embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.
  • oligonucleotide refers to a relatively short polynucleotide of less than 20 bases, including, without limitation, single-stranded deoxyribonucleotides, single- or double- stranded ribonucleotides, R A:DNA hybrids and double-stranded DNAs. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
  • Attenuated or attenuation refers to elimination or reduction of the natural virulence of a bacterium in a particular host organism, such as an avian or a mammal.
  • An "attenuated” bacterium or strain of bacteria is attenuated in virulence toward at least one species of subject or host organism that is susceptible to infection and disease by a virulent form of the bacterium or strain of the bacterium.
  • Recombinant as applied to a polynucleotide, means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
  • a recombinant bacterium is a bacterial cell comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original bacterial construct.
  • Typical recombinant or genetic engineering steps to generate a recombinant bacterium as referred to herein include bacteriophage transduction or homologous recombination, among other known techniques described in the art.
  • heterologous means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
  • “Naturally occurring” means a sequence found in nature and not synthetically prepared or modified.
  • modification refers to any deliberately inserted change in a nucleic acid or protein sequence, such as a deletion of all or part of the TCA enzyme-encoding sequences, or an insertion of a sequence into the TCA enzyme-encoding sequences that disrupts the translation of the nucleic acid sequence into the enzyme. Such disruption can be caused by insertion of a heterologous sequence into the TCA enzyme-encoding sequences.
  • the phrase "deficient in” means that the bacterium contains a modification designed to reduce, inhibit or ablate expression of the encoded TCA enzyme.
  • inflammasome activation refers to a specific immune response that is associated with inflammatory cell death and release of key effector cytokines that mediate adaptive immunity.
  • Animal or “subject” as used herein means a mammalian animal, including a human male or female, a veterinary or farm animal, e.g., horses, livestock, cattle, pigs, etc., a domestic animal or pet, e.g., dogs, cats; and animals normally used for clinical research, such as primates, rabbits, and rodents.
  • the subject of these methods and compositions is a human.
  • Animal as used herein is also meant to include other non- mammals or animal species that are commonly infected by Salmonella, such as avians or fowl that are used as food products, can be carriers for Salmonella, and are often the transmitters of the bacterium to humans.
  • a vector is meant an entity that delivers a heterologous molecule to cells, either for therapeutic or vaccine purposes.
  • a vector may include any genetic element including, without limitation, naked DNA, a phage, transposon, cosmid, episome, plasmid, or a virus.
  • Vectors are generated using the techniques and sequences provided herein, described in the examples, and in conjunction with techniques known to those of skill in the art. Such techniques include conventional cloning techniques of cDNA such as those described in texts such as Green and Sambrook, Molecular Cloning: A Laboratory Manual. 4 th Edit, Cold
  • amino acid sequence of interest e.g. , a target microbial gene and/or a suitable cancer or tumor gene
  • operably linked include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • “Expression control sequences” include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient R A processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic m NA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • efficient R A processing signals such as splicing and polyadenylation (poly A) signals
  • sequences that stabilize cytoplasmic m NA sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • a great number of expression control sequences including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized in the construction of the compositions and performance of the methods described herein.
  • host cell as used herein may refer to the cell or cell line in which the recombinant vector is produced.
  • an immunogenic composition is a composition to which a humoral (e.g., antibody) or cellular (e.g., a cytotoxic T cell) response, or, in one embodiment, an innate immune response, is mounted to a target gene product delivered by the immunogenic composition following delivery to a mammal or animal subject.
  • a humoral e.g., antibody
  • cellular e.g., a cytotoxic T cell
  • an innate immune response e.g., an innate immune response
  • therapeutic reagent or “regimen” is meant any type of treatment employed in the treatment or prevention of microbial infections or cancers with or without solid tumors, including, without limitation, chemotherapeutic pharmaceuticals, biological response modifiers, radiation, diet, vitamin therapy, hormone therapies, gene therapy, surgical resection, etc.
  • sequence identity refers to the residues in the two sequences that are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over the full- length of an open reading frame of a gene, protein, subunit, or enzyme, or a fragment of at least about 100 to 500 nucleotides, is desired. However, identity among smaller fragments, e.g. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.
  • percent sequence identity may be readily determined for amino acid sequences, over the full-length of a protein, or a fragment thereof.
  • a fragment is at least about 8 amino acids in length, 20 amino acids in length, 50 amino acids in length, 100 amino acids in length, and may be up to about 700 or more amino acids.
  • percent identity among the TCA enzymes, acn or icd, in other Salmonella strains and serovars can be about 75%, 80%, 85%, 90%, 95% or over 99%. In other embodiments, the percent identities of the selected genes/enzymes can be lower than 75%.
  • Identity is readily determined using such algorithms and computer programs as are defined herein at default settings. Preferably, such identity is over the full length of the protein, enzyme, subunit, or over a fragment of at least about 8 amino acids in length. However, identity may be based upon shorter regions, where suited to the use to which the identical gene product is being put. Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs, such as "Clustal W", accessible through Web Servers on the internet. Alternatively, Vector NTI® utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above.
  • polynucleotide sequences can be compared using Fasta, a program in GCG Version 6.1.
  • Fasta provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.
  • percent sequence identity between nucleic acid sequences can be determined using Fasta with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
  • programs are available for performing amino acid alignments. Generally, these programs are used at default settings, although one of skill in the art can alter these settings as needed.
  • one of skill in the art can utilize another algorithm or computer program that provides at least the level of identity or alignment as that provided by the referenced algorithms and programs.
  • sequences can also be defined as the ability to hybridized to the complement of a selected sequence, under stringent conditions. See, e.g., commonly used texts for the definitions of stringency, e.g., Green and Sambrook, cited above; or see, e.g., US Patent No. 8,974,798.
  • the term "cancer” refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • the cancer is an epithelial cancer.
  • the cancer can include, without limitation, breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, and multidrug resistant cancer, or subtypes and stages thereof.
  • the cancer is an "early stage” cancer.
  • the cancer is a "late stage” cancer.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • a Salmonella bacterium e.g., a Salmonella enterica Typhimurium bacterium ⁇ Stm
  • a Salmonella enterica Typhimurium bacterium ⁇ Stm that is genetically modified to be deficient or functionally deficient in a naturally occurring gene encoding a selected bacterial TCA enzyme.
  • Stm is used for convenience. It should be understood that other embodiments of the compositions and methods described herein using Stm may employ any suitable Salmonella strain or serovar in place of Stm. In one
  • the selected TCA enzyme gene is the gene encoding aconitase.
  • the selected gene is the gene encoding the TCA enzyme isocitrate dehydrogenase.
  • the absence or non-function of one or both of these two enzymes, but not all Salmonella TCA enzymes induce the bacteria to have elevated levels of cell death.
  • deficiencies in these enzymes induce the production of certain cytokines from macrophages and dendritic cells in the subject infected by the modified Stm.
  • IL-18 is generated, which is an indicator of the stimulation of the innate immune response.
  • the sequences of these genes and their locations in the Stm chromosome are known and accessible to those of skill in the art.
  • the bacterium selected for such modification may be a wildtype Salmonella enterica Typhimurium, such as strain 1344 and any other strain.
  • a wildtype Salmonella enterica Typhimurium which is attenuated by artificial methods prior to deletion of the selected TCA enzyme may be employed.
  • the attenuation of the Salmonella strain is suitable for the animal for which it is intended, e.g., an avian species, a livestock animal, e.g., pig, cow, etc, or a human.
  • the bacterium selected for such modification is an already attenuated strain of Salmonella enterica Typhimurium. As demonstrated in Example 10, one such attenuated strain is the aroA mutant Stm bacteria 116 . Many other wildtype and attenuated Stm bacteria are publically known and available from university or commercial collections of bacterial strains. It is anticipated that any attenuated Stm will be useful in generating the Stm modified to be deficient in aconitase or isocitrate dehydrogenase. The selection of the particular Stm strain is not anticipated to be a limitation of this invention.
  • a mutation may be inserted into the gene that renders it non-functional.
  • the gene may be deleted in whole or part and replaced with another sequence, such as a sequence encoding an antibiotic resistance gene.
  • the selection of the antibiotic resistance gene is conventional; it may be selected from among a wide number of such genes, including, without limitation, kanamycin, chloramphenicol, as well as others listed in public databases, such as the on-line ARDB-Antibiotic Resistance Genes Database.
  • the antibiotic resistance gene is present in a cassette or plasmid vector under the operable control of a suitable promoter.
  • the promoter is constitutive, such as that used in the examples.
  • other promoters may be selected from among a wide number in the art.
  • the promoter can be a prokaryotic promoter, for example, a Salmonella promoter, which directs expression of the target antigen in the Salmonella.
  • promoters are well known including the htrA promoter, the nirB promoter, the ssaH promoter, the ompR promoter, and any other Salmonella or other bacterial promoter that is upregulated when Salmonella is taken up by mammalian cells.
  • the promoter can be a eukaryotic promoter, such as the cytomegalovirus promoter.
  • cytomegalovirus promoter Numerous promoters are known in the art for such use and have been suggested for other Salmonella vector compositions; see e.g., US 6585975.
  • such a deletion/insertion may be engineered using a conventional technique, such as homologous recombination, transposon insertion or bacteriophage transduction. All of these techniques are known in the art; see for example the references cited herein, in the examples and at the end of this specification, all of which are incorporated by reference.
  • Such genetically modified Stm are found, as demonstrated herein to induce in an animal, upon infection, the release of inflammatory cytokines from macrophages or dendritic cells and an increase in the animal's innate immune response to the bacterium.
  • the Stm bacterium which is deficient in acnB or icdA, and optionally attenuated may be additionally genetically modified to further comprise a "target gene" inserted in the bacterial chromosome. See, for example the description of Example 11.
  • the target gene is a Salmonella gene, e.g., another gene from Salmonella enterica serovar Typhimurium (Stm), inserted in the chromosome at a non- naturally occurring site to enable the Salmonella to deliver larger amounts of the antigen encoded by the target gene than it would deliver in an unmodified bacterium.
  • Salmonella gene may be operatively associated with a stronger or more powerful promoter than exists in the naturally occurring, non-modified Stm or the duplicate occurrence of the Stm target gene in the chromosome allows for at least double the production of the encoded Stm antigen.
  • the target gene is a heterologous gene, e.g., a gene from another infectious microorganism.
  • the heterologous gene may be selected from bacteria or viruses or other infectious microorganisms that would be susceptible to the increase in innate immunity caused by the modified Stm described herein when administered to an animal or other subject. Therefore, in certain embodiments, the heterologous gene is one that encodes an antigen from another bacteria, fungi, parasitic microorganisms or multicellular parasites which infect human and non-human vertebrates.
  • the heterologous target gene is inlA,fdeC, cagA, IcrV, or B. anthracis Protective Antigen, among others.
  • the target gene selected may be the bacterial internalin gene, inlA of Listeria monocytogenes.
  • the target gene may encode a bacterial adhesin, fdeC of Enteropathogenic E. coli.
  • the target gene may encode the gene encoding the bacterial virulence factor cagA from Helicobacter pylori.
  • Still other targets may be selected from bacterial pathogens include pathogenic gram-positive cocci include pneumococci; staphylococci; and streptococci.
  • Pathogenic gram-negative cocci include meningococcus; gonococcus.
  • Pathogenic enteric gram-negative bacilli include enterobacteriaceae; pseudomonas, acinetobacteria and eikenella; melioidosis; salmonella; shigella; haemophilus; moraxella; H. ducreyi (which causes chancroid); brucella; Franisella tularensis (which causes tularemia); yersinia (pasteurella); streptobacillus moniliformis and spirillum; Gram-positive bacilli include listeria
  • Pathogenic anaerobic bacteria Diseases caused by pathogenic anaerobic bacteria include tetanus; botulism; other Clostridia; tuberculosis; leprosy; and other mycobacteria.
  • Pathogenic spirochetal diseases include syphilis; treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.
  • Other infections caused by higher pathogen bacteria and pathogenic fungi include actinomycosis; nocardiosis; cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis;
  • Rickettsial infections include Typhus fever, Rocky Mountain spotted fever, Q fever, and Rickettsialpox.
  • mycoplasma and chlamydial infections include: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal chlamydial infections.
  • Pathogenic eukaryotes encompass pathogenic protozoans and helminths and infections produced thereby include: amebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans; Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis;
  • Target genes are available also from the organisms and/or toxins produced thereby which have been identified by the Centers for Disease Control [(CDC), Department of Health and Human Services, USA], as agents which have potential for use in biological attacks.
  • these biological agents include, Bacillus anthracis (anthrax), Clostridium botulinum and its toxin (botulism), Yersinia pestis (plague), variola major (smallpox), Francisella tularensis (tularemia), and viral hemorrhagic fever, all of which are currently classified as Category A agents; Coxiella burnetii (Q fever); Brucella species (brucellosis), Burkholderia mallei (glanders), Ricinus communis and its toxin (ricin toxin), Clostridium perfringens and its toxin (epsilon toxin), Staphylococcus species and their toxins (enterotoxin B), all of which are currently classified as Category B agents
  • the heterologous gene is a gene from a mammalian tumor or cancer cell.
  • the heterologous gene may be selected from a tumor or cancer cell that would be susceptible to attack from the increase in innate immunity caused by the modified Stm described herein when administered to a subject with that cancer or tumor. Therefore, in certain embodiments, the heterologous gene is a gene encoding a cancer antigen, such as a full-length, wild-type cancer-specific antigens or mutated cancer-specific antigens or cancer-associated antigens.
  • Cancer-specific antigens are those epitopes and proteins found on a selected specific cancer or tumor cell, and not on all cancer cells.
  • Cancer-associated antigens are antigens that may be associated with more than one cancer or tumor cell type.
  • Exemplary cancer-specific antigens can include, without limitation, 707-AP, alpha (a)- fetoprotein, ART -4, BAGE; b-catenin/m, b-catenin/mutated Bcr-abl, CAMEL, CAP-1, mCASP-8, CDC27m, CDK4/m, CEA, CT, Cyp-B, MAGE-B2, MAGE-B1, ELF2M, ETV6- AML1, G250, GAGE, GnT-V, GplOO, HAGE, HER-2/neu , HPV-E7, HSP70-2M HST-2, hTERT, iCE , KIAA0205, LAGE, LDLR/FUT, MAGE , MART-1, MC1R, MUC1, MUM-1, - 2, -3, PI 5, pi 90 minor bcr-abl.
  • tumor or cancer genes encode VEGFR1, VEGFR2, MAGE -A 1, MUC-1, Thymosin ⁇ , EGFR, Her-2/neu, MAGE-3, Survivin, Heparanase 1, Heparanase 2, and CEA, among others.
  • Still other suitable antigens are those listed 117 , and incorporated by reference herein. See, also, texts identifying suitable antigens, such as Scott and Renner, in Encyclopedia of life Sciences 2001 Eds., John Wiley & Sons, Ltd.
  • heterologous antigens in the Stm bacterium such as the aroA acnB strain discussed in Example 11 is achieved by using a vector, which contains a suitable promoter, such as the constitutive Salmonella enterica rpsM promoter in operative association with DNA encoding the heterologous antigen.
  • a suitable promoter such as the constitutive Salmonella enterica rpsM promoter
  • Any of the promoters identified above or available and known to be useful in Salmonella may also abe selected to express the heterologous gene.
  • the resulting vector or cassette can be inserted onto the Salmonella chromosome at any site within the Stm genome or onto a specific site, for example one targeted by a transposon. As exemplified below, such a cassette can be inserted onto the att7 «7 site, which can be specifically targeted by transposition of the Tn7 transposon.
  • Insertion at other sites in the Stm chromosome can occur by use of other known transposons and methods available and known to those of skill in the art.
  • compositions or pharmaceutical compositions which contain the modified Stm bacteria described above.
  • the compositions comprising the modified Stm bacterium described above may be further associated with a pharmaceutically acceptable carrier for in vivo delivery.
  • the term "pharmaceutically acceptable carrier” or “diluent” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans or other mammals, or animals, such as avian species.
  • the diluent is saline or buffered saline.
  • Such pharmaceutically acceptable carriers suitable for use in such a composition are well known to those of skill in the art.
  • Such carriers include, without limitation, and depending upon pH adjustments, buffered water, buffered saline, such as 0.8% saline, phosphate buffer, 0.3% glycine, hyaluronic acid, alcoholic/aqueous solutions, emulsions or suspensions.
  • buffered water buffered saline, such as 0.8% saline, phosphate buffer, 0.3% glycine, hyaluronic acid, alcoholic/aqueous solutions, emulsions or suspensions.
  • Other conventionally employed diluents, adjuvants and excipients may be added in accordance with conventional techniques.
  • the pharmaceutical compositions can also contain a mild adjuvant, such as an aluminum salt, e.g., aluminum hydroxide or aluminum phosphate, aqueous suspensions of aluminum and magnesium hydroxides, liposomes, and oil in water emulsions.
  • aluminum salt e.g., aluminum hydroxide or aluminum phosphate
  • Carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusting agents may also be employed. Buffers include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid, e.g., citrates, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, trimethanmine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like may also be provided in the pharmaceutical carriers. These compositions are not limited by the selection of the carrier. The preparation of these pharmaceutically acceptable compositions, from the above-described components, having appropriate pH isotonicity, stability and other conventional characteristics is within the skill of the art.
  • the composition containing the attenuated and modified Salmonella described herein may be a composition suitable to be added to a foodstuff.
  • compositions described herein and particularly exemplified in the examples, below, are further useful in a variety of therapeutic or vaccinal methods.
  • Salmonella strain e.g., Salmonella enterica Typhimurium
  • Salmonella enterica Typhimurium involves administering a modified Stm or modified, attenuated Stm composition to an animal in need thereof.
  • the animal receiving this compositions is avian, e.g., particularly avians that are used for food, e.g., fowl, chicken, ducks, etc.
  • the subject receiving the Salmonella is a mammal susceptible to Salmonella infection, e.g., a human or livestock animal.
  • compositions described herein can be used to treat or prevent Salmonella infection in a subject, e.g., avians, livestock or humans.
  • a subject e.g., avians, livestock or humans.
  • the embodiment without the heterologous gene may be most appropriate, e.g., a suitably attenuated, acnB or z ' c ⁇ 5L4-deficient Stm.
  • compositions described herein can be employed in a method of inducing the innate immune response of an animal against a microbial infection or cancer comprising by administering a composition containing the modified TCA enzyme-deficient Stm carrying a heterologous gene to an animal in need thereof.
  • These uses would include the treatment or prevention of the infectious diseases or treatment of cancers/tumor from which the target heterologous gene originated, i.e., a pathogen or cancer identified above, or selected by the attending physician or medical personal.
  • the mode of administration and dosage may be selected by one of skill in the art, e.g., a veterinarian or physician.
  • the mode of administration be any suitable route: oral, subcutaneous injection, intravenous injection, intramuscular injection, mucosal, intra-arterial, intraperitoneal, parenteral, intradermal, transdermal, nasal, vaginal, or rectal or inhalation routes, among others.
  • oral refers to administration of a compound or composition to a subject by a route or mode along the alimentary canal, such as by swallowing liquid or solid forms of a composition from the mouth, administration of a composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a composition, and rectal administration, e.g., using suppositories that release a live bacterial vaccine strain described herein to the lower intestinal tract of the alimentary canal.
  • the doses may be administered as a single dose, multiple doses over a selected time gap, via prime/boosting protocols, etc.
  • administration can be by gavage in a carrier.
  • the bacteria can be added to gel beads and mixed with feed.
  • the bacteria can be sprayed onto the feathers or skin of the animals and inhaled. It is likely also that the bacteria can be lyophilized or otherwise treated and mixed with feed.
  • exemplary dosages may be 5 x 10 6 -5 x 10 10 colony forming units, e.g., 5 x 10 s colony forming units, or 5-1000 ⁇ g, e.g., 100 ⁇ g, antigen, or 10 5 -10 6 bacteria in 100 microliters for, e.g., chicken.
  • the selection of the doses and modes of administration and dosage regimens may be selected by one of skill in the art based on the subject to be vaccinated, the physical attributes of the subject, the virulence of the Salmonella strain, the route of administration, and other common factors.
  • a method of generating a Salmonella vaccine comprises deleting a naturally occurring gene encoding a TCA enzyme selected from aconitase or isocitrate dehydrogenase from the chromosome of a wildtype or attenuated Salmonella bacterium.
  • a TCA enzyme selected from aconitase or isocitrate dehydrogenase from the chromosome of a wildtype or attenuated Salmonella bacterium.
  • a method of generating a composition that induces the innate immune response against a target heterologous gene comprises deleting a naturally occurring gene encoding a TCA enzyme selected from aconitase or isocitrate dehydrogenase from the chromosome of a wildtype or attenuated Salmonella enterica Typhimurium bacterium and inserting into the bacterial genome a heterologous target gene from another infectious microorganism or from a cancer or tumor cell under the operative control of a suitable promoter or other known vector components.
  • TCA enzyme selected from aconitase or isocitrate dehydrogenase from the chromosome of a wildtype or attenuated Salmonella enterica Typhimurium bacterium
  • aroA acnB Salmonella strain to express heterologous genes from Listeria monocytogenes, Enteropathogenic E. coli, and Helicobacter pylori. See, e.g., the generation and use of the aroA acnB Salmonella strain to express heterologous murine Vascular Endothelial Growth Factor Receptor 2, VEGFR2, from the constitutive rpsM promoter as an anti -tumor vaccine construct.
  • the invention is now described with reference to the following examples.
  • the examples below illustrate how the inventors screened a Salmonella Typhimurium transposon library to identify bacterial factors that limit NLRP3 inflammasome activation.
  • absence of the Salmonella TCA enzyme aconitase induced rapid NLRP3 inflammasome activation.
  • This inflammasome activation correlated with elevated levels of bacterial citrate, and required mitochondrial ROS and bacterial citrate synthase.
  • Salmonella lacking aconitase displayed NLRP3- and caspase-1/11-dependent attenuation of virulence, and induced elevated serum IL-18 in wild-type mice.
  • Targeted deletion strains used in this study were made on the SL1344 strain background of S. enterica Typhimurium in which both subunits of flagellin, fliC and fljB, had been deleted through clean deletion of both genes (fliCfljB) or clean deletion of fliC and a kanamycin cassette insertion into fljB (fliCfljB ::kan) using standard methods 22 . When necessary, clean deletions were generated using the FRT recombinase 22 .
  • fliCfljBsseCacnB :kan,fliCfljBsseCicdA::kan,fliCfljBsseCaceA::kan,fliCfljBsseCfumA::kan.
  • TCA cycle mutations were moved into a fliCfljBsopEBE2 : :cat background.
  • sopEBElv.cat mutant strain was generated by sequential lamda re -mediated deletion of sopB and sopE2, followed by generation of an unmarked deletion of sopE by use of P22 lysate from SL1344 AsopE: :pSW245 61 , kindly provided by Sebastian Winter and Andreas Baumler (University of California Davis, Davis, CA). Bacteria were routinely grown at 37°C. For infection of cultured cells, bacteria were grown shaking overnight at 37°C in LB medium. Bacteria were diluted in LB containing 300 mM NaCl. Bacteria were grown standing for 3 hours to induce SPI-1 expression 58 . For mouse infections, bacteria were grown overnight with aeration at 37°C and diluted in PBS.
  • a nested random priming method 81 was used to determine the location of transposon insertion with the first round of PCR using a TetA primer (ACCTTTGGTCACCAACGCTTTTCC; SEQ ID NO: 5) together with a random primer (GTTTCCCAGTCACGATCNNNNNNN; SEQ ID NO: 6) at low stringency, followed by a second higher stringency PCR using a universal primer
  • C57BL/6 (B6) mice were from NCI, 129S6/SvE mice were from Taconic. Knockout mice used in these studies were on the B6 background and have been previously described: Caspl'-Caspll 1 - 50 , Asc ⁇ 91 , Nlrc ⁇ ' 53 , Asc ⁇ Nlrc ⁇ 13 , Nlrp ' 91 , Caspl ⁇ 12 and Caspll ⁇ 105 provided by Tiffany Horng and Junying Yuan (Harvard University, Boston, MA). Animals were maintained in accordance with the guidelines of the University of Pennsylvania
  • Bone marrow cells were grown at 37°C in a humidified incubator in DMEM containing HEPES, 10% FCS, and 30% L929 supernatant for 7-8 days. Differentiated BMDMs were replated into 24-, 48- or 96-well dishes 16-20 hours prior to infection. MCAT bone marrow was provided by A. Philip West and Gerald Shadel (Yale University, New Haven, CT). Bacterial strains described above were harvested, washed three times with DMEM, resuspended and added to the cells at an MOI of 20: 1. Bacteria were spun onto the cells at 1000 RPM for 5 minutes, and infected cells placed in a humidified tissue culture incubator at 37°C for 1 hour.
  • Gentamicin was added to the cells 1 hour post-infection to a final concentration of 100 ⁇ g/mL, and the cells placed in the incubator until harvesting.
  • 50 ng/mL LPS used in indicated experiments was E. coli 055 :B5 (Sigma).
  • 2.5 mM ATP (Sigma) used in indicated experiments remained in well for 4 hours post-addition.
  • BMDMs were pretreated with 50 ng/mL LPS 4 hours prior to infection for indicated conditions. 10 ⁇ MitoQ (kindly provided by Narayan Avadhani and Satish Srinavasan, University of Pennsylvania, Philadelphia, PA) or vehicle control (DMSO) was added to cells one hour post-LPS treatment and remained for the duration of the experiment.
  • BMDMs were seeded into 96-well plates at a density of 7x10 4 cells per well. The following day, culture medium was replaced with fresh DMEM. Cells were infected as described above and supernatants harvested at 4 hours post-infection. Lactate dehydrogenase release was quantified using the Cytotox96 Assay Kit (Clonetech) according to the manufacturer's instructions.
  • BMDMs were seeded into 48-well plates at a density of 1.5xl0 5 cells per well.
  • Cells were pretreated with E. coli LPS (Sigma) for 3 hours prior to bacterial infection as described above, and supernatants were harvested 4 hours post-infection.
  • Release of proinflammatory cytokines was measured by enzyme-linked immunosorbent assay (ELISA) using capture and detection antibodies against IL- ⁇ (eBioscience), IL-6 (BD Biosciences), and IL- 12 (BD Biosciences).
  • BMDMs were seeded into 24-well plates at a density of 3xl0 5 cells per well, and infected with bacteria as described above. 3 hours post-infection, cells were lysed in 20 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X- 100, and 1 mM EDTA. Lysates were mixed with protein loading buffer, boiled, centrifuged, and 20% of the total cell lysate loaded onto 4%-12% NuPAGE gels (Invitrogen). Proteins were transferred to PVDF membrane (Millipore) and blotted with rabbit anti-mouse caspase-1 antibody (Santa Cruz).
  • Mitochondrial superoxide (0 2 " ) was measured by using the mitochondrial O2 " indicator, MitoSOX Red (molecular probes; Invitrogen). Briefly, Nlrc4 ' BMDMs were grown as described above and seeded into 35mm plastic petri dishes with glass coverslip bottoms. Infections were performed as described above. 1 ⁇ g LPS was added to indicated plate 6 hours prior to imaging. BMDMs were loaded with 5 ⁇ MitoSOX Red for 30 minutes. Residual dye was removed by washing and dishes were mounted in an open perfusion microincubator and imaged by confocal microscopy.
  • Bacterial cultures were shaking overnight at 37°C in LB medium. Bacteria were diluted in LB containing 300 niM NaCl. Bacteria were grown standing to OD 0.5. Bacteria were immediately transferred to cold conical tubes and spun down at 4,000 rpm for 5 minutes at 4C. Bacterial pellets were washed with 0.8% saline solution, then pelleted. Metabolites were extracted with ice-cold 80% methanol and centrifuged for 10 minutes at 4C. D 2 7-myristic acid was used as an internal reference standard. TCA metabolites were reduced with sodium borodeuteride and deuterated standards added as previously described 62 .
  • mice were intraperitoneally infected with 5x10 2 bacteria for C57BL/6 mice, and lxl O 3 or lxlO 4 bacteria for 129S6/SvE mice.
  • IACUC Institutional Animal Care and Use Committee
  • mice were intraperitoneally infected with 5x10 2 bacteria for C57BL/6 mice, and lxl O 3 or lxlO 4 bacteria for 129S6/SvE mice.
  • C57BL/6 mice, or isogenic Nlrp3 ' or Caspl ⁇ ' Caspll '1' mice were fasted overnight and intragastrically inoculated with 2x10 7 bacteria.
  • BMDMs bone marrow-derived macrophages
  • isocitrate lyase encoded by aceA
  • aceA which generates glyoxylate from isocitrate in the glyoxylate cycle
  • the NLRP3 inflammasome can respond to the pore-forming activities of diverse bacterial secretion systems 8 ' 36 ' 69 .
  • Salmonella expresses two such systems, SPI-1 and SPI-2, yet paradoxically, in the absence of flagellin and NL C4, NLRP3 inflammasome activation does not occur in bone marrow-derived macrophages (BMDMs) until 12-16 hours post-infection 9 ' 72 .
  • BMDMs bone marrow-derived macrophages
  • Candidate mutants were identified by increased LDH release relative to the fliCfljB-dsfici t parental strain 4 hours post-infection of immortalized Nlrc4 ' macrophages (Figs. 1A).
  • Candidate mutants were rescreened in triplicate in primary Nlrc4 ⁇ ' ⁇ BMDMs and a secondary screen was performed in Caspl ' ' ' Caspll ' ' ' BMDMs to confirm that LDH release was indeed inflammasome-dependent (a representative group of five of initial candidate mutants is shown) (Fig. IB).
  • Candidate mutants were further tested for NLRC4-independent release of the caspase- 1 -dependent cytokine IL- ⁇ ⁇ (Fig. 1C), and normal secretion of caspase-1 -independent cytokines such as IL-6 or IL-12 (Fig. ID and data not shown).
  • sequencing the transposon junction of a subset of candidate mutants identified four genes ⁇ acnB, bcfB, rcsD, and melB) that had previously been isolated in a genome-wide screen for genes involved in Salmonella persistence 56 , consistent with the possibility that modulating inflammasome activation might promote persistent infection.
  • EXAMPLE 4 Deletion of Salmonella aconitase, isocitrate lysase, or isocitrate
  • dehydrogenase induces rapid NLRC4-independent NLRP3 inflammasome activation
  • Two genes initially identified by Lawley et ol. in a genome-wide screen for genes required for long term persistence, acnB and icdA 56 encode the TCA cycle enzymes aconitase and isocitrate dehydrogenase, respectively.
  • Aconitase converts citrate to isocitrate, while isocitrate dehydrogenase subsequently converts isocitrate to alpha-ketoglutarate. This suggested that the TCA cycle of intracellular Salmonella might be involved in modulating inflammasome activation of infected cells.
  • isocitrate lyase a component of the glyoxylate cycle pathway that converts isocitrate to glyoxylate, also contributes to persistent infection of multiple bacterial pathogens, including Mycobacterium tuberculosis, Salmonella, Pseudomonas, and Burkholderia species 27 ' 60 ' 70 101 .
  • Mycobacterium tuberculosis Salmonella, Pseudomonas, and Burkholderia species 27 ' 60 ' 70 101 .
  • its potential role in modulating inflammasome activation is unknown.
  • Nlrp3 ' and Caspl ⁇ ' Caspll '1' macrophages showed minimal levels of cytotoxicity in response to infection by acnB-, icdA-, and ace ⁇ -deficient bacteria, suggesting that the absence of Stm aconitase, isocitrate dehydrogenase, or isocitrate lyase induces NL P3 inflammasome activation. Consistently, caspase-1 processing was observed in cell lysates following infection of B6 and Nlrc4 ⁇ / ⁇ but not Nlrp3 ' BMDMs with acnB, aceA, or icdA mutants (Fig. 2D).
  • acnB, aceA, or icdA mutants induced significantly elevated IL-1 ⁇ secretion by B6 and Nlrc4 ' BMDMs, but not Nlrp3 ⁇ / ⁇ BMDMs, suggesting that NLRP3 activation was responsible for the increased production of IL-1 ⁇ (Fig. 2E).
  • secretion of caspase-1 -independent cytokines and expression of pro-IL- ⁇ itself was unaffected by the Stm TCA cycle genes, indicating that deletion of Stm TCA cycle genes did not impact TLR signaling per se (Fig. 2F and data not shown).
  • Intracellular bacteria can activate the AIM2 inflammasome via release of bacterial DNA into the cytosol 82 ' 88 ' 106 .
  • Aim2 ⁇ ⁇ BMDMs had no defect in inflammasome activation in response to acnB, icdA, or aceA mutants, suggesting that AIM2 was not involved in this response (data not shown).
  • NL P3 can participate in non-canonical inflammasome activation in response to Gram-negative bacteria by a mechanism involving caspase- l l 10 ' 34 ' 46 ' 86 .
  • Caspl 1 ' ' ' BMDMs had no defect in either LDH (Fig. 3A) or IL- ⁇ secretion (Fig.
  • EXAMPLE 6 NLRP3 inflammasome activation in response to Salmonella TCA cycle mutants requires the bacterial SPI-1 T3SS
  • NLRP3 inflammasome can be activated by the T3SS of other bacteria
  • the SPI-1 T3SS promotes bacterial invasion into non- phagocytic cells 30 ' 31 ' 58
  • the SPI-2 T3SS is upregulated within the SCV and is required for intracellular replication within host macrophages 17 ' 35 .
  • SipB the integral membrane component of the SPI-1 translocon, but not SseC, the integral membrane component of the SPI-2 translocon
  • SseC the integral membrane component of the SPI-2 translocon
  • caspase-1 - independent cytokines were unaffected by the presence or absence of SipB (Fig. 4C).
  • the defect in inflammasome activation caused by sipB mutation was not due to reduced numbers of intracellular bacteria, as infection with a five-fold higher dose of szpfi-deficient bacteria was performed in order to ensure intracellular levels of WT and isogenic sipB mutant bacteria (data not shown).
  • the SPI-2 T3SS plays a role in a caspase-1/11-dependent Salmonella-mduced late death 9 ' 77 .
  • ROS reactive oxygen species
  • MCAT Mitochondrial catalase
  • EXAMPLE 8 NLRP3 inflammasome activation by Stm TCA cycle mutants requires bacterial citrate synthase
  • inflammasome activation we sought to eliminate citrate production through the TCA cycle in the acnB, icdA, or aceA mutant strains by additionally deleting gltA, which encodes citrate synthase. Intriguingly, deletion of citrate synthase significantly reduced cytotoxicity and release of IL- ⁇ caused by acnB and icdA mutants (Fig. 6B and 6C). Lack of citrate synthase did not reduce inflammasome activation caused by aceA (data not shown), perhaps because deletion of aceA did not result in as high of an increase in citrate levels as the other two mutations. Together, these data suggest that excess buildup of bacterially-derived citrate triggers inflammasome activation in response to intracellular Salmonella.
  • NLRP3 inflammasome activation triggered by altered Stm TCA cycle metabolism could also promote anti-bacterial defense in vivo.
  • Aconitase-deficient Stm had no defect in virulence following intraperitoneal (i.p.) infection of C57BL/6 mice, and showed equivalent levels of spleen and liver colonization as parental fliCfljB bacteria (Fig. 7A and B). Mice infected with icdA mutant bacteria had significantly reduced tissue burdens in the liver and spleen following intraperitoneal infection, perhaps because of an overall reduced growth rate of the icdA mutant (data not shown).
  • acnB mutant Stm exhibited significantly reduced splenic tissue burden 6 days post-intragastric inoculation compared with fliCfljB- fected mice (Fig. 7C). Intriguingly, despite these lower burdens, serum IL-18 levels of ac «i?-infected C57BL/6 mice were significantly elevated in comparison with mice infected by fliCfljB Stm (Fig. 7E), consistent with enhanced in vitro inflammasome responses against ac «i?-deficient Stm.
  • Nlrp3 ' mice did not exhibit statistically significant differences in either CFU or serum IL-18 between the acnB and fliCfljB strains (Fig. 7D and F). Nlrp3 ' mice also succumbed to acnB mutant infection in higher numbers than B6 mice, providing further support for the role of NLRP3 in inflammasome activation in vivo in the context of infection by aconitase-deficient Stm.
  • EXAMPLE 1 0 AcnB contributes to persistent Salmonella infection in vivo
  • Nrampl encodes a lysosomal transporter that exports divalent metal cations from the SCV, and limits Salmonella intracellular
  • mice which express functional NRAMP1 protein 103 .
  • Intraperitoneal infection of 129 mice with 1000 CFU of WT Stm results in a chronic infection in 129 mice 76 .
  • MNNs mesenteric lymph nodes
  • spleens spleens
  • livers of parental ⁇ fliCfljB spleens
  • ac «i?-infected mice in the early stages of persistent infection, at days 7 and 21 post-infection (Fig. 8A-C and data not shown).
  • acwfi-deficient bacteria had a significant competitive disadvantage at both 7 and 21 days post-infection in the spleens and livers, but not in the MLNs (Fig. 8D and data not shown).
  • 129S6/SvE mice survived an elevated infectious dose (10,000 CFU i.p.) of acwS-deficient Stm, but succumbed over 2 to 3 weeks to infection by isogenic acwfi-sufficient bacteria (Fig. 8E).
  • BMDMs bone marrow derived lymph nodes
  • this inflammasome activation correlated with elevated levels of bacterial citrate, and was abrogated upon deletion of citrate synthase (gltA), supporting a role for citrate in the triggering of the NLRP3 inflammasome.
  • this inflammasome activation was dependent on both the genes encoding bacterial citrate synthase and the SPI-1 effector proteins that mediate Stm invasion.
  • the innate immune system can detect intracellular bacteria not only through sensing of bacterial proteins and virulence activities, but also by combining sensing of bacterial metabolites with sensing of virulence activity.
  • acnB and icdA are active mechanisms of evading inflammasome activation. These enzymes normally function in a fundamental biosynthetic pathway.
  • acnB is not essential for either intra- macrophage replication or virulence in the context of certain routes of in vivo infection.
  • the Salmonella genome contains two aconitase enzymes, but deletion of acnB alone is sufficient to mediate inflammasome activation.
  • dynamic regulation of AcnB functions in vivo to promote immune evasion.
  • icdA and acnB were not required for intracellular replication of Stm in vitro, these mutants were attenuated during in vivo infection: icdA was attenuated following intraperitoneal infection of NrampS B6 mice. There may be a metabolic requirement for icdA during in vivo infection.
  • acnB was dispensable for intraperitoneal infection of C57BL/6 mice, acnB- deficient bacteria were significantly attenuated following oral infection of B6 mice.
  • acnB mutant bacteria also had a defect during persistent infection in NramplR 129S6/SvE mice in both single and competitive infection, indicating that absence of acnB impacts in vivo bacterial virulence.
  • mice infected with acwfi-deficient Stm displayed significantly higher levels of serum IL-18 than mice infected with the parental acwfi-sufficient bacteria, indicating that Salmonella lacking acnB induce elevated inflammasome activation in vivo.
  • Altered bacterial citrate production may lead to altered production of other bacterial metabolites, which may themselves be the proximal triggers of NL P3 activation.
  • mitochondrial cardiolipin was recently reported to be a direct ligand for NLRP3 41 .
  • Cardiolipin is also a constituent phospholipid of bacterial membranes, and cardiolipin metabolism may be altered as a consequence of TCA cycle dysregulation. Future studies will determine whether infected macrophages respond directly to elevated cytoplasmic levels of citrate or whether other bacterial metabolites are involved.
  • vita-PAMPs Signatures of bacterial viability, termed vita-PAMPs or PAMPs per vita produced by metabolically active microbes, have been proposed to constitute an additional level of innate immune sensing, together with the classical pathogen associated molecular patterns or PAMPs as originally formulated by Janeway 44 ' 87 102 .
  • Bacterial mRNA 45 87 and cyclic di-nucleotides 110 function as vita-PAMPs during bacterial infection.
  • Recent studies have revealed that production of butyrate by intestinal bacteria can impact inflammation by control of intestinal regulatory T cell populations 1 15 ' 93 .
  • Our data provide evidence that intracellular bacterial metabolites serve as another class of vita-PAMP that signal inflammasome activation.
  • ROS plays a role both in the activation of the inflammasome complex itself, as well as in upregulation of NLRP3 inflammasome components 2 .
  • NLRP3 inflammasome activation appears to mobilize different cellular sources of OS for different stimuli, as LPS+ATP induces NLRP3 inflammasome activation through a pathway involving NOX2 (encoded by gp91 phox ), rather than mitochondrial ROS 65 ' 78 .
  • Previous studies have demonstrated a requirement for mitochondrial ROS in antimicrobial killing downstream of TLR signaling during Salmonella infection 108 .
  • icdA and acnB were not required for intracellular replication of Stm in vitro, these mutants were attenuated during in vivo infection: icdA was attenuated following intraperitoneal infection of Nramp s B6 mice, although it is not currently clear whether this relates to a metabolic requirement for icdA during in vivo infection. Interestingly, while acnB was dispensable for intraperitoneal infection of C57BL/6 mice, acwfi-deficient bacteria were significantly attenuated following oral infection of B6 mice.
  • acnB mutant bacteria also had a defect during persistent infection in Nrampl R 129S6/SvE mice in both single and competitive infection, indicating that absence of acnB impacts in vivo bacterial virulence.
  • mice infected with acwfi-deficient Stm displayed significantly higher levels of serum IL-18 than mice infected with the parental acwfi-sufficient bacteria, indicating that Salmonella lacking acnB induce elevated inflammasome activation in vivo.
  • Caspl ⁇ ' ⁇ Caspll ⁇ ' ⁇ and Nlrp3 ' mice mice showed significantly reduced production of IL-18 in response to acnB Stm.
  • EXAMPLE 1 1 Method To Generate Improved Salmonella Vaccine Strains
  • acwfi-deficient Stm induced elevated inflammatory responses in vivo, and are cleared more rapidly following infection.
  • acnB- deficient Stm induce elevated levels of a specific immune response, termed 'inflammasome activation' that is associated with inflammatory cell death and release of key effector cytokines that mediate adaptive immunity.
  • this protocol is an example of generating a useful and improved Salmonella vaccine using Stm strains lacking the aroA gene as attenuated vaccine strains.
  • the immunogenicity of aro -deficient Salmonella is notably poor.
  • Engineered to be deficient in acnB or icdA, it the attenuated, double mutant strain induces innate immunity in an animal to which it is administered and function as an improved vaccine.
  • mice To determine whether deletion of acnB enhances the efficacy of vaccination, we orally immunize mice with aroA single mutant bacteria, or aroA acnB double-mutant bacteria. This is done by feeding of these bacterial mutant strains to mice (10 9 organisms per mouse).
  • Induction of adaptive immunity is assessed by measuring Salmonella-specific CD4 and CD8 T cell responses on day 7 post- infection, antibody responses at day 21 post-infection, and protection against a lethal Salmonella challenge.
  • EXAMPLE 12 Method To Utilize Aroa AcnB Salmonella As Vaccine Delivery Vehicle Or Platform
  • a combined aroA acnB strain should enable the generation of robust immune responses against heterologous antigens that are expressed in Salmonella.
  • heterologous antigens from organisms for which there are no or only poor vaccines, as well as tumor antigens serve as particularly attractive targets.
  • Expression of heterologous antigens in the aroA acnB strain background is achieved by using a constitutive Salmonella enterica promoter, such as rpsM, and insertion of the rpsM promoter and DNA encoding the heterologous antigen together onto the Salmonella chromosome at a specific site, termed the attTn7 site.
  • the site is specifically targeted by transposition of the Tn7 transposon, and insertion of the Tn7 transposon into this site does not disrupt any other Salmonella genes.
  • aroA acnB Salmonella strains that individually express these particular genes. As described above, we then vaccinate mice with these strains by orogastric inoculation. Subsequent to vaccination, the mice will be exposed to lethal challenge with the respective microorganisms. We anticipate that aroA acnB strains that express heterologous antigens will provide robust immune protection against infection by
  • aroA acnB strain An extension of using the aroA acnB strain as a platform for vaccination against intestinal bacterial pathogens is the use of aroA acnB strain as a platform for vaccination against tumor antigens.
  • the first tumor antigen that we express is murine Vascular Endothelial Growth Factor Receptor 2, VEGFR2. This antigen is expressed from the constitutive rpsM promoter. This receptor is upregulated on many different tumors, and therefore provides an attractive target for antitumor vaccination.
  • mice Following vaccination with VEGFR2-expressing or control bacterial strains, mice are challenged with B16F10 tumor cells in a flank tumor melanoma model. Protection is evaluated by assessment of tumor burden and levels of lung tumor metastases.
  • EXAMPLE 1 3 Vaccination Challenge Studies
  • modified Stm deleted in the acnB gene i.e., SEQ ID NO: 1 was deleted
  • an attenuated modified Stm deleted in the icdA gene i.e., SEQ ID NO: 3 was deleted
  • Salmonella vaccine candidates in chicken were employed as Salmonella vaccine candidates in chicken.
  • Group 1 Day old chicken were divided into groups.
  • Each group of chickens was challenged 3 weeks after immunization with a chicken strain of Salmonella Typhimurium (ST). Two days after challenge, the chicken were examined to determine if and the extent to which the vaccinations protected the chicken from organ (liver) invasion (i.e., whether ST bacteria can be detected in the livers of the chicken without enrichment - accuracy down to 10 3 bacteria). Table 1 demonstrates the results.
  • Salmonella typhimurium directs the translocation of Sip proteins into the host cell. Mol Microbiol 24:747-756.
  • the Res phosphorelay system is specific to enteric pathogens/commensals and activates ydel, a gene important for persistent Salmonella infection of mice. Mol Microbiol 62:883-894.
  • L-asparaginase II produced by Salmonella typhimurium inhibits T cell responses and mediates virulence. Cell Host & Microbe 12:791-798.
  • Virulence determinants from a cystic fibrosis isolate of Pseudomonas aeruginosa include isocitrate lyase. Microbiology 154:1616-1627.
  • T-POP TnlO derivative
  • Salmonella vaccine vectors Microb Pathog 58: 17-28.

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Abstract

An attenuated Salmonella bacterium deficient in the naturally occurring gene that encodes one or both of the TCA enzymes aconitase or isocitrate dehydrogenase induces the innate immune response of an animal to which it is administered. The bacterium, when manipulated to express a target gene from another microorganism or a cancer or tumor, also can induce the innate immune response of a vaccinated animal to the target microorganism or cancer/tumor cell. The manipulated bacterium is useful as both a component for a Salmonella vaccine and as heterologous gene delivery platform to induce immune responses against other diseases.

Description

COM POSITIONS AN D M ETHODS US I NG MODI FI ED SALMONELLA
STATEMENT OF GOVERNMENT INTEREST
This invention was made with government support under Grant No. All 05346 awarded by the National Institutes of Health. The government has certain rights in the invention.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM
Applicant hereby incorporates by reference the Sequence Listing material filed in electronic form herewith. This file is labeled UPN-14-7023PCT_ST25.txt", was created on March 17, 2015, and is 18 KB in size.
BACKGROUND OF THE I NVENTION
Microbial infection triggers assembly of inflammasome complexes that promote caspase-1 -dependent antimicrobial responses. Inflammasome assembly is mediated by members of the Nucleotide binding domain-Leucine-Rich Repeat (NLR) protein family that respond to cytosolic bacterial products or disruption of cellular processes. Flagellin injected into host cells by invading Salmonella induces inflammasome activation through Nucleotide binding domain Leucine Rich Repeat Family Card Domain Containing 4 (NLRC4), while Nucleotide binding domain Leucine Rich Repeat Family Pyrin Domain Containing 3 (NLRP3) is required for inflammasome activation in response to multiple stimuli, including microbial infection, tissue damage, and metabolic dysregulation, through mechanisms that remain poorly understood. During systemic infection, Salmonella avoids NLRC4 inflammasome activation by downregulating flagellin expression. Macrophages exhibit delayed NLRP3 inflammasome activation following Salmonella infection, suggesting that Salmonella may evade or prevent the rapid activation of the NLRP3 inflammasome.
Pattern recognition receptors (PRRs) that detect and respond to evolutionarily conserved microbial structures such as lipopolysaccharide (LPS) or peptidoglycan, as well as pathogen-specific virulence activities, are critical for host immune defense71' 102. To promote infection, microbial pathogens inject virulence factors into the cytosol of infected cells to disrupt or modulate critical host physiological processes19. During this process, contamination of the target cell cytosol by microbial components triggers cytosolic PRRs of the Nucleotide binding domain-Leucine-Rich Repeat (NLR) family52. Diverse NLRs respond to a variety of endogenous and exogenous signals associated with infection, tissue stress or damage. For example, NL C4 responds to microbial products such as bacterial flagellin or structurally- related specialized secretion system components that are injected into the cytosol of infected cells during infection by bacterial pathogens including Pseudomonas, Legionella, and Salmonella spp.72'75'97 NLRs recruit pro-caspase-1 to multiprotein complexes termed inflammasomes, where pro-caspase-1 is processed and activated, leading to cleavage and secretion of caspase- 1 -dependent cytokines66'67, as well as pyroptosis, a caspase-1 -dependent pro-inflammatory cell death5.
Inflammasome activation and subsequent production of caspase-1 -dependent cytokines is important for both innate and adaptive antimicrobial responses63, as IL-1 family cytokines released upon inflammasome activation promote neutrophil migration to infected tissues and drive TH17 and TH1 responses against mucosal pathogens16'40. How pathogens evade inflammasome activation, and whether persistent bacterial pathogens evade or suppress inflammasome activation in order to establish or maintain persistence remains poorly understood.
Salmonella enterica species cause a range of disease from severe gastroenteritis to persistent systemic infection4. Salmonella enterica serovar Typhimurium {Stm) invades host cells by means of a type III secretion system (T3SS) encoded on Salmonella pathogenicity island I (SPI-1)18'57. Salmonella subsequently replicates within a Salmonella-containing vacuole (SCV) that is established and maintained by the activity of a second T3SS, encoded on a second pathogenicity island, SPI-217'35. Intestinal inflammation during Stm infection is triggered by NLRC4-dependent responses to Stm flagellin, accompanied by caspase- 1- dependent cytokine secretion and pyroptosis29. Activity of a SPI-1 effector protein, SopE, also contributes to SPI-1 -dependent inflammasome activation in intestinal epithelial cells79.
Within the inflamed intestine, specialized adaptations allow Stm to resist mucosal antimicrobial defenses84'99'109. However, flagellin expression is downregulated at systemic sites20'21, and enforced flagellin expression enhances NLRC4 activation and bacterial clearance, indicating that inflammasome activation in response to bacterial flagellin is detrimental for Stm replication during systemic infection73'95 .
NLRP3 responds to a wide variety of structurally unrelated molecules and activities, including extracellular ATP, bacterial pore-forming proteins, bacterial nucleic acids, crystals, and unsaturated fatty acids39'45'64'68 107. While ATP, crystals, and the Yersinia T3SS all induce rapid formation of an NLRP3 inflammasome that leads to caspase-1 activation within 1-2 hours8'64'68, Stm induces delayed activation of a non-canonical NLRP3 inflammasome 12-16 hours post-infection9. This non-canonical NLRP3 inflammasome is independent of the activities of the SPI-1 T3SS and instead is regulated by caspase-11 and TLR4-dependent production of type I interferon10'34'86.
NLRs detect exogenous and endogenous molecules that serve as indicators of infection or tissue stress. Together with the NAIP proteins, NLRC4 detects the cytosolic presence of bacterial flagellin and inner rod proteins of bacterial T3SSs, leading to rapid inflammasome activation and pyroptosis48 113. Downregulation of flagellin expression or inactivating the genes encoding flagellin enables pathogens to evaded NLRC4 detection14'20'73. Pathogens also utilize active mechanisms of inflammasome suppression, either by downregulating NLRC4, or by targeting other host pathways, such as autophagy, that regulate inflammasome
activation36'83. The pathogenic Yersinia species possess several distinct effector-mediated mechanisms for inflammasome modulation8'54. The activation of the AIM2 inflammasome in response to Mycobacteria is blocked by the ESX system of virulent mycobacteria91.
Collectively these studies indicate that inflammasome activation is a target of bacterial immune evasion strategies.
The NLRP3 inflammasome responds to a wide variety of structurally and chemically unrelated signals, and, along with NLRC4, contributes to host defense against Stm infection9. NLRP3 induces delayed inflammasome activation in response to Stm independently of the SPI-1 and SPI-2 T3SSs via a non-canonical pathway involving a TLR4-TRIF-IFN signaling pathway that requires caspase-1110'34'86.
SUMMARY OF THE INVENTION
In one aspect, an attenuated Salmonella, e.g., Salmonella enterica Typhimurium bacterium is provided that is deficient in the gene encoding one or both of the bacterial Citric Acid Cycle (TCA) enzymes, aconitase and isocitrate dehydrogenase. In one embodiment, an antibiotic resistance gene is inserted in the bacterium in place of the naturally occurring, deleted gene. In another embodiment, a heterologous gene is inserted in the bacterial chromosome.
In another aspect, a composition is provided comprising attenuated Salmonella, e.g.,
Salmonella enterica Typhimurium bacterium deficient in the gene encoding one of the bacterial TCA enzymes selected from aconitase and isocitrate dehydrogenase and a pharmaceutically acceptable carrier. In another aspect, a composition is provided comprising attenuated Salmonella, e.g., Salmonella enterica Typhimurium bacterium deficient in the gene encoding one of the bacterial TCA enzymes selected from aconitase and isocitrate dehydrogenase and further containing a heterologous gene.
In another aspect, a method for inducing the innate immune response of an animal against infection by a Salmonella strain, e.g., Salmonella enterica Typhimurium is provided that comprises administering to an animal in need thereof a composition containing an attenuated Salmonella enterica Typhimurium bacterium that is deficient in the gene encoding one of the bacterial TCA enzymes aconitase or isocitrate dehydrogenase.
In still another aspect, a method of inducing the innate immune response of an animal against a microbial infection or cancer comprises administering to an animal in need thereof a composition containing an attenuated Salmonella strain, e.g., Salmonella enterica
Typhimurium bacterium deficient in the gene encoding one of the bacterial TCA enzymes selected from aconitase and isocitrate dehydrogenase and further containing a heterologous gene. The heterologous gene is from another infectious microorganism or from a cancer cell or tumor cell.
In still a further aspect, a method of generating a Salmonella vaccine comprises deleting a naturally occurring gene encoding a TCA enzyme selected from aconitase or isocitrate dehydrogenase from the chromosome of a wildtype or attenuated Salmonella strain, e.g., Salmonella enterica Typhimurium bacterium. In certain embodiments, the method employs bacteriophage transduction or homologous recombination to effect the deletion or replacement.
In another aspect, a method of generating a composition that induces the innate immune response against a target gene comprises deleting a naturally occurring gene encoding a TCA enzyme selected from aconitase or isocitrate dehydrogenase from the chromosome of a wildtype or attenuated Salmonella strain, e.g., Salmonella enterica Typhimurium bacterium and inserting into the bacterial genome a heterologous target gene from another infectious microorganism or from a cancer or tumor cell. In certain embodiments, the method employs bacteriophage transduction or homologous recombination to effect the deletion or insertion.
Other aspects and advantages of these compositions and methods are described further in the following detailed description of the preferred embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 A are data generated from in vitro screens for bacterial mutants that fail to inhibit inflammasome activation. fliCfljB Tnl0d::tet library was grown in 96-well format under SPI-1 inducing conditions and screened for ability to induce LDH release in immortalized Nlrc4 ''' macrophages. FIG. 1 A shows two bar graphs providing data representative of a plate (left graph) from indicated screen (right graph). LPS+ATP is a positive control and fliCfljB parent strain is a negative control. All experiments other than initial screen were performed in triplicate and are representative of at least 3 independent experiments. * /><0.05.
FIG. IB provides data generated from infecting B6, Nlrc4' and Casp Casp ll'1' bone marrow derived macrophages (BMDMs) in triplicate with initial bacterial mutants that exhibited elevated lactate dehydrogenase (LDH) release in primary screen. Supernatants were harvested 4 hours post- infection, and assayed for LDH release as descried in FIG. 1A. All experiments other than initial screen were performed in triplicate and are representative of at least 3 independent experiments. * /><0.05.
FIG. 1C is a bar graph showing IL-Ιβ released from B6, N/rc "A and Caspl'1' Caspl l'1'
BMDMs was measured by ELISA 4 hours post-infection. All experiments other than initial screen were performed in triplicate and are representative of at least 3 independent experiments. * /><0.05.
FIG. ID shows bars graph data generated from measuring IL-6 released from infected cells 4 hours post-infection as a control cytokine. All experiments other than initial screen were performed in triplicate and are representative of at least 3 independent experiments. * /X0.05.
FIG. 2A is a schematic of S. Typhimurium TCA cycle.
FIG. 2B shows that deletion of Salmonella citrate or isocitrate TCA cycle enzymes induces NL P3 inflammasome activation. FIG. 2B. is a graph showing data from the infection of Casp 1''' Casp 11''' BMDMs with Stm carrying targeted deletions in indicated TCA cycle genes. The cells were lysed, bacteria diluted, and intracellular bacterial CFUs determined at 1, 8 and 24 hours post-infection. All data are representative of at least three independently performed experiments. * /><0.05.
FIG. 2C is a bar graph showing that targeted mutations in specific core TCA cycle genes lead to NLRP3- and caspase-l/caspase-11 -dependent cell death. LPS+ATP was used as a positive control for cell death, and fliCfljB used as a negative control (parent strain). All data are representative of at least three independently performed experiments. B6 - C57BL/6. * p<0.05.
FIG. 2D is a Western blot for active caspase-1 (plO) that was performed on lysates from B6, Nlrp3' , and Nlrc4' BMDMs 3 hours post-infection with indicated bacterial mutant strains, β-actin was blotted for as a loading control. Molecular weight markers in kD are indicated at left. All data are representative of at least three independently performed experiments. UI - Uninfected, B6 - C57BL/6. * / 0.05.
FIG. 2E are bar graphs showing supernatants from B6, Nlrc4' , Nlrp3' and Caspl''' Caspll'1' BMDMs infected with TCA mutants for 4 hours and analyzed for IL-Ιβ measured by ELISA. All data are representative of at least three independently performed experiments. B6 - C57BL/6. * /?<0.05.
FIG. 2F are bar graphs showing that IL-6 released from infected cells was measured as a control cytokine. All data are representative of at least three independently performed experiments. B6 - C57BL/6. * / 0.05.
FIG. 3 A provides data showing that deletion of bacterial TCA genes activates the canonical NL P3 inflammasome. FIG. 3A is a bar graph showing data generated from infecting B6, Caspl'1', Caspll'1' and Caspl'''CaspH''' BMDMs with bacterial TCA cycle mutants. Cell death was measured 4 hours post-infection by LDH release assay. LPS+ATP was used as a positive control and fliCfljB (parent strain for indicated mutants) was used as a negative control. All experiments were performed three independent times in triplicate, and data from representative experiments are shown. * /?<0.05.
FIG. 3B is a bar graph showing data from supernatants from infected cells analyzed 4 hours post- infection by ELISA for IL-lp. Experiments and data were performed and shown as described for FIG. 3A.
FIG. 3C is a bar graph showing data from supernatants from infected cells analyzed 4 hours post-infection by ELISA for IL-6. Experiments and data were performed and shown as described for FIG. 3A.
FIG. 4A provides data showing that NLRP3 inflammasome activation in response to Salmonella TCA cycle mutants requires the bacterial SPI-1 T3SS. FIG. 4A is a bar graph showing that supernatants of Nlrc4~/~ BMDMs were analyzed 4 hours post-infection with indicated bacterial strains for LDH release. All experiments were performed three independent times in triplicate and representative data are shown. * p<0.05, ** / θ.01. FIG. 4B is a bar graph showing that supematants of Nlrc4~ ' BMDMs were analyzed 4 hours post-infection with indicated bacterial strains for IL-Ιβ release. All experiments were performed three independent times in triplicate and representative data are shown. * p<0.05, ** /K0.01.
FIG. 4C is a bar graph showing that supematants of N/rc "A BMDMs were analyzed 4 hours post-infection with indicated bacterial strains for IL-12 release. All experiments were performed three independent times in triplicate and representative data are shown. * /?<0.05, ** /K0.01.
FIG. 4D is a bar graph showing data generated when N/rc "A BMDMs were infected with indicated parental bacterial strains or isogenic sseC mutants and assayed 4, 8 and 20 hours post-infection for cytotoxicity by LDH release. All experiments were performed three independent times in triplicate and representative data are shown.
Figure imgf000008_0001
FIG. 4E is a bar graph providing data generated by infecting Nlrc4~/~ BMDMs with parental or sopEBE2 mutant bacterial strains and assaying supematants 4 hours post- infection for cell death by LDH release by ELISA. All experiments were performed three independent times in triplicate and representative data are shown. * /?<0.05, ** /?<0.01.
FIG. 4F is a bar graph providing data generated by infecting Nlrc4~/~ BMDMs with parental or sopEBE2 mutant bacterial strains and assaying supematants 4 hours post-infection for IL-Ιβ release by ELISA. All experiments were performed three independent times in triplicate and representative data are shown. * /?<0.05, ** /?<0.01.
FIG. 4G is a bar graph providing data generated by infecting Nlrc4~/~ BMDMs with parental or sopEBE2 mutant bacterial strains and assaying supematants 4 hours post-infection for IL-12 release by ELISA. All experiments were performed three independent times in triplicate and representative data are shown. * / 0.05, ** / θ.01.
FIG. 5A provides data that Salmonella TCA cycle mutants trigger NLRP3
inflammasome activation through mitochondrial OS. FIG. 5 A shows a photograph and bar graph resulting from production of mitochondrial superoxide in Sim-infected Nlrc4~/~ BMDMs assayed four hours post-infection with aconitase, i.e., acnB strain. fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 μηι. * p<0.05.
FIG. 5B shows a photograph and bar graph resulting from production of mitochondrial superoxide in Sim-infected Nlrc4' BMDMs assayed four hours post- infection with aconitase, i.e., icdA strain. fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 μηι. * /?<0.05.
FIG. 5C is a bar graph showing B6 and MCAT BMDMs infected with indicated bacterial strains, for which cell death was assayed 4 hours post-infection. fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 μηι. * /?<0.05.
FIG. 5D is a bar graph showing IL-Ιβ in supernatants of B6 and MCAT BMDMs infected as described in FIG. 5C and measured by IA A. fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 μηι. * /?<0.05.
FIG. 5E is a bar graph showing IL-12 in supernatants of B6 and MCAT BMDMs infected as described in FIG. 5C and measured by I A. fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 μηι. * /?<0.05.
FIG. 5F is a bar graph showing data generated when Nlrc4~/~ BMDMs were pretreated with either vehicle control or ΙΟηΜ MitoQ 3 hours prior to infection. Cell death was assayed 4 hours post- infection by measuring release of LDH in cell supernatants. fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 μηι. * / 0.05.
FIG. 5G is a bar graph showing data when supernatants of samples treated with vehicle control or MitoQ as described in FIG. 5F were assayed for IL-Ιβ.
FIG. 5H is a bar graph showing data when supernatants of samples treated with vehicle control or MitoQ as described in FIG. 5F were assayed for IL-12. fliCfljB is the parent strain background for all indicated bacterial mutants. All experiments were performed three independent times in triplicate and representative data are shown. Scale bars, 20 μηι. * p<0.05.
FIG. 6A is a schematic with bar graphs for each component, showing that NLRP3 inflammasome activation by Salmonella aconitase and isocitrate dehydrogenase mutants correlates with excess bacterial citrate production and requires citrate synthase.
FIG. 6B is a bar graph showing data from the analysis of bacterial metabolites from bacteria grown under SPI-I inducing conditions using GC-MS analysis. Nlrc4' BMDMs were infected with indicated bacterial mutants. Supernatants were assayed 4 hours post-infection for levels of LDH release. fliCfljB is the parent strain background for all indicated bacterial mutants. Experiments were performed with four independently grown bacterial cultures for each strain. * p< 0.05.
FIG. 6C is is a bar graph showing data from the analysis of bacterial metabolites from bacteria grown under SPI-I inducing conditions using GC-MS analysis. Nlrc4' BMDMs were infected with indicated bacterial mutants. Supernatants were assayed 4 hours post-infection for levels of IL-Ιβ. fliCfljB is the parent strain background for all indicated bacterial mutants. Experiments were performed with four independently grown bacterial cultures for each strain. * /?< 0.05.
FIG. 6D is a bar graph showing data from the analysis of bacterial metabolites from bacteria grown under SPI-I inducing conditions using GC-MS analysis. Nlrc4' BMDMs were infected with indicated bacterial mutants. Supernatants were assayed 4 hours post-infection for levels of IL- 12. fliCfljB is the parent strain background for all indicated bacterial mutants. Experiments were performed with four independently grown bacterial cultures for each strain. * /?< 0.05.
FIG. 7A is a graph showing deletion of bacterial TCA cycle aconitase leads to a decrease in bacterial virulence. The data is generated from infecting C57BL/6 mice i.p. with 5x102 CFU. Spleens were harvested on days 3 and 5, and CFUs were enumerated by plating dilutions of tissue homogenates on selective plates. Dashed lines indicate the limit of detection. Mann-Whitney U test and unpaired two-tailed Student's t test were used to determine statistical significance for mouse CFU and cytokine data, respectively. Bars represent geometric means for CFU, and arithmetic mean for cytokine data. * /?<0.05, ** p<0.0l , * ** / 0.001. Data are representative of two independently performed experiments.
FIG. 7B is a graph showing the data from the experiment of FIG. 7A, but livers were harvested on days 3 and 5, and CFUs were enumerated by plating dilutions of tissue homogenates on selective plates. Data are representative of two independently performed experiments. Symbols are the same as described in FIG. 7A.
FIG. 7C is a graph showing data generated from infecting C57BL/6 and Caspl'1' Caspl l'1' mice with 2x107 CFU orally. Seven (7) days post-infection spleens were harvested for bacterial enumeration. Mice that succumbed to infection prior are designated with an 'X'. Data are representative of pooled data from two to three independent experiments, otherwise symbols and data are as described in FIG. 7A.
FIG. 7D is a graph showing data generated from the experiment of FIG. 7C, except that 7 days post-infection serum was harvested for IL-18 ELISAs. Symbols are described in FIGs. 7A and 7C. Data are representative of pooled data from two to three independent experiments.
FIG. 7E is a graph showing data generated from infecting C57BL/6 and Nlrp3' mice with 2x107 CFU orally. Seven (7) days post- infection spleens were harvested for bacterial enumeration. Symbols are described in FIGs. 7A and 7C. Data are representative of pooled data from two to three independent experiments.
FIG. 7F is a graph showing data generate from the experiment of FIG. 7E, except that 7 days post-infection serum was harvested serum for IL-18 ELISAs. Symbols are described in FIGs. 7A and 7C. Data are representative of pooled data from two to three independent experiments.
FIG. 8 A shows that deletion of bacterial TCA cycle aconitase leads to a defect in bacterial persistence. 129S6/SvE mice were infected with lxl O3 CFU of bacteria i.p., and mesenteric lymph nodes (MLNs were harvested on days 21 and 60, and CFUs enumerated by plating dilutions of tissue homogenates. In FIG. 8A and the following related figures, dashed lines indicate limit of detection. Mann- Whitney U test and unpaired two-tailed Student's t test were used to determine statistical significance for mouse CFU and cytokine data, respectively. Bars represent geometric means for CFU, and arithmetic mean for cytokine data. * /?<0.05, ** / O.01. Data are pooled from two independently performed experiments, except competitive index and survival curve studies, which indicate individual experiments.
FIG. 8B provides data from the experiment of FIG. 8A, except that spleens were harvested on days 21 and 60, and CFUs enumerated by plating dilutions of tissue
homogenates.
FIG. 8C provides data from the experiment of FIG. 8A, except that livers were harvested on days 21 and 60, and CFUs enumerated by plating dilutions of tissue
homogenates.
FIG. 8D provides data from an experiment in which 129S6/SvE mice were co-infected with lxl O3 CFU of each indicated bacterial strain i.p., and MLNs, spleens (left) and livers (right) were harvested on day 21 and competitive index (CI) was determined by plating tissue homogenates on different selective plates.
FIG. 8E provides data from an experiment in whichl29S6/SvE mice were infected i.p. with lxl O4 CFU of fliClfljB or isogenic acnB mutant bacterial strains and survival of the mice was monitored over 21 days. FIG. 8F provides data from an experiment in whichl29S6/SvE mice were infected with lxlO4 CFU fliCfljB or isogenic acnB mutant bacteria, and 7 days post-infection CFU per gram of spleens from mice was determined by plating tissue homogenates.
FIG. 8G provides data from the experiment of FIG. 8F except that 7 days post- infection CFU per gram of livers from mice was determined by plating tissue homogenates.
FIG. 8H provides data from the experiment of FIG. 8F except that serum levels of IL- 18 from mice were assayed at day 7 post-infection.
FIG. 81 provides data from the experiment of FIG. 8F except that serum levels of IL-6 from mice were assayed at day 7 post-infection.
DETAILED DESCRIPTION OF THE INVENTION
The inventors determined that Salmonella lacking certain TCA enzymes trigger NLRP3 inflammasome activation in infected macrophages, leading to elevated inflammatory responses and reduced virulence. Stm genes that modulate NLRP3 inflammasome activation during infection were identified. Four of the genes identified in the examples below {acnB, bcfB, rcsD, and melB) were previously found in a genome-wide screen for Salmonella persistence genes56, consistent with the possibility that modulating inflammasome activation might promote long-term systemic infection. bcfB encodes a fimbrial chaperone, rcsD encodes a member of a two-component system previously found to contribute to persistent infection and regulate resistance to antimicrobial peptide responses23'26, while melB encodes a symporter of melibiose and monovalent cations. acnB, encodes the enzyme aconitase, which mediates conversion of citrate to isocitrate as a key step in the TCA cycle. Interestingly, several other TCA cycle enzymes contribute to persistent infection by Salmonella, as well as a number of other bacterial pathogens ' ' ' .
As demonstrated in the examples below, Stm mutants deficient in aconitase or isocitrate dehydrogenase, but not other TCA cycle enzymes, induce rapid canonical NLRP3 inflammasome activation in BMDMs. The inventors have identified a link between mitochondrial Reactive Oxygen Species (ROS) and NLRP3 inflammasome activation in response to alteration of Salmonella citrate and isocitrate metabolism. These findings provide evidence that while the Salmonella TCA cycle may be dispensable for intracellular replication, it may enable Salmonella to evade the NLRP3 inflammasome by limiting the production of bacterial citrate. These data provide evidence that modulation of inflammasome activation is important for long-term bacterial persistence as well as in acute systemic infection, and that targeting such bacterial factors provides a broadly applicable strategy to enhance antibacterial immune defense.
These data also support the use of genetically modified Salmonella as a vaccine delivery platform by generating AcnB or /c<5L4-deficient mutants recombinantly expressing an epitope of interest and/or the AcnB and IcdA mutant salmonella being used to stimulate an immunogenic response against Salmonella itself.
Altogether these data indicate that acwfi-deficient Stm trigger inflammasome- dependent immune responses that contribute to antibacterial immune defense, Stm can limit adaptive immune responses through a variety of mechanisms including the induction of host nitric oxide as well as production of bacterial L-asparaginase51'90. As inflammasome activation leads to release of IL-Ιβ and IL-18, which contribute to TH1 and TH17
responses ' ' , infection with TCA cycle mutants overcomes these bacterial inhibitory mechanisms to prime S¾w-specific adaptive responses that promote control of bacterial infection. A long-standing challenge of anti-bacterial vaccine design has been to produce attenuated live vaccines that retain immunogenicity104. Interestingly, commonly used adjuvants that contain alum in human vaccine preparations trigger the NLRP3 inflammasome for efficient priming of T cell responses25. Thus, in addition to revealing a novel aspect of interactions between intracellular bacterial pathogens and host macrophages, the inventors have provided a strategy to induce robust adaptive cellular immune responses by employing strains of Salmonella that trigger robust NLRP3 inflammasome activation in vivo.
To our knowledge, this work describes the first identification of Salmonella genes that modulate NLRP3 inflammasome activation, and the first demonstration that bacterial TCA cycle enzymes impact activation of the NLRP3 inflammasome. Our data further provide evidence that Stm lacking the glyoxylate shunt enzyme isocitrate lyase also induce NLRP3 inflammasome activation in infected macrophages. Given the requirement of isocitrate lyase for persistence of bacterial pathogens such as M. tuberculosis, Pseudomonas, Burkholderia, and Salmonella ' ' ' and the overlap between the genes found in the inflammasome screen and the genes found in the Salmonella persistence screen, these data demonstrate that modulation of inflammasome activation is important for long-term bacterial persistence as well as in acute systemic infection, and that targeting such bacterial factors may provide a broadly applicable strategy to enhance antibacterial immune defense.
I. Definitions Unless defined otherwise in this specification, all scientific and technical terms used herein have the same meaning as commonly understood by to a person of skill in the fields of biology, biotechnology and molecular biology and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. However, for clarity, the following terms are defined as follows:
Salmonella as used herein refers to any strain of Salmonella, including any strain of Salmonella enterica, including Salmonella enterica serovar Typhimurium. The serovars of S. enterica that may be used as the attenuated bacterium of the live compositions described in accordance with various embodiments herein include, without
limitation, Salmonella enterica serovar Typhimurium ("^.typhimurium "), Salmonella montevideo, Salmonella enterica serovar Typhi ("S. typhi"), Salmonella enterica serovar, Paratyphi B ("S. paratyphi 13"), Salmonella enterica serovar Paratyphi C ("S. paratyphi C"), Salmonella enterica serovar Hadar ("S. hadar"), Salmonella enterica serovar Enteriditis ("S. enter iditis "), Salmonella enterica serovar Kentucky ("S. Kentucky"), Salmonella enterica serovar In/antis ("S. infantis"), Salmonella enterica serovar Pullorurn ("S. pullorum"), Salmonella enterica serovar Gallinarum ("S. gallinarum "), Salmonella enterica serovar Muenchen ("S. muenchen "), Salmonella enterica serovar Anaturn ("S. anatum "), Salmonella enterica serovar Dublin ("S. dublin"), Salmonella enterica serovar Derby ("S. derby"), Salmonella enterica serovar Choleraesuis var. kunzendorff'S. cholerae kunzendorf), and Salmonella enterica serovar minnesota (S. minnesota), among other known strains. Stm refers to Salmonella enterica serovar Typhimurium.
fliCfljB refers to Salmonella flagellins.
AcnB refers to aconitase or aconitate hydratase 2 (citrate hydro-lyase 2), which is a TCA enzyme found in Salmonella. In one embodiment, the nucleotide sequence of this enzyme is found at the chromosome locus of wildtype Salmonella strain SL1344 at tag 0159. In another embodiment, the nucleic acid sequence of the gene acn and the amino acid sequence of the acnB enzyme in Salmonella enterica Subsp. enterica serovar Typhimurium is found in the KEGG database at the Entry No. SL1344 0159 SEQ ID NOs: 1 and 2 respectively. Other publically available database entries for this gene and enzyme are found for the same or other Salmonella strains in the NCBI database, e.g., under GI378698137 or
11763877, among others. Sequences for the analogous aconitase gene and encoded enzyme in other Salmonella species are expected to be highly homologous or share percent identity for SEQ ID NOs: 1-2 of from about 75 % to over 99%. IcdA refers to isocitrate dehydrogenase, which is a TCA enzyme found in Salmonella. In one embodiment, the nucleotide sequence of the gene icdA and the amino acid sequence of the icdA enzyme in Salmonella enterica Subsp. enterica serovar Typhimurium is found in the KEGG database at the Entry No. SL1344 1176 SEQ ID NOs: 3 and 4, respectively. Other publically available database entries for this gene and enzyme are found for the same or other Salmonella strains in the NCBl database, e.g., under GI378699131or 11764650, among others. Sequences for the analogous isocitrate dehydrogenase gene and encoded enzyme in other Salmonella species are expected to be highly homologous or share percent identity for SEQ ID NOs: 3 or 4 of from about 75 % to over 99%.
NL P3 refers to Nucleotide binding domain Leucine Rich Repeat Family Pyrin
Domain Containing 3;
NLRC4 refers to Nucleotide binding domain Leucine Rich Repeat Family Card Domain Containing 4;
NRAMPl refers to Natural Resistance Associated Macrophage Protein 1 ;
AceA refers to isocitrate lyase;
Caspl refers to caspase-1 ;
Caspl 1 refers to caspase-11 ;
SPI-1 refers to Salmonella Pathogenicity Island 1 ;
SPI-2 refers to Salmonella Pathogenicity Island 2;
BMDM refers to bone marrow derived macrophages;
MLN refers to mesenteric lymph nodes.
The terms "nucleic acid sequence", "polynucleotide," when used in singular or plural form, generally refers to any nucleic acid sequence, polyribonucleotide or
polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or include single- and double-stranded regions. In addition, the term
"polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term "polynucleotide" specifically includes cDNAs. The term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases. In general, the term "polynucleotide" embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.
The term "oligonucleotide" refers to a relatively short polynucleotide of less than 20 bases, including, without limitation, single-stranded deoxyribonucleotides, single- or double- stranded ribonucleotides, R A:DNA hybrids and double-stranded DNAs. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
As used herein, "attenuated" or "attenuation" refers to elimination or reduction of the natural virulence of a bacterium in a particular host organism, such as an avian or a mammal. An "attenuated" bacterium or strain of bacteria is attenuated in virulence toward at least one species of subject or host organism that is susceptible to infection and disease by a virulent form of the bacterium or strain of the bacterium.
"Recombinant", as applied to a polynucleotide, means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant bacterium is a bacterial cell comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original bacterial construct. Typical recombinant or genetic engineering steps to generate a recombinant bacterium as referred to herein include bacteriophage transduction or homologous recombination, among other known techniques described in the art.
Typically, "heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
"Naturally occurring" means a sequence found in nature and not synthetically prepared or modified.
The terms "modification" refers to any deliberately inserted change in a nucleic acid or protein sequence, such as a deletion of all or part of the TCA enzyme-encoding sequences, or an insertion of a sequence into the TCA enzyme-encoding sequences that disrupts the translation of the nucleic acid sequence into the enzyme. Such disruption can be caused by insertion of a heterologous sequence into the TCA enzyme-encoding sequences. According to this invention, the phrase "deficient in" means that the bacterium contains a modification designed to reduce, inhibit or ablate expression of the encoded TCA enzyme.
The term "inflammasome activation" refers to a specific immune response that is associated with inflammatory cell death and release of key effector cytokines that mediate adaptive immunity.
"Animal" or "subject" as used herein means a mammalian animal, including a human male or female, a veterinary or farm animal, e.g., horses, livestock, cattle, pigs, etc., a domestic animal or pet, e.g., dogs, cats; and animals normally used for clinical research, such as primates, rabbits, and rodents. In one embodiment, the subject of these methods and compositions is a human. "Animal" as used herein is also meant to include other non- mammals or animal species that are commonly infected by Salmonella, such as avians or fowl that are used as food products, can be carriers for Salmonella, and are often the transmitters of the bacterium to humans.
By "vector" is meant an entity that delivers a heterologous molecule to cells, either for therapeutic or vaccine purposes. As used herein, a vector may include any genetic element including, without limitation, naked DNA, a phage, transposon, cosmid, episome, plasmid, or a virus. Vectors are generated using the techniques and sequences provided herein, described in the examples, and in conjunction with techniques known to those of skill in the art. Such techniques include conventional cloning techniques of cDNA such as those described in texts such as Green and Sambrook, Molecular Cloning: A Laboratory Manual. 4th Edit, Cold
Spring Harbor Laboratory Press, 2012, use of overlapping oligonucleotide sequences of the Salmonella genomes, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence.
By "minigene" or "expression cassette" is meant the combination of a selected heterologous gene or nucleic acid sequence of interest (e.g. , a target microbial gene and/or a suitable cancer or tumor gene) and the operably linked regulatory elements necessary to drive translation, transcription and/or expression of the gene product in the host cell in vivo or in vitro. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
"Expression control sequences" include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient R A processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic m NA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized in the construction of the compositions and performance of the methods described herein.
By "host cell" as used herein may refer to the cell or cell line in which the recombinant vector is produced.
As used herein, an immunogenic composition is a composition to which a humoral (e.g., antibody) or cellular (e.g., a cytotoxic T cell) response, or, in one embodiment, an innate immune response, is mounted to a target gene product delivered by the immunogenic composition following delivery to a mammal or animal subject.
By "therapeutic reagent" or "regimen" is meant any type of treatment employed in the treatment or prevention of microbial infections or cancers with or without solid tumors, including, without limitation, chemotherapeutic pharmaceuticals, biological response modifiers, radiation, diet, vitamin therapy, hormone therapies, gene therapy, surgical resection, etc.
The term "percent sequence identity" or "identical" in the context of nucleic acid sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over the full- length of an open reading frame of a gene, protein, subunit, or enzyme, or a fragment of at least about 100 to 500 nucleotides, is desired. However, identity among smaller fragments, e.g. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired. Similarly, "percent sequence identity" may be readily determined for amino acid sequences, over the full-length of a protein, or a fragment thereof. Suitably, a fragment is at least about 8 amino acids in length, 20 amino acids in length, 50 amino acids in length, 100 amino acids in length, and may be up to about 700 or more amino acids. As described herein, the percent identity among the TCA enzymes, acn or icd, in other Salmonella strains and serovars can be about 75%, 80%, 85%, 90%, 95% or over 99%. In other embodiments, the percent identities of the selected genes/enzymes can be lower than 75%.
Identity is readily determined using such algorithms and computer programs as are defined herein at default settings. Preferably, such identity is over the full length of the protein, enzyme, subunit, or over a fragment of at least about 8 amino acids in length. However, identity may be based upon shorter regions, where suited to the use to which the identical gene product is being put. Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs, such as "Clustal W", accessible through Web Servers on the internet. Alternatively, Vector NTI® utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta, a program in GCG Version 6.1. Fasta provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using Fasta with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference. Similarly programs are available for performing amino acid alignments. Generally, these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program that provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. The similarities between the sequences can also be defined as the ability to hybridized to the complement of a selected sequence, under stringent conditions. See, e.g., commonly used texts for the definitions of stringency, e.g., Green and Sambrook, cited above; or see, e.g., US Patent No. 8,974,798.
As used in the compositions and methods described herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. In one embodiment, the cancer is an epithelial cancer. In various embodiments of the methods and compositions described herein, the cancer can include, without limitation, breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, and multidrug resistant cancer, or subtypes and stages thereof. In still an alternative embodiment, the cancer is an "early stage" cancer. In still another embodiment, the cancer is a "late stage" cancer.
The term "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
As used herein, the term "about" is defined as a variability of 10% from the reference given, unless otherwise specified. It should be understood that while various embodiments in the specification are presented using "comprising" language, under various circumstances, a related embodiment is also be described using "consisting of or "consisting essentially of language. Throughout this specification, the words "comprise", "comprises", and
"comprising" are to be interpreted inclusively rather than exclusively. The words
"consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively. The terms "a" or "an" refers to one or more, for example, "an immunogenic composition" is understood to represent one or more such compositions. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.
One skilled in the art may readily reproduce the compositions and methods described herein by use of the elements described herein, which are publicly available from conventional sources.
II. Compositions
In one aspect, there is provided a Salmonella bacterium, e.g., a Salmonella enterica Typhimurium bacterium {Stm), that is genetically modified to be deficient or functionally deficient in a naturally occurring gene encoding a selected bacterial TCA enzyme.
Throughout this specification, the reference Stm is used for convenience. It should be understood that other embodiments of the compositions and methods described herein using Stm may employ any suitable Salmonella strain or serovar in place of Stm. In one
embodiment of the Salmonella bacterium the selected TCA enzyme gene is the gene encoding aconitase. In another embodiment, the selected gene is the gene encoding the TCA enzyme isocitrate dehydrogenase. As shown in the examples below, the absence or non-function of one or both of these two enzymes, but not all Salmonella TCA enzymes, induce the bacteria to have elevated levels of cell death. Further, deficiencies in these enzymes induce the production of certain cytokines from macrophages and dendritic cells in the subject infected by the modified Stm. As one example, IL-18 is generated, which is an indicator of the stimulation of the innate immune response. The sequences of these genes and their locations in the Stm chromosome are known and accessible to those of skill in the art.
To generate a TCA enzyme deficient Salmonella bacterium, one may genetically engineer a deletion of the naturally occurring gene encoding the selected TCA enzyme in the bacterium's genome. In one embodiment, the bacterium selected for such modification may be a wildtype Salmonella enterica Typhimurium, such as strain 1344 and any other strain. In another embodiment, a wildtype Salmonella enterica Typhimurium which is attenuated by artificial methods prior to deletion of the selected TCA enzyme may be employed. In one embodiment, the attenuation of the Salmonella strain is suitable for the animal for which it is intended, e.g., an avian species, a livestock animal, e.g., pig, cow, etc, or a human. In another embodiment, the bacterium selected for such modification is an already attenuated strain of Salmonella enterica Typhimurium. As demonstrated in Example 10, one such attenuated strain is the aroA mutant Stm bacteria116. Many other wildtype and attenuated Stm bacteria are publically known and available from university or commercial collections of bacterial strains. It is anticipated that any attenuated Stm will be useful in generating the Stm modified to be deficient in aconitase or isocitrate dehydrogenase. The selection of the particular Stm strain is not anticipated to be a limitation of this invention.
In addition to a complete deletion of the gene encoding the selected Stm TCA enzyme, a mutation may be inserted into the gene that renders it non-functional. In still another embodiment, the gene may be deleted in whole or part and replaced with another sequence, such as a sequence encoding an antibiotic resistance gene. The selection of the antibiotic resistance gene is conventional; it may be selected from among a wide number of such genes, including, without limitation, kanamycin, chloramphenicol, as well as others listed in public databases, such as the on-line ARDB-Antibiotic Resistance Genes Database.
In certain embodiments, the antibiotic resistance gene is present in a cassette or plasmid vector under the operable control of a suitable promoter. In one embodiment, the promoter is constitutive, such as that used in the examples. However, other promoters may be selected from among a wide number in the art. For example, the promoter can be a prokaryotic promoter, for example, a Salmonella promoter, which directs expression of the target antigen in the Salmonella. Examples of such promoters are well known including the htrA promoter, the nirB promoter, the ssaH promoter, the ompR promoter, and any other Salmonella or other bacterial promoter that is upregulated when Salmonella is taken up by mammalian cells. Alternatively, the promoter can be a eukaryotic promoter, such as the cytomegalovirus promoter. Numerous promoters are known in the art for such use and have been suggested for other Salmonella vector compositions; see e.g., US 6585975.
As demonstrated in the examples below, particularly Examples 1 -4, such a deletion/insertion may be engineered using a conventional technique, such as homologous recombination, transposon insertion or bacteriophage transduction. All of these techniques are known in the art; see for example the references cited herein, in the examples and at the end of this specification, all of which are incorporated by reference. Such genetically modified Stm are found, as demonstrated herein to induce in an animal, upon infection, the release of inflammatory cytokines from macrophages or dendritic cells and an increase in the animal's innate immune response to the bacterium.
In yet a further aspect of the invention, the Stm bacterium which is deficient in acnB or icdA, and optionally attenuated, may be additionally genetically modified to further comprise a "target gene" inserted in the bacterial chromosome. See, for example the description of Example 11.
In one embodiment, the target gene is a Salmonella gene, e.g., another gene from Salmonella enterica serovar Typhimurium (Stm), inserted in the chromosome at a non- naturally occurring site to enable the Salmonella to deliver larger amounts of the antigen encoded by the target gene than it would deliver in an unmodified bacterium. As another example, the Salmonella gene may be operatively associated with a stronger or more powerful promoter than exists in the naturally occurring, non-modified Stm or the duplicate occurrence of the Stm target gene in the chromosome allows for at least double the production of the encoded Stm antigen.
In yet another embodiment, the target gene is a heterologous gene, e.g., a gene from another infectious microorganism. For example, the heterologous gene may be selected from bacteria or viruses or other infectious microorganisms that would be susceptible to the increase in innate immunity caused by the modified Stm described herein when administered to an animal or other subject. Therefore, in certain embodiments, the heterologous gene is one that encodes an antigen from another bacteria, fungi, parasitic microorganisms or multicellular parasites which infect human and non-human vertebrates. In certain embodiments, the heterologous target gene is inlA,fdeC, cagA, IcrV, or B. anthracis Protective Antigen, among others. As demonstrated in Example 11 , in one embodiment, the target gene selected may be the bacterial internalin gene, inlA of Listeria monocytogenes. In another embodiment, the target gene may encode a bacterial adhesin, fdeC of Enteropathogenic E. coli. In still another embodiment, the target gene may encode the gene encoding the bacterial virulence factor cagA from Helicobacter pylori. Still other targets may be selected from bacterial pathogens include pathogenic gram-positive cocci include pneumococci; staphylococci; and streptococci.
Pathogenic gram-negative cocci include meningococcus; gonococcus. Pathogenic enteric gram-negative bacilli include enterobacteriaceae; pseudomonas, acinetobacteria and eikenella; melioidosis; salmonella; shigella; haemophilus; moraxella; H. ducreyi (which causes chancroid); brucella; Franisella tularensis (which causes tularemia); yersinia (pasteurella); streptobacillus moniliformis and spirillum; Gram-positive bacilli include listeria
monocytogenes; erysipelothrix rhusiopathiae; Corynebacterium diphtheria (diphtheria); cholera; B. anthracis (anthrax); donovanosis (granuloma inguinale); and bartonellosis.
Diseases caused by pathogenic anaerobic bacteria include tetanus; botulism; other Clostridia; tuberculosis; leprosy; and other mycobacteria. Pathogenic spirochetal diseases include syphilis; treponematoses: yaws, pinta and endemic syphilis; and leptospirosis. Other infections caused by higher pathogen bacteria and pathogenic fungi include actinomycosis; nocardiosis; cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis;
candidiasis, aspergillosis, and mucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis, torulopsosis, mycetoma and chromomycosis; and dermatophytosis. Rickettsial infections include Typhus fever, Rocky Mountain spotted fever, Q fever, and Rickettsialpox. Examples of mycoplasma and chlamydial infections include: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal chlamydial infections. Pathogenic eukaryotes encompass pathogenic protozoans and helminths and infections produced thereby include: amebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans; Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis;
schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm) infections.
Many target genes are available also from the organisms and/or toxins produced thereby which have been identified by the Centers for Disease Control [(CDC), Department of Health and Human Services, USA], as agents which have potential for use in biological attacks. For example, some of these biological agents, include, Bacillus anthracis (anthrax), Clostridium botulinum and its toxin (botulism), Yersinia pestis (plague), variola major (smallpox), Francisella tularensis (tularemia), and viral hemorrhagic fever, all of which are currently classified as Category A agents; Coxiella burnetii (Q fever); Brucella species (brucellosis), Burkholderia mallei (glanders), Ricinus communis and its toxin (ricin toxin), Clostridium perfringens and its toxin (epsilon toxin), Staphylococcus species and their toxins (enterotoxin B), all of which are currently classified as Category B agents; and Nipan virus and hantaviruses, which are currently classified as Category C agents. In addition, target genes from other organisms, which are so classified or differently classified, may be identified and/or used for such a purpose in the future.
In another embodiment, the heterologous gene is a gene from a mammalian tumor or cancer cell. For example, the heterologous gene may be selected from a tumor or cancer cell that would be susceptible to attack from the increase in innate immunity caused by the modified Stm described herein when administered to a subject with that cancer or tumor. Therefore, in certain embodiments, the heterologous gene is a gene encoding a cancer antigen, such as a full-length, wild-type cancer-specific antigens or mutated cancer-specific antigens or cancer-associated antigens. Cancer-specific antigens are those epitopes and proteins found on a selected specific cancer or tumor cell, and not on all cancer cells. Cancer-associated antigens are antigens that may be associated with more than one cancer or tumor cell type. Exemplary cancer-specific antigens can include, without limitation, 707-AP, alpha (a)- fetoprotein, ART -4, BAGE; b-catenin/m, b-catenin/mutated Bcr-abl, CAMEL, CAP-1, mCASP-8, CDC27m, CDK4/m, CEA, CT, Cyp-B, MAGE-B2, MAGE-B1, ELF2M, ETV6- AML1, G250, GAGE, GnT-V, GplOO, HAGE, HER-2/neu , HPV-E7, HSP70-2M HST-2, hTERT, iCE , KIAA0205, LAGE, LDLR/FUT, MAGE , MART-1, MC1R, MUC1, MUM-1, - 2, -3, PI 5, pi 90 minor bcr-abl. Still other suitable tumor or cancer genes encode VEGFR1, VEGFR2, MAGE -A 1, MUC-1, Thymosin βΐ, EGFR, Her-2/neu, MAGE-3, Survivin, Heparanase 1, Heparanase 2, and CEA, among others. Still other suitable antigens are those listed117, and incorporated by reference herein. See, also, texts identifying suitable antigens, such as Scott and Renner, in Encyclopedia of life Sciences 2001 Eds., John Wiley & Sons, Ltd.
Expression of heterologous antigens in the Stm bacterium, such as the aroA acnB strain discussed in Example 11 is achieved by using a vector, which contains a suitable promoter, such as the constitutive Salmonella enterica rpsM promoter in operative association with DNA encoding the heterologous antigen. Any of the promoters identified above or available and known to be useful in Salmonella may also abe selected to express the heterologous gene. The resulting vector or cassette can be inserted onto the Salmonella chromosome at any site within the Stm genome or onto a specific site, for example one targeted by a transposon. As exemplified below, such a cassette can be inserted onto the att7«7 site, which can be specifically targeted by transposition of the Tn7 transposon.
Insertion at other sites in the Stm chromosome can occur by use of other known transposons and methods available and known to those of skill in the art.
Such attenuated, acnB or z'c<5L4-deficient Stm bacterium carrying the heterologous gene can, upon infection, deliver the heterologous gene to the subject, while stimulating innate immunity and thus generate protection in the subject against the microorganism or tumor cell from where the target gene originates. Thus, as still other aspects, are compositions or pharmaceutical compositions which contain the modified Stm bacteria described above. The compositions comprising the modified Stm bacterium described above may be further associated with a pharmaceutically acceptable carrier for in vivo delivery. As used herein the term "pharmaceutically acceptable carrier" or "diluent" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans or other mammals, or animals, such as avian species. In one embodiment, the diluent is saline or buffered saline. Such pharmaceutically acceptable carriers suitable for use in such a composition are well known to those of skill in the art. Such carriers include, without limitation, and depending upon pH adjustments, buffered water, buffered saline, such as 0.8% saline, phosphate buffer, 0.3% glycine, hyaluronic acid, alcoholic/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents, adjuvants and excipients, may be added in accordance with conventional techniques. Optionally, the pharmaceutical compositions can also contain a mild adjuvant, such as an aluminum salt, e.g., aluminum hydroxide or aluminum phosphate, aqueous suspensions of aluminum and magnesium hydroxides, liposomes, and oil in water emulsions.
Carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusting agents may also be employed. Buffers include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid, e.g., citrates, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, trimethanmine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like may also be provided in the pharmaceutical carriers. These compositions are not limited by the selection of the carrier. The preparation of these pharmaceutically acceptable compositions, from the above-described components, having appropriate pH isotonicity, stability and other conventional characteristics is within the skill of the art. See, e.g., texts such as Remington: The Science and Practice of Pharmacy, 22nd ed, Lippincott Williams & Wilkins, publ., 2012; and The Handbook of Pharmaceutical Excipients, 7th edit., eds. R. C. Rowe et al, Pharmaceutical Press, 2012. According to certain methods, where the intended subject for administration is a chicken, or other animal, the composition containing the attenuated and modified Salmonella described herein may be a composition suitable to be added to a foodstuff.
Other suitable known components of the vectors and compositions are those found in other Salmonella vaccine or vehicle compositions. See for example, US Patent No. 7,115,269; International Patent Application Publication No. WO2013/177291 ; and publications such as McSorley et al, 1997 :Inf. Immun., 65(1): 171-178, among others.
III. Methods
The compositions described herein and particularly exemplified in the examples, below, are further useful in a variety of therapeutic or vaccinal methods. In one aspect, a method for inducing the innate immune response of an animal against infection by a
Salmonella strain, e.g., Salmonella enterica Typhimurium, involves administering a modified Stm or modified, attenuated Stm composition to an animal in need thereof. In one
embodiment, the animal receiving this compositions is avian, e.g., particularly avians that are used for food, e.g., fowl, chicken, ducks, etc. In another embodiment, the subject receiving the Salmonella is a mammal susceptible to Salmonella infection, e.g., a human or livestock animal.
In one aspect, the compositions described herein can be used to treat or prevent Salmonella infection in a subject, e.g., avians, livestock or humans. For this purpose, the embodiment without the heterologous gene may be most appropriate, e.g., a suitably attenuated, acnB or z'c<5L4-deficient Stm.
In still another aspect, the compositions described herein can be employed in a method of inducing the innate immune response of an animal against a microbial infection or cancer comprising by administering a composition containing the modified TCA enzyme-deficient Stm carrying a heterologous gene to an animal in need thereof. These uses would include the treatment or prevention of the infectious diseases or treatment of cancers/tumor from which the target heterologous gene originated, i.e., a pathogen or cancer identified above, or selected by the attending physician or medical personal.
Depending upon the subject selected, type of animal, size, weight, general health, etc., and purpose of treatment, the mode of administration and dosage may be selected by one of skill in the art, e.g., a veterinarian or physician. For example, the mode of administration be any suitable route: oral, subcutaneous injection, intravenous injection, intramuscular injection, mucosal, intra-arterial, intraperitoneal, parenteral, intradermal, transdermal, nasal, vaginal, or rectal or inhalation routes, among others. The term "oral" refers to administration of a compound or composition to a subject by a route or mode along the alimentary canal, such as by swallowing liquid or solid forms of a composition from the mouth, administration of a composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a composition, and rectal administration, e.g., using suppositories that release a live bacterial vaccine strain described herein to the lower intestinal tract of the alimentary canal. The doses may be administered as a single dose, multiple doses over a selected time gap, via prime/boosting protocols, etc.
When administered as an animal vaccine, e.g., a vaccine for chicken, for example, administration can be by gavage in a carrier. Alternatively, the bacteria can be added to gel beads and mixed with feed. Still alternatively, the bacteria can be sprayed onto the feathers or skin of the animals and inhaled. It is likely also that the bacteria can be lyophilized or otherwise treated and mixed with feed.
Similarly the selection of suitable dosages will be within the skill of the art depending upon the subject and course of treatment. Exemplary dosages may be 5 x 106-5 x 1010 colony forming units, e.g., 5 x 10s colony forming units, or 5-1000 μg, e.g., 100 μg, antigen, or 105-106 bacteria in 100 microliters for, e.g., chicken. The selection of the doses and modes of administration and dosage regimens may be selected by one of skill in the art based on the subject to be vaccinated, the physical attributes of the subject, the virulence of the Salmonella strain, the route of administration, and other common factors.
In yet a further aspect, a method of generating a Salmonella vaccine is also provided, which comprises deleting a naturally occurring gene encoding a TCA enzyme selected from aconitase or isocitrate dehydrogenase from the chromosome of a wildtype or attenuated Salmonella bacterium. As described above and in the examples, using the teachings of this invention to modify or inactivate the acnB or isdA gene, one of skill in the art may select any known methodology to generate the modified bacterium, e.g., using one or more of bacteriophage transduction or homologous recombination or other recombinant methods to effect the deletion or replacement. See e.g., the generation and use in Example 10 of the aroA acnB double-mutant Stn bacteria.
In a final aspect, a method of generating a composition that induces the innate immune response against a target heterologous gene comprises deleting a naturally occurring gene encoding a TCA enzyme selected from aconitase or isocitrate dehydrogenase from the chromosome of a wildtype or attenuated Salmonella enterica Typhimurium bacterium and inserting into the bacterial genome a heterologous target gene from another infectious microorganism or from a cancer or tumor cell under the operative control of a suitable promoter or other known vector components. Again, conventional techniques are well-known for this use. See, e.g., the generation and use of the aroA acnB Salmonella strain to express heterologous genes from Listeria monocytogenes, Enteropathogenic E. coli, and Helicobacter pylori. See, e.g., the generation and use of the aroA acnB Salmonella strain to express heterologous murine Vascular Endothelial Growth Factor Receptor 2, VEGFR2, from the constitutive rpsM promoter as an anti -tumor vaccine construct.
IV. Examples
The invention is now described with reference to the following examples. The examples below illustrate how the inventors screened a Salmonella Typhimurium transposon library to identify bacterial factors that limit NLRP3 inflammasome activation. Surprisingly, absence of the Salmonella TCA enzyme aconitase induced rapid NLRP3 inflammasome activation. This inflammasome activation correlated with elevated levels of bacterial citrate, and required mitochondrial ROS and bacterial citrate synthase. Importantly, Salmonella lacking aconitase displayed NLRP3- and caspase-1/11-dependent attenuation of virulence, and induced elevated serum IL-18 in wild-type mice. Together, these data link Salmonella genes controlling oxidative metabolism to inflammasome activation and suggest that NLRP3 inflammasome evasion promotes systemic Salmonella virulence. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations that become evident as a result of the teaching provided herein.
EXAMPLE 1 : Materials and Methods
A. Bacterial strains and infection conditions
Targeted deletion strains used in this study were made on the SL1344 strain background of S. enterica Typhimurium in which both subunits of flagellin, fliC and fljB, had been deleted through clean deletion of both genes (fliCfljB) or clean deletion of fliC and a kanamycin cassette insertion into fljB (fliCfljB ::kan) using standard methods22. When necessary, clean deletions were generated using the FRT recombinase22. Isogenic mutants were made on the fliCfljB strain background: fliCfljBacnB::kan,fliCfljBicdA::kan,fliCfljBaceA::kan, fliCfljBfumA::kan,fliCfljBsipB::cat,fliCfljBacnB::kansipB::cat,fliCfljBicdA::kansipB::cat, fliCfljBaceA: . kansipB: :cat, fliCfljBfumA : . kansipB: :cat, fliCfljBsseC: :kan,
fliCfljBsseCacnB::kan,fliCfljBsseCicdA::kan,fliCfljBsseCaceA::kan,fliCfljBsseCfumA::kan. TCA cycle mutations were moved into a fliCfljBsopEBE2 : :cat background. The sopEBElv.cat mutant strain was generated by sequential lamda re -mediated deletion of sopB and sopE2, followed by generation of an unmarked deletion of sopE by use of P22 lysate from SL1344 AsopE: :pSW24561, kindly provided by Sebastian Winter and Andreas Baumler (University of California Davis, Davis, CA). Bacteria were routinely grown at 37°C. For infection of cultured cells, bacteria were grown shaking overnight at 37°C in LB medium. Bacteria were diluted in LB containing 300 mM NaCl. Bacteria were grown standing for 3 hours to induce SPI-1 expression58. For mouse infections, bacteria were grown overnight with aeration at 37°C and diluted in PBS.
B. Genomic DNA isolation, PC and transposon insertion sequencing
Random transposition of Tn/0d::7¾ into Salmonella chromosome was achieved by use of a modified low-specificity transposase47 and T-POP85, provided by Eric Kofoid and John Roth (University of California Davis, Davis, CA). Genomic DNA was isolated from individual transposon mutants by phenol-chloroform extraction. A nested random priming method81 was used to determine the location of transposon insertion with the first round of PCR using a TetA primer (ACCTTTGGTCACCAACGCTTTTCC; SEQ ID NO: 5) together with a random primer (GTTTCCCAGTCACGATCNNNNNNNNN; SEQ ID NO: 6) at low stringency, followed by a second higher stringency PCR using a universal primer
(GACAAGATGTGTATCCACCTTACC; SEQ ID NO: 7) together with a primer containing the 5 ' defined sequence of the random primer (GTTTCCCAGTCACGATC; SEQ ID NO: 8). PCR reactions were purified and sequenced using the universal primer.
C. Mice and Macrophage infections
C57BL/6 (B6) mice were from NCI, 129S6/SvE mice were from Taconic. Knockout mice used in these studies were on the B6 background and have been previously described: Caspl'-Caspll1- 50 , Asc^ 91 , Nlrc^' 53 , Asc^Nlrc^ 13 , Nlrp ' 91 , Caspl^ 12 and Caspll^ 105 provided by Tiffany Horng and Junying Yuan (Harvard University, Boston, MA). Animals were maintained in accordance with the guidelines of the University of Pennsylvania
Institutional Animal Use and Care Committee. Bone marrow cells were grown at 37°C in a humidified incubator in DMEM containing HEPES, 10% FCS, and 30% L929 supernatant for 7-8 days. Differentiated BMDMs were replated into 24-, 48- or 96-well dishes 16-20 hours prior to infection. MCAT bone marrow was provided by A. Philip West and Gerald Shadel (Yale University, New Haven, CT). Bacterial strains described above were harvested, washed three times with DMEM, resuspended and added to the cells at an MOI of 20: 1. Bacteria were spun onto the cells at 1000 RPM for 5 minutes, and infected cells placed in a humidified tissue culture incubator at 37°C for 1 hour. Gentamicin was added to the cells 1 hour post-infection to a final concentration of 100 μg/mL, and the cells placed in the incubator until harvesting. 50 ng/mL LPS used in indicated experiments was E. coli 055 :B5 (Sigma). 2.5 mM ATP (Sigma) used in indicated experiments remained in well for 4 hours post-addition. For experiments utilizing MitoQ, BMDMs were pretreated with 50 ng/mL LPS 4 hours prior to infection for indicated conditions. 10 μΜ MitoQ (kindly provided by Narayan Avadhani and Satish Srinavasan, University of Pennsylvania, Philadelphia, PA) or vehicle control (DMSO) was added to cells one hour post-LPS treatment and remained for the duration of the experiment.
D. Cell death assays
BMDMs were seeded into 96-well plates at a density of 7x104 cells per well. The following day, culture medium was replaced with fresh DMEM. Cells were infected as described above and supernatants harvested at 4 hours post-infection. Lactate dehydrogenase release was quantified using the Cytotox96 Assay Kit (Clonetech) according to the manufacturer's instructions.
E. Cytokine production
BMDMs were seeded into 48-well plates at a density of 1.5xl05 cells per well. Cells were pretreated with E. coli LPS (Sigma) for 3 hours prior to bacterial infection as described above, and supernatants were harvested 4 hours post-infection. Release of proinflammatory cytokines was measured by enzyme-linked immunosorbent assay (ELISA) using capture and detection antibodies against IL-Ιβ (eBioscience), IL-6 (BD Biosciences), and IL- 12 (BD Biosciences).
F. Western blotting and antibodies
BMDMs were seeded into 24-well plates at a density of 3xl05 cells per well, and infected with bacteria as described above. 3 hours post-infection, cells were lysed in 20 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X- 100, and 1 mM EDTA. Lysates were mixed with protein loading buffer, boiled, centrifuged, and 20% of the total cell lysate loaded onto 4%-12% NuPAGE gels (Invitrogen). Proteins were transferred to PVDF membrane (Millipore) and blotted with rabbit anti-mouse caspase-1 antibody (Santa Cruz
Biotechnology). Secondary antibody was goat anti-rabbit HRP (Jackson Immunoresearch). The membrane was exposed on radiographical film using SuperSignal West Pico kit (Pierce).
G. Measurement of mitochondrial superoxide Mitochondrial superoxide (02 " ) was measured by using the mitochondrial O2" indicator, MitoSOX Red (molecular probes; Invitrogen). Briefly, Nlrc4' BMDMs were grown as described above and seeded into 35mm plastic petri dishes with glass coverslip bottoms. Infections were performed as described above. 1 μg LPS was added to indicated plate 6 hours prior to imaging. BMDMs were loaded with 5 μΜ MitoSOX Red for 30 minutes. Residual dye was removed by washing and dishes were mounted in an open perfusion microincubator and imaged by confocal microscopy. Confocal images were obtained using a Leica SP5-2 inverted confocal microscope at 561 nm excitation with a 63X oil objective. Images were analyzed by masking the perinuclear region of the cells and the mean MitoSOX Red fluorescence was quantified using Leica Application Suite software.
H. Metabolite analysis
Bacterial cultures were shaking overnight at 37°C in LB medium. Bacteria were diluted in LB containing 300 niM NaCl. Bacteria were grown standing to OD 0.5. Bacteria were immediately transferred to cold conical tubes and spun down at 4,000 rpm for 5 minutes at 4C. Bacterial pellets were washed with 0.8% saline solution, then pelleted. Metabolites were extracted with ice-cold 80% methanol and centrifuged for 10 minutes at 4C. D27-myristic acid was used as an internal reference standard. TCA metabolites were reduced with sodium borodeuteride and deuterated standards added as previously described62. Dried samples were resuspended in 30 μΐ anhydrous pyridine and added to GC-MS autoinjector vials containing 70 μΐ N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide derivatization reagent. The samples were incubated at 70°C for 1 hour, after which aliquots of 1 μΐ were injected for analysis. GC- MS data were collected on an Agilent 5975C series GC/MSD system (Agilent Technologies) operating in election ionization mode (70 eV) and selected ion monitoring. Quantified metabolites were normalized relative to cell number as described previously62.
H. Mouse infections
Mice were housed in accordance with National Institutes of Health (NIH)- and
University of Pennsylvania-approved guidelines, and all studies involving mice were performed in accordance with approved University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) protocols and procedures. Eight- to ten-week-old age- and sex- matched mice were intraperitoneally infected with 5x102 bacteria for C57BL/6 mice, and lxl O3 or lxlO4 bacteria for 129S6/SvE mice. For oral infections, C57BL/6 mice, or isogenic Nlrp3' or Caspl^'Caspll'1' mice were fasted overnight and intragastrically inoculated with 2x107 bacteria. For single infection experiments, unmarked fliCfljB, acnB::kan, or icdA::kan were used. For competition infections, 1x10 unmarked fliCfljB with 1x10 fliCfljB::kan, or lxlO3 unmarked fliCfljB with lxlO3 marked fliCfljBacnB : :kan were mixed 1 :1 and injected intraperitoneally. Mice were sacrificed, and the tissues and sera harvested at indicated times post-infection. Bacterial load was determined by plating dilutions of tissue homogenates on appropriate selective plates. For competitive index experiments, identical dilutions of homogenates were plated on both streptomycin and kanamycin plates, and CI was calculated in accordance with previously described methods6.
I. Statistical analysis
Kaplan-Meier curves were used to plot survival of infected mice. Plotting of data and statistical analysis was performed using Graphpad Prism 5.0 software. Statistical significance is indicated in respective figure legends was determined by unpaired two-tailed Student's t test or Mann- Whitney U test, as indicated. Error bars in all figures represent SEM.
EXAMPLE 2: Identifying Negative Regulators Of NLRP3 Inflammasome Activation
We therefore hypothesized that Stm might evade or prevent rapid activation of a canonical NLRP3 inflammasome, and that this evasion might contribute to systemic bacterial virulence. Several bacterial and viral pathogens evade NLRP3 inflammasome activation33'98, but whether Salmonella is capable of doing so is unknown.
To identify potential negative regulators of NLRP3 inflammasome activation, we generated and screened a transposon library of flagellin-deficient Stm mutants for elevated inflammasome activation in NL C4-deficient bone marrow-derived macrophages (BMDMs). Sequencing of candidate hits identified acnB, the gene encoding the TCA cycle enzyme aconitase, which converts citrate to isocitrate, as well as several other genes that had been previously isolated in a screen for Salmonella genes that are required for persistent Salmonella infection in vivo56. Intriguingly, isocitrate lyase (encoded by aceA), which generates glyoxylate from isocitrate in the glyoxylate cycle, contributes to persistent but not acute infection by Salmonella as well as Mycobacterium tuberculosis21,10.
To test the potential role of Salmonella TCA cycle metabolism in inflammasome modulation, we generated targeted deletions in acnB as well as genes encoding other TCA cycle enzymes. Notably, deletion of aconitase, isocitrate lyase, or isocitrate dehydrogenase (icdA), but not other TCA enzymes, induced rapid NLRP3 -dependent inflammasome activation in S¾m-infected macrophages, suggesting that activity of these enzymes limits NLRP3 inflammasome activation by intracellular Salmonella. Moreover, aconitase-deficient Salmonella exhibited a defect in acute systemic virulence following oral administration, and were deficient in their ability to persist in a chronic infection. These findings define the first genes that mediate NLRP3 inflammasome evasion by Salmonella and suggest that inflammasome evasion contributes to persistence of bacterial pathogens. Our data further suggest that sensing of bacterial metabolites may provide an additional level of innate immune recognition, and that regulation of metabolite production by intracellular pathogens represents a pathogen immune evasion strategy.
EXAMPLE 3: Identification of Salmonella genes that modulate inflammasome activation
The NLRP3 inflammasome can respond to the pore-forming activities of diverse bacterial secretion systems8'36'69. Salmonella expresses two such systems, SPI-1 and SPI-2, yet paradoxically, in the absence of flagellin and NL C4, NLRP3 inflammasome activation does not occur in bone marrow-derived macrophages (BMDMs) until 12-16 hours post-infection9'72. We therefore considered the possibility that Stm might evade or inhibit rapid SPI-1 - or SPI-2- dependent NLRP3 inflammasome activation and devised a screen to identify such modulators.
We first generated a library of individual Tn/0<i::7¾-based transposon insertions85 in a flagellin-deficient (fliCfljB) strain of Salmonella enterica serovar Typhimurium {Stm).
Candidate mutants were identified by increased LDH release relative to the fliCfljB-dsfici t parental strain 4 hours post-infection of immortalized Nlrc4' macrophages (Figs. 1A).
Candidate mutants were rescreened in triplicate in primary Nlrc4~'~ BMDMs and a secondary screen was performed in Caspl'''Caspll''' BMDMs to confirm that LDH release was indeed inflammasome-dependent (a representative group of five of initial candidate mutants is shown) (Fig. IB).
Candidate mutants were further tested for NLRC4-independent release of the caspase- 1 -dependent cytokine IL-Ι β (Fig. 1C), and normal secretion of caspase-1 -independent cytokines such as IL-6 or IL-12 (Fig. ID and data not shown). Intriguingly, sequencing the transposon junction of a subset of candidate mutants identified four genes {acnB, bcfB, rcsD, and melB) that had previously been isolated in a genome-wide screen for genes involved in Salmonella persistence56, consistent with the possibility that modulating inflammasome activation might promote persistent infection.
EXAMPLE 4: Deletion of Salmonella aconitase, isocitrate lysase, or isocitrate
dehydrogenase induces rapid NLRC4-independent NLRP3 inflammasome activation Two genes initially identified by Lawley et ol. in a genome-wide screen for genes required for long term persistence, acnB and icdA56, encode the TCA cycle enzymes aconitase and isocitrate dehydrogenase, respectively. Aconitase converts citrate to isocitrate, while isocitrate dehydrogenase subsequently converts isocitrate to alpha-ketoglutarate. This suggested that the TCA cycle of intracellular Salmonella might be involved in modulating inflammasome activation of infected cells. Interestingly, isocitrate lyase, a component of the glyoxylate cycle pathway that converts isocitrate to glyoxylate, also contributes to persistent infection of multiple bacterial pathogens, including Mycobacterium tuberculosis, Salmonella, Pseudomonas, and Burkholderia species27'60'70 101. However, its potential role in modulating inflammasome activation is unknown. To further assess the role of TCA or glyoxylate cycle genes in modulation of inflammasome activation, we generated in-frame deletions in icdA, sucAB,fumA, mdhA, and aceA, which encode isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, fumarase, malate dehydrogenase, and isocitrate lyase, respectively (Fig. 2A). Consistent with findings that the TCA cycle is dispensable for macrophage replication per se7, invasion and replication of Stm in Caspl^'Caspll'1' BMDMs were unaffected by deletion of TCA cycle genes (Fig. 2B).
Each of these mutants were capable of growth in rich medium, although as expected, icdA, sucAB and mdhA had growth defects in minimal medium with either glucose or glycerol as carbon sources (data not shown). Intriguingly, infection of either B6 or Nlrc4 ~'~ BMDMs with acnB, icdA, and aceA but not sucAB, mdhA, or fumA mutants resulted in greatly increased LDH release in comparison to BMDMs infected by the parental wild-type strain (Fig. 2C and data not shown). In contrast, Nlrp3' and Caspl^'Caspll'1' macrophages showed minimal levels of cytotoxicity in response to infection by acnB-, icdA-, and ace^-deficient bacteria, suggesting that the absence of Stm aconitase, isocitrate dehydrogenase, or isocitrate lyase induces NL P3 inflammasome activation. Consistently, caspase-1 processing was observed in cell lysates following infection of B6 and Nlrc4~/~ but not Nlrp3' BMDMs with acnB, aceA, or icdA mutants (Fig. 2D). Moreover, acnB, aceA, or icdA mutants induced significantly elevated IL-1 β secretion by B6 and Nlrc4' BMDMs, but not Nlrp3~/~ BMDMs, suggesting that NLRP3 activation was responsible for the increased production of IL-1 β (Fig. 2E). Importantly, secretion of caspase-1 -independent cytokines and expression of pro-IL-Ιβ itself was unaffected by the Stm TCA cycle genes, indicating that deletion of Stm TCA cycle genes did not impact TLR signaling per se (Fig. 2F and data not shown).
The residual IL-Ιβ secretion observed following infection of Nlrp3' BMDMs is likely due to activation of the NLRC4 inflammasome by the SPI-1 inner rod protein PrgJ (Miao et al, 2010b), as Nlrc^'Nlrp^' and Nlrc^Asc^ BMDMs did not exhibit LDH release or IL-Ιβ secretion (data not shown). EXAMPLE 5: Salmonella aconitase, isocitrate lysase, or isocitrate dehydrogenase mutants induce activation of a canonical NLRP3 inflammasome
Intracellular bacteria can activate the AIM2 inflammasome via release of bacterial DNA into the cytosol82'88'106. However, Aim2~ ~ BMDMs had no defect in inflammasome activation in response to acnB, icdA, or aceA mutants, suggesting that AIM2 was not involved in this response (data not shown). NL P3 can participate in non-canonical inflammasome activation in response to Gram-negative bacteria by a mechanism involving caspase- l l10'34'46'86. However, Caspl 1''' BMDMs had no defect in either LDH (Fig. 3A) or IL-Ιβ secretion (Fig. 3B) in response to infection by acnB, icdA, or aceA, whereas Caspl''' BMDMs were as defective as Caspl'1' Caspl l'1' BMDMs, indicating that these mutants activated a caspase-1- and NLRP3-dependent canonical inflammasome. Caspase-1 -independent cytokines were unaltered in response to infection by TCA cycle mutants or in the absence of caspase-1 and/or 11 , again indicating a lack of a direct role for Stm TCA cycle genes in modulating TLR signaling (Fig. 3C).
EXAMPLE 6: NLRP3 inflammasome activation in response to Salmonella TCA cycle mutants requires the bacterial SPI-1 T3SS
As the NLRP3 inflammasome can be activated by the T3SS of other bacteria, we next sought to test whether the rapid NLRP3 inflammasome activation induced by Stm TCA mutants involved T3SS activity. The SPI-1 T3SS promotes bacterial invasion into non- phagocytic cells30'31'58, whereas the SPI-2 T3SS is upregulated within the SCV and is required for intracellular replication within host macrophages17'35. Intriguingly, SipB, the integral membrane component of the SPI-1 translocon, but not SseC, the integral membrane component of the SPI-2 translocon, was required for LDH release and IL-Ιβ secretion in response to infection by the TCA cycle mutants (Fig. 4A and 4B). Importantly, caspase-1 - independent cytokines were unaffected by the presence or absence of SipB (Fig. 4C). The defect in inflammasome activation caused by sipB mutation was not due to reduced numbers of intracellular bacteria, as infection with a five-fold higher dose of szpfi-deficient bacteria was performed in order to ensure intracellular levels of WT and isogenic sipB mutant bacteria (data not shown). The SPI-2 T3SS plays a role in a caspase-1/11-dependent Salmonella-mduced late death9'77.
We therefore investigated the possibility that SPI-2 might play a role in later cell death induced by these TCA cycle mutants. No differences in cell death were detected between parental TCA cycle mutants and the isogenic sseC mutant strains over a 20-hour infection time-course, indicating that SPI-2 does not contribute to inflammasome activation by Stm TCA cycle mutants at either early or later timepoints (Fig. 4D). Altogether, these data suggested that either a signal from the SPI-1 T3SS or the activity of the SPI-1 T3SS in combination with disruption of specific TCA cycle genes triggers the NLRP3 inflammasome in response to Stm infection. In order to distinguish between these possibilities, we generated acnB, icdA, and aceA mutant strains that also had combined deficiency in three SPI-1 secreted effector proteins, SopE, SopE2, and SopB. Deletion of these genes greatly reduces Salmonella invasion of epithelial cells, but does not otherwise compromise the activity of the SPI-1 secretion system itself11'114.
Intriguingly, while each individual effector had a minimal effect on NLRP3 inflammasome activation (data not shown), the combined additional deletion of sopE, sopB, and sopE2 significantly reduced cytotoxicity and IL-Ιβ secretion in response to infection by acnB, icdA, or aceA mutant Salmonella (Fig. 4E and 4F). This was the case despite equivalent levels of bacterial uptake by macrophages as well as equivalent secretion of caspase-1- independent cytokines (Fig. 4G and data not shown).
These data suggest that a combined signal from SPI-1 -mediated disruption of host actin and a metabolic signal due to alteration of the Stm TCA cycle are responsible for inflammasome activation by the TCA cycle mutant bacterial strains. Interestingly, SopE triggers inflammasome activation in stromal cells and in macrophage cell lines independently of flagellin37'79, but the inflammasomes involved in this response have not been defined, and we did not observe a role for SopE alone in inflammasome activation in primary BMDMs. EXAMPLE 7: Stm TCA cycle mutants trigger NLRP3 inflammasome activation through mitochondrial ROS
Multiple models have been proposed for how the NL P3 inflammasome is activated in response to a large number of disparate stimuli55. Along with potassium efflux, reactive oxygen species (ROS) are a key regulatory signal for NLRP3 inflammasome
activation24'38'65'92 115. Mitochondrial ROS are reported to trigger NLRP3 inflammasome activation in response to uric acid and silica crystals65.
Notably, both the icdA and acnB mutant strains induced significantly elevated levels of mitochondrial ROS production than the isogenic parental fliCfljB strain, as measured by MitoSox staining of infected cells (Fig. 5A and 5B). Recent studies have demonstrated that ROS itself is not the direct signal for NLRP3 inflammasome activation, and indeed, LPS treatment of BMDMs triggers significant levels of ROS, but by itself is insufficient for robust inflammasome activation41'80.
We therefore asked whether this elevated mitochondrial ROS contributed to inflammasome activation during infection by Stm TCA mutants. Mitochondrial catalase (MCAT) transgenic mice overexpress human catalase targeted to the mitochondria, and therefore generate lower levels of mitochondrial hydrogen peroxide than wild-type mice59'89 108. Intriguingly, MCAT BMDMs failed to induce elevated LDH or IL-Ιβ secretion in response to infection by acnB, icdA, or aceA mutants compared to isogenic fliCfljB Stm, indicating that inflammasome activation by Stm TCA cycle mutants was limited by reduction of mitochondrial ROS (Fig. 5C and 5D).
Importantly, NLRP3 inflammasome activation was unaffected in response to
LPS+ATP, indicating that BMDMs from MCAT mice do not have a global defect in NLRP3 inflammasome activation. Furthermore, production of the inflammasome-independent cytokine IL-12 and expression of NLRP3 protein were unaffected in MCAT BMDMs, demonstrating that the effect of MCAT on the NLRP3 inflammasome was related to activation, and not due to reduced expression of inflammasome components (Fig. 5E and data not shown).
In accordance with our previous observations in Nlrp3' BMDMs, residual inflammasome activation in MCAT cells was likely due to an NLRC4-dependent response to the PrgJ protein. To test this, and to rule out a possible developmental effect of catalase overexpression, we acutely depleted mitochondrial ROS by treating cells with the mitochondrial-specific ROS scavenger MitoQ after the cells were primed with LPS43'94. Notably, treatment of Nlrc4' BMDMs with MitoQ prior to infection significantly reduced release of LDH and IL-Ιβ in response to Stm TCA cycle mutants, but did not affect secretion of inflammasome-independent cytokines or the response to LPS+ATP (Fig. 5F-H). Together, these data demonstrate a requirement for mitochondrial ROS in NLRP3 inflammasome activation triggered by Stm TCA cycle mutants.
EXAMPLE 8: NLRP3 inflammasome activation by Stm TCA cycle mutants requires bacterial citrate synthase
We next wanted to determine how deletion of TCA cycle genes involved in citrate or isocitrate metabolism might induce inflammasome activation. Accumulation of excess citrate in cells lacking aconitase or isocitrate dehydrogenase can inhibit phospho-fiructokinase, a rate- limiting enzyme of glycolysis100. Thus, deletion of aconitase or isocitrate dehydrogenase can result in growth inhibition coupled with export of excess citrate ' . However, whether deficiencies in these genes cause citrate accumulation in Stm has not been investigated.
Notably, profiling of TCA cycle metabolite levels in the acnB, icdA, aceA,fumA, and parental wild-type {fliCfljB) strains revealed that aconitase, isocitrate dehydrogenase or isocitrate lyase mutants all exhibited elevated levels of citrate, although relative citrate levels were most substantially increased in acnB and icdA mutants (Fig. 6A).
To test the possibility that excess citrate might be responsible for NLRP3
inflammasome activation, we sought to eliminate citrate production through the TCA cycle in the acnB, icdA, or aceA mutant strains by additionally deleting gltA, which encodes citrate synthase. Intriguingly, deletion of citrate synthase significantly reduced cytotoxicity and release of IL-Ιβ caused by acnB and icdA mutants (Fig. 6B and 6C). Lack of citrate synthase did not reduce inflammasome activation caused by aceA (data not shown), perhaps because deletion of aceA did not result in as high of an increase in citrate levels as the other two mutations. Together, these data suggest that excess buildup of bacterially-derived citrate triggers inflammasome activation in response to intracellular Salmonella.
EXAMPLE 9: AcnB limits inflammasome-dependent anti-bacterial immune responses during acute infection
Systemic NL C4 inflammasome activation in response to Salmonella flagellin promotes anti-bacterial immune defense and enhances in vivo bacterial clearance73. We therefore sought to test whether NLRP3 inflammasome activation triggered by altered Stm TCA cycle metabolism could also promote anti-bacterial defense in vivo. Aconitase-deficient Stm had no defect in virulence following intraperitoneal (i.p.) infection of C57BL/6 mice, and showed equivalent levels of spleen and liver colonization as parental fliCfljB bacteria (Fig. 7A and B). Mice infected with icdA mutant bacteria had significantly reduced tissue burdens in the liver and spleen following intraperitoneal infection, perhaps because of an overall reduced growth rate of the icdA mutant (data not shown).
We therefore utilized the acnB strain for subsequent studies. Although acnB deficiency did not result in loss of virulence following intraperitoneal infection, acnB mutant Stm exhibited significantly reduced splenic tissue burden 6 days post-intragastric inoculation compared with fliCfljB- fected mice (Fig. 7C). Intriguingly, despite these lower burdens, serum IL-18 levels of ac«i?-infected C57BL/6 mice were significantly elevated in comparison with mice infected by fliCfljB Stm (Fig. 7E), consistent with enhanced in vitro inflammasome responses against ac«i?-deficient Stm. Critically, the virulence defect of the acnB mutant was abrogated in Caspl 'Casp 11" mice, coinciding with significantly reduced serum IL-18 levels in Caspl ' Caspl 1~'~ mice infected with either bacterial strain. Nlrp3' mice also exhibited reduced serum IL-18 in response to the acnB mutant, and IL-18 production was not significantly different from Nlrp3' mice infected with fliCfljB bacteria, indicating that NLRP3 plays a functionally important in vivo role in IL-18, production in response to acnB infection (Fig. 7F).
Notably, Nlrp3' mice did not exhibit statistically significant differences in either CFU or serum IL-18 between the acnB and fliCfljB strains (Fig. 7D and F). Nlrp3' mice also succumbed to acnB mutant infection in higher numbers than B6 mice, providing further support for the role of NLRP3 in inflammasome activation in vivo in the context of infection by aconitase-deficient Stm.
EXAMPLE 1 0: AcnB contributes to persistent Salmonella infection in vivo
Given the observation that isocitrate lyase contributes to persistence of various bacterial pathogens27'60'70 101, we sought to test the role of aconitase in promoting Salmonella persistence. Stm infection of B6 mice results in acute lethal systemic infection due to an inactivating mutation in Nrampl in B6 mice103. Nrampl encodes a lysosomal transporter that exports divalent metal cations from the SCV, and limits Salmonella intracellular
replication28'42.
To test the role of AcnB in chronic Stm infection, we utilized 129S6/SvE (129) mice, which express functional NRAMP1 protein103. Intraperitoneal infection of 129 mice with 1000 CFU of WT Stm results in a chronic infection in 129 mice76. Importantly, under these conditions, we observed similar bacterial burdens in the mesenteric lymph nodes (MLNs), spleens, and livers of parental {fliCfljB)- or ac«i?-infected mice in the early stages of persistent infection, at days 7 and 21 post-infection (Fig. 8A-C and data not shown). However, at later times post-infection (day 60), bacterial burdens in the spleens of ac«i?-infected mice were significantly lower compared to mice infected with isogenic aconitase-sufficient bacteria, although bacterial numbers in the livers were not significantly different at this time (Fig. 8B and C).
In competitive infection in which 129S6/SvE mice were infected with equal numbers of fliCfljB and acwfi-deficient Stm, acwfi-deficient bacteria had a significant competitive disadvantage at both 7 and 21 days post-infection in the spleens and livers, but not in the MLNs (Fig. 8D and data not shown). Finally, 129S6/SvE mice survived an elevated infectious dose (10,000 CFU i.p.) of acwS-deficient Stm, but succumbed over 2 to 3 weeks to infection by isogenic acwfi-sufficient bacteria (Fig. 8E). Interestingly, day 7 bacterial burdens in 129S6/SvE mice infected with the higher dose were similar in spleens and livers, although bacterial numbers were statistically lower in the spleens of acwfi-infected mice (Fig. 8F). There was no difference in the bacterial burdens at this timepoint in the livers between infected mouse groups (Fig. 8G).
Importantly, similar to our findings in oral infection of B6 mice, acwfi-infected mice exhibited significantly higher levels of serum IL-18 compared with fliCfljB -infected animals, whereas IL-6 levels were equivalent (Fig. 8H and I). Altogether, these data indicate that infection with acwfi-deficient Salmonella triggers inflammasome activation in vivo.
DISCUSSION OF THE RESULTS OF EXAMPLES 1 -10
In the above examples, the inventors demonstrated that Stm mutants deficient in aconitase or isocitrate dehydrogenase, but not other TCA cycle enzymes, induce rapid canonical NLRP3 inflammasome activation in bone marrow derived lymph nodes (BMDMs). Notably, this inflammasome activation correlated with elevated levels of bacterial citrate, and was abrogated upon deletion of citrate synthase (gltA), supporting a role for citrate in the triggering of the NLRP3 inflammasome. Surprisingly, this inflammasome activation was dependent on both the genes encoding bacterial citrate synthase and the SPI-1 effector proteins that mediate Stm invasion. Thus, the innate immune system can detect intracellular bacteria not only through sensing of bacterial proteins and virulence activities, but also by combining sensing of bacterial metabolites with sensing of virulence activity.
Regulating levels of citrate production by acnB and icdA during infection is likely an active mechanism of evading inflammasome activation. These enzymes normally function in a fundamental biosynthetic pathway. However, acnB is not essential for either intra- macrophage replication or virulence in the context of certain routes of in vivo infection.
Intriguingly, the Salmonella genome contains two aconitase enzymes, but deletion of acnB alone is sufficient to mediate inflammasome activation. Thus dynamic regulation of AcnB functions in vivo to promote immune evasion.
Although icdA and acnB were not required for intracellular replication of Stm in vitro, these mutants were attenuated during in vivo infection: icdA was attenuated following intraperitoneal infection of NrampS B6 mice. There may be a metabolic requirement for icdA during in vivo infection. Interestingly, while acnB was dispensable for intraperitoneal infection of C57BL/6 mice, acnB- deficient bacteria were significantly attenuated following oral infection of B6 mice. acnB mutant bacteria also had a defect during persistent infection in NramplR 129S6/SvE mice in both single and competitive infection, indicating that absence of acnB impacts in vivo bacterial virulence. Notably, despite having reduced bacterial tissue burdens, mice infected with acwfi-deficient Stm displayed significantly higher levels of serum IL-18 than mice infected with the parental acwfi-sufficient bacteria, indicating that Salmonella lacking acnB induce elevated inflammasome activation in vivo.
Altered bacterial citrate production may lead to altered production of other bacterial metabolites, which may themselves be the proximal triggers of NL P3 activation. For example, mitochondrial cardiolipin was recently reported to be a direct ligand for NLRP341. Cardiolipin is also a constituent phospholipid of bacterial membranes, and cardiolipin metabolism may be altered as a consequence of TCA cycle dysregulation. Future studies will determine whether infected macrophages respond directly to elevated cytoplasmic levels of citrate or whether other bacterial metabolites are involved. Signatures of bacterial viability, termed vita-PAMPs or PAMPs per vita produced by metabolically active microbes, have been proposed to constitute an additional level of innate immune sensing, together with the classical pathogen associated molecular patterns or PAMPs as originally formulated by Janeway44'87 102. Bacterial mRNA 45 87 and cyclic di-nucleotides110 function as vita-PAMPs during bacterial infection. Recent studies have revealed that production of butyrate by intestinal bacteria can impact inflammation by control of intestinal regulatory T cell populations1 15'93. Our data provide evidence that intracellular bacterial metabolites serve as another class of vita-PAMP that signal inflammasome activation.
What is the mechanism by which aconitase- or isocitrate dehydrogenase-deficient Stm induce rapid activation of the NLRP3 inflammasome? This inflammasome activation is unlikely to be due to lysis of bacteria and cytosolic release of bacterial nucleic acids as it is independent of AIM2. It is also dependent solely on caspase-1, indicating that it does not mobilize a non-canonical caspase-11 inflammasome. Interestingly, both acnB and icdA mutants induced elevated levels of mitochondrial ROS production from infected macrophages, and limiting mROS production by either genetic or pharmacological methods eliminated inflammasome activation in response to TCA mutant Stm. Our data therefore support a link between core metabolic processes of intracellular bacteria and mitochondria in regulating inflammasome activation.
The precise role and sources of ROS in inflammasome activation in response to different stimuli remains mysterious55. ROS plays a role both in the activation of the inflammasome complex itself, as well as in upregulation of NLRP3 inflammasome components2. Notably, NLRP3 inflammasome activation appears to mobilize different cellular sources of OS for different stimuli, as LPS+ATP induces NLRP3 inflammasome activation through a pathway involving NOX2 (encoded by gp91phox), rather than mitochondrial ROS65'78. Previous studies have demonstrated a requirement for mitochondrial ROS in antimicrobial killing downstream of TLR signaling during Salmonella infection108. Our studies now provide a link between mitochondrial ROS and NLRP3 inflammasome activation in response to alteration of Salmonella citrate metabolism. These findings provide evidence that while the Salmonella TCA cycle is dispensable for intracellular replication, it enables Salmonella to evade the NLRP3 inflammasome by limiting the production of bacterial citrate.
Although icdA and acnB were not required for intracellular replication of Stm in vitro, these mutants were attenuated during in vivo infection: icdA was attenuated following intraperitoneal infection of Nramps B6 mice, although it is not currently clear whether this relates to a metabolic requirement for icdA during in vivo infection. Interestingly, while acnB was dispensable for intraperitoneal infection of C57BL/6 mice, acwfi-deficient bacteria were significantly attenuated following oral infection of B6 mice. acnB mutant bacteria also had a defect during persistent infection in NramplR 129S6/SvE mice in both single and competitive infection, indicating that absence of acnB impacts in vivo bacterial virulence. Notably, despite having reduced bacterial tissue burdens, mice infected with acwfi-deficient Stm displayed significantly higher levels of serum IL-18 than mice infected with the parental acwfi-sufficient bacteria, indicating that Salmonella lacking acnB induce elevated inflammasome activation in vivo. Indeed, Caspl~'~Caspll~'~ and Nlrp3' mice mice showed significantly reduced production of IL-18 in response to acnB Stm.
EXAMPLE 1 1 : Method To Generate Improved Salmonella Vaccine Strains
The above examples demonstrated that Salmonella enterica serovar Typhimurium (Stm) strains lacking or functionally deleted in aconitase (acnB) or the functionally related gene isocitrate dehydrogenase (icdA) induce elevated inflammatory responses in vitro111. The examples further demonstrated that acwfi-deficient Stm induced elevated inflammatory responses in vivo, and are cleared more rapidly following infection. In particular, acnB- deficient Stm induce elevated levels of a specific immune response, termed 'inflammasome activation' that is associated with inflammatory cell death and release of key effector cytokines that mediate adaptive immunity. Because deletion of acnB confers increased immunogenicity on Stm strains, this protocol is an example of generating a useful and improved Salmonella vaccine using Stm strains lacking the aroA gene as attenuated vaccine strains. Alone, the immunogenicity of aro -deficient Salmonella is notably poor. Engineered to be deficient in acnB or icdA, it the attenuated, double mutant strain induces innate immunity in an animal to which it is administered and function as an improved vaccine.
In order to construct an aroA acnB double mutant Stm strain, we first generated P22 transducing phage lysates from acnBv.Km mutant Stm strains which we had previously constructed by homologous recombination of Kanamycin resistance into the acnB locus. aroA mutant bacteria have been previously described116. We performed phage transduction of the acnBv.Km mutation into the aroA strain background, selected for Km1 bacteria, and verified the presence of the acnB deletion by PC .
To determine whether deletion of acnB enhances the efficacy of vaccination, we orally immunize mice with aroA single mutant bacteria, or aroA acnB double-mutant bacteria. This is done by feeding of these bacterial mutant strains to mice (109 organisms per mouse).
Induction of adaptive immunity is assessed by measuring Salmonella-specific CD4 and CD8 T cell responses on day 7 post- infection, antibody responses at day 21 post-infection, and protection against a lethal Salmonella challenge.
EXAMPLE 12: Method To Utilize Aroa AcnB Salmonella As Vaccine Delivery Vehicle Or Platform
Based on the anticipated results that acnB confers increased immunogenicity to the aroA bacterial strains, a combined aroA acnB strain should enable the generation of robust immune responses against heterologous antigens that are expressed in Salmonella. In particular, heterologous antigens from organisms for which there are no or only poor vaccines, as well as tumor antigens serve as particularly attractive targets. Expression of heterologous antigens in the aroA acnB strain background is achieved by using a constitutive Salmonella enterica promoter, such as rpsM, and insertion of the rpsM promoter and DNA encoding the heterologous antigen together onto the Salmonella chromosome at a specific site, termed the attTn7 site. The site is specifically targeted by transposition of the Tn7 transposon, and insertion of the Tn7 transposon into this site does not disrupt any other Salmonella genes.
We test the ability of the aroA acnB Salmonella strain to induce immune protection against several enteric bacterial pathogens, notably Listeria monocytogenes, Enteropathogenic E. coli, and Helicobacter pylori. In the case of Listeria, we utilize the bacterial internalin gene, inlA. In the case of pathogenic E. coli species, we utilize a bacterial adhesin, iieC. In the case of Helicobacter, we utilize the gene encoding the bacterial virulence factor cagA.
Specifically, we construct aroA acnB Salmonella strains that individually express these particular genes. As described above, we then vaccinate mice with these strains by orogastric inoculation. Subsequent to vaccination, the mice will be exposed to lethal challenge with the respective microorganisms. We anticipate that aroA acnB strains that express heterologous antigens will provide robust immune protection against infection by
microorganisms that naturally express these antigens.
An extension of using the aroA acnB strain as a platform for vaccination against intestinal bacterial pathogens is the use of aroA acnB strain as a platform for vaccination against tumor antigens. We utilize the same method of tumor antigen expression in the aroA acnB strain as described above, namely, the att7«7 site for insertion by miniTn7 phage. The first tumor antigen that we express is murine Vascular Endothelial Growth Factor Receptor 2, VEGFR2. This antigen is expressed from the constitutive rpsM promoter. This receptor is upregulated on many different tumors, and therefore provides an attractive target for antitumor vaccination. Following vaccination with VEGFR2-expressing or control bacterial strains, mice are challenged with B16F10 tumor cells in a flank tumor melanoma model. Protection is evaluated by assessment of tumor burden and levels of lung tumor metastases. EXAMPLE 1 3: Vaccination Challenge Studies
Attenuated, modified Stm deleted in the acnB gene (i.e., SEQ ID NO: 1 was deleted) and an attenuated modified Stm deleted in the icdA gene (i.e., SEQ ID NO: 3 was deleted) as described above were employed as Salmonella vaccine candidates in chicken.
Experiment A
Day old chicken were divided into groups. Group 1 (n=5) was a non-immunized control challenge. Group 2 (n=5) was orally vaccinated by gavage with about 105 to 106 bacteria in 100 microliters saline of wild-type Stm. Groups 3 through 5 (n=5 for each group) were orally vaccinated by gavage with about 105 to 106 bacteria in 100 microliters saline of fliCfliB (Group 3), the attenuated modified, SEQ ID NO: 1 -deleted Stm (Group 4), and the attenuated modified, SEQ ID NO: 3-deleted Stm (Group 5).
Each group of chickens was challenged 3 weeks after immunization with a chicken strain of Salmonella Typhimurium (ST). Two days after challenge, the chicken were examined to determine if and the extent to which the vaccinations protected the chicken from organ (liver) invasion (i.e., whether ST bacteria can be detected in the livers of the chicken without enrichment - accuracy down to 103 bacteria). Table 1 demonstrates the results.
TABLE 1
Figure imgf000045_0001
Experiment B
In another experiment, the same protocol was used to immunize five similar groups of day old chicken, were divided into groups. All groups had 5 chickens. Groups 1 to 5 were vaccinated as described in the first experiment. Each group of chickens was challenged 3 weeks after immunization with a chicken strain of Salmonella Typhimurium (ST). In this experiment, the chicken were examined seven days after challenge for cecal (intestinal) colonization by the chicken ST (i.e., with bacterial enrichment). Table 2 demonstrates the results.
TABLE 2
Figure imgf000045_0002
Experiment C
In another experiment, the same vaccination protocol was employed as for Experiment A. However, bacterial enrichment was added when the organ invasion was calculated. With bacterial enrichment, the results were as described in Table 3. TABLE 3
Figure imgf000046_0001
Experiment D
In another experiment, the same vaccination protocol was employed as for Experiment B. However, bacterial enrichment was added when cecal colonization was calculated. With bacterial enrichment, the results were as described in Table 4.
TABLE 4
Figure imgf000046_0002
It should be noted that due to the two different techniques to test for organ invasion, it is possible to find 0 chickens positive, but still find cfu after bacterial enrichment. These results demonstrate that both Stm attenuated mutants reduced Salmonella colonization of chicken and thus work well as vaccines.
Each and every patent, patent application, including US provisional application NO. 61/954,269 and non-patent publications, including publications listed herein, and each and every publically available nucleic acid, amino acid, peptide and vector sequence, cited throughout the disclosure, is expressly incorporated herein by reference in its entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention are devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include such embodiments and equivalent variations.
TABLE 1
(Sequence Listing Free Text)
The following information is provided for sequences containing free text under numeric identifier <223>.
Figure imgf000047_0001
PUBLICATIONS
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Claims

CLAIMS:
1. An attenuated, genetically modified Salmonella bacterium deficient in the naturally occurring gene encoding a bacterial TCA enzyme aconitase (acn) or isocitrate dehydrogenase (icd).
2. The bacterium according to claim 1, having a complete or partial deletion of the naturally occurring acn or icd gene encoding the selected TCA enzyme in the bacterium's genome that reduces, inhibits or ablates the function of the encoded gene.
3. The bacterium according to claim 1, comprising an antibiotic resistance gene.
4. The bacterium according to claim 4, wherein the antibiotic resistance gene is present in the position of the wholly or partially deleted gene encoding the selected TCA enzyme in the bacterial chromosome.
5. The bacterium according to claim 1, which is a strain of Salmonella enterica.
6. The bacterium according to claim 5, which is the strain Salmonella enterica serovar Typhimurium.
7. The bacterium according to claim 6, which is the strain Salmonella enterica serovar Typhimurium aroA.
8. The bacterium according to claim 6, which is the strain Salmonella enterica serovar Typhimurium, strain SL1344.
9. The bacterium according to claim 1 , which is a wildtype Salmonella attenuated prior to deletion of the selected TCA enzyme.
10. The bacterium according to any of claims 1 to 9, produced via bacteriophage transduction or homologous recombination.
11. The bacterium according to any of claims 1 to 9, wherein the genetic modification is the deletion or functional deletion of the Salmonella aconitase gene SEQ ID NO: 1 of Salmonella enterica Typhimurium, strain SL1344 or of a homologous or identical sequence of an acn gene of another Salmonella.
12. The bacterium according to claim 11, wherein the acn gene has a sequence that shares from 75 to 99% sequence identity with SEQ ID NO: 1.
13. The bacterium according to any of claims 1 to 9, wherein the genetic modification is the deletion or functional deletion of the Salmonella isocitrate dehydrogenase (icd) gene SEQ ID NO: 3 of Salmonella enterica Typhimurium, strain SL1344 or of a homologous or identical sequence of an icd gene of another Salmonella strain.
14. The bacterium according to claim 13, wherein the icd gene has a sequence that shares from 75 to 99% sequence identity with SEQ ID NO: 1.
15. The bacterium according to claim 1, wherein the genetic modification is the deletion or functional deletion of both acn and icd genes.
16. The bacterium according to claim 1 , that induces in an animal, upon infection, the release of inflammatory cytokines from macrophages or dendritic cells and an increase in the animal's innate immune response to the bacterium.
17. The bacterium according to claim 1 or 15, further comprising a target gene inserted in the bacterial chromosome.
18. The bacterium according to claim 17, wherein the target gene is a heterologous gene from another infectious microorganism.
19. The bacterium according to claim 18, wherein the target gene is a heterologous gene from a mammalian tumor or cancer cell.
20. The bacterium according to claim 19, wherein the target gene is a Salmonella gene located at a site different from that in naturally occurring Salmonella or under the control of a non-naturally occurring promoter.
21. The bacterium of claim 1 in a pharmaceutically acceptable carrier.
22. A composition comprising a bacterium according to claim 21 and an optional adjuvant.
23. A method of inducing the innate immune response of an animal against infection by a Salmonella strain comprising administering the bacterium of any of claims 1 -9 or 15 or the composition of claim 21 to an animal in need thereof.
24. The method according to claim 23, wherein said animal is avian or mammalian.
25. The method according to claim 23, wherein said animal is a livestock animal used for human consumption.
26. The method according to claim 24, wherein the animal is a human.
27. A composition comprising the bacterium of any of claims 1 to 9 or 15 for use in inducing the innate immune response of an animal against infection by Salmonella.
28. A method of inducing the innate immune response of an animal against a microbial infection or cancer comprising administering a composition comprising a bacterium of any of claims 1 to 9 or 15, and further comprising a target gene inserted in the bacterial chromosome, to an animal or mammal in need thereof.
29. The method according to claim 28, wherein the target gene is a heterologous gene from another infectious microorganism.
30. The method according to claim 28, wherein the target gene is a heterologous gene from a mammalian tumor or cancer cell.
31. A method of generating a Salmonella vaccine comprising modifying a naturally occurring gene encoding a TCA enzyme aconitase or isocitrate dehydrogenase in a wildtype or attenuated Salmonella bacterium to reduce, inhibit or ablate the function of the encoded gene.
32. The method according to claim 30, wherein said modification comprises a whole or partial deletion of the gene.
33. The method according to claim 30, wherein said modification comprises inserting into the gene sequence another sequence that disrupts the translation of the gene into the encoded enzyme.
34. The method according to claims 32, comprising replacing the deleted or partially deleted TCA enzyme-encoding gene with, or inserting into the TCA enzyme-encoding gene sequence, an antibiotic resistance gene.
35. The method according to claim 32, comprising replacing the deleted or partially deleted TCA enzyme-encoding gene with, or inserting into the TCA enzyme-encoding gene sequence, another selected gene.
36. The method according to any one of claims claim 31 to 35, comprising using bacteriophage transduction or homologous recombination to effect the deletion or replacement.
37. A method of generating a composition that induces the innate immune response against a target gene comprising modifying a naturally occurring gene encoding a TCA enzyme aconitase or isocitrate dehydrogenase in the chromosome of a wildtype or attenuated Salmonella bacterium and inserting into the bacterial genome a heterologous target gene from another infectious microorganism or from a cancer or tumor cell.
38. The method according to claim 37, wherein said modification comprises a whole or partial deletion of the gene.
39. The method according to claim 37, wherein said modification comprises a whole or partial deletion of both the acn and icd genes.
40. The method according to claim 37, wherein said modification comprises inserting into the TCA enzyme- encoding gene sequence the heterologous target gene that disrupts the translation of the TCA enzyme- encoding gene into the encoded enzyme.
41. The method according to any of claims 37 to 40, comprising using bacteriophage transduction or homologous recombination to effect the deletion or insertion.
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