Tuberculosis (TB) remains the leading cause of bacterial disease-related death and is among the t... more Tuberculosis (TB) remains the leading cause of bacterial disease-related death and is among the top 10 overall causes of death worldwide. The complex nature of this infectious lung disease has proven difficult to treat, and significant research efforts are now evaluating the feasibility of host-directed, adjunctive therapies.
Tetraspanins are a family of membrane proteins regulating cell morphology, motility, fusion, and ... more Tetraspanins are a family of membrane proteins regulating cell morphology, motility, fusion, and signaling. CD151 is expressed on T cells that stabilizes the immune synapse during antigen recognition, engages in integrin signaling, and primes T cell activation. We have recently demonstrated that CD151 expression leads to increased proliferation and activation in human CD4 + T cells. Given its important functional role, we hypothesized that CD151 may be elevated on M. tb -specific effector CD4 + T cells. We assessed CD151 on ex vivo PBMCs from PPD+ donors (Purified Protein Derivative from M. tb ) stimulated with M. tb H37Rv whole cell lysate. CD151 + frequencies were elevated on IFN-γ + (52 ± 6% vs. 17 ± 2%, P + (74 ± 6% vs. 17 ± 2%, P + T cells compared to overall baseline levels. To assess the functionality of CD151 on M. tb -specific CD4 + T cells, we established an ex vivo culture model with M. tb -infected monocyte-derived macrophages (MDMs) in the presence of autologous PPD+ or...
The global antimicrobial resistance crisis poses a significant threat to humankind in the coming ... more The global antimicrobial resistance crisis poses a significant threat to humankind in the coming decades. Challenges associated with the development of novel antibiotics underscore the urgent need to develop alternative treatment strategies to combat bacterial infections. Host-directed therapy is a promising new therapeutic strategy that aims to boost the host immune response to bacteria rather than target the pathogen itself, thereby circumventing the development of antibiotic resistance. However, host-directed therapy depends on the identification of druggable host targets or proteins with key functions in antibacterial defense. Protein Kinase R (PKR) is a well-characterized human kinase with established roles in cancer, metabolic disorders, neurodegeneration, and antiviral defense. However, its role in antibacterial defense has been surprisingly underappreciated. Although the canonical role of PKR is to inhibit protein translation during viral infection, this kinase senses and re...
Tuberculosis (TB) is a deadly infectious lung disease caused by the pathogenic bacterium Mycobact... more Tuberculosis (TB) is a deadly infectious lung disease caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). The identification of macrophage signaling proteins exploited by Mtb during infection will enable the development of alternative host-directed therapies (HDT) for TB. HDT strategies will boost host immunity to restrict the intracellular replication of Mtb and therefore hold promise to overcome antimicrobial resistance, a growing crisis in TB therapy. Protein Kinase R (PKR) is a key host sensor that functions in the cellular antiviral response. However, its role in defense against intracellular bacterial pathogens is not clearly defined. Herein, we demonstrate that expression and activation of PKR is upregulated in macrophages infected with Mtb. Immunological profiling of human THP-1 macrophages that overexpress PKR (THP-PKR) showed increased production of IP-10 and reduced production of IL-6, two cytokines that are reported to activate and inhibit IFNγ-dependent...
Mycobacterium tuberculosis (Mtb) kills infected macrophages by inhibiting apoptosis and promoting... more Mycobacterium tuberculosis (Mtb) kills infected macrophages by inhibiting apoptosis and promoting necrosis. The tuberculosis necrotizing toxin (TNT) is a secreted nicotinamide adenine dinucleotide (NAD) glycohydrolase that induces necrosis in infected macrophages. Here, we show that NAD depletion by TNT activates RIPK3 and MLKL, key mediators of necroptosis. Notably, Mtb bypasses the canonical necroptosis pathway since neither TNF-α nor RIPK1 are required for macrophage death. Macrophage necroptosis is associated with depolarized mitochondria and impaired ATP synthesis, known hallmarks of Mtb-induced cell death. These results identify TNT as the main trigger of necroptosis in Mtb-infected macrophages. Surprisingly, NAD depletion itself was sufficient to trigger necroptosis in a RIPK3- and MLKL-dependent manner by inhibiting the NAD salvage pathway in THP-1 cells or by TNT expression in Jurkat T cells. These findings suggest avenues for host-directed therapies to treat tuberculosis a...
Differentiation of circulating monocytes into tissue-bound or tissue-resident macrophages is a cr... more Differentiation of circulating monocytes into tissue-bound or tissue-resident macrophages is a critical regulatory process affecting host defense and inflammation. However, the regulatory signaling pathways that control the differentiation of monocytes into specific and distinct functional macrophage subsets are poorly understood. Herein, we demonstrate that monocyte-to-macrophage differentiation is controlled by the Protein Phosphatase, Mg/Mn-dependent 1A (PPM1A). Genetic manipulation experiments demonstrated that overexpression of PPM1A attenuated the macrophage differentiation program, while knockdown of PPM1A expression accelerated the ability of monocytes to differentiate into macrophages. We identify imiquimod and Pam3CSK4 as two Toll-like receptor agonists that induce PPM1A expression, and show that increased expression of PPM1A at the onset of differentiation impairs cellular adherence, reduces expression of inflammatory (M1) macrophage-specific markers, and inhibits the pro...
The ability to suppress host macrophage apoptosis is essential for M. tuberculosis (Mtb) to repli... more The ability to suppress host macrophage apoptosis is essential for M. tuberculosis (Mtb) to replicate intracellularly while protecting it from antibiotic treatment. We recently described that Mtb infection upregulated expression of the host phosphatase PPM1A, which impairs the antibacterial response of macrophages. Here we establish PPM1A as a checkpoint target used by Mtb to suppress macrophage apoptosis. Overproduction of PPM1A suppressed apoptosis of Mtb-infected macrophages by a mechanism that involves inactivation of the c-Jun N-terminal kinase (JNK). Targeted depletion of PPM1A by shRNA or inhibition of PPM1A activity by sanguinarine restored JNK activation, resulting in increased apoptosis of Mtb-infected macrophages. We also demonstrate that activation of JNK by subtoxic concentrations of anisomycin induced selective apoptotic killing of Mtb-infected human macrophages, which was completely blocked in the presence of a specific JNK inhibitor. Finally, selective killing of Mtb...
Antimicrobial agents and chemotherapy, Oct 1, 2016
Copper (Cu) ions are likely the most important immunological metal-related toxin utilized in cont... more Copper (Cu) ions are likely the most important immunological metal-related toxin utilized in controlling bacterial infections. Impairment of bacterial Cu resistance reduces viability within the host. Thus, pharmacological enhancement of Cu-mediated antibacterial toxicity may lead to novel strategies in drug discovery and development. Screening for Cu toxicity-enhancing antibacterial molecules identified 8-hydroxyquinoline (8HQ) to be a potent Cu-dependent bactericidal inhibitor of Mycobacterium tuberculosis The MIC of 8HQ in the presence of Cu was 0.16 μM for replicating and nonreplicating M. tuberculosis cells. We found 8HQ's activity to be dependent on the presence of extracellular Cu and to be related to an increase in cell-associated labile Cu ions. Both findings are consistent with 8HQ acting as a Cu ionophore. Accordingly, we identified the 1:1 complex of 8HQ and Cu to be its active form, with Zn, Fe, or Mn neither enhancing nor reducing its Cu-specific action. This is rem...
Assay and drug development technologies, Jan 21, 2016
In the last 40 years, only a single new antituberculosis drug was FDA approved. New tools that im... more In the last 40 years, only a single new antituberculosis drug was FDA approved. New tools that improve the drug development process will be essential to accelerate the development of next-generation antituberculosis drugs. The drug development process seems to be hampered by the inefficient transition of initially promising hits to candidate compounds that are effective in vivo. In this study, we introduce an inexpensive, rapid, and BSL-2 compatible infection model using macrophage-passaged Mycobacterium tuberculosis (Mtb) that forms densely packed Mtb/macrophage aggregate structures suitable for drug efficacy testing. Susceptibility to antituberculosis drugs determined with this Mtb/macrophage aggregate model differed from commonly used in vitro broth-grown single-cell Mtb cultures. Importantly, altered drug susceptibility correlated well with the reported ability of the respective drugs to generate high tissue and cerebrospinal fluid concentrations relative to their serum concentr...
Co-infection with HIV-1 and Mycobacterium tuberculosis (Mtb) is a major public health issue. Whil... more Co-infection with HIV-1 and Mycobacterium tuberculosis (Mtb) is a major public health issue. While some research has described how each pathogen accelerates the course of infection of the other pathogen by compromising the immune system, very little is known about the molecular biology of HIV-1/Mtb co-infection at the host cell level. This is somewhat surprising, as both pathogens are known to replicate and persist in macrophages. We here identify Protein Phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) as a molecular link between Mtb infection and increased HIV-1 susceptibility of macrophages. We demonstrate that both Mtb and HIV-1 infection induce the expression of PPM1A in primary human monocyte/macrophages and THP-1 cells. Genetic manipulation studies revealed that increased PPMA1 expression rendered THP-1 cells highly susceptible to HIV-1 infection, while depletion of PPM1A rendered them relatively resistant to HIV-1 infection. At the same time, increased PPM1A expression abrogated ...
Proceedings of the National Academy of Sciences, 2014
Significance The mechanisms that enable Mycobacterium tuberculosis , the causative agent of tuber... more Significance The mechanisms that enable Mycobacterium tuberculosis , the causative agent of tuberculosis, to resist drug treatment and survive the immune response are poorly understood. In this study we discovered that M. tuberculosis produces the protein channel protein with necrosis-inducing toxin (CpnT), which forms a channel in the outer membrane and releases a toxic domain into the extracellular milieu. This toxin has no similarity to known bacterial toxins and kills eukaryotic cells by necrosis, suggesting that it is required for escape of M. tuberculosis from macrophages and for dissemination. The channel domain of CpnT is used for uptake of nutrients across the outer membrane. Taken together, CpnT is a protein with functions in two fundamental processes in M. tuberculosis physiology: nutrient acquisition and control of host cell death.
The dynamic and coordinated exchange of multiple GTPases between the cytosol and the phagosome me... more The dynamic and coordinated exchange of multiple GTPases between the cytosol and the phagosome membrane represents a critical process during phagosome biogenesis. In particular, acquisition of Rab7 is crucial for progression to the stage where formation of phagolysosomes is observed. Optimal Rab7 effector function requires its conversion to the GTP-bound form where it becomes activated. In light of this regulatory node, the GDP/GTP switch on the Rab7 molecule represents a tractable event to dissect the control of phagosome maturation by intracellular pathogen or their products. Direct measurement of Rab7 activation requires 32P-GTP binding to renatured Rab7 recovered by pull downs and resolved by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and autoradiography. Here, we describe a novel, alternative, nonradioactive assay to measure Rab7 activity which takes advantage of the specific binding of activated (GTP bound) Rab7 to its effector RILP (Rab7 interacting lysosomal protein). Active Rab7 bound to immobilized recombinant RILP on latex beads can be detected quantitatively by either classical Western blotting or flow cytometry.
The increased incidence of tuberculosis (TB) gave impetus for the increased interest in the study... more The increased incidence of tuberculosis (TB) gave impetus for the increased interest in the study of mycobacterial genetics, which culminated in the publication of the full genome sequence of many mycobacterial strains. Since then, many genes and open reading frames of unknown function have been described and the expression of their encoded proteins is critical toward understanding the pathogenesis of TB and developing therapeutic and preventive strategies. Therefore there is an increased need for highly efficient methods for cloning of mycobacterial genes, as the limited cloning flexibility of current Escherichia coli-mycobacteria shuttle vectors remains a frequent impediment in genetic manipulation of mycobacteria. In order to overcome this limitation, we have converted representative extrachromosomal and integrative vectors into multiple destination mycobacterial vectors for one-step and restriction enzyme-free recombination cloning methodology that uses in vitro site-specific recombination. We provide several examples that highlight the potential of recombination cloning for gene expression in slow and fast-growing mycobacteria. Thus, a gene of interest can be transferred by simple recombination into our mycobacterial destination vectors, which serve a multitude of functional genomic studies.
Adaptive gene expression in prokaryotes is mediated by protein kinases and phosphatases. These re... more Adaptive gene expression in prokaryotes is mediated by protein kinases and phosphatases. These regulatory proteins mediate phosphorylation of histidine or aspartate in two-component systems and serine/threonine or tyrosine in eukaryotic and eukaryote-like protein kinase systems. The genome sequence of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, does not possess a defined tyrosine kinase. Nevertheless, it encodes for protein tyrosine phosphatases. Here, we report that Map1985, is a functional low-molecular tyrosine phosphatase that is secreted intracellularly upon macrophage infection. This finding suggests that Map1985 might contribute to the pathogenesis of Mycobacterium avium subsp. paratuberculosis by dephosphorylating essential macrophage signaling and/or adaptor molecules.
Tuberculosis (TB) remains the leading cause of bacterial disease-related death and is among the t... more Tuberculosis (TB) remains the leading cause of bacterial disease-related death and is among the top 10 overall causes of death worldwide. The complex nature of this infectious lung disease has proven difficult to treat, and significant research efforts are now evaluating the feasibility of host-directed, adjunctive therapies.
Tetraspanins are a family of membrane proteins regulating cell morphology, motility, fusion, and ... more Tetraspanins are a family of membrane proteins regulating cell morphology, motility, fusion, and signaling. CD151 is expressed on T cells that stabilizes the immune synapse during antigen recognition, engages in integrin signaling, and primes T cell activation. We have recently demonstrated that CD151 expression leads to increased proliferation and activation in human CD4 + T cells. Given its important functional role, we hypothesized that CD151 may be elevated on M. tb -specific effector CD4 + T cells. We assessed CD151 on ex vivo PBMCs from PPD+ donors (Purified Protein Derivative from M. tb ) stimulated with M. tb H37Rv whole cell lysate. CD151 + frequencies were elevated on IFN-γ + (52 ± 6% vs. 17 ± 2%, P + (74 ± 6% vs. 17 ± 2%, P + T cells compared to overall baseline levels. To assess the functionality of CD151 on M. tb -specific CD4 + T cells, we established an ex vivo culture model with M. tb -infected monocyte-derived macrophages (MDMs) in the presence of autologous PPD+ or...
The global antimicrobial resistance crisis poses a significant threat to humankind in the coming ... more The global antimicrobial resistance crisis poses a significant threat to humankind in the coming decades. Challenges associated with the development of novel antibiotics underscore the urgent need to develop alternative treatment strategies to combat bacterial infections. Host-directed therapy is a promising new therapeutic strategy that aims to boost the host immune response to bacteria rather than target the pathogen itself, thereby circumventing the development of antibiotic resistance. However, host-directed therapy depends on the identification of druggable host targets or proteins with key functions in antibacterial defense. Protein Kinase R (PKR) is a well-characterized human kinase with established roles in cancer, metabolic disorders, neurodegeneration, and antiviral defense. However, its role in antibacterial defense has been surprisingly underappreciated. Although the canonical role of PKR is to inhibit protein translation during viral infection, this kinase senses and re...
Tuberculosis (TB) is a deadly infectious lung disease caused by the pathogenic bacterium Mycobact... more Tuberculosis (TB) is a deadly infectious lung disease caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). The identification of macrophage signaling proteins exploited by Mtb during infection will enable the development of alternative host-directed therapies (HDT) for TB. HDT strategies will boost host immunity to restrict the intracellular replication of Mtb and therefore hold promise to overcome antimicrobial resistance, a growing crisis in TB therapy. Protein Kinase R (PKR) is a key host sensor that functions in the cellular antiviral response. However, its role in defense against intracellular bacterial pathogens is not clearly defined. Herein, we demonstrate that expression and activation of PKR is upregulated in macrophages infected with Mtb. Immunological profiling of human THP-1 macrophages that overexpress PKR (THP-PKR) showed increased production of IP-10 and reduced production of IL-6, two cytokines that are reported to activate and inhibit IFNγ-dependent...
Mycobacterium tuberculosis (Mtb) kills infected macrophages by inhibiting apoptosis and promoting... more Mycobacterium tuberculosis (Mtb) kills infected macrophages by inhibiting apoptosis and promoting necrosis. The tuberculosis necrotizing toxin (TNT) is a secreted nicotinamide adenine dinucleotide (NAD) glycohydrolase that induces necrosis in infected macrophages. Here, we show that NAD depletion by TNT activates RIPK3 and MLKL, key mediators of necroptosis. Notably, Mtb bypasses the canonical necroptosis pathway since neither TNF-α nor RIPK1 are required for macrophage death. Macrophage necroptosis is associated with depolarized mitochondria and impaired ATP synthesis, known hallmarks of Mtb-induced cell death. These results identify TNT as the main trigger of necroptosis in Mtb-infected macrophages. Surprisingly, NAD depletion itself was sufficient to trigger necroptosis in a RIPK3- and MLKL-dependent manner by inhibiting the NAD salvage pathway in THP-1 cells or by TNT expression in Jurkat T cells. These findings suggest avenues for host-directed therapies to treat tuberculosis a...
Differentiation of circulating monocytes into tissue-bound or tissue-resident macrophages is a cr... more Differentiation of circulating monocytes into tissue-bound or tissue-resident macrophages is a critical regulatory process affecting host defense and inflammation. However, the regulatory signaling pathways that control the differentiation of monocytes into specific and distinct functional macrophage subsets are poorly understood. Herein, we demonstrate that monocyte-to-macrophage differentiation is controlled by the Protein Phosphatase, Mg/Mn-dependent 1A (PPM1A). Genetic manipulation experiments demonstrated that overexpression of PPM1A attenuated the macrophage differentiation program, while knockdown of PPM1A expression accelerated the ability of monocytes to differentiate into macrophages. We identify imiquimod and Pam3CSK4 as two Toll-like receptor agonists that induce PPM1A expression, and show that increased expression of PPM1A at the onset of differentiation impairs cellular adherence, reduces expression of inflammatory (M1) macrophage-specific markers, and inhibits the pro...
The ability to suppress host macrophage apoptosis is essential for M. tuberculosis (Mtb) to repli... more The ability to suppress host macrophage apoptosis is essential for M. tuberculosis (Mtb) to replicate intracellularly while protecting it from antibiotic treatment. We recently described that Mtb infection upregulated expression of the host phosphatase PPM1A, which impairs the antibacterial response of macrophages. Here we establish PPM1A as a checkpoint target used by Mtb to suppress macrophage apoptosis. Overproduction of PPM1A suppressed apoptosis of Mtb-infected macrophages by a mechanism that involves inactivation of the c-Jun N-terminal kinase (JNK). Targeted depletion of PPM1A by shRNA or inhibition of PPM1A activity by sanguinarine restored JNK activation, resulting in increased apoptosis of Mtb-infected macrophages. We also demonstrate that activation of JNK by subtoxic concentrations of anisomycin induced selective apoptotic killing of Mtb-infected human macrophages, which was completely blocked in the presence of a specific JNK inhibitor. Finally, selective killing of Mtb...
Antimicrobial agents and chemotherapy, Oct 1, 2016
Copper (Cu) ions are likely the most important immunological metal-related toxin utilized in cont... more Copper (Cu) ions are likely the most important immunological metal-related toxin utilized in controlling bacterial infections. Impairment of bacterial Cu resistance reduces viability within the host. Thus, pharmacological enhancement of Cu-mediated antibacterial toxicity may lead to novel strategies in drug discovery and development. Screening for Cu toxicity-enhancing antibacterial molecules identified 8-hydroxyquinoline (8HQ) to be a potent Cu-dependent bactericidal inhibitor of Mycobacterium tuberculosis The MIC of 8HQ in the presence of Cu was 0.16 μM for replicating and nonreplicating M. tuberculosis cells. We found 8HQ's activity to be dependent on the presence of extracellular Cu and to be related to an increase in cell-associated labile Cu ions. Both findings are consistent with 8HQ acting as a Cu ionophore. Accordingly, we identified the 1:1 complex of 8HQ and Cu to be its active form, with Zn, Fe, or Mn neither enhancing nor reducing its Cu-specific action. This is rem...
Assay and drug development technologies, Jan 21, 2016
In the last 40 years, only a single new antituberculosis drug was FDA approved. New tools that im... more In the last 40 years, only a single new antituberculosis drug was FDA approved. New tools that improve the drug development process will be essential to accelerate the development of next-generation antituberculosis drugs. The drug development process seems to be hampered by the inefficient transition of initially promising hits to candidate compounds that are effective in vivo. In this study, we introduce an inexpensive, rapid, and BSL-2 compatible infection model using macrophage-passaged Mycobacterium tuberculosis (Mtb) that forms densely packed Mtb/macrophage aggregate structures suitable for drug efficacy testing. Susceptibility to antituberculosis drugs determined with this Mtb/macrophage aggregate model differed from commonly used in vitro broth-grown single-cell Mtb cultures. Importantly, altered drug susceptibility correlated well with the reported ability of the respective drugs to generate high tissue and cerebrospinal fluid concentrations relative to their serum concentr...
Co-infection with HIV-1 and Mycobacterium tuberculosis (Mtb) is a major public health issue. Whil... more Co-infection with HIV-1 and Mycobacterium tuberculosis (Mtb) is a major public health issue. While some research has described how each pathogen accelerates the course of infection of the other pathogen by compromising the immune system, very little is known about the molecular biology of HIV-1/Mtb co-infection at the host cell level. This is somewhat surprising, as both pathogens are known to replicate and persist in macrophages. We here identify Protein Phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) as a molecular link between Mtb infection and increased HIV-1 susceptibility of macrophages. We demonstrate that both Mtb and HIV-1 infection induce the expression of PPM1A in primary human monocyte/macrophages and THP-1 cells. Genetic manipulation studies revealed that increased PPMA1 expression rendered THP-1 cells highly susceptible to HIV-1 infection, while depletion of PPM1A rendered them relatively resistant to HIV-1 infection. At the same time, increased PPM1A expression abrogated ...
Proceedings of the National Academy of Sciences, 2014
Significance The mechanisms that enable Mycobacterium tuberculosis , the causative agent of tuber... more Significance The mechanisms that enable Mycobacterium tuberculosis , the causative agent of tuberculosis, to resist drug treatment and survive the immune response are poorly understood. In this study we discovered that M. tuberculosis produces the protein channel protein with necrosis-inducing toxin (CpnT), which forms a channel in the outer membrane and releases a toxic domain into the extracellular milieu. This toxin has no similarity to known bacterial toxins and kills eukaryotic cells by necrosis, suggesting that it is required for escape of M. tuberculosis from macrophages and for dissemination. The channel domain of CpnT is used for uptake of nutrients across the outer membrane. Taken together, CpnT is a protein with functions in two fundamental processes in M. tuberculosis physiology: nutrient acquisition and control of host cell death.
The dynamic and coordinated exchange of multiple GTPases between the cytosol and the phagosome me... more The dynamic and coordinated exchange of multiple GTPases between the cytosol and the phagosome membrane represents a critical process during phagosome biogenesis. In particular, acquisition of Rab7 is crucial for progression to the stage where formation of phagolysosomes is observed. Optimal Rab7 effector function requires its conversion to the GTP-bound form where it becomes activated. In light of this regulatory node, the GDP/GTP switch on the Rab7 molecule represents a tractable event to dissect the control of phagosome maturation by intracellular pathogen or their products. Direct measurement of Rab7 activation requires 32P-GTP binding to renatured Rab7 recovered by pull downs and resolved by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and autoradiography. Here, we describe a novel, alternative, nonradioactive assay to measure Rab7 activity which takes advantage of the specific binding of activated (GTP bound) Rab7 to its effector RILP (Rab7 interacting lysosomal protein). Active Rab7 bound to immobilized recombinant RILP on latex beads can be detected quantitatively by either classical Western blotting or flow cytometry.
The increased incidence of tuberculosis (TB) gave impetus for the increased interest in the study... more The increased incidence of tuberculosis (TB) gave impetus for the increased interest in the study of mycobacterial genetics, which culminated in the publication of the full genome sequence of many mycobacterial strains. Since then, many genes and open reading frames of unknown function have been described and the expression of their encoded proteins is critical toward understanding the pathogenesis of TB and developing therapeutic and preventive strategies. Therefore there is an increased need for highly efficient methods for cloning of mycobacterial genes, as the limited cloning flexibility of current Escherichia coli-mycobacteria shuttle vectors remains a frequent impediment in genetic manipulation of mycobacteria. In order to overcome this limitation, we have converted representative extrachromosomal and integrative vectors into multiple destination mycobacterial vectors for one-step and restriction enzyme-free recombination cloning methodology that uses in vitro site-specific recombination. We provide several examples that highlight the potential of recombination cloning for gene expression in slow and fast-growing mycobacteria. Thus, a gene of interest can be transferred by simple recombination into our mycobacterial destination vectors, which serve a multitude of functional genomic studies.
Adaptive gene expression in prokaryotes is mediated by protein kinases and phosphatases. These re... more Adaptive gene expression in prokaryotes is mediated by protein kinases and phosphatases. These regulatory proteins mediate phosphorylation of histidine or aspartate in two-component systems and serine/threonine or tyrosine in eukaryotic and eukaryote-like protein kinase systems. The genome sequence of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, does not possess a defined tyrosine kinase. Nevertheless, it encodes for protein tyrosine phosphatases. Here, we report that Map1985, is a functional low-molecular tyrosine phosphatase that is secreted intracellularly upon macrophage infection. This finding suggests that Map1985 might contribute to the pathogenesis of Mycobacterium avium subsp. paratuberculosis by dephosphorylating essential macrophage signaling and/or adaptor molecules.
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