WO2001055398A1 - Burkholderia toxins - Google Patents

Abstract

A novel composition comprising toxins produced from Burkholderia species is described and is effective in inhibiting nematode growth.

Description

TITLE OF THE INVENTION

Burkholderia Toxins

This application claims benefit of an earlier filed Provisional application serial no. 60/178,029 filed on January 26, 2000.

Field of the invention

This invention relates to toxins produced from Burkholderia sp . and methods of use of such toxins as therapeutic compositions and as agricultural agents .

INTRODUCTION

Two closely related bacteria of the genus Burkholderia, B . mallei , the causative agent of human and equine glanders, and B. pseudomallei , which causes melioidosis, have been recognized as potential biowarfare agents . Relatively little is known about the molecular events involved in the pathogenesis caused by either of these two microorganisms.

Burkholderia mallei and B . pseudomallei are related by their nucleotide sequence similarity and by the etiology of the diseases they cause (Neubauer and Meyer 1997, Revue Internationale des Service de Sante des Forces Ar ee, 70, 258-65; Woods, DeShazer et al . 1999, Microb. Infect. 1-7, 1-5). The clinical manifestations of both infections range from pyoderma to fatal septicemia. Bacteria have been isolated from every organ in severely infected individuals , including the brain (Asche 1991, Today's Life Science, June: 34-40). Human infections with either organism frequently result in septic-pneumonia, and can be fatal even under aggressive antibiotic therapies (Neubauer and Meyer 1997, supra; Alibek and Handelman 1999, Biohazard. New York, Random House; Dance 1999, In Tropical Infectious Diseases: principles, pathogens and practice . R. L. Geurrant et al . Philadelphia, Churchill Livingstone. 430-437; Woods, DeShazer et al . 1999, supra) .

Burkholderia mallei is an obligate pathogen that has been eliminated from North America and some parts of the world by quarantine and slaughter of infected horses (Neubauer and Meyer 1997, supra) . In contrast, Burkholderia pseudomallei is a soil saprophyte endemic in southeast Asia and Australia (for review see Yabuuchi and Arakawa 1993, Microbiol . Immunol. 37, 823-836; Dance 1999, supra; Woods, DeShazer et al . 1999, supra). Typically, fatal human infections with B . pseudomallei correlate with host risk factors including chronic alcoholism or diabetes . The relationship between these conditions and the defects in host defense leading to a fatal clinical outcome is unknown. The saprophytic life-style and the existence of known risk factors suggest that B . pseudomallei is an opportunistic human pathogen. Despite the association of severe disease with compromised hosts, both bacterial species have shown they are highly infectious in humans if aerosol exposure occurs (Neubauer and Meyer 1997, supra; Alibek and Handelman 1999, supra) . Person-to-person spread has thus far been rare (McCormick, Sexton et al . 1975, Annal . Int. Med. 83, 512-513; Kunakorn, Jayanetra et al . 1991, Lancet 337, 1290-1) . Intensive study of the molecular pathogenesis mechanisms employed by potential bio- warfare agents may lead to more effective countermeasures, including both therapeutic treatments and protective vaccines . Recently, the human pathogen, Pseudomonaε aeruginosa, was shown to use an overlapping set of virulence factors to cause disease in mice, plants, insects, and nematodes (Rahme, Stevens et al . 1995, Science 268, 1899-1902; Tan, Maha an-Miklos et al .

1999, Proc. Natl . Acad. Sci . USA 96, 715-20). Use of this discovery has led to the development of non- vertebrate genetic model systems to screen for bacterial virulence factors , and defense mechanisms in the animal host (Mahajan-Miklos, Tan et al . 1999, supra; Tan, Mahajan-Miklos et al . 1999, supra; Tan, Rahme et al . 1999, Proc. Natl. Acad. Sci. USA 96, 2408-2413). Because non-vertebrate models have few ethical or cost constraints as subjects for experimentation and are genetically tractable themselves (C. elegans and Arabidopsis thaliana) , extensive in vivo mutant screens have been developed. Many genes that would not have otherwise been identifiable as pathogenesis factors have been discovered by using lower-order hosts (Mahajan-Miklos, Tan et al . 1999, Cell 96, 47-56; Tan, Mahajan-Miklos et al. 1999, supra; Tan, Rahme et al . 1999, supra). Importantly, host targets and resistance factors can also be identified and characterized in these systems (Darby, Cosma et al . 1999, Proc. Natl. Acad. Sci. USA 96, 152002-7) . This report documents the development and characterization of the first nematode- Burkholderia model for identifying vertebrate virulence factors. SUMMARY

We investigated a non-mammalian host model system for fitness in genetic screening for virulence- attenuating mutations in the potential biowarfare agents Burkholderia pseudomallei and B. mallei . We determined that Burkholderia pseudomallei is able to cause disease-like symptoms and kill the nematode Caenorhabidi tis elegans . Analysis of killing in the surrogate disease model with B . pseudomallei mutants indicated that killing did not require lipopolysacharride (LPS) O-antigen, aminoglycoside/macrolide efflux pumping, type II pathway secreted exo-enzymes, or motility. Burkholderia thailandensis and some strains of B. cepacia also killed nematodes . Manipulation of the nematode host genotype suggests that the neuromuscular intoxication caused by both B. pseudomallei and B. thailandensis acts in part through a disruption of normal Ca⁺² signal transduction. Both species produce a UV sensitive, gamma-irradiation resistant, limited diffusion, paralytic agent as a part of their nematode pathogenic mechanism. The results of this investigation suggest that killing by B . pseudomallei is an active process in C. elegans, i.e. the killing was not due to starvation, and the C. elegans model might be useful for the identification of vertebrate animal virulence factors in B. pseudomallei .

Therefore, the present invention relates to a composition comprising Burkholderia vertebrate virulence factors or toxins . The factors or toxins can be isolated from one or more species of

Burkholderia and combined to produce an effective vertebrate vaccine against Burkholderia infections and to treat a nematode infection in vertebrates . These toxin compositions responsible for killing nematodes can also be utilized in agricultural settings to combat nematodes which cause yield losses to farmers .

Therefore, it is an object of the present invention to provide a vaccine against Burkholderia bacteria comprising toxins produced from Burkholderia pseudomallei , B . mallei , B . cepacia , and/or B. thailandensis in an amount effective to elicit protective antibodies in an animal against said bacteria and a pharmaceutically acceptable diluent, carrier, or excipient. It is another object of the present invention to provide a method for preparing a vaccine against Burkholderia comprising isolating toxins produced from Burkholderia species.

It is yet another object of the present invention to provide a composition for inhibiting nematode growth in a subject or plant, the composition comprising toxins produced from Burkholderia species, such as Burkholderia thailandensis .

We have found that the Burkholeria toxins have a Ca⁺² channel blocking activity and since most of the signalling pathways in nematodes and mammals are conserved, the toxins my be useful as Ca⁺² channel agonists .

Further objects and advantages of the present invention will be clear from the description that follows .

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1) Kinetic analysis of nematode killing by Burkholderia sp. Values plotted represent averages from 3 analyses . Standard Deviation of the mean values (SD) is shown (error bars) . Open squares represent N2 animals fed B . vietnamiensis FCO 369 (negative control) , solid triangles, solid line are B. pseudomallei 1026b, open triangles are B. pseudomallei NCTC 4845, solid triangles, dashed line, are B. thailandensis E264, and open circles are nematodes that have been starved - no food during the experiment .

Figure 2) Live and dead bacteria kill nematodes at different rates. Depicts a killing analysis of multi-staged N2 animals feeding upon gamma irradiated or live E264. N=2. Data are mean values still moving ± SD(error bars) . The killing kinetic for live E264 is indicated in solid lines, while that of gamma- irradiated E264 is depicted in dashed line.

Figure 3A and 3B C. elegans genotype modulates survival. (3A) egl -9 vs E264 and PAO on MYOB. Comparison of the relative pathogenicity of P. aeruginosa PAO and B. thailandensis E264, and the effect of two alleles of the egl -9 gene (wild- type(N2), and (n571) ) Values plotted represent averages from 3 independent analyses . Open diamonds represent nematodes fed E. coli OP50. Wild-type N2 Bristol animals are depicted by diamonds . Solid diamond, dashed line represents animals fed upon MYOB grown P. aeruginosa PAO, while solid diamond, solid lines are MYOB grown B. thailandensis E264. Open circle, dashed line represent C. elegans egl -9 (n571) homozygotes . Open circles, solid line represents egl- 9 (n571) /E264 values. SD is shown as error bars. L4 animals were used for each assay. While egl-9 (n586) homozygous animals are not depicted, they performed similar to the egl-9 (n571 ) homozygotes, confirming the results of Darby et.al. (Darby, Cosma et al . 1999, supra) . Notice that PAO has little killing activity under MYOB media relative to E264. Under BHI media PAO kills wild-type, but not egl -9 mutants; E264 kills both egl -9 mutants and wild-type, although egl -9 mutations enhance survivorship. (3B) Ca⁺² signal transduction mutations affects survivorship vs. E264. The differential effect upon survivorship of L-type Ca⁺² channel genotype. Values plotted represent mean values from four triple-point analyses. Filled diamond, solid lines = egl-1.9 (n2368) , and open diamond, solid lines indicates egl-19 (n582 ) . Open circles, solid line represent unc-36 (n251) homozygotes, while wild-type N2 is indicated by filled triangles, solid line, unc-43 (n498) homozygous animals are depicted by solid squares, solid line. Because many of the mutant lines grew more slowly than the rest of the lines analyzed, developmental synchronization of all of the lines for simultaneous assay was impossible; therefore, 40-100 L4 /young adult animals were hand picked for each assay. The LT50's were calculated from these data (see procedures) .

DETAILED DESCRIPTION

The present invention relates to toxins from Burkholderia species capable of producing disease-like symptoms in nematodes.

To establish a non-vertebrate host model system for studying pathogenic bacteria, a plate mortality assay was used. The assay utilized a standard C. elegans solid growth medium that had the usual food source (E. coli ) substituted with a potential pathogen. The outcome was determined by identifying the 'victor' . If the nematodes were unaffected, within a few days, they devoured the lawn of bacteria and thousands of progeny nematodes were visible on the plate. However, if the bacteria kill the nematodes, few if any of the progenitors transferred to the pathogenic lawn survived. Intermediate pathogenesis contained modest nematode population growth.

Four strains of Burkholderia pseudomallei ; 4 strains of B. mallei ; 7 strains of the B . cepacia complex; a single strain each of B. thailandensis , B. cocovenenans , B . pyrrocinia, and Ralstonia pickettei were tested for their ability to kill nematodes . Many strains killed the nematodes or only allowed a modest population growth relative to the E. coli OP50 negative control strain. Under MYOB medium conditions B. pseudomallei , B . thailandensis, and B . cepacia species were nematocidal, but not the B. mallei isolates. The B . pyrrocinia, B . cocovenenans, and R . pickettei isolates had a weak effect upon population growth. The strain-specific differential in population size suggested variable killing. An LT50 assay (Tan, Rahme et al . 1999, supra) was employed to explore nematode killing. The LT50 is the calculated time at which 50% of the nematode population was observed to cease movement (see Materials and Methods below) . Differences in LT50 times represent different rates of killing. The genus Burkholderia is continuously expanding and it is highly likely that other species and strains of Burkholderia not tested here would contain similar toxins. The level of toxicity of these bacteria can be determined by using the assay described above and calculating the LT50 for each bacterial toxin. Any toxin described herein or later discovered can be used as an antihelminthic to treat nematode infections as long as an LT50 is measured wherein the rate of production of offspring is not faster than the rate they die such that population growth does not occur.

The toxins can be prepared from any strain of Burkholderia bacteria, including but not limited to, Burkholderia pseudomallei , B . mallei , B . cepacia, B . thailandensis, B . cocovenenans, B . pyrrocinia, and Ralstonia pickettei .

In addition to wild type Burkholderia bacteria, mutants of these organisms may be useful, such as those which have reduced side effects in treated subjects or in which the toxin is inactivated or attenuated. UV inactivated B . pseudomallei and B. thailandensis were no longer nematocidal. In marked contrast, gamma-irradiated lawns of B. thailandensis and B . pseudomallei were nematocidal. The toxins can, for example be attenuated by UV inactivation, heat, protease, acid or base treatments, to name a few.

In one embodiment, this invention relates to a method for isolating and purifying the nematode toxins from Burkholderia . A crude toxin extract can be added to soil as a disinfectant prior to planting for use as crop preservative/protectant or after planting as a treatment for plants which are infested with nematodes . The crude extract can comprise complete bacteria whole or disrupted, chopped or powdered, can be lyophilized, or attached to another particle for ease in transfer to a plant. In a preferred embodiment, toxin extract from B. thailandensis is used since it is not a human pathogen. The extract can be applied in an amount sufficient to effect the desired result. For further purification of the toxin, cells and membrane fragments are removed from the solution by methods known in the art such as centrifugation, differential centrifugation, filtration, microfiltration, ultrafiltration, however, ultracentrifugation is preferable for removing the small membrane fragments and the solution is subjected ion-exchange chromatography. Other processes might include isoelectric focusing, gel chromatography. Following disruption, the toxin may be separated from the cellular debris by any technique suitable for separation of particles in complex mixtures. The toxins may then be purified by well known isolation techniques. Suitable techniques for purification include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, electrophoresis, immunoadsorption, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, liquid chromatography (LC) , high performance LC (HPLC) , fast performance LC (FPLC) , hydroxylapatite chromatography and lectin chromatography. Anion exchangers include diethyla inoethyl (DEAE) {-OCH2CH2N

+H(CH2CH3)2); quaternary aminoethyl (QAE) {- OCH2CH2N+(C2H5)-CH2CHOH-CH3}; and quaternary ammonium (Q) {-OCH2CHOH-CH3CHOH-CH2N+(CH3)C3} . Such functional groups are bound to various supports, each support varying in particle size, but also vary with respect to the support material. Examples of support material include: Monobeads , 10 urn bead of hydrophilic polystyrene/divinylbenzene{i . e . , Mono Q (Pharmacia)}, Minibeads, 3 urn bead of a hydrophilic polymer {i.e., Mini Q (Pharmacia)}, 15 & 30 urn monodispersed hydrophilized rigid, polystyrene/divinylbenzene beads {i.e., Q (Pharmacia)} Sepharose, 34-50 urn highly crosslinked agarose beads {i.e., HiTrap Q (Pharmacia) and Econo-Pac High Q (Bio-Rad) } Sepharose Fast Flow, 90 urn agarose beads {i.e., QSepharose Fast Flow

(Pharmacia)}, Sepharose Big Beads, 100-300 urn agarose beads {i.e., QSepharose Big Beads (Pharmacia)}.

The chloride ion (Cl^") is the counterion of choice for anion exchange chromatography, with the choice of buffer dependent on the required pH interval. While Tris has a an effective buffering range of 7.6 to 8.0, other buffers which may be used include: N-methyl-diethanolamine (pH 8.0-8.5), diethanolamine (pH 8.4-8.8), 1, 3-diamino-propane (pH 8.5-9.0), ethanolamine (pH 9.0-9.5), and potentially piperazine (pH 9.5-9.8). These buffers are used at a low concentration, usually 20mM, but could be as high as 50 M.

Other columns or methods may be used as long as they maintain native structure of the toxins so that immunogenicity and function is intact, allow large volumes of a dilute protein solution to be loaded and concentrated, the buffers are biologically compatible, the method is rapid in order minimize degradation of product and few processing steps required.

It is preferable that each column be dedicated to a specific strain of Burkholderia . The optimal toxin concentration in the final product would be approximately 10 doses per ml. But the range could be as low as 0.1 dose per ml up to much higher levels of 5000 doses per ml as long as solubility is maintained, i.e. concentration not too high to cause precipitation and not too low may make filtration too costly and time consuming. If toxin concentration is too low then it must be concentrated by centrifugal size-exclusion filtration (mw cutoff of 10000 to 100,000 more preferably 30,000 mw cutoff) .

Once the toxin is isolated, it can be subjected to sequence analysis in order to indentify both DNA and amino acid sequence. In another embodiment, the present invention relates to a recombinant DNA molecule that includes a vector and a DNA sequence as described above . The vector can take the form of a plasmid, phage, cosmid, YAC, eukaryotic expression vector such as a DNA vector, Pichia pastoris, or a virus vector such as for example, baculovirus vectors, retroviral vectors or adenoviral vectors, and others known in the art. The cloned gene may optionally be placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences, or sequences which may be inducible and/or cell type- specific. Suitable promoters will be known to a person with ordinary skill in the art. The expression construct will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. In one embodiment, the expression vector can be expressed in one or more plant cells of a plant such that the toxin is produced in an effort to reduce the ability of the nematode to harm the plant.

Introduction of the nucleic acid molecules or vectors into a host cell to produce a transformed host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al . , Basic Methods In Molecular Biology (1986) , Current Protocols in

Molecular Biology, Ausubel, F. M. et al . (Eds), Wiley S Sons, Inc. or Sherman et al . , 1986, Methods in Yeast Genetics . Cold Spring Harbor Laboratory Press, New York. Transformations into yeast are typically carried out according to the method of Van Solingen et al . , 1977, J. Bact . , 130, 946 and Hsiao et al . 1979, Proc Natl Acad Sci USA 76, 3829-3833. All documents cited herein supra and infra are hereby incorporated in their entirety by referece thereto. In a further embodiment, the present invention relates to host cells stably transformed or transfected with the above-described recombinant DNA constructs. The host cell can be prokaryotic (for example, bacterial) , lower eukaryotic (for example, yeast or insect) or higher eukaryotic (for example, all mammals, including but not limited to rat and human) and plant cells derived from agronomical or horticultural species including, monocot, dicot, and gymnosperm species as well as nonvascular plants.

Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences which are compatible with the designated host are used. Among prokaryotic hosts, E. coli is most frequently used. Expression control sequences for prokaryotes include promoters, optionally containing operator portions, and ribosome binding sites. Transfer vectors compatible with prokaryotic hosts are commonly derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers. These markers may be used to obtain successful transformants by selection. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning. Volumes I and II (D. N. Glover ed. 1985) for general cloning methods. The DNA sequence can be present in the vector operably linked to a sequence encoding an IgG molecule, an adjuvant, a carrier, or an agent for aid in purification of the toxin, such as glutathione S-transferase, or a series of histidine residues also known as a histidine tag. The recombinant molecule can be suitable for transfecting eukaryotic cells, for example, mammalian cells and yeast cells in culture systems. Saccharomyces cerevisiae, Saccharomyces carlsbergensis , and Pichia pastoris are the most commonly used yeast hosts, and are convenient fungal hosts. Control sequences for yeast vectors are known in the art. Mammalian and plant cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC) , such as HEK293 cells , and NIH 3T3 cells, to name a few. Suitable mammalian and plant promoters are also known in the art and include viral promoters such as that from SV40, Rous sarcoma virus (RSV) , adenoviruε (ADV) , bovine papilloma virus (BPV) , and cytomegalovirus (CMV) and for plants, 35S Cauliflower Mosaic Virus (CaMV 35S) , mannopine synthase (mas) and octopine synthase (ocs) . When expressed in a transgenic plant, DNA sequences under the control of these promoters are found at relatively low or moderate levels and are expressed fairly evenly (i.e. constitutively) throughout the plant. See, for example, van der Zall, et al., 1991, Plant Mol . Biol.16, 983; Ohl et al . , 1990, Cell 2, 837). Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolic enzymes, such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate. Mammalian cells may also require terminator sequences and poly A addition sequences; enhancer sequences which increase expression may also be included, and sequences which cause amplification of the gene may also be desirable. These sequences are known in the art. The transformed or transfected host cells can be used as a source of DNA sequences described above . When the recombinant molecule takes the form of an expression system, the transformed or transfected cells can be used as a source of the protein described below. If the toxin is a lipid, or associated with a lipid, extraction procedures for lipids are known in the art such as chloroform extraction, thin layer chromatography to separate by hydrophobicity and others (Ruiz-Gutierrez, V., Perez-Camino, MC, 2000, J. Chromatrogr 885, 321- 341; Lipid Analysis: a practical approach. Richard John Hamilton and Shiela Hamilton, ed. IRI Press at Ocford University Press, 1992; Lipids . Helmut K. Mangold, ed. Boca Raton, Fl . CRC Press, 1984). Additionally, it is possible that a carbohydrate moeity is associated with the toxin or the lipid and may be extracted along with the toxin and important for the toxin activity.

The vectors of the present invention can be used to transform plant cells . The constructs of this invention are further manipulated to include genes coding for plant selectable markers such as enzymes providing for production of a compound identifiable by color change such as GUS (beta-glucuronidase) , or by luminescence, such as luciferase. The transformed cells are then cultivated under conditions appropriate for the regeneration of plants, resulting in production of transgenic plants. See, e.g., Gelvin, S. B. ans Schilperoort , R. A., eds . Plant Molecular Biology Manual, Second Edition, Suppl. 1 (1995) Kluwer Academic Publishers, Boston Mass., U.S.A. As used herein, the term transgenic plants includes plants that contain either DNA or RNA which does not naturally occur in the wild type plant or known variants . Trangenic plants include those into which isolated nucleic acids have been introduced and their decscendents, produced from seed, vegetative propagation, cell, tissue or protoplast culture, or the like wherein such alteration is maintained. Seed can be obtained from the regenerated plant or from a cross between the regenerated plant and a suitable plant of the same species. Alternatively, the plant can be vegetatively propagated. The construct containing the nucleic acid encoding the toxins of the present invention are particulary useful for controlling nematode attack. Nematodes are primitive eukaryotic root parasites. These small worms live in the soil where they puncture plant roots and suck the cellular contents, weakening the plant and providing an entry point for pathogenic fungi and bacteria. When the construct is expressed in a transgenic plant as described above, the nematode toxin is produced, so that, under nematode attack, the toxin is expressed locally when and where it is required. This mechanism would be an efficient and cost-effective improvement in nematode control which presently consists of applying pesticides to the soil for two or three weeks . A polypeptide or amino acid sequence derived from the DNA sequences mentioned above, refers to a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 2-5 amino acids, and more preferably at least 8-10 amino acids, and even more preferably at least 11-15 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence .

A "biologically active derivative thereof" is a toxin that is modified by amino acid deletion, addition, substitution, or truncation, or that has been chemically derivatized, but that nonetheless functions in the same manner as the wild type toxin. The term "fragment" is meant to refer to any polypeptide subset . Fragments can be prepared by subjecting Burkholderia toxins to the action of any one of a number of commonly available proteases, such as trypsin, chymotrypsin or pepsin, or to chemical cleavage agents, such as cyanogen bromide. The term "variant" is meant to refer to a molecule substantially similar in structure and function to either the entire toxin or to a fragment thereof. A protein or peptide is said to be 'substantially similar' if both molecules have substantially similar amino acid sequences, preferably greater than about 80% sequence identity, or if the three-dimensional backbone structures of the molecules are superimposable, regardless of the level of identity between the amino acid sequences. Thus, provided that two molecules possess similar activity, they are considered variants as that term is used herein even if the structure of one of the molecules is not found in the other, or if the sequences of amino acid residues are not identical. The term 'analog' is meant to refer to a protein that differs structurally from the wild type toxin, but possesses similar activity.

A recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence; it may be generated in any manner, including for example, chemical synthesis, or expression of a recombinant expression system. In addition the polypeptide can be fused to other proteins or polypeptides which increase its antigenicity, such as adjuvants for example. As noted above, the methods of the present invention are suitable for production of any polypeptide of any length, via insertion of the above- described nucleic acid molecules or vectors into a host cell and expression of the nucleotide sequence encoding the polypeptide of interest by the host cell. Once transformed host cells have been obtained, the cells may be cultivated under any physiologically compatible conditions of pH and temperature, in any suitable nutrient medium containing assimilable sources of carbon, nitrogen and essential minerals that support host cell growth. Recombinant polypeptide-producing cultivation conditions will vary according to the type of vector used to transform the host cells. For example, certain expression vectors comprise regulatory regions which require cell growth at certain temperatures, or addition of certain chemicals or inducing agents to the cell growth medium, to initiate the gene expression resulting in the production of the recombinant polypeptide. Thus, the term "recombinant polypeptide-producing conditions, " as used herein, is not meant to be limited to any one set of cultivation conditions. Appropriate culture media and conditions for the above-described host cells and vectors are well-known in the art. Following its production in the host cells, the polypeptide of interest may be isolated by several techniques. To liberate the polypeptide of interest from the host cells, the cells are lysed or ruptured. This lysis may be accomplished by contacting the cells with a hypotonic solution, by treatment with a cell wall-disrupting enzyme such as lysozyme, by sonication, by treatment with high pressure, or by a combination of the above methods. Other methods of cell disruption and lysis that are known to one of ordinary skill may also be used. Following disruption, the polypeptide may be separated from the cellular debris by any technique suitable for separation of particles in complex mixtures. The polypeptide may then be purified by well known isolation techniques . Suitable techniques for purification include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, electrophoresis, immunoadsorption, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, liquid chromatography (LC) , high performance LC (HPLC) , fast performance LC (FPLC) , hydroxylapatite chromatography and lectin chromatography .

The recombinant or fusion protein can be used as a diagnostic tool and in a method for producing antibodies against the toxin, detectably labeled and unlabeled. The transformed host cells can be used to analyze the effectiveness of drugs and agents which inhibit toxin function, such as host proteins or chemically derived agents or natural or synthetic drugs and other proteins which may interact with the cell to down-regulate or alter the expression of the toxin, or its cofactors .

In another embodiment, the present invention relates to monoclonal or polyclonal antibodies specific for the above-described toxins . For instance, an antibody can be raised against a toxin described above, or against a portion thereof of at least 10 amino acids, perferrably, 11-15 amino acids. Persons with ordinary skill in the art using standard methodology can raise monoclonal and polyclonal antibodies to the toxin (or polypeptide) of the present invention, or a unique portion thereof. Material and methods for producing antibodies are well known in the art (see for example Goding, in, Monoclonal Antibodies: Principles and Practice, Chapter 4, 1986) . If the toxin is a lipid, micelles formed using the lipid, once inactivated, can be used for raising antibodies against the toxin.

The level of expression of the toxin, can be detected at several levels. Using standard methodology well known in the art, assays for the detection and quantitation of RNA can be designed, and include northern hybridization assays, in si tu hybridization assays, and PCR assays, among others. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning , Volumes I and II (D. N. Glover ed. 1985) , or Current Protocols in Molecular Biology, Ausubel, F. M. et al . (Eds), Wiley & Sons, Inc. for general description of methods for nucleic acid hybridization. Polynucleotide probes for the detection of RNA can be designed from the derived sequence. For example, RNA isolated from samples can be coated onto a surface such as a nitrocellulose membrane and prepared for northern hybridization. In the case of in situ hybridization of biopsy samples for example, the tissue sample can be prepared for hybridization by standard methods known in the art and hybridized with polynucleotide sequences which specifically recognize toxin RNA. The presence of a hybrid formed between the sample RNA and the polynucleotide can be detected by any method known in the art such as radiochemistry, or immunochemistry, to name a few.

One of skill in the art may find it desirable to prepare probes that are fairly long and/or encompass regions of the amino acid sequence which would have a high degree of redundancy in the corresponding nucleic acid sequences. In other cases, it may be desirable to use two sets of probes simultaneously, each to a different region of the gene. While the exact length of any probe employed is not critical, typical probe sequences are no greater than 500 nucleotides, even more typically they are no greater than 250 nucleotides; they may be no greater than 100 nucleotides, and also may be no greater than 75 nucleotides in length. Longer probe sequences may be necessary to encompass unique polynucleotide regions with differences sufficient to allow related target sequences to be distinguished. For this reason, probes are preferably from about 10 to about 100 nucleotides in length and more preferably from about 20 to about 50 nucleotides.

The DNA sequence of the toxin can be used to design primers for use in the detection of toxin using the polymerase chain reaction (PCR) or reverse transciption PCR (RT-PCR) . The primers can specifically bind to the cDNA produced by reverse transcription of toxin RNA, for the purpose of detecting the presence, absence, or quantifying the amount of RNA by comparison to a standard. The primers can be any length ranging from 7-40 nucleotides, preferably 10-15 nucleotides, most preferably 18-25 nucleotides homologous or complementary to a region of the toxin sequence. Reagents and controls necessary for PCR or RT-PCR reactions are well known in the art. The amplified products can then be analyzed for the presence or absence of toxin sequences, for example by gel fractionation, by radiochemistry, and immunochemical techniques. This method is advantageous since it requires a small number of cells. Once the toxin is detected, a determination whether the cell is overexpressing or underexpressing the toxin can be made by comparison to the results obtained from a normal cell using the same method. Decreased toxin may be an indication of reduced virulence of the infecting bacteria, or an indication that tissue- specific or site-specific expression of the gene is reduced. In another embodiment, the present invention relates to a diagnostic kit for the detection of toxin RNA in cells, said kit comprising a package unit having one or more containers of toxin oligonucleotide primers for detection of toxin by PCR or RT-PCR or toxin polynucleotides for the detection of toxin RNA in cells by in si tu hybridization or northern analysis, and in some kits including containers of various reagents used for the method desired. The kit may also contain one or more of the following items : polymerization enzymes, buffers, instructions, controls, detection labels. Kits may include containers of reagents mixed together in suitable proportions for performing the methods in accordance with the invention. Reagent containers preferably contain reagents in unit quantities that obviate measuring steps when performing the subject methods. In a further embodiment, the present invention provides a method for identifying and quantifying the level of toxin present in a particular biological sample. Any of a variety of methods which are capable of identifying (or quantifying) the level of toxin in a sample can be used for this purpose .

Diagnostic assays to detect toxin may comprise a biopsy or in si tu assay of cells from an organ or tissue sections, as well as an aspirate of cells from a tumour or normal tissue. In addition, assays may be conducted upon cellular extracts from organs, tissues, cells, urine, or serum or blood or any other body fluid or extract. When assaying a biopsy, the assay will comprise, contacting the sample to be assayed with a toxin ligand or substrate, natural or synthetic, or an antibody, polyclonal or monoclonal, which recognizes toxin, or antiserum capable of detecting toxin, and detecting the complex formed between toxin present in the sample and the toxin ligand, substrate, or antibody added.

Toxin ligands or anti-toxin antibodies, or fragments of ligand and antibodies capable of detecting toxin may be labeled using any of a variety of labels and methods of labeling for use in diagnosis and prognosis of disease associated with Burkholderia . Examples of types of labels which can be used in the present invention include, but are not limited to, enzyme labels, radioisotopic labels, non-radioactive isotopic labels, and chemiluminescent labels.

Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5- steroid isomerase, yeast-alcohol dehydrogenase, alpha- glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase, etc .

Examples of suitable radioisotopic labels include ³H, ^llxIn, ¹²⁵I, ³²P, ³⁵S, ¹⁴C, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ^1XC, ¹⁹F, ¹²³I, etc. Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, ⁴⁶Fe, etc.

Examples of suitable fluorescent labels include a ¹⁵²Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycodyanin label, an allophycocyanin label, a fluorescamine label, etc.

Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, etc.

Those of ordinary skill in the art will know of other suitable labels which may be employed in accordance with the present invention. The binding of these labels to ligands and to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Typical techniques are described by Kennedy, J. H. , et al . , 1976 { Clin . Chim. Acta 70, 1-31), and Schurs, A. H. W. M. , et al . 1977 { Clin . Chim Acta 81, 1-40). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, and others, all of which are incorporated by reference herein.

The detection of the antibodies (or fragments of antibodies) of the present invention can be improved through the use of carriers . Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides , agaroses , and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to toxin. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Those skilled in the art will note many other suitable carriers for binding monoclonal antibody, or will be able to ascertain the same by use of routine experimentation. The ligands or antibodies, or fragments of antibodies or ligands of toxin discussed above may be used to quantitatively or qualitatively detect the presence of toxin. Such detection may be accomplished using any of a variety of immunoassays known to persons of ordinary skill in the art such as radioimmunoassays, i munometic assays, etc. Using standard methodology well known in the art, a diagnostic assay can be constucted by coating on a surface (i.e. a solid support) for example, a microtitration plate or a membrane (e.g. nitrocelluolose membrane) , antibodies specific for toxin or a portion of toxin, and contacting it with a sample from a person suspected of having a toxin related disease. The presence of a resulting complex formed between toxin in the sample and antibodies specific therefor can be detected by any of the known detection methods common in the art such as fluorescent antibody spectroscopy or colorimetry. A good description of a radioimmune assay may be found in Laboratory Technicaies and Biochemistry in Molecular Biology, by Work, T.S., et al . North Holland Publishing Company, N.Y. (1978) , incorporated by reference herein. Sandwich assays are described by Wide at pages 199-206 of Radioimmune Assay Method, edited by Kirkham and Hunter, E. & S. Livingstone, Edinburgh, 1970.

The diagnostic methods of this invention are predictive of patients suffering from infection with Burkholderia . The toxins can be used to identify inhibitors of toxin activity. Natural and synthetic agents and drugs can be discovered which result in a reduction or elimination of toxin activity. Knowledge of the mechanism of action of the inhibitor is not necessary as long as a decrease in the activity of toxin is detected. Inhibitors may include agents or drugs which either bind or sequester toxin substrate (s) or cofactor(s), or inhibit the toxin itself, directly, for example by irreversible binding of the agent or drug to toxin, or indirectly, for example by introducing an agent which binds the toxin substrate. Agents or drugs related to this invention may result in partial or complete inhibition of toxin activity. Inhibitors of toxin may be used in the treatment or amelioration of conditions related to Burkholderia infection.

Agents which decrease toxin RNA include, but are not limited to, one or more ribozymes capable of digesting toxin RNA, or antisense oligonucleotides capable of hybridizing to toxin RNA such that the translation of toxin RNA is inhibited or reduced resulting in a decrease in the level of toxin. These antisense oligonucleotides can be administered as DNA, as DNA entrapped in proteoliposomes containing viral envelope receptor proteins (Kanoda, Y. et al . , 1989, Science 243, 375) or as part of a vector which can be expressed in the target cell such that the antisense DNA or RNA is made . Vectors which are expressed in particular cell types are known in the art, for example, for the mammary gland, please see Furth, (1997) { J. Mammary Gland Biol . Neopl . 2, 373) for examples of conditional control of gene expression in the mammary gland. Alternatively, the DNA can be injected along with a carrier. A carrier can be a protein such as a cytokine, for example interleukin 2, or polylysine-glycoprotein carrier. Such carrier proteins and vectors and methods of using same are known in the art. In addition, the DNA could be coated onto tiny gold beads and said beads introduced into the skin with, for example, a gene gun (Ulmer, J. B. et al . , 1993, Science 259, 1745).

Alternatively, antibodies, or compounds capable of reducing or inhibiting toxin, that is reducing or inhibiting either the expression, production or activity of toxin, such as antagonists, can be provided as an isolated and substantially purified protein, or as part of an expression vector capable of being expressed in the target cell such that the toxin-reducing or inhibiting agent is produced. In addition, co-factors such as various ions, i.e. Ca²⁺ or factors which affect the stability of the toxin can be administered to modulate the expression and function of toxin. These formulations can be administered by standard routes. In general, the combinations may be administered by the topical, transdermal, intraperitoneal, oral, rectal, or parenteral (e.g. intravenous, subcutaneous, or intramuscular) route. In addition, toxin-inhibiting compounds may be incorporated into biodegradable polymers being implanted in the vicinity of where drug delivery is desired or implanted so that the toxin-inhibiting compound is slowly released systemically. The biodegradable polymers and their use are described, for example, in detail in Brem et al . (1991) J.

Neurosurg. 74, 441-446. These compounds are intended to be provided to recipient subjects in an amount sufficient to effect the inhibition of toxin. Similarly, agents which are capable of negatively affecting the expression, production, stability or function of toxin, are intended to be provided to recipient subjects in an amount sufficient to effect the inhibition of toxin. An amount is said to be sufficient to "effect" the inhibition or induction of toxin if the dosage, route of administration, etc. of the agent are sufficient to influence such a response. In providing a patient with agents which modulate the expression or function of toxin to a recipient patient, the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of agent which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient) , although a lower or higher dosage may be administered.

A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient. The compounds of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences [16th ed. , Osol, A. ed., Mack Easton PA. (1980)]. In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the above-described compounds together with a suitable amount of carrier vehicle. Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb the compounds . The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the method of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the compounds of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacrylate) - microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions . Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980) .

The present invention also provides kits for use in the diagnostic or therapeutic methods described above. Kits according to this aspect of the invention may comprise one or more containers, such as vials, tubes, ampules, bottles and the like, which may comprise one or more of the compositions of the invention. The kits of the invention may comprise one or more of the following components, one or more compounds or compositions of the invention, and one or more excipient, diluent, or adjuvant.

In another embodiment, the present invention describes a Burkholderia strain which contains a deletion or mutation of the toxin gene. A mutant strain can be used to characterize the toxin and as a possible therapeutic for neuromuscular disease if the mutant toxin serves as an antagonist of toxin activity.

In one embodiment, the present invention relates to a vaccine for protection against Burkholderia pathogens . The vaccine comprises toxins isolated from such bacteria or a fraction of the bacteria containing the toxin. The vaccine can be prepared by isolating toxins using methods described above. One or more isolated toxin is prepared for administration to mammals by methods known in the art, which can include, deactivating, filtering to sterilize the solution, diluting the solution, adding an adjuvant and stabilizing the solution. As such, in one embodiment, a composition of the present invention can include one or more toxin and one or more adjuvants or carriers . Adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, Freund's adjuvant, other bacterial cell wall components, aluminum-based salts, calcium-based salts, silica, polynucleotides, toxoids, serum proteins, viral coat proteins, other bacterial-derived preparations, gamma interferon, block copolymer adjuvants, such as Hunter's Titermax adjuvant (CytRx™, Inc. Norcross, GA) , Ribi adjuvants (availabe from Ribi ImmunoChem Research, Inc. Hamilton, MO), and saponins and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark) .

Carriers are typically compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, oils, esters, and glycols .

The vaccine can be lyophilized to produce a vaccine against Burkholderia bacteria in a dried form for ease in transportation and storage. The dried compositions can be used for oral delivery. Toxins can also be mixed with a pharmaceutically acceptable excipient, such as an isotonic buffer that is tolerated by the organism to be administered the vaccine. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer, and Tris buffer, while examples of preservatives include thimerosal, m- or 0-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non- liquid formulation, the excipient can comprise, for example, dextrose, human serum albumin, and/or preservatives to which sterile water or saline can be added prior to administration.

Further, the vaccine may be prepared in the form of a mixed vaccine which contains the toxins described above and at least one other antigen as long as the added antigen does not interfere with the effectiveness of the vaccine and the side effects and adverse reactions are not increased additively or synergistically. The vaccine can be associated with chemical moieties which may improve the vaccine's solubility, absorption, biological half life, etc.

The moieties may alternatively decrease the toxicity of the vaccine, eliminate or attenuate any undesirable side effect of the vaccine, etc. Moieties capable of mediating such effects are disclosed in Remington 's Pharmaceutical Sciences (1980) . Procedures for coupling such moeities to a molecule are well known in the art .

The vaccine may be stored in a sealed vial, ampule or the like. The present vaccine can generally be administered in the form of a spray for intranasal administration, or by nose drops, inhalants, swabs on tonsils, or a capsule, liquid, suspension or elixirs for oral administration. In the case where the vaccine is in a dried form, the vaccine is dissolved or suspended in sterilized distilled water before administration .

Generally, the vaccine may be administered orally, subcutaneously, intradermally or intramuscularly but preferably intranasally or orally in a dose effective for the production of neutralizing antibody and resulting in protection from infection or disease. The vaccine may be in the form of single dose preparations or in multi-dose flasks which can be used for mass vaccination programs. Reference is made to Remington 's Pharmaceutical Sciences, Mack Publising Co., Easton, PA, Osol (ed.) (1980); and New Trends and Developments in Vaccines, Voller et al . (eds.), University Park Press, Baltimore, MD. (1978) , for methods of preparing and using vaccines . Acceptable protocols to administer compositions in an effective manner include individual dose size, number of doses, frequencey of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A preferred single dose of a toxin composition is from about 1.0 pg/kg to about 10/kg (body weight of recipient) grams. Boosters are preferably administered when the immune response of an organism is no longer being effectively modulated. Such compositions can be administered from about two weeks to several years after the original administration. A preferred administration schedule is one in which from about 1 pg to about 10 grams of a composition per kg body weight of the organism is adminsitered from about one to about four times over a time period of from about one month to about 6 months .

In another embodiment, the present invention relates to a method of reducing Burkholderia infection symptoms in a patient by administering to said patient an effective amount of toxin antibodies, including those made in humans, either polyclonal or combinations of monoclonals to toxins, as described above. When providing a patient with toxin antibodies, the dosage administered will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of the above compounds which is in the range of from about 1 pg/kg to 500 mg/kg (body weight of patient) , although a lower or higher dosage may be administered.

The present invention also provides a kit comprising a pharmaceutical (for prophylaxis i.e. a vaccine or for therapy i.e. a therapeutic) as described above in a container preferably a pre-filled syringe or glass vial/ampoule with printed instructions on or accompanying the container concerning the administration of the pharmaceutical to a patient to prevent or treat conditions caused by gram-negative bacterial infections .

Described below are examples of the present invention which are provided only for illustrative purposes, and not to limit the scope of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in this art which are obvious to those skilled in the art are within the spirit and scope of the present invention.

The following methods and materials were used in the examples below.

Strains/maintenance : Bacterial strains were maintained on LB agar, Burkholderia mallei was maintained on LBG (LB + 4% glycerol) . Burkholderia pseudomallei mutants were maintained on LB supplemented with streptomycin (100 ug/ml) and tetracycline at (50 ug/ml) . Overnight cultures were grown in LB or LBG at 37°C. Bacillus anthracis and B . subtilis were cultured on BHI liquid medium, and BHI plates. N2 Bristol C. elegans were maintained by growth on E. coli OP50 spotted onto standard C. elegans growth agar (MYOB) . Population growth occurred at 23°C. Standard protocols for growth, growth medium and manipulation of C. elegans are described in (Riddle, Blumenthal et al . , 1997. C. eleαans II . Plainview, NY, Cold Spring Harbor Press) . DA 1032 (avr-14; avr-15) was kindly provided by L. Avery (UTSW (Dallas) ) . The following mutant strains were obtained from CGC (C. elegans Genetics Center, UM (St. Paul)): BC23, BC177, CB55, CB251, CB408, CB904, CB1072, CB1091, CBllll, CB1112, CB1141, CB1152, DA695, DA823, DA939, DA944, DA945, DA1031, DA1034, DA1035, DA1051, DA1055, KP1097, MT1092, MT1201, MT1212, MT1216, MT2426, MT2598, MT2605, MT3198, MT6129, PR1158.

Nematocidal activity assays : Bacteria were grown aerobically overnight in 2-3 mL of LB liquid cultures and spotted (10-20 uL) onto the C. elegans growth medium: MYOB, NG, NGM (NG+0.15M Sorbitol), PG or PGM (PG+ 0.15M Sorbitol) . Animals were hand transferred onto the spots and monitored according to the procedure of Tan et al . (Tan, Rahme et al . 1999, supra) . Growth of the nematode populations was compared to growth on E. coli OP50. The positive control for nematode pathogenesis was P. aeruginosa PA14 (Mahajan-Miklos, Tan et al . 1999, supra; Tan, Rahme et al . 1999, supra).

Killing kinetic assays and LT50 calculation: Analysis was performed blind to animal genotype, using hundreds of hyprochlorite-synchronized juvenile (L4 stage) animals ina time course experiment (Tan, Rahme et al . , 1999, supra). Three 6-well culture plates were prepared by spreading of overnight bacterial cultures with a Q-tip into each of the 6 wells. After overnight growth at 37°C, the uniform lawns of the bacterial strain to be analyzed were allowed to cool to ca. 23°C. Synchronized animals were inoculated into each well of the test plates in 3 intervals; the first plate was started at 9 a.m (0 hr) , the second at 12 pm (3 hr) , and the last at 3 pm (6 hr) . Starvation was assessed by inoculating synchronized L4 animals into foodless MYOB wells and observing the "ratio still moving" over the course of the 30 hr experiment (number moving/total number) . All assays involving B. pseudomallei were performed under BSL-3 conditions with a microscope-equipped camera in a biological safety cabinet and a video monitor for remote viewing. Rather than scoring loss of reflex-action as a marker for nematode death, death was confirmed by high-power (120 x) visual inspection. The percent still moving was determined by averaging the movement ratios and multiplying them by 100, for each time point among at least three experiments. The LT50 was calculated from the mean percent still moving values using a nonlinear logistic, 3 parameter regression of the form y= a/(l+ (x/x₀)^b), where a and b are parameters fit to the curve and xO is the LT50. The calculations were made using SigmaPlot (Version 4.0, SigmaPlot) and Excel (Version 5/95, Microsoft) for the PC.

Food source switching: Approximately 20 L4 stage animals (L4s) were transferred to B . pseudomallei 1026b, NCTC 4845, B. thailandensis E264, or E. coli OP50, and allowed to feed for 4 hr or 8 hr. Individuals were then transferred back onto E. coli and the resulting population was counted at day 4. L4s were examined at the time of transfer back onto E.coli to note whether they had stopped pharyngeal pumping. "Starvation" was assessed by nematode transfer to empty MYOB for 4 or 8 hours, followed by transfer back to OP50 for further monitoring. To minimize the carry-over of pathogenic bacteria, the experiments were also performed in parallel with plates supplemented with tetracycline at 20 ug/mL. The bacterial food source for these plates was E. coli DH5α carrying the plasmid pBR322. N=3. The data from these plates were averages into the data in Table 3. Because the tetracycline kills the Burkholderia species outside and inside of the nematodes, the tetracycline experiment animal-counts reflect the action of the toxin, and the data without tetracycline presumably reflects the action of both bacterial proliferation and intoxication.

Egg-laying inhibition: Egg laying inhibition was monitored using homozygous egl-19 (n2368) host animals. The (n2368) allele expresses an egl-c (constituative egg laying) phenotype, as such it retains very few eggs in its uterus . Because few eggs are retained by adults, accumulation of the eggs upon a plate is easy to monitor. One hundred ul of saturated overnight cultures of E264, OP50 or 1026b, were spotted onto MYOB agar and spread out to confluency with a sterile cotton swab. The plates were incubated overnight at 37°C, allowed to cool to room temperature, and then were seeded with 10 adults per plate. The number of embryos sown was counted at 4 and 25 hours. Two plates per bacterial species were examined in parallel. The experiment was performed two times . The average number ± SD of embryos per plate is shown in Table 3. Toxin diffusion: lOOul from LB overnight cultures of B . pseudomallei 1026b, B . thailandensis E264 , E. coli OP50, and P. aeruginosa PA14 were spotted either onto 100 mm MYOB agar plates (100 mm PGM agar plates for PA14), or MYOB (PGM) overlaid with a 0.2 u NYTRAN

(Schleicher and Scheull (through Midwest Scientific (St. Louis)) filter disc. After incubating the spotted plates at 37°C overnight, the plates were allowed to cool to room temperature. The plates containing the bacterial lawn supported upon the filters had the filters lifted from the plates . E. coli OP50 grown N2 Bristol C. elegans were washed with 10 ml sterile M9 + 10 mM MgCl₂ and collected by low speed centrufigation four times. The animals were counted by measuring the number of animals in 20 ul of the resuspension. About 20 of the washed multi-stage animals were spotted either into the bacterial lawns or onto the plates that had the lawns filter lifted, and mortality was monitored at 6, and 24 hours.

UV/gamma inactivation: Aerobic overnight bacterial cultures were spotted onto 12 MYOB agar plates (PGM for PA14) per species. The inoculated plates were allowed to grow overnight at 37°C. The following day, the plates were inverted upon a UV transilluminator (mix 260-280 nm) for 0 seconds, 6 sec, 39 sec, 1 min, 10 min, and 60 min or exposed to radioactive ⁵⁴Co (~ 23,333 RAD/min) for the same time increments. Two plates were exposed in parallel. The irradiated plates were allowed to cool to room temperature and then a P200 pipette tip was stabbed into the lawn of bacteria. The lawn stab was rinsed into 1 ml of LB liquid medium and serially diluted (logs of 10) to a ratio of 1:1 X 10^"6. Colonies were counted after overnight growth at 37°C. After the plate stab, 3 L4- staged N2 Bristol C. elegans were transferred onto each plate (Day = 4) . Any threshold to the radiation doses was tested by extending the UV exposure to 120 minutes , and the gamma irradiation exposures were confirmed at 130 min (~3xl0⁶ rads), and 385 min (~9xl0⁶ rads) . All increased exposures resulted in no change to the experimental outcomes while visibly discoloring the petri dishes containing the inactivated bacteria. The 1.4xl0⁶ rad plates were tested 3 times, the 3xl0⁶ rad and 9xlO^δ rad plates were checked two times.

Toxin killing kinetic: 100 ul from saturated aerobic overnight bacterial cultures of B . thailandensis E264 were spotted onto 4 (60 mm) MYOB agar plates, and spread out to confluency with a sterile cotton swab. After overnight growth at 37°C, the plates were allowed to cool to room temperature (~23°C) . Two plates were selected and exposed to radioactive ⁵⁴Co at a dose of 3xl0⁶ rads. The bacterial lawns were next stabbed with a P200 pipette tip. The lawn stab was rinsed into 1 mL of LB liquid medium and serially diluted (logs of 10) to a ratio of 1:1 x 10^"6. Colonies were counted after overnight growth at 37°C. The next day, following conformation of the gamma inactivation, approximately 50 multistaged N2 C. elegans were placed into the lawns. The ratio still moving was monitored at 24 and 48 hours after introduction in parallel for both the live and inactivated lawns . The plates were incubated at room temperature over the course of the experiment. The experiment was performed twice, the data are reported mean values ± the SD.

Food choice: Numerous L4-stage N2 Bristol animals were placed in various positions on a series of plates that contained E. coli and B. thailandensis E264 (or B. pseudomallei 1026b) . Three plates were prepared, each contained a thin longitudinal band of bacterial lawn of E. coli and E264 (or 1026b) . Three zones were demarcated: Zone A, bounded by the edge of the plate and the stripe of E. coli OP50; Zone B, the space between the E. coli and the B . thailandensis E264 (or 1026b) ; and Zone C, the space between the outer- edge of the E264 (1026b) and the edge of the petri- dish. Approximately 20 hypochlorite-synchronized L4 animals were placed into Zone A on plate 1, the midpoint of Zone B on plate 2, and Zone C on plate 3. Six hours later, the location and disposition of the feeding nematodes were determined.

Example 1

Establising a model system: To establish a non- vertebrate host model system for studying pathogenic bacteria, candidate hosts must be screened for their pathogenesis phenotype. Tan et al . (Tan, Mahajan- Miklos et al . 1999, supra) evaluated C. elegans as a potential model host for P. aeruginosa by a simple plate assay. The plate mortality assay utilized a standard C. elegans solid growth medium that had the usual food source {E. coli ) substituted with a potential pathogen. The outcome was determined by identifying the "victor" . If the nematodes were unaffected, within a few days, they devoured the lawn of bacteria and thousands of progeny nematodes were visible on the plate. However, if the bacteria kill the nematodes, few if any of the progenitors transferred to the pathogenic lawn survived.

Intermediate pathogenesis contained modest nematode population growth. Example 2

Burkholderia species kill nematodes: Four strains of Burkholderia pseudomallei ; 4 strains of B . mallei ; 7 strains of the B . cepacia complex; a single strain each of B. thailandensis , B . cocovenenans , B. pyrrocinia , and Ralstonia pickettei were tested for their ability to kill nematodes . Many strains killed the nematodes or only allowed a modest population growth relative to the E. coli OP50 negative control strain (Table 1) . Under MYOB medium conditions B. pseudomallei , B . thailandensis, and B . cepacia species were nematocidal, but not the B . mallei isolates. The B . pyrrocinia , B. cocovenenans, and R . pickettei isolates had a weak effect upon population growth (Table 1) .

TABLE 1 STRAIN MYOB PGM REFERENCE/SOURCE E. coli OP50 & DH5α +++++ ++++ A. Golden, NIH Bethesda

P. aeruginosa PA14 ++ 0 (Tan, Rahme et al., 1999) M. W. Tan, MGH

B. cepacia ATCC 25416 0 ATCC, D. DeShazer USAMRIID (genomovar I)

B. multivorans c5568 +++++ ++ D. DeShazer USAMRIID (genomovar II)

B. cepacia K56-2 ++ D. DeShazer USAMRIID (genomovar III)

B. cepacia Fco 362 0 + D. DeShazer USAMRIID (genomovar IV)

B. vietnamiensis Fco 369 +++++ ++ D. DeShazer USAMRIID (genomovar V) B. cepacia ATCC 27515 0 0 ATCC, D. Waag USAMRIID

B. cepacia ATCC 35130 + 0 ATCC, D. Waag USAMRIID TABLE 1 Continued STRAIN MYOB PGM REFERENCE/SOURCE

B. cocovenenans +++ ATCC

ATCC 33664

B. pyrrocinia +++ ATCC ATCC 15958

R. pickettei

ATCC 27512 +++++ ++ ATCC

B. thailandensis E264 0 0 Brett, DeShazer et al. 1998 D. DeShazer, USAMRIID

B. pseudomallei E203 0 0 Brett, DeShazer et al. 1997 D. DeShazer, USAMRIID

B. pseudomallei 316c 0 0 Brett, DeShazer et al. 1997 D. DeShazer, USAMRIID

B. pseudomallei + 0 D. Waag, USAMRIID

NCTC 4845

B. pseudomallei 1026b 0 Brett, DeShazer et al. 1997 D. DeShazer, USAMRIID

DD503= 1026b Δ 0 Moore, DeShazer et al. 1999

(amrR- opr A) ;rpsL D. DeShazer, USAMRIID

SRM117=1026b 0 DeShazer, Brett et al. 1998

(gsp::Tn5-OT182) D. DeShazer, USAMRIID

DD213= 1026b 0 DeShazer, Brett et al. 1999

(gspD::Tn5-OT182) D. DeShazer, USAMRIID

MM36=1026b 0 DeShazer, Brett et al. 1997

(fliC::Tn5-OT182) D. DeShazer, USAMRIID

B. mallei NCTC 10229 +++++ D. Waag USAMRIID B. mallei NCTC 10260 +++++ D. Waag USAMRIID B. mallei ATCC 23344 +++++ Fritz, Vogel et al. 1999 ATCC, D. Waag USAMRIID

Interestingly, many, but not all of the tested strains exhibited enhanced killing relative to MYOB medium under the high osmolarity conditions of PGM medium (Table 1). Phenazines, super-oxide generating small molecule toxins, are produced by P. aeruginosa PA14 grown under PGM agar conditions (Mahajan-Miklos, Tan et al . 1999, supra) many of the strains tested may have a similar killing mechanism under PGM agar growth conditions. Only the genomevar IV B . cepacia, Fco 362, did not kill nematodes as well under PGM agar conditions (Table 1) .

Some of the pathogenic strains supported small or modest nematode population growth, while others allowed no growth (Table 1) . The strain-specific differential in population size suggested variable killing. An LT50 assay (Tan, Rahme et al . 1999, supra) was employed to explore nematode killing further. The LT50 is the calculated time at which 50% of the nematode population was observed to cease movement (see procedures) . Differences in LT50 times represent different rates of killing. The kinetics of killing by

B . pseudomallei and B . thailandensis strains are shown in Figure 1 and summarized in Table 2. The L4 stage

C. elegans LT50 for B . pseudomallei strains ranged between 16 and 23 hr of feeding. The B . thailandensis strain E264 killed most efficiently with an LT50 time of about 10 hours. Because the B . vietnamiensis strain Fco 369 was found not to kill nematodes (Table 1) it was used as a more suitable 'non-pathogenic' negative control for LT50 analysis.

Table 2

Strain LT50

(hr of feeding ±SD

E. coli OP50 ND

P. aeruginosa UCBPP-PA14 >30 (MYOB)*

Starvation >30

B. cepacia ATCC 25416 13.5 ± 3.5

B. cepacia K56-2 >30

B. cepacia Fco 362 19.9±2 . 7

B. thailandensis E264 9.5+1.6

B. pseudomallei 1026b 17.7+1.7

B. pseudomallei NCTC 4845 19.8±0.8 Example 3

Characterized B . pseudomallei mutations have little effect on nematode killing: B . pseudomallei knock-out mutations affecting aminoglycoside and macrolide efflux pumping, DD503, (DeShazer, Brett et al . 1998, Mol. Microbiol. 30, 1081-100) , LPS O-antigen , SRM117, (DeShazer, Brett et al . 1998, supra), general protein secretion machinery (gspD) , DD213, (DeShazer, Brett et al. 1999, J. Bacteriol. 181, 4661-4), and the flagella (fliC), MM36, (DeShazer, Brett et al . 1997, J.

Bacteriol. 179, 2116-2125), showed little effect upon the ability to kill L4 stage C. elegans (Table 1) . Detailed LT50 analysis of the mutant strains verses wild-type N2 C. elegans confirmed the initial observation (data not shown) .

Nematode death syndrome : Observations of the phenotype of dying nematodes suggested that killing by B. pseudomallei and B . thailandensis was an active process. Nematode pathogenesis appeared to affect locomotor functions as evidenced by the rapid onset of lethargy. Locomotion visibly decreased after exposure for as little as 1 hr, and the rate of foraging (side- to-side feeding head movement) was similarly affected in the same time-frame. Pharyngeal pumping was affected by more prolonged feeding, and after 4 hr, approximately 50% of the nematode population had stopped pumping (Table 3) . In addition, nematodes fed on lawns of B . pseudomallei and B . thailandensis exhibited an egg-laying deficient { egl-d) phenotype. Nearly all L4 staged nematodes that fed on B. pseudomallei (or B . thailandensis) became 'bags-of- worms ' early in adult-hood. The ability of the pathogens to inhibit egg laying was measurable. Both B . pseudomallei 1026b and B . thailandensis E264 were able to inhibit egg-laying in adults homozygous for a constituative egg-laying mutation. The egg-laying inhibition conditioned a more than 40% decrease in accumulated eggs within four hours. Over the next 20 hours of feeding few additional eggs were sown (Table 3) . ' Bags-of-worms ' are the result of internal hatching of retained embryos (Riddle, Blumenthal et al. 1997, supra) .

Table 3

Starting 4 hrs 4 hrs 4 hrs 8 hrs 8 hrs 24 hrs

Food switch % mean# switch % mean

Strain #animals pumping eggs #animals pumping # eggs

E. coli >1000 94(±5) 75(±2) >1000 92 430(±330)

B. thailendensis 4(±5) 37(±8) 45(±1) 0(±0) 18 37(±5)

E264 3(±2) 55(±5) 48(±1) 2(±2) 23 48(±1)

B. pseudomallei 73(±52) ND ND 35(±42) ND ND

NCTC 4845

Starvation >1000 96(±6) ND ND ND ND

Even after the nematodes were returned to the E. coli lawn killing was observed. L4 stage N2 Bristol animals fed on B . pseudomallei 1026b, NCTC 4845 or B. thailandensis E264 for longer than 8 hr did not recover once transferred back onto E. coli (OP 50) plates (Table 3). Feeding for shorter intervals allowed a stochastic chance of recovery that appeared to be strain-specific (Table 3) . Although 50% of the nematodes stopped pumping at 4 hr, this phenotype did not correlate with animal survival to reproduction. Approximately 80% of the nematodes had stopped pharyngeal pumping by 8 hr (Table 3) .

Every life-stage of C. elegans was susceptible to B. pseudomallei and B . thailandensis except unhatched embryos (data not shown) . L3 stage and dauer (a developmentally arrested life stage) animals survived longer than L4/adult animals, probably because the less developed (or arrested) L3/dauer animals fed less than the voracious L4/adults (Riddle, Blumenthal et al . 1997, supra) . Dead nematodes were often found lying coiled. Embryos sown in lawns of E264,

NCTC4845, and 1026b did hatch but the Ll did not survive. Starved C. elegans is able to arrest its development at the Ll stage as a response to the lack of available food (Riddle, Blumenthal et al . 1997, supra) . The E264 and 1026b fed Lls were not simply starvation arrested as they also did not recover once they were transferred back to OP50.

Curiously, nematodes killed in the lawn of bacteria took on a ghostly and hollow 'shell-like' appearance approximately 48 hr after the L4s were first introduced. The nematode shells induced by Burkholderia strains 1026b, NCTC 4845 and E264 were termed 'chalk-mark ghosts'. Chalk-mark ghosts appeared to have no discernable internal cell structures. Often the ghosts eroded to a mere outline of where a nematode died in the bacterial lawn. Although some ghosts were formed by adults that died and subsequently became 'bags-of-worms ' , ghosts also were found from animals too young to have 'bagged' . Examination of many chalk-mark ghosts found that every life stage became a ghost after dying on the Burkholderia lawn. Attempts to isolate the ghosts for more detailed characterization were unsuccessful.

Example 4

Nematode killing exploration: The relatively rapid death rate (10-18 hr) and the death syndrome's phenotypic components suggested that the nematocidal killing observed may be mediated by, or in concert with, intoxication. Bacteria producing a toxin may exhibit cell-free killing. Conditioned culture medium may kill, and or dead bacteria may kill as well as live bacteria. Bacterial cell-free nematode toxicity was not detected (data not shown) . Bacterial culture filtrates from B. pseudomallei and B . thailandensis liquid (LB and MYOB) , as well as 'conditioned' solid (MYOB) agar, did not kill C. elegans . In contrast, when P. aeruginosa PA14 was grown on a 0.2 u filter on PGM agar and the filter was lifted from the PGM agar plate, the 'conditioned' agar killed nematodes in a cell-free manner, confirming the results of Mahajan- Milikos et al . (Mahajan-Miklos, Tan et al . 1999, supra) .

While neither the B . pseudomallei nor B . thailandensis isolates tested possessed a diffusable nematocidal agent, it was possible that B. pseudomallei and B. thailandensis exhibited cell-associated toxicity. However, UV inactivated B . pseudomallei and B. thailandensis were no longer nematocidal (Table 4) . In marked contrast, gamma-irradiated lawns of B. thailandensis and B . pseudomallei were nematocidal. Lawns irradiated with as much as 9xl0⁶ rads retained the ability to kill nematodes and suppress population growth (data not shown) even though no colony forming units were recoverable from any of the 1.4xl0⁶ rad (60 minutes) or larger irradiation doses. Although irradiated lawns killed the nematodes, killing analysis showed wild-type N2 Bristol nematodes died 2- 4 times more quickly when fed live bacterial lawns than gamma irradiation killed bacterial lawns (Figure 2) . Living bacteria are not essential for pathogenesis (killing indicated by line A) , however, living/proliferating bacteria make up a large portion, roughly half, of the killing activity (indicated by B) . These data suggest that the nematode killing process consists of an intoxication mechanism plus additional mechanisms dependent upon living bacteria. The skew toward the slower killing relative to Figure 1 is a reflection of the mixed multi-staged nematode population assayed.

Table 4

Strain UV 60 mm ^MCo =1.4xl0⁶ rads Ratio

Inactivated nematode count nematode count UV/rad

Dav 4 Dav 4

E.co OP50 1000's 1000's 1.0 (E= 1.0)

B thailandensis Ε26A 137+7.1 1.5+0.7 91.3

B pseudomallei 1026b 143±41 1.5+0.7 95.3

P aeruginosa PA14 2.5+3.5 4±1.4 0.63 (E=1.0)

Recoverable CFU= 0 Recoverable CFU= 0

C. eleσans respond to environmental stimuli . Nematodes exhibit aversion to high-salt environments and a preference for high-sugar environments. In addition, they tend to migrate within a temperature gradient to the temperature at which they were reared (Dusenbery, Sheridan et al . 1975, Genetics 80, 297-309). Further, Jones and Candido (1999, J Exp. Zool . 284, 147-57) found that C. elegans would stop feeding in response to environmental intoxicants, such as ethanol, methanol, heavy-metals and the toxic pthalimide fungicide captan (Jones and Candido 1999, supra) . L4 stage N2 Bristol animals exhibited no aversion to eating B. thailandensis or B . pseudomallei over E. coli (Table 5) . Behavioral 'choice' between 1026b and E264 was monitored similarly; the nematodes fed randomly, (i.e., also without aversion (B. pseudomallei data not shown) ) . If the E264 or 1026b were intoxicating the animals, or were otherwise a generally bad food source, the nematodes did not indicate behaviorally that they were able to sense any toxicity or nutritional deficiency.

Table 5 Starting position: Frequency in Frequency in E. coli OP50 @ 6hrs B. th. E264 @ 6hrs Ύ²

Start by OP50 0.59+0.17 (E= 1.0) 0.37+0.23(E=0) 8:p=«0.005

Start between 0.1 1±0.02 (E=0.5) 0.88±O.O2(E=0.5) 13.6:p=«0.005 Start by E264 0.16+0.05 (E=0) 0.84±0.05(E=1.0) 1.08:p= -0.50 Example 5

Host genotype modulates survival. Darby et al . demonstrated that host mutations in C. elegans conferred resistance to a neuromuscular paralysis phenotype conditioned by the PAO isolate of P. aeruginosa when grown upon BHI medium (Darby, Cosma et al. 1999, Proc. Natl Acad. Sci. USA 96, 152002-7). Since the death phenotype conditioned by B. pseudomallei and B . thailandensis resembled that of P. aeruginosa, we determined the affect of egl-9 mutations upon host survival. Further, since the toxicity of the pathogens appeared to affect the nematodes neuromuscular signaling, we screened a variety of characterized C. elegans mutations in neural response pathways looking for mutations that would modify positively (or negatively) nematode host survivorship. Figure 3 depicts the results.

Loss-of-function mutations in the egl-9 gene ( (n571) , and(n586)), whose product is of unknown function, enhanced nematode survival but did not provide qualitative resistance to either B. thailandensis or B . pseudomallei grown upon MYOB agar (Figure 3A and data not shown) . P. aeruginosa PAO did not kill wild- type C. elegans N2 or egl -9 mutants under the MYOB agar conditions assayed. For a direct comparison, BHI medium was utilized to grow B. thailandensis E264, B . pseudomallei 1026b, and P. aeruginosa PAO. While the egl -9 mutations did confer resistance to PAO paralytic-killing under BHI medium, confirming the results of Darby et al . (Darby, Cosma et al . 1999, supra), the mutant animals, however, still succumbed to both the E264 and 1026b with about the same kinetics as that of MYOB medium grown strains (data not shown) . LT50 analysis showed that egl -9 (n571) homozygotes lived more than two times longer than wild-type N2 under MYOB conditions (Table 6) .

Table 6

Genotype % WT LT50 ± SD Wild-type N2 9.5 + 1.6 (100%) egl-9(n571) 214 ± 12 egl-19(n2368)sd 287 ± 19 egl-19(αd695)sd 248 ± 12 egl-19(n582) 41 ± 2.0 egl-19(n2368); unc-36(e251) 72 ± 33 egl-I9(αd695); unc-36(e251) 190 ± 14 egl-19(n582); unc-36(e251) 13 ± 6.0 unc-36(e251) 34 ± 4.0 unc-43(n498)sd 269 ± 10 unc-43(e408) 228 ± 1.0 unc-43(n498nl 179) 233 ± 4.0 unc-43(n498n!186) 205 ± 25

Interestingly, mutations that disrupted normal Ca .+⁺2 signal transduction also had an affect upon nematode survival (Figure 3B and Table 6) . Loss-of-function mutations affecting L-type voltage gated Ca⁺² channels subunits expressed in the body-wall muscle { unc-36) and the neuromuscular junction (egl-19) , encoded by the egl -19 (n582) , and unc-36(n251) alleles, decreased nematode survivorship in homozygotes. Conversely, gain-of-function mutations affecting egl -19, (n2368) sd, and (ad695) sd, enhanced homozygote survivorship (Figure 3B, Table 6 and data not shown) . Double-mutant analysis suggests that unc-36 If ; egl- 19 If double-mutant animals were more sensitive to the pathogenic Burkholderia sp. than the single mutants alone, and that the egl-19 (gf) alleles could partially mitigate the negative affect of an unc-36 (If ) allele (MOST sensitive unc-36 (n251 ) ; egl -19 (n582) > unc- 36 (n251 ) ≥ egl-19 (n582) > unc-36 (n251 ) ; egl -19 (n2368) > wild-type > unc-36 (n251 ) ; egl-19 (ad695) ≥ egl- 19 (n2368) LEAST sensitive) (Figure 3B, Table 6 and data not shown. Mutations in the unc-2 gene { unc- 2(e55)), which also encodes a similar L-type voltage- gated Ca⁺² channel subunit involved in neurotransmitter adaptation (Schafer and Kenyon 1995, Nature 375, 73- 78), had little effect upon survivorship (data not shown) .

The C. elegans L-type Ca⁺² channel encoded by the egl - 19 and unc-36 genes provide the Ca⁺² necessary for signal transduction processes which are mediated in part by the calcium and calmodulin dependant protein kinase II (CaMKII) gene, unc-43 (Rongo and Kaplan 1999, Nature 402, 195-197). Because egl -19 and unc-43 act in the same neuromuscular signaling pathway, mutations in the unc-43 gene were also examined. We tested five unc-43 alleles in a killing assay against B . thailandensis E264 and calculated the LT50s for each of the alleles (Figure 3B, Table 6 and data not shown) . Homozygotes for the gain-of-function unc-43 allele {n498) had an LT50 that was almost three times longer than wild-type animals (Table 6) . Animals homozygous for the null allele, unc-43 { e408) , also exhibited an extended LT50 time relative to wild type, but the extension was less dramatic (Table 6) . Homozygotes for hypomorphic unc-43 alleles { {n498nll 79) and {n498nll86) ) behaved similarly to the null allele homozygotes (Table 6). Because many of the single and double mutant combinations tested are paralyzed it was important to confirm that the accelerated Burkholderia sp. induced paralytic death was due to the presence of the specified alleles and not simply a consequence of already being paralyzed. To asses the specificity of the interaction we examined the LT50 of another highly paralyzed mutant animal, unc-22 { If) homozygotes (Moerman and Baillie 1979, Genetics 91, 95-104). Animals homozygous for two different mutant alleles of the unc-22 gene { unc-22 { s7) and unc-22 { sl 77) ) stopped twitching at a rate similar to wild-type N2 Bristol animals (data not shown) .

DISCUSSION

Lower-order hosts to model pathogenesis . The objective of this study was to establish a lower-order model for identification of important virulence factors in pathogenic bacteria. The data shown here suggest that the disease phenotype observed in nematode hosts after exposure to B . pseudomallei may be valuable for investigating the pathogenesis of these bacteria, especially since few of its virulence determinants are understood. Many members of genus Burkholderia tested were able to kill nematodes (Table 1) .

The finding that B . mallei was not pathogenic in this surrogate model system suggests that nematode killing by this genus may not represent general correlates of animal virulence. However, the observed lack of pathogenesis by B . mallei in nematodes is not surprising, as this organism has no known environmental reservoir. While B . mallei was not directly amenable to study in the surrogate model, B. pseudomallei appeared to be.

All B. pseudomallei , B . thailandensis and B . cepacia species had strains capable of killing nematodes. However, pathogenicity in nematodes may not correlate directly with pathogenicity in mammals as the most nematocidal strain tested was B . thailandensis . Burkholderia thailandensis , like B. pseudomallei , is a soil saprophyte, often co-isolated from the same environmental sample (Brett, DeShazer et al . 1997, supra; Brett, DeShazer et al . 1998, supra). Classically, arabinose assimilation has been a discriminator of B . pseudomallei 'like' species virulence. Arabinose non-assimilators are much more virulent than arabinose assimilators . Recently, Brett et al . (Brett, DeShazer et al . 1997, supra; Brett, DeShazer et al . 1998, supra) has suggested that arabinose assimilators belong to species other than B. pseudomallei , the designation for one of those species was B. thailandensis .

B . thailandensis strains are markedly reduced in mammal virulence when compared to B . pseudomallei . The hamster LD50 for B . pseudomallei is less than 10 bacteria in 48 hours (DeShazer, Brett et al . 1997, supra) . The hamster LD50 of B . thailandensis is 105 times higher than B . pseudomallei (Brett, DeShazer et al. 1997, supra; Brett, DeShazer et al . 1998, supra), it does, however, still kill hamsters. While clearly not as adept at animal killing as B. pseudomallei , B. thailandensis possess some characteritics that suggest it may represent an intermediate form of pathogen. Harley et al . (Harley, Dance et al . 1998, Microbios . 96, 71-93) showed that B . thailandensis, like B. pseudomallei and B . mallei , is capable of invading and multiplying inside eukaryotic cells. Brett et al

(Brett, DeShazer et al . 1997, supra), showed B. thailandensis is the most cytotoxic of the related species. Further, there is at least one report of a human clinical infection with an arabinose assimilating 'B. pseudomal lei-like' strain,

(Lertpatanasuwan, Sermsi et al . 1999, Clin. Infect. Dis . 28, 927-8). Combining these reports with the results presented here suggests that arabinose assimilating B . pseudomallei 'like' strains deserve more intensive study.

Nematode killing is an active process . Genetic analysis using available B . pseudomallei mutants suggested that killling is an active process.

Nematode killing did not depend upon secreted exo- enzyme proteases, Upases or other 'digestive' enzymes because B . pseudomallei harboring gspD insertion mutations (DD213 and C21) and wild-type 1026b killed equally (Table 1 and data not shown) . The role of other secretion systems needs to be investigated. Any role of type III and type IV secretion systems in B. pseudomallei veterbrate pathogenesis has not been reported. B . pseudomallei mutants in a RND-class efflux transporter (DD503) killed as well as wild-type bacteria, suggesting that nematode killing is not due to the export of bacterial products such as aminoglycoside or macrolide antibiotic compounds (Table 1 and data not shown) . Streptomyces avermi tilis, another soil saprophyte species, produces potent antihelminthic macrolides called avermectins (Ormond 1983, U.S. Patent no. 4,412,991). Avermectin resistance conferring mutants have been isolated and characterized in C. elegans, { avr-14 ; avr-15) (Dent, Smith et al . 2000, supra). Pathogenic Burkholderia sp. kill avermectin resistant mutant C. elegans at the same rate as wild-type animals (A.L. O'Quinn and J.A. Jeddeloh, unpublished data) , also positing against the action of known anti-helmintic macrolides.

Burkholderia pseudomallei harboring mutations in LPS O-antigen synthesis (SRM117), which are modestly attenuated in Sryian hamsters, guinea pigs and diabetic infant rats, (Bryan, Wong et al . 1994, Can J. Infect. Dis . 5, 170-8; DeShazer, Brett et al . 1998, supra) , induce nearly wild-type pathogenesis in nematodes (A.L. O'Quinn and J.A. Jeddeloh, Table 1 and data not shown) . Motility has a negligible role in nematode pathogenesis because the flic knock-out strain, (MM36) kills as fast as the 1026b parent strain (A.L. O'Quinn and J.A. Jeddeloh, Table 1 and data not shown) .

Theoretically, nematode killing could exist along a continuum of relevance; ranging from an active process initiated by the bacteria, to a passive process manifested by starvation. Under some culture conditions, bacteria of the genus Yersinia inhibit growth of C elegans in a manner that suggests the animals are unable to feed normally (Creg Darby, personal communication) . Starvation caused little observable mortality under the 30 hr window during the LT50 assays (Figure 1) . Starvation among nematodes increases foraging and pharyngeal-pumping behaviors (Avery and Horvitz 1990, J. Exp. Zool . 253, 263-70). The data presented here suggest that both of these processes were inhibited by B. pseudomallei (Table 3), arguing against a non-specific, starvation-based lethal mechanism and positing some active aspect to the nematode killing observed.

Feeding on B . pseudomallei or B. thailandensis for long intervals ensured death even when the animals were returned to a nonpathogenic E. coli lawn (Table 3) . Feeding for 4 hr was long enough to substantially compromise the population-size attainable at day four, as well as make 50% of the nematodes stop feeding and laying eggs (Table 3). Feeding cessation, however, did not correlate with whether or not the animals survived to breed. Survivorship was further reduced at longer feeding intervals (Table 3). The loss of survivorship at the extended feeding interval suggests that either a lethal dose of some type of toxin was delivered and/or that longer feeding ensured that the pathogen multiplicity of infection became high enough to establish an infection with a lethal outcome.

Further evidence of the specific nature of the B. pseudomallei-nematode interaction is presented in Table 4. UV-inactivated bacteria were no longer able to kill nematodes . While bacterial lawns inactivated with radioactive cobolt continued to kill C. elegans . The photo-sensitive, radiation resistant nematode killing activity had no apparent upper ceiling as lawns inactivated with 9xl0⁶ rads continued to kill nematodes (data not shown) . The nematode death phenotype conditioned by gamma-killed bacteria was consistent with that of live bacteria. Irradiated bacteria continued to inhibit locomotion, feeding and egg-laying behaviors. However, there was one noticeable difference. The animals killed by irradiated bacteria appeared less constipated than those killed by live bacteria. Bacterial population growth within the C. elegans intestinal tract is correlated with pathogenesis in P. aeruginosa (Tan, Rahme et al . 1999, supra). Analogously, irradiated bacteria killed more slowly than live bacteria (Figure 3 ) . Combining the results of these observations suggests that nematode pathogenesis by B. thailandensis and B. pseudomallei involve an intoxication mechanism plus additional factors that depend upon living bacteria for delivery. The simplest interpretation would be that living/ proliferating bacteria deliver more of the toxin. Because gamma-irradiation inactivated B . thailandensis E264 , and B . pseudomallei 1026b kills nematodes, the feasibility of using irradiated plate lawns to purify and characterize the toxic moiety (s) is under exploration.

Nematode killing by intoxication ? An advantage to using C. elegans over cultured cells to model pathogenesis is that as an animal, it can sense and respond to environmental stimuli with observable changes in behavior (Dusenbery, Sheridan et al . 1975, Genetics 80, 297-309; Hedgecock and Russell 1975, Proc. Natl. Acad. Sci. USA 72, 4061-65; Wolinsky and Way 1990, Behav. Genet. 20, 169-89) . C. elegans ceases feeding in response to environmental intoxicants in a dose dependant fashion, and resumes feeding when the intoxicant is removed, (Jones and Candido 1999, J. Exp. Zool. 284, 147-57).

A similar pattern of feeding cessation behavior was observed after feeding the nematodes B . pseudomallei or B. thailandensis . Feeding cessation occurred in -50% of the nematode population within 4 hr (Table 3) . Feeding cessation as a behavioral response to feeding on pathogens seems to indicate that the nematodes were able to 'sense' the apparently harmful nature of the food source, and subsequently 'decided' not to eat more. Because the close relative B . pyrrocinia produces the potent fungicide pyrrolnitrin (Hammer, Hill et al . 1991 , Applied and Environmental Microbiology 63, 2147-2154; Hammer, Burd et al . 1999, FEMS Microbiol. Lett. 180, 39-44), and B . pyrrocinia has little nematocidal activity (Table 1) , it is unlikely the nematode feeding cessation induced by B. pseudomallei or B. thailandensis is mediated through bacterial production of anti-fungal/antibiotic products .

Another way to explore the ability of the nematodes to perceive any apparent toxicity of B . pseudomallei or B . thailandensis was to allow the nematodes to 'choose' the food source; aversion to pathogenic bacteria may reflect a perception of toxicity by the nematodes . The food choice experiments indicated that the animals were not averse to feeding on B. pseudomallei or B . thailandensis (Table 5). A tantalizing alternative hypothesis regarding the mechanism of feeding cessation exists that may unify both observations.

According to this alternative hypothesis, the animals cannot 'sense' the pathogens to be a bad food source or intoxicating. In this model, feeding cessation, as well as loss of locomotor activity and egg-laying, are consequences of a bacterial pathogenesis factor which affects either the neurons controlling these actions or the ability of the muscles to respond to normal neuronal stimuli. Under this hypothesis, there is no, or little, cellular perception of the acute toxicity. C. elegans - Burkholderia species interaction involves paralytic killing. The functional impairment of the locomotor, pharyngeal, and vulval muscles in C. elegans suggest that B . pseudomallei and B. thailandensis may employ some type of neurotoxin or paralytic agent as a part of their nematode pathogenic mechanism. Conflicts exist in the B . pseudomallei literature regarding the presence of toxins, which may be due to the differences in the assays and cell-types used to detect them (Nigg, Heckly et al . 1958, Proc. Soc. Exp. Biol. Med. 89, 17-20; Brett, DeShazer et al . 1997, supra; Haase, Janzen et al . 1997, J. Med. Microbiol. 46, 557-63; Haussler, Nimtz et al . 1998, Infect. Immun. 66, 1588-93). Early toxicological investigations of B . pseudomallei suggest it may possess a neurotoxic/paralytic activity (Nigg, Heckly et al . 1958, supra) . Several examples of neurotoxicicity are documented among human and animal melioidosis cases (Nigg, Heckly et al . 1958, supra; Narita 1982, National Institute of Animal Health Quarterly 22, 170-179; Woods, Currie et al . 1992, Clin. Inf. Dis . 15, 163-6; Smith, Angus et al . 1997, Infect. Immun. 65, 4319-21).

Loss of regulated L-type voltage gated Ca⁺² channel activity could result in paralysis both by inhibition of signaling pre-synaptically, as well as post- synaptically. Voltage gated Ca⁺² channels mediate both signaling processes (Lee, Loebel et al . 1997, EMBO 16, 6066-76) . Further, pathogenesis by uropathogenic E. coli , has been recently shown to utilize a pathogenesis mechanism involving host L-type Ca⁺² channels, (Uhlen, Laestadius et al . 2000, Nature 405, 694-697), suggesting that attack by prokaryotes upon eukaryotic second messenger signaling pathways may be a general pathogenesis strategy.

Nematode paralysis and death caused by Burkholderia sp. may result from a toxin mediated inability to restore Ca⁺² membrane potentials. Perhaps the action of the gain-of-function alleles in the egl -19 L-type Ca⁺² channel subunit, and the unc-43 CaMKII, partially impedes the establishment and perception of bacterially preturbed changes in Ca⁺² signals, resulting in a slowing of killing/paralysis. Whatever the specific mechanism, double mutant analysis (Figure 3B and Table 6) suggests that both the body wall muscles { unc-36) and the neuromuscular junction { egl - 19) are targeted by the bacteria.

Both the egl-19 gene and egl-9 genes appear to be expressed in a similar spectrum of nematode cell types (Lee, Loebel et al . 1997, supra; Darby, Cos a et al . 1999, supra) . egl -9 is a protein of unknown function. Loss-of-function mutations in the egl -9 gene have the same phenotype as gain-of-function mutations in the egl-19 gene and the unc-43 gene with respect to Burkholderia-species survival enhancement. Perhaps egl-9 is interacting with egl-19 or unc-43 as a mediator, or modulator of their activity. Double mutants would provide a clue and may define another genetic function for egl -9, outside of its effect upon egg-laying (egl= egg-laying deficient) ; double mutant construction is underway.

Claims

What is claimed is:

1. A composition comprising toxin from Burkholderia species .

2. The composition according to claim 1 wherein said Burkholderia is chosen from the group consisting essentially of: B. pseudomallei, B. thailandensis, B . cepacia, and B. mallei .

3. An antihelmenthic composition comprising the composition according to claim 1.

4. An antihelmenthic composition comprising the composition according to claim 2.

5. A composition comprising purified and isolated toxin from Burkholderia chosen from the group consisting essentially of B. pseudomallei, B. thailandensis , B. mallei , and B. cepacia .

6. A vaccine for protection against B. pseudomallei comprising a composition comprising a toxin according to claim 5.

7. An antihelminthic treatment for reducing symptoms from helminthic infections from nematodes, said method comprising administering to a patient in need of such treatment an effective amount of B. thailandensis toxin in a pharmaceutically acceptable excipient.

8. A pharmaceutical composition comprising at least one toxin from a Burkholderia species and a pharmaceutically acceptable excipient.

9. A kit comprising a composition according to claim 8 in a container with printed instructions on or accompanying the container concerning the administration of the composition to a patient to protect against or treat conditions caused by Burkholderia

9. A nematode growth inhibitor comprising the composition according to claim 1.

10. A nematode toxin according to claim 9 wherein said Burkholderia is B. thailandensis .

11. A method for combatting nematodes in soil, comprising blending into the soil an effective amount of the composition according to claim 10.

12. A method for combatting nematodes in a plant, comprising applying the composition of claim 1 onto said plant.

13. Isolated DNA encoding a nematode toxin from Burkholderia .

14. The DNA according to claim 13 wherein said Burkholderia is B. thailandensis .

15. A vector comprising the isolated DNA of claim 14

16. A plant cell comprising the vector of claim

15

17. A plant comprising the plant cell of claim 16.

18. A seed of a plant of claim 17.

19. A tissue culture of the plant of claim 17.

20. A plant expressing a B. thailandensis nematode toxin.