AU2001250037A1 - Methods for screening and identifying pathogen virulence factors - Google Patents

Description

METHODS FOR SCREENING AND IDENTIFYING PATHOGEN NTRIIT,ENCE FACTORS

Background of the Invention The invention relates to screening methods for identifying pathogen virulence factors and for identifying drugs that inhibit pathogen infections.

Microbial pathogens use a variety of complex strategies to subvert host cellular functions to ensure their multiplication and survival. Some pathogens that have co-evolved or have had a long-standing association with their hosts utilize finely tuned host-specific strategies to establish a pathogenic relationship. During infection, pathogens encounter different conditions, and respond by expressing virulence factors that are appropriate for the particular environment, host, or both.

Although antibiotics have been effective tools in treating infectious disease, the emergence of drug resistant pathogens is becoming problematic in the clinical setting. New antibiotic or antipathogenic molecules are therefore needed to combat such drug resistant pathogens. Accordingly, there is a need in the art for screening methods aimed not only at identifying and characterizing potential antipathogenic agents, but also for identifying and characterizing the virulence factors that enable pathogens to infect and debilitate their hosts.

Summary of the Invention We have discovered that the microbial pathogen, Salmonella typhimurium, establishes a long-lasting, persistent infection in the nematode, Caenorhabditis elegans. This discovery enables simple screening methods for identifying the interplay between environmental and host signals (e.g., host-dependent or host- independent signals) and physiological pathogenic pathways that control or regulate genes responsible for establishing a persistent infection, as in the colonization of the gut of the nematode. In one aspect, the invention features an isolated nematode persistently infected with an isolated pathogen. Preferably, the nematode is C. elegans (such as a one-day old adult hermaphrodite worm or an L4 larval stage worm). In preferred embodiments, the isolated pathogen expresses a detectable marker; or colonizes the intestine of the nematode. In still other preferred embodiments, the pathogen is Salmonella (such as Salmonella typhimurium strain SL1344 or strain LT2).

In another aspect, the invention features a method of screening for a virulence factor that enables a pathogen to develop a persistent infection in a nematode. The method generally involves the steps of: (a) exposing a nematode to a mutagenized pathogen, a pathogen expressing a gene not normally present in the pathogen, or a pathogen overexpressing a pathogen gene; (b) determining whether the mutant or otherwise altered pathogen persistently infects the nematode, where a reduction or enhancement of disease in the nematode relative to that caused by the non-mutagenized or otherwise altered pathogen indicates a mutation in a virulence factor or a virulence factor gene that enables the pathogen to develop a persistent infection in the nematode; and (c) using the mutation or virulence factor gene as a marker for identifying the virulence factor.

Preferably, the nematode utilized in the method of screening for a pathogenic virulence factor is Caenorhabditis elegans (such as a one-day old adult hermaphrodite worm or an L4 larval stage worm). In preferred embodiments, the mutated or otherwise altered pathogen used for identifying the virulence factor expresses a detectable marker. In other preferred embodiments, colonization of the intestine of the nematode by the mutated or otherwise altered pathogen is decreased. In still other preferred embodiments, the pathogen used is Salmonella (such as Salmonella typhimurium strain SL1344 or strain LT2).

In yet other preferred embodiments, the method utilizes a salmonellae/C elegans killing assay. In such methods, the mutated or otherwise altered pathogen has reduced or enhanced capacity to develop a persistent infection in C. elegans, causing less or more killing than the non-mutagenized or otherwise altered pathogen. In another aspect, the invention features a method of screening for a compound that inhibits a persistent pathogenic infection in a nematode. The method generally involves the steps of: (a) providing a nematode persistently infected with a pathogen; (b) contacting the persistently infected nematode with a test compound; and (c) determining whether the test compound inhibits the persistent infection in the nematode.

Preferably, the nematode utilized in the compound screening method is Caenorhabditis elegans (such as a one-day old adult hermaphrodite worm or an L4 larval stage worm). In preferred embodiments, the pathogen used in the compound screening assay expresses a detectable marker. In other preferred embodiments, colonization of the intestine of the nematode by the pathogen is reduced. In still other preferred embodiments, the pathogen used is Salmonella (such as Salmonella typhimurium strain SL1344 or strain LT2).

In yet other preferred embodiments, the test compound is provided in a compound library; is a small organic compound; or is a peptide, peptidomimetic, or an antibody or fragment thereof.

In other preferred embodiments, the compound screening method utilizes a salmonellae/C. elegans killing assay. In such methods, the pathogen that persistently infects C. elegans causes less killing in the presence of the test compound than in the absence of the test compound.

In another aspect, the invention features a method of screening for a virulence factor that enables a pathogen to develop a persistent infection in a nematode. The method generally involves the steps of: (a) exposing a nematode to a pathogen expressing a detectable marker, the pathogen being mutagenized expressing a gene not normally expressed by the pathogen, or overexpressing a pathogen gene; (b) determining whether the mutant pathogen persistently infects the nematode by measuring the level of detectable marker in the nematode, where a decrease or increase of the marker in the nematode relative to that caused by the non-mutagenized pathogen indicates a mutation in a virulence factor or a virulence factor gene that enables the pathogen to develop a persistent infection in the nematode; and (c) using the mutation or virulence factor gene as a marker for identifying the virulence factor.

In another aspect, the invention features a method of screening for a compound that inhibits a persistent pathogenic infection in a nematode. The method generally features the steps of: (a) providing a nematode persistently infected with a pathogen expressing a detectable marker; (b) contacting the persistently infected nematode with a test compound; and (c) determining whether the pathogen persistently infects the nematode by measuring the level of detectable marker in the nematode, where a decrease of the detectable marker in the nematode indicates that the test compound inhibits a persistent pathogenic infection in the nematode.

Exemplary pathogenic bacteria useful in the methods of the invention, as well as for producing persistently infected nematodes, include, without limitation, Aerobacter, Aeromonas, Acinetobacter, Agrobacterium, Bacillus, Bacteroides, Bartonella, Bordetella, Bortella, Borrelia, Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Citrobacter, Clostridium, Cornyebacterium, Enterobacter, Enterococcus, Escherichia, Francisella, Gardnerella, Haemophilus, Hafnia, Helicobacter, Klebsiella, Legionella, Listeria, Morganella, Moraxella, Mycobacterium, Neisseria, Pasteurella, Proteus, Providencia, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Stentorophomonas, Treponema, Xanthomonas, Vibrio, and Yersinia.

By "virulence factor" is meant a cellular component (e.g., a protein such as a transcription factor or a molecule) without which a pathogen (e.g., a bacterium) is incapable of causing disease or infection in a eukaryotic host organism (e.g., a nematode or mammal). Such components, for example, are involved in the adaptation of the bacteria to a host (e.g., a nematode host), establishment of a bacterial infection, maintenance of a bacterial infection, or generation of the damaging effects of the infection to the host organism. Further, the phrase includes components that act directly on host tissue, as well as components which regulate the activity or production of other pathogenesis factors.

By "inhibits a pathogen" is meant the ability of a test compound to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a pathogen-mediated disease or infection in a eukaryotic host organism. Preferably, such inhibition decreases pathogenicity by at least 5%, more preferably by at least 25%, and most preferably by at least 50% or more, as compared to symptoms in the absence of the test compound in any appropriate pathogenicity assay (for example, those assays described herein). In one particular example, inhibition may be measured by monitoring pathogenic symptoms in a nematode persistently infected with a salmonellae pathogen exposed to a test compound or extract, a decrease in the level of pathogenic symptoms relative to the level of symptoms in the host organism not exposed to the compound indicating compound-mediated inhibition of the salmonellae pathogen.

By "persistent infection" or "persistently infected" is meant an invasion or colonization of a host animal (e.g., nematode) by a pathogen (e.g., Salmonella) that is damaging to the host, where the size of the persistent pathogenic population that are associated with the host after the host has been transferred to a non-infectious environment remains at least 30%, preferably 50%, more preferably 80%, and most preferably 90%, or even 95% to 99%) of the size of the pathogenic population before the transfer of the host to a non-infectious environment. Such an infection also includes an increase in the numbers of the pathogenic population that are associated with the host when the host is first exposed to a relatively small number of the pathogen mixed with an excess of non-pathogenic bacteria after which the host is transferred to a non-infectious environment. A persistent infection is typically measured using a nematode feeding assay (as described herein) where bacteria are assayed for their ability to establish a long-lasting association in the worm intestine. By "detectable marker" is meant a gene whose expression may be assayed; such genes include, without limitation, β-glucuronidase (GUS), luciferase (LUC), chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), and β-galactosidase. By "isolated nematode" is meant a nematode that is purified from contaminating organisms and maintained in a culture. Exemplary nematodes (wild type or mutant), such as C. elegans, are obtained from publicly available sources or purified from the environment according to standard methods known in the art. By "isolated pathogen" is meant a microbial strain that has been cultured, through the actions of man, and that elicits a disease response in a host.

The present invention provides a number of advantages. For example, the invention facilitates the identification of novel targets and therapeutic approaches for preparing therapeutic agents active on virulence factors and genes that enable a pathogen to develop a long-lasting, persistent infection in its host organism.

The invention also provides long awaited advantages over a wide variety of standard screening methods used for distinguishing and evaluating the efficacy of a compound against salmonellae pathogens. In one particular example, the screening methods described herein allow for the simultaneous evaluation of host toxicity as well as anti-salmonellae potency in a simple in vivo screen. Moreover, the methods of the invention allow one to evaluate the ability of a compound to inhibit salmonellae pathogenesis, and, at the same time, to evaluate the ability of the compound to stimulate and strengthen a host's response to salmonellae pathogenic attack. Accordingly, the methods of the invention provide a straightforward means to identify compounds that are both safe for use in eukaryotic host organisms (i.e., compounds which do not adversely affect the normal development and physiology of the organism) and efficacious against pathogenic microbes that establish persistent infections in their hosts. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for anti-salmonellae pathogenic effect with high-volume throughput, high sensitivity, and low complexity. The methods are also relatively inexpensive to perform and enable the analysis of small quantities of active substances found in either purified or crude extract form. Furthermore, the methods disclosed herein provide a means for identifying compounds that have the capability of crossing eukaryotic cell membranes and which maintain therapeutic efficacy in an in vivo method of administration. In addition, the above-described methods of screening are suitable for both known and unknown compounds and compound libraries.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Detailed Description The drawings will first be described. Drawings

Fig. 1 A shows the mechanism C. elegans killing by S. typhimurium strain SL1344. Between 10 to 20 worms were placed on each plate and each assay consisted of two replicates. L4 stage (open circle) and 1-day-old adult hermaphrodite (closed triangle and circles) worms fed either on S. typhimurium SL1344 (closed triangle and open circle) or on E. coli OP50 (closed circle). The small bars represent the standard deviations and the results are representative of at least six independent experiments.

Fig. IB shows the results of a C. elegans shifting experiment and the percentages of dead worms after transfer to OP50-containing plates after feeding for 5 hours on SL1344 (open circle) or on OP50 (closed circle). The small bars represent the standard deviations and the results are representative of at least three independent experiments. The insert shows the percentages of dead worms after transfer to OP50-containing plates after feeding for 1, 3, or 5 hours on SL1344. The small bars found in the insert represent the standard deviations and the results are representative of at least two independent experiments.

Fig. 2 shows confocal images of bacterial colonization of the C. elegans intestine. The worms were fed o E. coli expressing GFP (E. co/t/GFP) (Panels A and B), S. typhimurium expressing GFP (S. typhimurium! 'GFP) (Panels C and D) for 72 hours, or on P. aeruginosa expressing GFP (P. aeruginosa/GFr') (Panels E and F) for 24 hours. In the transmission images (Panels A, C, and E), the intestine margins are noted with arrows. Merged images (B, D, and F) show the bacterial fluorescence (green channel) and the gut autofluorescence (red channel). The bars represent 50 μm.

Fig. 3 shows confocal images of bacterial colonization of the C. elegans intestine in a shifting experiment. The worms were fed on OP50 for 24 hours after feeding on E. coli expressing GFP (E. co/i/GFP)(panel A) or S. typhimurium expressing GFP (S. typhimurium/ GW)(panels B and C) for 5 hours. The merged images show the bacterial fluorescence (green channel) and the gut autofluorescence (red channel). The bars represent 50 μm.

Fig. 4A shows that S. typhimurium proliferates in the intestine of C. elegans. Several dilutions of S. typhimurium in E. coli were prepared on NG plates, and 10 worms were immediately place on the plates. Each assay consisted of two replicates. After 24 hours, the worms were transferred to new plates and the amount of Salmonella in the plates was determined by counting colony forming units (c.f.u.) on MacConkey agar plates. The small bars represent the standard deviations and the results are representative of at least two independent experiments.

Fig. 4B shows that S. typhimurium proliferates in the intestine of C. elegans. Seventy to eighty 1 -day-old adult hermaphrodite worms were placed on plates containing S. typhimurium expressing GFP (S. typhimurium/ 'GFP) and E. coli not expressing GFP in a ratio of 1 : 10,000. In addition, the same number of worms were placed on E. coli expressing GFP. After 5 hours the worms were washed in M9 buffer and transferred to E. coli not expressing GFP plates. Subsequently, every 24 hours, 10 worms were transferred to M9 buffer containing 1% Triton X-100, and then the worms were mechanically disrupted to count the bacteria present in the gut. Bacteria were subsequently diluted in 10 mM MgSO₄, plated on plates containing ampicillin, and the c.f.u. counted. The data represent the means ± S.D. of two independent experiments. Fig. 5A shows that worm killing requires direct interaction between live S. typhimurium and C. elegans. PA14 (open circle) were grown on 0.45 μm filters placed on PGS plates, and SL1344 were grown on either PGP (closed circle) or NG plates (square). Following growth of the bacteria, filters were removed, the plates exposed to UN light for 5 minutes to kill any possible contaminating bacteria, heat-killed OP50 added as a source of food, and 20 worms were placed on each plate. Small bars represent the standard deviations and the results are representative of at least two independent experiments.

Fig. 5B shows that worm killing requires direct interaction between live S. typhimurium and C. elegans. Twenty worms feeding on heat-killed SL1344 (closed circle) or SL1344 live (open circle) and heat-killed SL14028 (closed square) or live SL14028 (open square). The small bars represent the standard deviations and the results are representative of at least two independent experiments.

Below we describe experimental evidence demonstrating that Salmonella typhimurium causes disease in the nematode C. elegans by establishing a persistent infection, including a long-lasting association in the nematode intestine, and that C. elegans feeding on lawns of S. typhimurium eventually die over the course of a few days as a result of a pathogenic process. The salmonellae/C. elegans killing assay described herein therefore provides a useful system for identifying novel virulence factors responsible for a pathogen's ability to develop a persistent infection, as well as for identifying compounds that either inhibit pathogenicity of salmonellae, promote a host's resistance to the pathogen, or both. The following experimental examples are intended to illustrate, not limit, the scope of the claimed invention.

S. typhimurium Kills C. elegans by Establishing a Long-lasting Infection

To determine if S. typhimurium kills C. elegans, worms were fed on a lawn of bacteria grown on ΝG agar and the time to kill 50% of the worms (LT50) was calculated in six independent experiments. The reduced LT50 of worms fed with S. typhimurium (LT50 = 5.10 ±0.7 days) compared with the one obtained using worms fed with E. coli (LT50 = 9.86 ±0.9 days), indicated that the worms were killed by S. typhimurium (Fig 1 A).

In addition, the physiology of the worms fed with S. typhimurium was 5 different than worms feeding on E. coli OP50. As reported for P. aeruginosa infection (Tan et al., Proc. Natl. Acad. Sci. 96:2408-2413, 1999), the motility of the worms and the rate of pharyngeal pumping gradually declined, until the nematodes became immobile and died. In some cases, worms became laden with eggs, and at early times during the infections embryos hatched internally,

10 suggesting an egg-laying defect. To study if S. typhimurium was capable of preventing eggs from hatching, eggs were placed in plates containing S. typhimurium. We observed that 95% of the eggs hatched, indicating that S. typhimurium does not prevent hatching under these conditions.

Since S. typhimurium is known for establishing a long-standing

15 association or persistent infection with their hosts (Galan and Bliska, Annu. Rev. Cell Dev. Biol. 12:221-255, 1996), the bacterium was examined for its ability to colonize the worm intestine in an irreversible manner. The worms were fed with S. typhimurium for five hours, and then were transferred to plates containing E. coli. The results shown in Fig. IB indicate that after 5 hours of infection S.

20 typhimurium colonized the worm intestine, and that the worms died in the course of several days. The rate of killing in this shifting experiment was similar to those obtained when the C. elegans was in contact with S. typhimurium during the whole killing process. To study the time course of S. typhimurium infection, adult worms were allowed to feed on S. typhimurium for 1, 3, and 5 hours and then

25 transferred to plates containing E. coli. As shown in the insert presented in Fig. IB, the proportion of worms that survived after the transfer to E. coli plates was inversely proportional to the time spent feeding on SL1344. At 8 days, only 30% of the worms that had been transferred to the E. coli plates after feeding on SL1344 for 1 hour died. The proportion of dead worms after 8 days increased to

30 90 % when they were fed on SL1344 for 5 hours before being transferred. In contrast, when C. elegans was fed on Pseudomonas aeruginosa PA14 for 6 hours then transferred to E. coli, no killing was observed within the time frame of the experiment (60 hours) (or within the normal time frame of 100% killing by feeding constantly on PA14). This result is interpreted as indicating that PA14 does not establish a lethal infection within the parameters of the experiments 5 described for Salmonella. The Killing Mediated by S. typhimurium Correlates with Proliferation of the Bacteria in the Gut

To confirm that S. typhimurium killing involves an infectious process, we constructed a S. typhimurium strain expressing the Aeguorea victoria GFP. After

10 72 hours of feeding on S. typhimurium expressing GFP, the intestinal lumen was found to be distended and full of intact bacteria, suggesting that S. typhimurium may be proliferating in the gut (Fig. 2, Panels B and C). Although Pseudomonas aeruginosa PA14 is unable to establish a long-lasting infection, similar results were obtained when the worms were fed with PA14/GFP for 24 hours (Fig. 2,

15 Panels E and F). In contrast, no intact bacteria were observed when the worms were fed with the control E. coli DH5alpha/GFP strain and the lumen was not distended (Fig. 2, Panels A and B). The worms were also fed with S. typhimurium/ 'GFP or E. coli DH5alpha/GFP for only 5 hours and then transferred to E. coli plates. Fig. 3 (Panels A, B, and C) shows that no E. coli was found in 0 the C. elegans intestine after 24 hours, while S. typhimurium survived into the intestinal lumen. Intact S. typhimurium/GFF is shown at higher magnification (Fig. 3, Panel C).

To evaluate the colonization process of S. typhimurium, the amount of bacteria used in the feeding assays was reduced. Several dilutions of S. 5 typhimurium in E. coli were prepared on NG plates and the worms were immediately placed on the plates for feeding. The percentage f Salmonella was then determined by counting the presence of c.f.u. on MacConkey agar plates. The results of these feeding experiments showed that S. typhimurium diluted up to 1:1,000 was capable of killing C. elegans at levels similar to feeding on 100% S.

30 typhimurium, whereas a 1:10,000 dilution showed reduced virulence (Fig. 4A). Since 0.1% of S. typhimurium supported killing, this concentration of bacteria was used to carry out a chase experiment to quantify the amount of bacteria in the lumen of the intestine during the infection. The worms were exposed for five hours to 0.1% SL1344/GFP in OP50 or to DH5alpha/GFP, and then transferred to OP50 plates. Every 24 hours 10 worms were placed in a 1.5 ml centrifuge tube and disrupted by pressing them with a pellet pestle. Dilutions of bacteria were plated and the c.f.u. counted. The results showed that after 24 hours DH5alpha/GFP practically disappeared from the worm intestine, whereas SL1344/GFP proliferated, colonizing the worm intestine (Fig. 4B). Direct Interaction between S, typhimurium and C. elegans is Required for Killing P. aeruginosa has been shown to kill C. elegans by the secretion of diffusible toxins referred to as "fast killing" (Tan et al., Proc. Natl. Acad. Sci. 96:2408-2413, 1999 and Mahajan-Miklos et al., Cell 96: 47-56 1999). To determine if C. elegans killing by S. typhimurium is mediated by a diffusible toxin, a killing assay was performed under fast killing conditions, using sorbitol containing plates (PGS plates). It has been shown that L4 worms died much more rapidly than adults under the P. aeruginosa fast killing conditions (Tan et al., Proc. Natl. Acad. Sci. 96:2408-2413, 1999), therefore, L4 stage worms were used in these experiments. S. typhimurium and control P. aeruginosa and E. coli were grown on 0.45 μm filters placed on the plates. The filters containing the bacteria were removed and heat inactivated E. coli were place as a source of food to avoid starvation (Fig. 5 A). The results showed that the killing mediated by S. typhimurium under these conditions is not due to any diffusible toxin. Fig. 5B shows that C. elegans survived when fed heat-killed Salmonella. We have also fed the worms using the heat-killed and live S. typhimurium clinical isolated strain 14028. The results shown in Fig. 5B indicate that heat-killed SL14028 failed to kill C. elegans, and that live SL14028 killed the worms as found for SL1344. Materials and Methods

The above-described results were obtained using the following materials and methods.

Bacterial Strains, Plasmids, and Growth Conditions 5 The P. aeruginosa strain PA14 (Rahme et al., Science 268, 1899-1902,

1995), PA14/GFP strain (Tan et al., Proc Natl Acad Sci U S A 96, 715-720, 1999), S. typhimurium strain SL1344 (Hoiseth, Nature 291, 238-239, 1981), SL1344/GFP (this work), S. typhimurium clinical isolated strain 14028 (SL14028) were kindly provided by E. Hohman , Escherichia coli strain DH5alpha 10 (Bethesda Research Laboratories), DH5alpha/GFP strain (Tan et al., Proc Natl Acad Sci U S A 96, 715-720, 1999), and Escherichia coli strain OP50 (Brenner, Genetics 77, 71-94, 1974) were grown at 37 °C in Luria Broth (LB) media. The SL1344/GFP strain was made using the construct pSMC21-GFP (Bloemberg et al., Appl. Environ. Microbiol. 63:4543-51, 1997). 15 Maintenance of the Nematodes

The nematodes were maintained as hermaphrodites at 20 °C, grown on standard plates and fed with Escherichia coli strain OP50 as described by Sulston and Hodgkin (In: the Nematode Caenorhabditis elegans, ed., W.B. Wood, Cold Spring Harbor Lab. Press, Plainview, N.Y., pp. 587-606, 1988). Worms were 20 observed under a dissecting microscope (Leica MZ6).

Bacterial Infection Assays

The killing assays were conducted by spreading 10 μl of bacterial culture grown overnight in LB on nematode growth (NG; modified from nematode growth medium agar described in Sulston and Hodgkin (In: the Nematode 25 Caenorhabditis elegans, ed., W.B. Wood, Cold Spring Harbor Lab. Press, Plainview, N.Y., pp. 587-606, 1988); using 0.35% instead of 0.25% peptone or peptone-glucose-sorbitol (PGS; 1% Bacto-Peptone/1% NaCl/1% glucose/0.15 M sorbitol/1.7% Bacto-Agar) media for nematode killing assays (3.5 cm diameter plates). After spreading the bacterial culture, plates were incubated at 37 °C for 30 12 hours and then ten to twenty worms were placed on the assay plate, which was then incubated at 25 °C. When required, the bacteria were killed by heating at 60 °C for 90 minutes. The E. coli strain OP50 was used as a control for the assays. Worm mortality was scored over time, and a worm was considered dead when it failed to respond to touch. Any worms that died as a result of getting stuck to the wall of the plate were excluded from the analysis. The time to kill 50% of the nematodes (LT50) was calculated using the PRISM (version 2.00) computer program using the equation: Y=Bottom+(Top-

Bottom)/(l+10^Λ((LogΕC50-X)*HillSlope)), where X is the logarithm of days and Y is the average of killed worms; Y starts at Bottom and goes to Top with a sigmoid shape.

C. elegans Shifting Experiments

Eighty to one-hundred one-day-old adult hermaphrodite worms were seeded on bacteria lawns and allowed to feed. After 1, 3, or 5 hours, the worms were transferred to plates containing E. coli OP50. Before transfer, worms were washed two times in M9 buffer, transferred to plates containing OP50 for two hours and then transferred to new plates containing OP50. Every 24 hours the worms were transferred to new plates and the c.f.u. counted. To count the c.f.u, 10 worms were washed in M9 buffer and transferred to a 1.5 ml tube with M9 buffer containing 1% Triton X-100 where they were mechanically disrupted using a pellet pestle. Dilutions of bacteria were made in 10 mM MgSO₄, plated on ampicillin containing plates or in MacConkey agar base plates, and the c.f.u. counted.

Con focal Microscopy

The worms were seeded on DH5alpha GFP or SL1344/GFP lawns. After 5 hours, one half the total number of worms was transferred to plates containing E. coli OP50 as described above. For each time point, five worms were placed on a pad of 1% agar in PBS and 30 μM sodium azide in M9 buffer was used as anesthetic. The experiments were repeated at least four times and confocal imaging was performed using a Leica TCS SP confocal microscope. Composite images were assembled and edited using Adobe PhotoShop 5.0. Nematode Screening Systems For Identifying Virulence Factors Establishing Persistent Infections

Based on the results described above showing that Salmonella establishes a persistent infection in the nematode, C. elegans, we have developed methods for identifying virulence determinants important for establishing this type of infection. The screen, in general, utilizes the above-described pathogen/nematode killing assays and exploits the ability to screen thousands of randomly generated mutant pathogens. In addition to using wild type host worms in the screening assays, mutant worms that are constipated or defecation defective, such as aex-2 and unc-25, mutants that are grinding defectiye, such as phm-2 and eat-14, and specific ABC transporter mutants such as pgp-4 and mrp-1 may be utilized as well.

In general, a pathogen is assayed (using the method described herein) for its ability to establish a persistent infection in a nematode. Once identified, the pathogen, such as Salmonella typhimurium strain SL1344, is mutated according to standard methods known in the art and then subsequently evaluated for its ability to induce disease by establishing a persistent infection in the nematode host organism. A mutagenized pathogen found to have diminished ability for establishing a persistent infection is useful in the method of the invention. Such mutant pathogens are then used for identifying host-dependent or host- independent virulence factors responsible for establishing the persistent infection according to methods known in the art.

Other screening assays for identifying and characterizing virulence factors include constitutive or overexpression of putative virulence genes from Salmonella or other bacterial species. Examples would be constitutively expressing a putative transcription factor or repressor on a plasmid in the pathogen, such as Salmonella typhimurium strain SL1344, and testing these altered pathogens for enhanced or reduced virulence in C. elegans. Alternatively, a plasmid library of clones covering the entire Salmonella typhimurium (or other pathogen) genome, could be screened for enhancement or supression of Salmonella virulence in C. elegans. The results of these screens identify virulence factors, and subsequently provided valuable information as to their downstream targets.

The following is a working example of a virulence factor nematode screening system which utilizes the human clinical isolate S. typhimurium strain SL1344 identified as persistently infecting the gut of C. elegans. The advantage of using a nematode as a host for studying microbial pathogens is the relative simplicity of identifying non-pathogenic mutants in the nematode system.

In one preferred working example, in which survival is monitored, four to eight C. elegans worms (e.g.,one-day old adult hermaplirodites) are placed on a lawn of a mutagenized strain of a pathogen, and survival is monitored after approximately five hours according to the methods described herein. For example, a pathogen, such as Salmonella typhimurium strain SL1344, is mutated according to any standard procedure, e.g., standard in vivo or in vitro insertional/transposon mutagenesis methods (see, e.g., Kleckner et al., J Mol. Biol. 116: 125, 1977; Simon et al., Gene 80 (1): 161-169, 1989). Other methods are also available, e.g., chemical mutagenesis, or directed mutagenesis of DNA. After very few or no live worms are found on a plate seeded with wild-type, pathogenic S. typhimurium strain SL1344, whereas on a plate with mutagenized S. typhimurium strain SL1344, increased survival (e.g., as deteπnined by an increased LT₅₀) of the worms is observed. Thus, the ability of worms to grow in the presence of mutated S. typhimurium strain SL1344 is an indication that a gene responsible for pathogenicity has been inactivated. The positions of the inactivating mutations are then identified using standard methods, (e.g., by polymerase chain reaction and sequencing of insertion/transposon junctions or by mapping), leading to the cloning and identification of the mutated virulence factor(s) (e.g., by nucleotide sequencing).

In another working example, pathogenesis is assayed by monitoring colonization of the nematode intestine, two to eight C. elegans worms (e.g., L4 hermaphrodite larvae) are placed on a lawn of mutagenized Salmonella typhimurium strain LT2, prepared from a mutagenic library, expressing a detectable marker (e.g., GFP) for an appropriate period of time and then transferred to plates for feeding on non-pathogenic bacteria (e.g., E. cσtϊ/DH5alpha). Strain LT2 is mutated according to standard methods. After approximately five hours worms are examined for the presence of the pathogen in the intestine using confocal microscopy. Worms feeding on wild-type, pathogenic S. typhimurium strain LT2, will present bacteria growing and proliferating in the gut. Worms feeding on mutated LT2 that contain a mutation in a gene that enables the establishment of a persistent infection will present a reduced, non-proliferating population in the gut. Thus, the absence or reduced presence of mutated LT2 in the worm intestine is taken as an indication that a gene responsible for pathogenicity has been inactivated. The mutated virulence factor is then identified using standard methods. Compound Screening Assays

As discussed above, our experimental results demonstrated that virulence factors are involved in pathogenicity of the nematode, C. elegans. Based on this discovery we have also developed a screening procedure for identifying therapeutic compounds (e.g., anti-pathogenicity pharmaceuticals) which can be used to inhibit the ability of a pathogen to persistently infection. In general, the method involves screening any number of compounds for therapeutically- active agents by employing the Salmonellae/nematode killing system described herein. Based on our demonstration that these pathogens infect and kill C. elegans, it will be readily understood that a compound which interferes with the pathogenicity of a pathogen (e.g., a salmonellae pathogen) in a nematode also provides an effective therapeutic agent in a mammal (e.g., a human patient). Whereas most antibiotics currently in medical use are either bactericidal or bacteriostatic, thus favoring resistant strains or mutants, the compounds identified in the screening procedures described herein do not kill the bacteria or prevent their growth in vitro, but instead render them non-pathogenic. Moreover, since the screening procedures of the invention are performed in vivo, it is also unlikely that the identified compounds will be highly toxic to the host organism. Accordingly, the methods of the invention simplify the evaluation, identification, and development of active agents such as drugs for the treatment of pathogenic diseases caused by microbes that can establish a long lasting association or persistent infection with a nematode. In general, the chemical screening methods of the invention provide a straightforward means for selecting natural product extracts or compounds of interest from a large population which are further evaluated and condensed to a few active and selective materials. Constituents of this pool are then purified and evaluated in the methods of the invention to determine their anti-pathogenic activity.

Test Extracts and Compounds

In general, novel anti-pathogenic drugs are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. The screening method of the present invention is appropriate and useful for testing compounds from a variety of sources for possible anti-pathogenic activity. The initial screens may be performed using a diverse library of compounds, but the method is suitable for a variety of other compounds and compound libraries. Such compound libraries can be combinatorial libraries, natural product libraries, or other small molecule libraries. In addition, compounds from commercial sources can be tested, as well as commercially available analogs of identified inhibitors.

For example, those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi- synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods. In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their anti-pathogenic activity should be employed whenever possible.

When a crude extract is found to have anti-pathogenic activity, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having anti-pathogenic activity. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful agents for the treatment of pathogenicity are chemically modified according to methods known in the art. Since many of the compounds in libraries such as combinatorial and natural products libraries, as well as in natural products preparations, are not characterized, the screening methods of this invention provide novel compounds which are active as inhibitors or inducers in the particular screens, in addition to identifying known compounds which are active in the screens. Therefore, this invention includes such novel compounds, as well as the use of both novel and known compounds in pharmaceutical compositions and methods of treating. Exemplary High Throughput Screening Systems

To evaluate the efficacy of a molecule or compound in promoting host resistance to, or inhibiting pathogenicity of, a number of high throughput assays may be utilized. For example, to enable mass screening of large quantities of natural products, extracts, or compounds in an efficient and systematic fashion, Caenorhabditis elegans, (e.g., one-day-old adult hermaphrodite worms, an L4 hermaphrodite larvae or a mutant worm such as aex-2, unc-25,phm-2, eat-14, pgp-4, or mrp-1), are cultured in wells of a microtiter plate, facilitating the semiautomation of manipulations and full automation of data collection. As is discussed above, salmonellae pathogens establish a persistent infection, including a long-lasting association that kills C. elegans. If a salmonellae pathogen has diminished pathogenicity, then adult or L4 worms live, develop into adult hermaphrodites, and produce thousands of live progeny. Accordingly, if C. elegans is incubated with the pathogen, the worms will die, unless a compound is present to reduce pathogenicity. The presence of such live progeny is easily detected using a variety of methods, including visual screening with standard microscopes.

To evaluate the ability of a test compound or extract to promote a host's resistance to a pathogen or to repress pathogenicity of a pathogen, a test compound or extract is inoculated at an appropriate dosage into an appropriate agar medium seeded with an appropriate amount of an overnight culture of a pathogen, e.g., S. typhimurium strain LT2. If desired, various concentrations of the test compound or extract can be inoculated to assess dosage effect on both the host and the pathogen. Control wells are inoculated with non-pathogenic bacteria (negative control) or a pathogen in the absence of a test compound or extract (positive control). Plates are then incubated 24 hours at 37° C to facilitate the growth of the pathogen. Microtiter dishes are subsequently cooled to 25 °C, and two C. elegans L4 hermaphrodite larva expressing a detectable marker such as GFP are added to the plate and incubated at 25 °C, the upper limit for normal physiological integrity of C. elegans. At an appropriate time interval, e.g., five hours, wells are examined for surviving worms, the presence of progeny, or both, e.g., by visual screening or monitoring motion of worms using a motion detector, or monitoring the fluorescence of the nematodes. In another working example, the presence of a persistent infection in gut of C. elegans is carried out as follows. Media is prepared as described herein and a test compound or compound library is also added. On the tissue culture plate, approximately eight worms, at the one-day old adult stage, are placed on the lawn of pathogenic bacteria expressing a detectable markers such as GFP from a plate of OP50 E. coli. The plates are incubated at 25°C for 5 hours and then the worms are transferred to a plate of OP50 E. coli not expressing GFP. After 24 hours, the worms are examined by fluorsecent microcopy for the presence of the pathogen in the worm intestine. Each experimental condition is done in triplicate and repeated at least twice. Test compounds that reduce the presence the pathogen in the worm intestine are taken as being useful for treating microbial infection.

Comparative studies between treated and control worms (or larvae) are used to determine the relative efficacy of the test molecule or compound in promoting the host's resistance to the pathogen or inhibiting the establishment of a persistent infection. A test compound which effectively stimulates, boosts, enhances, increases, or promotes the host's resistance to the pathogen or which inhibits, inactivates, suppresses, represses, or controls pathogenicity of the pathogen, and does not significantly adversely affect the normal physiology, reproduction, or development of the worms is considered useful in the invention.

Use The methods of the invention provide a simple means for identifying virulence factors that enable a pathogen to establish a persistent infection in a nematode and compounds capable of either inhibiting pathogenicity or enhancing an organism's resistance capabilities to such pathogens. Accordingly, a chemical entity discovered to have medicinal value using the methods described herein are useful as either drugs, or as information for structural modification of existing anti-pathogenic compounds, e.g., by rational drug design.

For therapeutic uses, the compositions or agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a phaπnaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections which provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of an anti-pathogenic agent in a physio logically- acceptable carrier. In the context of treating a bacterial infection a "therapeutically effective amount" or "pharmaceutically effective amount" indicates an amount of an antibacterial agent, e.g., as disclosed for this invention, which has a therapeutic effect. This generally refers to the inhibition, to some extent, of the normal cellular functioning of bacterial cells (e.g., salmonellae cells) causing or contributing to a bacterial infection. The dose of antibacterial agent which is useful as a treatment is a "therapeutically effective amount." Thus, as used herein, a therapeutically effective amount means an amount of an antibacterial agent which produces the desired therapeutic effect as judged by clinical trial results, standard animal models of infection, or both. This amount can be routinely determined by one skilled in the art and will vary depending upon several factors, such as the particular bacterial strain involved and the particular antibacterial agent used. This amount can further depend on the patient's height, weight, sex, age, and renal and liver function or other medical history. For these purposes, a therapeutic effect is one which relieves to some extent one or more of the symptoms of the infection and includes curing an infection.

The compositions containing antibacterial agents of virulence factors or genes can be administered for prophylactic or therapeutic treatments, or both. In therapeutic applications, the compositions are administered to a patient already suffering from an infection from bacteria (similarly for infections by other microbes), in an amount sufficient to cure or at least partially arrest the symptoms of the infection. An amount adequate to accomplish this is defined as "therapeutically effective amount." Amounts effective for this use will depend on the severity and course of the infection, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. In prophylactic applications, compositions containing the compounds of the invention are administered to a patient susceptible to, or otherwise at risk of, a particular infection. Such an amount is defined to be a "prophylactically effective amount." In this use, the precise amounts again depend on the patient's state of health, weight, and the like. However, generally, a suitable effective dose will be in the range of Oil to 10000 milligrams (mg) per recipient per day, preferably in the range of 10-5000 mg per day. The desired dosage is preferably presented in one, two, three, four, or more subdoses administered at appropriate intervals throughout the day. These subdoses can be administered as unit dosage forms, for example, containing 5 to 1000 mg, preferably 10 to 100 mg of active ingredient per unit dosage form. Preferably, the compounds of the invention will be administered in amounts of between about 2.0 mg/kg to 25 mg/kg of patient body weight, between about one to four times per day.

Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E.W. Martin. The amount of the antipathogenic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the type of disease and extensiveness of the disease. Generally, amounts will be in the range of those used for other agents used in the treatment of other microbial diseases, although in certain instances lower amounts will be needed because of the increased specificity of the compound. A compound is administered at a dosage that inhibits microbial proliferation.

All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

What is claimed is:

Claims (39)

Claims

1. An isolated nematode persistently infected with an isolated pathogen.

2. The nematode of claim 1, wherein said pathogen expresses a detectable marker.

3. The nematode of claim 1, wherein said nematode is Caenorhabditis elegans.

4. The nematode of claim 1, wherein said nematode is a one-day old adult hermaphrodite or an L4 larval stage worm.

5. The nematode of claim 1, wherein said pathogen colonizes the intestine of said nematode.

6. The nematode of claim 1 , wherein said pathogen is Salmonella.

7. The nematode of claim 6, wherein said pathogen is Salmonellae typhimurium strain SL1344.

8. A method of screening for a virulence factor that enables a pathogen to develop a persistent infection in a nematode, comprising the steps of:

(a) exposing a nematode to a mutagenized pathogen;

(b) determining whether said mutant pathogen persistently infects said nematode, a reduction of disease in said nematode relative to that caused by the non-mutagenized pathogen indicating a mutation in a virulence factor that enables said pathogen to develop a persistent infection in said nematode; and

(c) using said mutation as a marker for identifying said virulence factor.

9. The method of claim 8, wherein said pathogen expresses a detectable marker.

10. The method of claim 8, wherein said nematode is a one_rday old adult hermaphrodite or an L4 larval stage worm.

11. The method of claim 8, wherein said pathogen colonizes the intestine of said nematode.

12. The method of claim 8, wherein said pathogen is Salmonella.

13. The method of claim 12, wherein said pathogen is Salmonella typhimurium strain SL1344.

14. The method of claim 8, wherein said nematode is C. elegans.

15. The method of claim 8, wherein said method utilizes a salmonellae/C. elegans killing assay.

16. The method of claim 15, wherein said mutated pathogen causes less C. elegans killing than the non-mutagenized pathogen.

17. A method of screening for a compound that inhibits a persistent pathogenic infection in a nematode, comprising the steps of: (a) providing a nematode persistently infected with a pathogen;

(b) contacting said persistently infected nematode with a test compound; and

(c) determining whether the test compound inhibits the pathogenicity of said pathogen in said persistently infected nematode.

18. The method of claim 17, wherein said pathogen expresses a detectable gene.

19. The nematode of claim 17, wherein said nematode is a one-day old adult hermaphrodite or an L4 larval stage worm.

20. The nematode of claim 17, wherein colonization of the intestine of said nematode by said mutated pathogen is decreased.

21. The method of claim 17, wherein said pathogen is Salmonella.

22. The method of claim 21, wherein said pathogen is Salmonella typhimurium strain SL1344.

23. The method of claim 17, wherein said nematode is C. elegans.

24. The method of claim 17, wherein said test compound is provided in a compound library.

25. The method of claim 17, wherein said test compound is a small organic compound.

26. The method of claim 17, wherein said test compound is a peptide, peptidomimetic, or an antibody or fragment thereof.

27. The method of claim 17, wherein said inhibition of pathogenicity is measured by a salmonellae/C. elegans killing assay.

28. The method of claim 27, wherein said pathogen causes less C. elegans killing in the presence of said test compound than in the absence of said test compound.

29. A method of screening for a virulence factor that enables a pathogen to develop a persistent infection in a nematode, comprising the steps of:

(a) exposing a nematode to a mutagenized pathogen expressing a detectable marker; (b) determining whether said mutant pathogen persistently: infects said nematode by measuring the level of detectable marker in said nematode, where a decrease of the marker in said nematode relative to that caused by the non- mutagenized pathogen indicates a mutation in a virulence factor that enables said pathogen to develop a persistent infection in said nematode; and (c) using said mutation as a marker for identifying said virulence factor.

30. A method of screening for a compound that inhibits a persistent pathogenic infection in a nematode, comprising the steps of:

(a) providing a nematode persistently infected with a pathogen expressing a detectable marker; (b) contacting said persistently infected nematode with a test compound; and

(c) determining whether said pathogen persistently infects said nematode by measuring the level of detectable marker in said nematode, where a decrease of the marker in said nematode indicates that said test compound inhibits a persistent pathogenic infection in the nematode.

31. A method of screening for a virulence factor that enables a pathogen to develop a persistent infection in a nematode, said method comprising:

(a) exposing a nematode to a pathogen expressing a gene not normally expressed by said pathogen; (b) determining whether said pathogen persistently infects said nematode by measuring the level of detectable marker in said nematode, where a decrease or increase of the marker in the nematode relative to that caused by the non-mutagenized pathogen indicates a mutation in a virulence factor or a virulence factor gene that enables the pathogen to develop a persistent infection in the nematode; and

(c) using gene expressed by said pathogen as a marker for identifying saidvirulence factor.

32. method of claim 31 , wherein said screen utilizes a salmonellae/ C. elegans killing assay.

33. The method of claim 31 , wherein said pathogen has a reduced or enhanced capacity to develop a persistent infection in C. elegans.

34. A method of screening for a virulence factor that enables a pathogen to develop a persistent infection in a nematode, said method comprising:

(a) exposing a nematode to a pathogen, wherein said pathogen overexpresses a pathogen gene ;

(b) determining whether the pathogen persistently infects the nematode by measuring the level of detectable marker in the nematode, where a decrease or increase of the marker in the nematode relative to that caused by the non-mutagenized pathogen indicates a mutation in a virulence factor or a virulence factor gene that enables the pathogen to develop a persistent infection in the nematode; and

(c) using the mutation or virulence factor gene as a marker for identifying the virulence factor.

35. The method of claim 34, wherein said screen utilizes a salmonellae/C. elegans killing assay.

36. The method of claim 34, wherein said pathogen has a reduced or enhanced capacity to develop a persistent infection in C. elegans.

37. A method of screening for a virulence factor that enables a pathogen to develop a persistent infection in a nematode, comprising:

(a) exposing a nematode to a pathogen expressing a detectable marker, the pathogen being mutagenized; (b) determining whether the mutant or otherwise altered pathogen persistently infects the nematode by measuring the level of detectable marker in the nematode, where a decrease or increase of the marker in the nematode relative to that caused by the non-mutagenized pathogen indicates a mutation in a virulence factor or a virulence factor gene that enables the pathogen to develop a persistent infection in the nematode; and

(c) using the mutation for identifying the virulence factor.

38. A method of screening for a virulence factor that enables a pathogen to develop a persistent infection in a nematode, comprising:

(a) exposing a nematode to a pathogen expressing a detectable marker, the pathogen expressing a gene not normally expressed by the pathogen;

(b) determining whether the mutant or otherwise altered pathogen persistently infects the nematode by measuring the level of detectable marker in the nematode, where a decrease or increase of the marker in the nematode relative to that caused by the non-mutagenized pathogen indicates a mutation in a virulence factor or a virulence factor gene that enables the pathogen to develop a persistent infection in the nematode; and

(c) using the gene expressed by said pathogen as a marker for identifying the virulence factor.

39. A method of screening for a virulence factor that enables a pathogen to develop a persistent infection in a nematode, comprising:

(a) exposing a nematode to a pathogen expressing a detectable marker, the pathogen overexpressing a pathogen gene; (b) determining whether the mutant or otherwise altered pathogen persistently infects the nematode by measuring the level of detectable marker in the nematode, where a decrease or increase of the marker in the nematode relative to that caused by the non-mutagenized pathogen indicates a mutation in a virulence factor or a virulence factor gene that enables the pathogen to develop a persistent infection in the nematode; and

(c) using the gene expressed by said pathogen as a marker for identifying the virulence factor.