JP2003528318A - Methods for screening and identifying pathogen virulence factors - Google Patents

Abstract

  (57) [Summary] Physiological virulence that regulates or regulates environmental and host signals (e.g., host-dependent or host-independent signals) and the genes responsible for establishing persistent infection as found in nematode gastrointestinal colonization. Disclosed are screening methods for identifying interactions between pathways.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION The present invention relates to screening methods for identifying pathogen virulence factors and screening methods for identifying drugs that inhibit pathogen infection.

Microbial pathogens use a variety of complex strategies to interfere with host cell function and ensure pathogen growth and survival. Some pathogens that have co-evolved with the host or have long co-existed with the host utilize a finely tuned, host-specific strategy to establish disease-causing relationships. During infection, pathogens encounter a variety of conditions and respond by expressing virulence factors appropriate for the particular environment, host, or both.

Although antibiotics have been effective tools in the treatment of infectious diseases, the emergence of drug resistant pathogens has become a problem in clinical settings. Therefore, new antibiotics or anti-pathogenic molecules are needed to combat such drug resistant pathogens. Therefore, there is a screening method aimed not only at identifying and characterizing potential anti-pathogenic agents, but also at identifying and characterizing virulence factors that allow pathogens to infect and debilitate the host. Required in the art.

SUMMARY OF THE INVENTION The present inventors have found that the microbial pathogen Salmonella typhimurium.
Found a long-term persistent infection in the nematode Caenorhabditis elegans. With this discovery, environmental and host signals (e.g., host-dependent or host-independent signals),
It enables a simple screening method to identify the interrelationships of the physiological pathogenic pathways that control or regulate the genes responsible for establishing a persistent infection as found in nematode colonization of the gastrointestinal tract.

In one aspect, the invention features an isolated nematode persistently infected with an isolated pathogen. Preferably, the nematode is C. elegans (eg, 1-day-old adult hermaphroditic nematodes or L4 larval stage nematodes). In a preferred embodiment, the isolated pathogen expresses a detectable marker or colonizes the gut of the nematode. In yet another preferred embodiment, the pathogen is Salmonella (e.g.,
Salmonella typhimurium SL1344 strain or LT2 strain).

In another aspect, the invention features a method of screening a virulence factor that allows a pathogen to cause a persistent infection in a nematode. The method generally comprises the steps of: (a) exposing the nematode to a mutagenized pathogen, a pathogen expressing a gene not normally present in the pathogen, or a pathogen overexpressing the pathogen gene. (b) a step of determining whether a mutant or otherwise modified pathogen persistently infects a nematode, a disease caused by a non-mutagenized pathogen or otherwise modified pathogen A disease in C. elegans that is reduced or promoted compared to C. elegans shows a virulence factor or a mutation in the virulence factor gene that allows the pathogen to cause a persistent infection in the nematode; (c) identify virulence factors Using the mutation or virulence factor gene as a marker for.

[0007] Preferably, the nematode utilized in the method of screening for a pathogen virulence factor is Caenorhabditis elegans (eg, a 1-day-old adult hermaphroditic nematode or
L4 larva stage nematodes). In a preferred embodiment, the mutant or otherwise modified pathogen used to identify the virulence factor expresses a detectable marker. In another preferred embodiment, nematode intestinal colonization by the mutant pathogen or otherwise modified pathogen is reduced. In yet another preferred embodiment, the pathogen used is Salmonella (eg S. typhimurium strain SL1344 or LT2 strain).

In yet another preferred embodiment, the method utilizes the Salmonella / C. Elegans killing assay. In such a method, the mutated pathogen or otherwise modified pathogen has a reduced or increased ability to produce a persistent infection in C. elegans, such that the non-mutagenized pathogen or otherwise. Causes less killing than or causes more killing than pathogens modified in.

[0009] In another aspect, the invention features a method of screening for compounds that inhibit persistent pathogen infection in nematodes. The method is generally characterized by the steps of: (a) providing a persistently infected nematode to the pathogen; (b) contacting the persistently infected nematode with a test compound. And (c) determining whether the test compound inhibits persistent infection in C. elegans.

Preferably, the nematode utilized in the compound screening method is Caenorhabditis elegans (eg, 1-day-old adult hermaphroditic nematode or L4 larval stage nematode).
). In a preferred embodiment, the pathogen used in the compound screening assay expresses a detectable marker. In another preferred embodiment, nematode intestinal colonization by the pathogen is reduced. In yet another preferred embodiment,
The pathogen used is Salmonella (e.g. S. typhimurium strain SL1344 or LT2.
It is a stock company.

In yet another preferred embodiment, the test compound is provided in a compound library, is a small organic compound, or is a peptide, peptidomimetic, or antibody or fragment thereof.

[0012] In another preferred embodiment, the compound screening method utilizes a Salmonella / C. Elegans killing assay. In such a method, 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 a virulence factor that allows a pathogen to cause a persistent infection in a nematode. The method generally includes the following steps: (a) the pathogen is mutagenized and the pathogen expresses a gene that is not normally expressed or overexpresses the pathogen gene, detectable Exposing the nematode to a pathogen expressing a different marker; (b) determining whether the mutant pathogen persistently infects the nematode by measuring the level of the detectable marker in the nematode. , A mutation in the virulence factor or virulence factor gene that allows a pathogen to cause a persistent infection in a nematode, which marker in the nematode is reduced or increased relative to the level of the marker caused by the non-mutagenized pathogen And (c) using the mutant or virulence factor gene as a marker to identify the virulence factor.

In another aspect, the invention features a method of screening for compounds that inhibit persistent pathogen infection in nematodes. The method generally comprises the following steps: (a) providing a nematode persistently infected with a pathogen expressing a detectable marker; (b) testing with a persistently infected nematode. Contacting the compound; and (c) determining whether the pathogen persistently infects the nematode by measuring the level of the detectable marker in the nematode, A step in which the reduction indicates that the test compound inhibits persistent pathogen infection in nematodes.

Exemplary pathogenic bacteria useful in the methods of the present invention and which provide persistently infected nematodes include Aerobacter, Aeromonas,
Acinetobacter genus (Acinetobacter), Agrobacterium genus (Agrobacterium),
Bacillus, Bacteroides, Bartonella
), Genus Bordetella, genus Bortella, genus Borrelia
, Brucella, Burkholderia, Burkholderia, Calymmatobacterium, Campylobacter, Citrobacter, Clostridium, Corynebacterium , Enterobacter, Enterococcus, Escherichia, Francis
sella), Gardnerella genus (Gardnerella), Haemophilus genus (Haemophilus), Hafnia genus (Hafnia), Helicobacter genus (Helicobacter), Klebsiella genus (Klebsiella)
, Legionella, Listeria, Morgane
lla), Moraxella (Moraxella), Mycobacterium (Mycobacterium), Neisseria (Neisseria), Pasteurella (Pasteurella), Proteus (Proteus),
Providencia (Providencia), Salmonella, Serratia (Serratia), Shigella (Shigella), Staphylococcus (Staphylococcus), Streptococcus (Streptococcus)
, Stentorophomonas (Stentorophomonas), Treponema (Treponema),
Xanthomonas, Vibrio, and Yersinia (Ye
rsinia), but is not limited thereto.

“Virulence factor” refers to the pathogen (eg, bacterium) without which a eukaryotic host organism
By a cellular component (eg, a protein such as a transcription factor or molecule) that is unable to cause a disease or infection in (eg, a nematode or mammal). Such components are involved, for example, in the adaptation of bacteria to the host (eg, nematode host), the establishment of bacterial infection, the maintenance of bacterial infection, or the development of deleterious effects of the infection on the host organism. In addition, this phrase includes components that act directly on the host tissue as well as components that regulate the activity or production of other disease causing factors.

By “inhibit a pathogen” is meant the ability of a test compound to reduce, prevent, attenuate, diminish, stop, or stabilize the development or progression of a disease or infection by a pathogen in a eukaryotic host organism. . Preferably such inhibition is
In any suitable pathogenicity assay (e.g., an assay described herein), virulence is at least 5%, more preferably at least 25%, most preferably as compared to symptoms in the absence of the test compound. Preferably it is reduced by at least 50% or more. In one particular example, inhibition can be measured by monitoring the pathogen-induced symptom in a nematode persistently infected with the Salmonella pathogen exposed to the test compound or extract and exposed to the compound. The reduced severity of the pathogen relative to the severity of the pathology in the host organism is indicative of inhibition of the Salmonella pathogen by the compound.

By “persistent infection” or “persistently infected” is meant that a host-harmful pathogen (eg, Salmonella) invades or colonizes a host animal (eg, nematode), where And the size of the persistent pathogen population that is symbiotic with the host after transfer of the host to the non-infectious environment is at least 30%, preferably 50% of the size of the pathogen population before transfer of the host to the non-infectious environment. , More preferably 80%, most preferably 90%, or even 95% to 99%. Such an infection is also symbiotic with the host when first exposed to the host with a relatively small number of pathogens mixed with an excess of non-pathogenic bacteria and subsequently transferred to a non-infectious environment. Includes an increase in the number of pathogen populations. Persistent infection is
It is generally measured using the nematode feeding assay (the assay described herein), where the bacteria are assayed for their ability to establish long-term symbiosis in the nematode intestine.

By “detectable marker” is meant a gene whose expression can be assayed. Such genes include β-glucuronidase (GUS), luciferase (LUC)
, Chloramphenicol transacetylase (CAT), green fluorescent protein (GFP)
, And β-galactosidase, but are not limited thereto.

“Isolated nematode” means a nematode purified from a contaminated organism and maintained in 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 well known in the art.

By “isolated pathogen” is meant a microbial strain that has been cultured by human manipulation and that elicits a disease response in the host.

The present invention provides many benefits. For example, the present invention facilitates the identification of novel targets and therapeutic approaches for preparing therapeutic agents active against virulence factors and genes that allow pathogens to cause long-term persistent infections in host organisms. To

The present invention also provides a long-awaited benefit over the wide variety of standard screening methods used to identify and assess the efficacy of compounds that resist Salmonella pathogens. In one particular example, the screening methods described herein allow for simple assessment of host toxicity as well as anti-Salmonella potency in a simple in vivo screen. In addition, the methods of the present invention can be used to assess the ability of compounds to inhibit Salmonella pathogenesis, as well as to assess the ability of compounds to stimulate and potentiate the host's response to Salmonella pathogen attack. You can

Thus, the method of the invention is safe to use in a eukaryotic host organism (ie a compound that does not adversely affect the normal development and physiological function of the organism) and establishes a persistent infection in the host. It provides a simple and unambiguous means for identifying compounds that are effective against pathogenic microorganisms. Further, the methods of the present invention provide a method of assaying virtually any number of compounds for anti-Salmonella pathogen efficacy with high throughput, high sensitivity, and low complexity. This method is also relatively inexpensive to perform and enable the analysis of small amounts of active substance found in either purified or crude extract form. In addition, the methods disclosed herein provide a means to identify compounds that have the ability to cross eukaryotic cell membranes and that retain therapeutic efficacy in in vivo methods of administration. Furthermore, the screening method described above is suitable for 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 of the invention and the claims.

DETAILED DESCRIPTION In the following, we show that S. typhimurium causes disease in the nematode C. elegans by establishing a persistent infection, including long-term symbiosis in the gut of the nematode, and finally Describe experimental evidence demonstrating that C. elegans, which feeds on the lawn of Salmonella typhimurium, dies within a few days as a result of the pathogenic process. Thus, the Salmonella / C. Elegans killing assay described herein is a useful system for identifying novel virulence factors responsible for the ability of pathogens to cause persistent infection, and to inhibit Salmonella virulence, or Provided are useful systems for identifying compounds that promote host resistance to pathogens, or both. The following experimental examples are intended to illustrate rather than limit the scope of the claimed invention.

C. elegans kills C. elegans by establishing long-term infection To determine whether C. elegans kills C. elegans
Time to feed the bacterial flora grown on NG agar and kill 50% of nematodes (LT50)
Was calculated in 6 independent experiments. LT50 obtained using nematodes fed E. coli (
(LT50 = 9.86 ± 0.9 days) decreased in LT50 (LT50) of nematodes feeding on Salmonella typhimurium.
= 5.10 ± 0.7 days), it was shown that nematodes were killed by Salmonella typhimurium (
(Figure 1A).

Furthermore, the physiological function of the nematodes that ingested Salmonella typhimurium was different from the nematodes that ingested E. coli OP50. As reported for P. aeruginosa infection (T.
An et al., Proc. Natl. Acad. Sci. 96: 2408-2413, 1999), nematode motility and the rate of pharyngeal pumping gradually decreased until the nematode became immobile or died. . In some cases, the nematode becomes lawn with,
The embryos hatched internally early in the infection (which suggests a defect in spawning). To study whether S. typhimurium could prevent eggs from hatching, eggs were placed on plates containing S. typhimurium. We have observed that 95% of the eggs hatch. This indicates that Salmonella typhimurium does not prevent hatching under these conditions.

Since Salmonella typhimurium is known to establish long-term symbiosis or persistent infection with the host (Galan and Bliska, Annu. Rev. Cell Dev. Biol. 12: 221-255, 1996),
Nematodes were examined for their ability to irreversibly colonize the intestine of the nematode. The nematodes were fed with Salmonella typhimurium for 5 hours and then transferred onto E. coli-containing plates. The results shown in FIG. 1B show that Salmonella typhimurium colonized the gut of the nematode 5 hours after infection and the nematode died within a few days. The killing rate in this migration experiment was almost the same as the killing rate obtained by contacting C. elegans and Salmonella typhimurium throughout the killing process. To study the time course of Salmonella typhimurium infection, adult nematodes were fed with Salmonella typhimurium for 1, 3, and 5 hours and then transferred onto E. coli-containing plates. As shown in the inset in FIG. 1B, the percentage of C. elegans surviving transfer to E. coli plates was inversely proportional to the time spent feeding SL1344. On day 8, only 30% of nematodes killed on E. coli plates after feeding SL1344 for 1 hour. When SL1344 was fed for 5 hours before transfer of nematodes, the proportion of dead nematodes after 8 days increased to 90%. In contrast, when C. elegans was fed P. aeruginosa PA14 for 6 hours and then transferred to E. coli, 100% killing was achieved within the time frame of the experiment (60 hours) (or by continuous feeding of PA14. (Within the normal time frame)
No killing was observed at. This result is interpreted to indicate that PA14 has not established a lethal infection within the experimental parameters mentioned for Salmonella.

Killing by Salmonella typhimurium correlates with growth of bacteria in the digestive tract. To confirm that killing of Salmonella typhimurium is accompanied by an infectious process, the present inventors
A Salmonella typhimurium strain expressing Aequorea victoria GFP was constructed.
After 72 hours of feeding GFP-expressing Salmonella typhimurium, the intestinal lumen was found to be swollen and filled with intact bacteria. This suggests that Salmonella typhimurium may be growing in the gastrointestinal tract (Figure 2, Panel B and Panel C). Pseudomonas aeruginosa PA14 cannot establish long-term infection, but PA14 / GFP in nematodes
Were fed for 24 hours with similar results (Figure 2, Panel E and Panel F).
. In contrast, no intact bacteria were observed when the nematodes were fed the control E. coli DH5α / GFP strain and the lumen was not swollen (Figure 2, Panel A and Panel B). Similarly, the nematode is fed with Salmonella typhimurium / GFP or Escherichia coli DH5α / GFP for 5 hours,
Then, it was transferred to an E. coli plate. FIG. 3 (panels A, B, and C) shows that after 24 hours, E. coli was not found in the intestine of C. elegans, while Salmonella typhimurium survived in the intestinal lumen. The intact state of Salmonella typhimurium / GFP is shown at high magnification (Figure 3, panel C).

To assess the colonization process of Salmonella typhimurium, the amount of bacteria used in the feeding assay was reduced. Several types of Salmonella typhimurium diluted with E. coli
Prepared on NG plates, nematodes were quickly placed on the plates for feeding. The proportion of Salmonella is then cfu on MacConkey agar plates.
Was determined by measuring the presence of. The result of these feeding experiments is 1: 1,00
It was shown that Salmonella typhimurium diluted to 0 was able to kill C. elegans at levels similar to 100% Salmonella typhimurium feeding, but the 1: 10,000 dilution showed reduced virulence (Figure 4A). .

Since killing was supported by 0.1% Salmonella typhimurium, this concentration of bacteria was used in a follow-up experiment to quantify the amount of bacteria in the intestinal lumen during infection. Nematodes were exposed to 0.1% SL1344 / GFP or DH5α / GFP diluted in OP50 for 5 hours and then transferred onto OP50 plates. Every 24 hours, 10 nematodes were placed in a 1.5 ml centrifuge tube and disrupted by pressing with a pellet pestle. Inoculate with the bacterial dilution and c
Counted .fu. The results showed that after 24 hours DH5α / GFP substantially disappeared from the nematode intestine, but SL1344 / GFP proliferated and colonized the nematode intestine (FIG. 4B).
.

Pseudomonas aeruginosa, which requires a direct interaction between Salmonella typhimurium and C. elegans for killing, is secreted by a diffusible toxin called "fast killing".
It has been shown to kill C. elegance (Tan et al., Proc. Natl. Acad. Sci. 96:24.
08-2413, 1999 and Mahajan-Miklos et al., Cell 96: 47-56, 1999). To determine whether C. elegans killing by Salmonella typhimurium was mediated by diffusible toxins, killing assays were performed under rapid killing conditions using sorbitol-containing plates (PGS plates). Under conditions of rapid killing of Pseudomonas aeruginosa, L4 nematodes have been shown to die much faster than adults (Tan et al., Proc. Natl. Acad. Sci. 96: 2408-2413.
, 1999). Therefore, L4 stage nematodes were used for these experiments. Salmonella typhimurium and control P. aeruginosa and E. coli were grown on 0.45 μm filters placed on plates. The filter containing the bacteria was removed and heat inactivated E. coli was added as a food source to avoid starvation (Fig. 5A). The results showed that the killing by S. typhimurium under these conditions was not due to diffusible toxin. Figure 5B
Shows that C. elegans survived when fed a heat killed Salmonella. We have also fed nematodes with heat killed and live Salmonella typhimurium clinical isolate 14028. The results shown in Figure 5B show that heat-killed SL14028 did not kill C. elegans and live SL140 as found in SL1344.
28 indicates that the nematode was killed.

Materials and Methods The above results were obtained using the following materials and methods.

Bacterial strains, plasmids, and growth conditions P. aeruginosa PA14 strain (Rahme et al., Science 268, 1899-1902, 1995), PA14 / GFP strain (Tan et al., Proc Natl Acad Sci USA 96, 715-720, 1999). ), Salmonella typhimurium SL1344 strain (Ho
iseth, Nature 291, 238-239, 1981), SL1344 / GFP (this study), Salmonella typhimurium clinical isolate 14028 (SL14028) was kindly provided by E. Hohmann. E. coli DH5α strain (Bethesda Research Laboratories
ories)), DH5α / GFP strain (Tan et al., Proc Natl Acad Sci USA 96, 715-720, 1999).
, And Escherichia coli OP50 strain (Brenner, Genetics 77, 71-94, 1974) with Luria broth (
LB) medium was grown at 37 ° C. The SL1344 / GFP strain was created using the construct pSMC21-GFP (Bloemberg et al. Appl. Environ. Microbiol. 63: 4543-51, 1997).

Maintenance of nematodes Sulston and Hodgkin ("The Nematode Caenorhabditis elegans", edited by WB Wood, Cold Sp)
ring Harbor Lab. Press, Plainview, NY, pages 587-606, 1988), nematodes were maintained as hermaphrodites at 20 ° C, grown on standard plates and fed with E. coli OP50 strain. Let Nematodes were observed under a dissecting microscope (Leica MZ6).

Bacterial Infection Assay 10 μl of bacterial culture grown overnight in LB was used for nematode growth (NG; Su
lston and Hodgkin ("Nematode Cenolabditis Elegance (the Nematode C
aenorhabditis elegans), WB Wood, Cold Spring Harbor Lab.Press, Plai
The killing assay was performed by smearing (modified from nematode growth medium agar described in Nview, NY, pp. 587-606, (1988)). Nematode killing assay (3.5 cm
For diameter plates) 0.35% peptone or peptone-glucose-sorbitol (PGS; 1% Bacto-peptone / 1% NaCl / 1% glucose /
0.15M sorbitol / 1.7% bacto-agar) medium was used. After smearing the bacterial cultures, the plates were incubated at 37 ° C for 12 hours, then 10-20 nematodes were placed on the assay plate and then incubated at 25 ° C. If necessary,
Bacteria were killed by heating at 60 ° C for 90 minutes. E. coli OP50 strain was used as a control for the assay. Nematode mortality was recorded over time and nematodes were considered dead when they became unresponsive to contact. Nematodes that died as a result of sticking to the wall of the plate were excluded from the analysis. The time to kill 50% of the nematodes (LT50) is calculated using the PRISM (version 2.00) computer program as follows:

[Equation 1] Y = Bottom + (Top-Bottom) / (1 + 10 ^ ((LogEC50-X) ^* HillSlope) (where X is the logarithm of the number of days, and Y is the average of killed nematodes; Y Starts from Bottom and progresses to Top in an S-shape).

C. elegans transfer experiment 80 to 100 1-day-old adult hermaphroditic nematodes were inoculated on the bacterial flora and fed. After 1, 3, or 5 hours, nematodes were transferred to E. coli OP50 containing plates. Prior to transfer, nematodes were washed twice with M9 buffer and transferred onto OP50 containing plates for 2 hours and then onto fresh OP50 containing plates. Every 24 hours, nematodes were transferred to new plates and cfu were counted. M9 of 10 nematodes to measure cfu
The cells were washed with buffer, transferred to a 1.5 ml tube containing M9 buffer containing 1% Triton X-100, and mechanically disrupted using a pellet pestle. Bacterial dilutions were made by dilution with 10 mM MgSO ₄ and plated on ampicillin containing plates or MacConkey agar base plates and cfu counted.

Confocal microscopic nematodes were inoculated on a lawn of DH5α / GFP or SL1344 / GFP. After 5 hours, half the total number of nematodes was transferred onto E. coli OP50 containing plates as described above. At each time point, 5 nematodes were placed on a pad of 1% agar in PBS and 30μ dissolved in M9 buffer.
M sodium azide was used as an anesthetic. The experiment was repeated at least 4 times and confocal imaging was performed using a Leica TCS SP confocal microscope. Adobe Pho
I used toShop5.0 to collect and edit the merged images.

Nematode Screening System for Identifying Virulence Factors That Establish Persistent Infection Based on the above results showing that Salmonella establishes a persistent infection in the nematode C. elegans, the inventors We have developed a method to identify the virulence determinants that are important in the establishment of infection in Escherichia coli. This screening generally involves
/ Utilize the ability to screen thousands of randomly generated mutant pathogens using the nematode killing assay. In addition to using wild-type host nematodes in screening assays, constipated or defecation-deficient mutant nematodes (e.g., aex-2 and unc-25), grinding Mutants defective in (eg phm-2 and eat-14) as well as specific ABC transporter mutants (eg pgp-4 and mrp-1) can also be used.

In general, pathogens are assayed (using the methods described herein) for their ability to establish a persistent infection in nematodes. Once identified, pathogens such as S. typhimurium strain SL1344 are mutated according to standard methods well known in the art and then evaluated for their ability to induce disease by establishing a persistent infection in the nematode host organism. To be done. Mutagenized pathogens found to have a diminished ability to establish a persistent infection are useful in the methods of the invention. Such mutant pathogens are then used to identify host-dependent or host-independent virulence factors responsible for establishing a persistent infection, according to methods well 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. For example, by constitutively expressing a putative transcription factor or repressor on a plasmid in a pathogen such as S. typhimurium SL1344 and testing these altered pathogens for increased or decreased virulence in C. elegans. is there. Or Salmonella typhimurium (or other pathogens)
A plasmid library of clones covering the entire genome of C. elegans can be screened for increased or suppressed Salmonella virulence in C. elegans. The results of these screens identified virulence factors and subsequently provided valuable information on downstream targets of virulence factors.

The following is a practical example of a virulence factor nematode screening system using the human clinical isolate S. typhimurium SL1344 strain confirmed to persistently infect the digestive tract of C. elegans. The advantage of using nematodes as hosts to study microbial pathogens is that it is relatively easy to identify non-pathogenic variants in the nematode system.

In one preferred practical example in which survival is monitored, 4-8 C. elegans nematodes (eg, 1-day-old adult hermaphrodites) are placed in a lawn of a pathogen mutagenized strain, and Survival is monitored after 5 hours according to the methods described herein. For example, any standard procedure (e.g., standard in vivo or in vitro insertion / transposon mutagenesis (e.g., Kleckner et al., J Mol. Biol. 116: 125, 1977; Simon et al., Gen.
e 80 (1): 161-169, 1989)), pathogens such as Salmonella typhimurium strain SL1344 are mutated. Other methods, such as chemical mutagenesis or DNA directed mutagenesis, can also be utilized. Then wild-type pathogenic Salmonella typhimurium SL134
No living nematodes are seen on the plate seeded with 4 strains at all, but increased nematode survival on plates seeded with mutagenized S. typhimurium SL1344 strains (e.g., LT ₅₀ Is confirmed). Therefore, the ability of C. elegans to grow in the presence of the mutant S. typhimurium SL1344 strain indicates that the gene responsible for pathogenicity has been inactivated. The position of the inactivating mutation is then identified using standard methods (e.g., by polymerase chain reaction and sequencing of the insertion / transposon junction, or by mapping), and one (e.g., by nucleotide sequencing) is identified. Or more mutant virulence factors are cloned and identified.

In another practical example where virulence is assayed by monitoring intestinal colonization of C. elegans, 2-8 C. elegans nematodes (eg, L4 hermaphroditic larvae) are mutated. Prepared from a mutagenic library and placed on a lawn of mutagenized S. typhimurium LT2 strain expressing a detectable marker (e.g., GFP) for a suitable period of time, and then a non-pathogenic bacterium (e.g., E. coli / DH5α) is transferred to a plate for feeding. The LT2 strain has been mutated according to standard methods. After about 5 hours,
Nematodes are examined for the presence of pathogens in the intestine using a confocal microscope. Nematodes feeding on the wild-type pathogenic Salmonella typhimurium strain LT2 show bacteria growing and multiplying in the digestive tract. C. elegans that ingested the mutant LT2, which contains a mutation in a gene that allows the establishment of a persistent infection, show a reduced non-proliferative population in the digestive tract. Therefore, the absence or low amount of the mutant LT2 in the intestine of the nematode is considered to indicate that the gene responsible for pathogenicity has been inactivated. Mutant virulence factors are then identified using standard methods.

Compound Screening Assay As described above, the results of our experiments demonstrate that virulence factors are involved in the virulence of the nematode C. elegans. Based on this finding, we also developed a screening procedure to identify therapeutic compounds (e.g., anti-pathogenic drugs) that can be used to inhibit the ability of pathogens to become persistently infected. . Generally, this method involves screening any number of compounds for therapeutically active agents by using the Salmonella / Nematode Killing System described herein. Based on our evidence that these pathogens infect and kill C. elegans, compounds that interfere with the pathogenicity of pathogens (e.g., Salmonella pathogens) in nematodes are also mammalian (e.g., , Human patients) will result in effective therapeutic agents. Most antibiotics currently used in medicine are either bactericidal or bacteriostatic, thus favoring resistant strains or mutants, while being identified by the screening procedures described herein. The compounds neither kill the bacteria nor prevent the growth of the bacteria in vitro, but instead render them avirulent. Moreover, since the screening procedure of the present invention is performed in vivo, it is unlikely that the identified compound will be highly toxic to the host organism.

Accordingly, the method of the present invention provides for the evaluation of active agents, such as drugs for the treatment of diseases by pathogens caused by microorganisms capable of establishing long-term symbiosis or persistent infection with nematodes, Simplify identification and development.

In general, the chemical screening methods of the present invention provide a simple and unambiguous means for selecting a natural product extract or compound of interest from a large population. These natural product extracts or compounds are further evaluated and concentrated to a select few active substances. The components of this pool are then purified and evaluated in the method of the invention for measuring antipathogenic activity.

Test Extracts and Compounds In general, new anti-pathogenic drugs are prepared according to methods well known in the art.
Identified from large or chemical libraries of natural products or synthetic (or semi-synthetic) extracts. The screening methods of the present invention are suitable and useful for testing compounds from various sources for potential antipathogenic activity. Although initial screening can be done with a diverse library of compounds, this method is suitable for a variety of other compounds and compound libraries. Such compound libraries may be combinatorial libraries, natural product libraries, or other small molecule libraries. In addition, compounds from commercial sources, as well as commercially available analogs of the identified inhibitors can be tested.

For example, one of ordinary skill in the art of drug discovery and development will understand that the exact source of the test extract or compound is not critical to one or more screening procedures of the invention. Thus, the methods described herein can be used to screen virtually any number of chemical extracts or compounds. 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 modifications of existing compounds. To perform random or directed synthesis (e.g., semisynthetic or total synthesis) of any number of compounds, including but not limited to sugar-, lipid-, peptide-, and nucleic acid-based compounds, a large number of Methods are also available. A library of synthetic compounds is available from Brandon Associates (Bra
ndon Associates (Merrimack, NH) and Aldrich Chemic
al) (Milwaukee, WI). Alternatively, a library of natural compounds in the form of bacterial, fungal, plant, and animal extracts is available from Biotics (Su
ssex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft.Pie
rce, FL), and Pharmamar USA (Cambridge)
, MA) from many sources. In addition, natural and synthetically produced libraries are made, if desired, according to methods well known in the art (eg, by standard extraction and fractionation methods).
Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

Furthermore, dereplication (eg taxonomic dereplication, biological dereplication,
And methods for chemical dereplication, or any combination thereof, or replicates or repeats of substances already known for antipathogenic activity.
Those skilled in drug discovery and development will readily understand that methods for elimination of at) should be used whenever possible.

If the crude extract is found to have anti-pathogenic activity, a further fractionation of the positive lead extract is necessary to isolate the chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the thorough characterization and identification of chemical entities within the crude extract with antipathogenic activity. Methods for fractionating and purifying such heterogeneous extracts are well known in the art. If desired, compounds shown to be useful agents for the treatment of pathogenicity are chemically modified according to methods well known in the art.

Since many of the compounds contained in the libraries (eg combinatorial and natural product libraries) as well as natural product preparations have not been characterized, the screening methods of the present invention allow known known actives in screening. In addition to identifying compounds, new compounds are obtained that are active as inhibitors or inducers in particular screens. Accordingly, the present invention includes such novel compounds, as well as the use of the novel and known compounds in pharmaceutical compositions and therapeutic methods.

Exemplary High Throughput Screening Systems Many high throughput assay methods can be used to assess the efficacy of a molecule or compound in promoting host resistance or inhibiting pathogenicity.

For example, microtiter plate facilitation that facilitates semi-automation of operations and full automation of data collection to allow efficient and systematic large-scale screening of large quantities of natural products, extracts, or compounds. In the well, Caenorhabditis elegans (e.g., 1-day-old adult hermaphroditic nematode, L4 hermaphroditic larva, or
Cultivation of mutant nematodes such as aex-2, unc-25, phm-2, eat-14, pgp-4, or mrp-1). As mentioned above, Salmonella pathogens are persistent infections that kill C. elegans.
Establish (including long-term symbiosis). When the pathogenicity of the Salmonella pathogen is diminished, the adult or L4 nematode survives and develops into a hermaphroditic adult, producing thousands of live offspring. Thus, when C. elegans is incubated with a pathogen, nematodes die unless a compound that diminishes the pathogenicity is present. The presence of such live offspring is readily detected using a variety of methods, including standard microscopic visual screening.

To assess the ability of a test compound or extract to promote host resistance to a pathogen or suppress pathogen virulence, an appropriate amount of pathogen (eg, S. typhimurium LT2 strain) overnight culture was inoculated. The appropriate agar medium is inoculated with the appropriate dose of test compound or extract. If desired, various concentrations of test compound or extract can be inoculated in order to evaluate dose effects on both the host and the pathogen. Control wells are inoculated with non-pathogenic bacteria (negative control) or in the absence of test compound or extract (positive control). The plates are then incubated at 37 ° C for 24 hours to promote growth of the pathogen. The microtiter dish is then cooled to 25 ° C and two C. elegans L4 hermaphroditic larvae expressing a detectable marker (e.g., GFP) are added to the plate and C.
Incubate at 25 ° C, the upper limit for obtaining normal and complete elegance physiology. Presence of living nematodes, progeny, at appropriate time intervals (e.g., 5 hours), for example, by visual screening or monitoring of nematode movement using a motion detector, or by monitoring nematode fluorescence. Examine the wells for, or both.

In another practical example, the presence of persistent infection in the digestive tract of C. elegans is performed as follows. Media is prepared as described herein and test compound or compound library is also added. Approximately 8 1-day-old adult stages in tissue culture plates
Nematodes are placed from OP50 E. coli plates on a lawn of pathogenic bacteria that express a detectable marker (eg, GFP). Incubate the plate at 25 ° C for 5 hours,
The nematodes are then transferred onto plates of OP50 E. coli that do not express GFP. 24 hours later,
Nematodes are examined by fluorescence microscopy for the presence of pathogens in the intestines of the nematodes. Repeat each experimental condition in triplicate and repeat at least twice. Test compounds that reduce the presence of pathogens in the intestine of nematodes are considered to be useful in treating microbial infections.

Comparative studies of treated and control nematodes (or larvae) are used to determine the relative efficacy of test molecules or compounds in promoting host resistance to pathogens or inhibiting establishment of persistent infection. Effectively stimulates, increases, enhances, enhances, or promotes host resistance to a pathogen, or inhibits, inactivates, blocks, suppresses, or suppresses the pathogenicity of a pathogen, but Test compounds that do not significantly adversely affect normal physiology, reproduction, or development are considered useful in the invention.

Uses The methods of the present invention determine the virulence factors that allow pathogens to establish persistent infections in nematodes, and compounds that are capable of inhibiting pathogenicity or the ability of an organism's resistance to such pathogens. It provides a simple means to identify compounds that can be potentiated. Thus, a chemical entity discovered to have pharmaceutical value using the methods described herein may be a drug or of structural modification of an existing anti-pathogenic compound, for example by rational drug design. It is useful as information for

For therapeutic use, the compositions or agents identified using the methods disclosed herein may be administered systemically, eg, in a pharmaceutically acceptable buffer such as saline. Dissolved and prescribed. Preferred routes of administration include, for example, subcutaneous, intravenous, intraperitoneal, intramuscular, or intradermal injections, which deliver a continuous and continuous concentration of drug to the patient. Treatment of human patients or other animals is carried out with a therapeutically effective amount of the anti-pathogenic agent dissolved in a physiologically acceptable carrier.
With respect to the treatment of bacterial infections, a "therapeutically effective amount" or "pharmaceutically effective amount" refers to the amount of an antibacterial agent that has a therapeutic effect (eg, the antibacterial agents disclosed herein).
This generally means to some extent inhibit normal cell function of bacterial cells (eg, Salmonella cells) that cause or contribute to bacterial infection. A therapeutically useful dose of an antimicrobial agent is a "therapeutically effective amount." Thus, a therapeutically effective amount as used herein means an amount of antimicrobial agent that 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 of ordinary skill in the art and will depend on several factors such as the particular bacterial strain involved and the particular antimicrobial agent used. This amount may be further influenced by the height, weight, sex, age of the patient, and renal and liver function, or other medical history. For this purpose, the therapeutic effect is
The effect of alleviating one or more symptoms of an infection to some extent, including the cure of the infection.

The composition comprising the virulence factor or gene antimicrobial agent can be administered for prophylactic and / or therapeutic treatments. In therapeutic use, the composition is administered to a patient already suffering from a bacterial infection (as well as infections by other microorganisms) in an amount sufficient to cure, or at least partially subdue, the symptoms of the infection. To be done. An amount adequate to accomplish this is defined as "therapeutically effective amount." The effective amount for this use will depend on the severity and course of the infection, previous treatment regimen, patient health and response to the drug, and the judgment of the treating physician. In prophylactic applications, compositions containing the compounds of the invention are administered to patients susceptible to or at risk of a particular infection. Such an amount is defined to be a "prophylactically effective amount." Also in this use, the exact amount will depend on the patient's health, weight, etc. However, in general, a suitable effective amount is in the range of 0.1 milligram to 10,000 milligrams (mg) / recipient / day, preferably 10 mg to 5000 mg / day. The desired dose is preferably given in 1, 2, 3, 4 or more subdose administered at appropriate intervals per day. These subdoses may be, for example, 5-100 per unit dosage form.
It can be administered as a unit dosage form containing 0 mg, preferably 10-100 mg of active ingredient. Preferably, the compound of the invention is in an amount of about 2.0 mg to 25 mg per kg body weight of the patient.
It is administered about 1 to 4 times a day.

Suitable carriers and formulations thereof are described, for example, in "Remington's Pharmaceutical Sciences" by EW Martin.
The amount of anti-pathogenic agent administered will depend on the mode of administration, the age and weight of the patient, and the type of disease and the extent of disease. In general, the amount will be in the range of that used for other agents used in the treatment of other microbial diseases, although in certain cases lesser amounts may be given due to the increased specificity of the compound. Needed. The compound is administered at a dose that inhibits the growth of microorganisms.

All publications and patents mentioned herein are hereby incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. Be incorporated.

From the foregoing description, those skilled in the art can easily understand the essential features of the present invention, and make various modifications and changes to the present invention to meet various usages and conditions. be able to. Accordingly, other aspects are also within the claims.

[Brief description of drawings]

FIG. 1A shows the killing mechanism of C. elegans by Salmonella typhimurium SL1344 strain. Ten to 20 nematodes were placed on each plate and each assay consisted of 2 replicates. L4 stage (white circles) and 1-day-old adult hermaphrodites (black triangles and black circles) nematodes were fed with Salmonella typhimurium SL1344 (black triangles and white circles) or Escherichia coli OP50 (black circles). Small bars indicate standard deviation and results are representative of at least 6 independent experiments. Figure 1B shows the results of a C. elegans migration experiment, and SL1344 (open circles) or OP50.
(Black circles) shows the percentage of nematodes that died after feeding on OP50-containing plates after feeding for 5 hours. Small bars indicate standard deviation and results are representative of at least 3 independent experiments. The inset shows O1 after feeding SL1344 for 1, 3, or 5 hours.
The percentage of dead nematodes after transfer onto P50 containing plates is shown. The small bar found in the inset indicates standard deviation and the results are representative of at least 2 independent experiments.

FIG. 2 shows confocal images of bacterial colonization of the intestine of C. elegans. C. elegans are fed with GFP-expressing E. coli (E. coli / GFP) (panels A and B), GFP-expressing S. typhimurium (S. typhi / GFP) (panels C and D) for 72 hours, or Pseudomonas aeruginosa expressing GFP (Pseudomonas aeruginosa / GFP) (panel E and panel F) was fed for 24 hours. In the transmission images (panel A, panel C, and panel E), the end of the intestine is indicated by an arrow. Inset images (B, D, and F) show bacterial fluorescence (green channel) and gut autofluorescence (red channel). The bar indicates 50 μm.

FIG. 3 shows confocal images of bacterial colonization of the intestine of C. elegans in a migration experiment. Nematodes were fed with GFP-expressing E. coli (E. coli / GFP) (panel A) or GFP-expressing S. typhimurium (S. typhimurium / GFP) (panels B and C) for 5 hours, followed by 24 OP50s. Feeded for hours. Inset images show bacterial fluorescence (green channel) and gastrointestinal autofluorescence (red channel). The bar indicates 50 μm.

FIG. 4A shows that Salmonella typhimurium grows in the intestine of C. elegans. Several S. typhimurium dilutions diluted with E. coli were prepared on NG plates and 10 nematodes were quickly placed on the plates. Each assay was repeated twice. After 24 hours, nematodes were transferred to new plates and the amount of Salmonella in the plates was determined by counting colony forming units (cfu) on MacConkey agar plates. Small bars indicate standard deviation and results are representative of at least 2 independent experiments. FIG. 4B shows that Salmonella typhimurium grows in the intestine of C. elegans. 70-80 1-day-old adult hermaphroditic nematodes were placed on plates containing a 1: 10,000 ratio of GFP-expressing Salmonella typhimurium (S. typhimurium / GFP) and non-GFP-expressing E. coli. Furthermore, an equal number of nematodes were placed on E. coli expressing GFP. After 5 hours, the nematodes were washed with M9 buffer and transferred onto a plate of Escherichia coli not expressing GFP. Every 24 hours thereafter, 10 nematodes were transferred to M9 buffer containing 1% Triton X-100, then
It was mechanically disrupted to count the bacteria present in the digestive tract. Then 10mM bacteria
Diluted with MgSO ₄ , placed on a plate containing ampicillin and counted for cfu. Data represent the mean ± SD of 2 independent experiments.

FIG. 5A shows that nematode killing requires a direct interaction between live Salmonella typhimurium and C. elegans. PA14 (open circles) were grown on 0.45 μm filters placed on PGS plates and SL1344 was grown on either PGP (filled circles) or NG plates (squares). After bacterial growth, filters were removed, plates were exposed to UV light for 5 minutes to kill all potentially contaminating bacteria, heat-killed OP50 was added as a food source, and 20 nematodes were added. Placed on each plate. Small bars indicate standard deviation and results are representative of at least 2 independent experiments. Figure 5B shows that nematode killing requires a direct interaction between live Salmonella typhimurium and C. elegans. Heat killed SL1344 (black circles) or live SL in 20 nematodes
1344 (open circle) and heat killed SL14028 (black square) or living SL14028 (white square)
Was fed. Small bars indicate standard deviation and results are representative of at least 2 independent experiments.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C12Q 1/02 G01N 33/15 Z G01N 33/15 33/569 Z 33/569 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL , IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Invention Person Yogay Peter S. 44 F-Term (Reference) 2B045 AA40 BA11 BB50 CB21 FB04 4B063 QA07 QQ05 QQ91 QR74 QR75 QS03 QS10 QX02 4C084 AA02 AA17 MA01 ZB352, Cambridge Three Street, Cambridge, Mass., USA

Claims (39)

[Claims] 1. An isolated nematode persistently infected with the isolated pathogen. 2. The nematode according to claim 1, wherein the pathogen expresses a detectable marker. 3. Caenorhabditis elegan
The nematode according to claim 1, which is s). 4. The nematode according to claim 1, which is a 1-day-old adult hermaphroditic or L4 larval stage nematode. 5. The nematode according to claim 1, wherein the pathogen colonizes the intestine of the nematode. 6. The nematode according to claim 1, wherein the pathogen is Salmonella. 7. The pathogen is Salmonella typhimurium SL1344.
The nematode according to claim 6, which is a strain. 8. A method of screening for a virulence factor that allows a pathogen to cause a persistent infection in a nematode, comprising: (a) mutating the pathogen. Exposing the worm; (b) determining whether the mutant pathogen persistently infects the nematode, wherein the reduced disease in the worm is compared to the disease caused by the non-mutagenized pathogen. , Showing a mutation in a virulence factor that allows the pathogen to cause a persistent infection in the nematode; and (c) using the mutation as a marker to identify the virulence factor. 9. The method of claim 8, wherein the pathogen expresses a detectable marker. 10. The method according to claim 8, wherein the nematode is a 1-day-old adult hermaphroditic or L4 larval stage nematode. 11. The method of claim 8, wherein the pathogen colonizes the intestine of a nematode. 12. The method of claim 8, wherein the pathogen is Salmonella. 13. The method according to claim 12, wherein the pathogen is Salmonella typhimurium SL1344 strain. 14. The method of claim 8, wherein the nematode is C. elegans. 15. The method of claim 8, which utilizes a Salmonella / C. Elegans killing assay. 16. The method of claim 15, wherein the mutant pathogen causes less C. elegans killing than the non-mutagenized pathogen. 17. A method for screening a compound that inhibits persistent infection of a pathogen in a nematode, the method comprising the steps of: (a) providing a nematode persistently infected with the pathogen; Contacting the test compound with a persistently infected nematode; and (c) determining whether the test compound inhibits the pathogenicity of the pathogen in the persistently infected nematode. 18. The method of claim 17, wherein the pathogen expresses a detectable gene. 19. The nematode according to claim 17, which is a 1-day-old adult hermaphroditic or L4 larval stage nematode. 20. The nematode of claim 17, wherein colonization of the gut of the nematode by the mutant pathogen is reduced. 21. The method of claim 17, wherein the pathogen is Salmonella. 22. The method according to claim 21, wherein the pathogen is Salmonella typhimurium SL1344 strain. 23. The method of claim 17, wherein the nematode is C. elegans. 24. The test compound is provided internally in a compound library.
17 Method described. 25. The method of claim 17, wherein the test compound is a small organic compound. 26. The method of claim 17, wherein the test compound is a peptide, peptidomimetic, or antibody or fragment thereof. 27. The method of claim 17, wherein the inhibition of pathogenicity is measured by Salmonella / C. Elegans killing assay. 28. The method of claim 27, wherein the pathogen causes C. elegans killing in the presence of the test compound less than in the absence of the test compound. 29. A method of screening for virulence factors that allows a pathogen to cause persistent infection in nematodes, the method comprising the steps of: (a) mutagenesis expressing a detectable marker. Exposing the nematode to the pathogen; (b) determining whether the mutant pathogen persistently infects the nematode by measuring the level of a detectable marker in the nematode. A reduced marker in the nematode relative to the level of the marker caused by the uninduced pathogen, showing a mutation in the virulence factor that allows the pathogen to cause a persistent infection in the nematode; and (c) using the mutation as a marker to identify the virulence factor. 30. A method for screening a compound that inhibits persistent infection of a pathogen in a nematode, comprising the steps of: (a) providing a nematode persistently infected with a pathogen expressing a detectable marker. (B) contacting the test compound with the persistently infected nematode; and (c) measuring the level of a detectable marker in the nematode to maintain the pathogen persisting in the nematode. Determining whether to be infected, and a decrease in the marker in the nematode indicating that the test compound inhibits persistent pathogen infection in the nematode. 31. A method of screening for virulence factors that enable a pathogen to cause a persistent infection in a nematode, comprising the steps of: (a) a pathogen expressing a gene not normally expressed by the pathogen. Exposing the nematode to the nematode; (b) determining whether the pathogen persistently infects the nematode by measuring the level of a detectable marker in the nematode, which is mutagenized. A marker in the nematode that is decreased or increased relative to the level of the marker caused by the non-pathogen, exhibits a virulence factor or a mutation in the virulence factor gene that allows the pathogen to cause a persistent infection in the nematode; and (c) using a gene expressed by the pathogen as a marker to identify the virulence factor. 32. The method of claim 31, wherein the screening utilizes the Salmonella / C. Elegans killing assay. 33. The method of claim 31, wherein the ability of the pathogen to cause a persistent infection in C. elegans is reduced or increased. 34. A method of screening for a virulence factor that allows a pathogen to cause a persistent infection in a nematode, the method comprising the steps of: (a) a pathogen overexpressing a pathogen gene; (B) determining whether the pathogen persistently infects the nematode by measuring the level of the detectable marker in the nematode, and the marker caused by the non-mutagenized pathogen. A marker in the nematode that is decreased or increased relative to the level of virulence factor or a mutation in the virulence factor gene that allows the pathogen to cause a persistent infection in the nematode; and (c) the virulence factor. Using the mutation or virulence factor gene as a marker to identify. 35. The method of claim 34, wherein the screening utilizes a Salmonella / C. Elegans killing assay. 36. The method of claim 34, wherein the ability of the pathogen to cause a persistent infection in C. elegans is reduced or increased. 37. A method of screening for virulence factors that allows a pathogen to cause persistent infection in a nematode, the method comprising the steps of: (a) a marker being mutagenized, detectable To the pathogen expressing the marker,
Exposing the nematode; (b) determining whether the mutant or otherwise modified pathogen persistently infects the nematode by measuring the level of a detectable marker in the nematode. A marker within the nematode that is present in the nematode that is reduced or increased relative to the level of the marker caused by the non-mutagenized pathogen is a virulence factor or virulence factor gene that enables the pathogen to cause a persistent infection in the nematode. Demonstrating a mutation; and (c) using the mutation to identify virulence factors. 38. A method of screening for a virulence factor that allows a pathogen to cause a persistent infection in a nematode, the method comprising the steps of: (a) expressing a detectable marker, which is normally associated with the pathogen. Exposing the nematode to a pathogen expressing a non-expressed gene; (b) the mutant or otherwise modified pathogen is detected in the nematode by measuring the level of a detectable marker in the nematode. Marker in the nematode, which is reduced or increased compared to the level of the marker caused by the non-mutagenized pathogen, causes the pathogen to cause persistent infection in the nematode. A virulence factor or a mutation in the virulence factor gene that enables the Step using a gene that is expressed by the bulk. 39. A method of screening for a virulence factor that allows a pathogen to cause a persistent infection in a nematode, the method comprising the steps of: (a) expressing a detectable marker and expressing the pathogen gene. Exposing the nematode to an overexpressing pathogen; (b) whether the mutant or otherwise modified pathogen persistently infects the nematode by measuring levels of detectable markers in the nematode. A marker within the nematode that is at the stage of determining whether the pathogen causes a persistent infection in the nematode, which is decreased or increased relative to the level of the marker caused by the non-mutagenized pathogen. Factor or virulence factor gene mutation; and (c) expression by the pathogen as a marker to identify virulence factors. Stage to use that gene.