The art of serendipity: killing of Caenorhabditis elegans by human pathogens as a model of bacterial and fungal pathogenesis

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1 Review The art of serendipity: killing of Caenorhabditis elegans by human pathogens as a model of bacterial and fungal pathogenesis Eleftherios Mylonakis, Frederick M Ausubel, Robin Jian Tang and Stephen B Calderwood CONTENTS Methodology for killing assays Other bacteria Host response Five-year view Expert opinion Key issues References Affiliations Author for correspondence Harvard Medical School, Tel.: emylonakis@partners.org KEYWORDS: AIDS, Burkholderia pseudomallei, Caenorhabditis elegans, Cryptococcus neoformans, Enterococcus faecalis, HIV, MAPkinase, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens The nematode worm, Caenorhabditis elegans, has been used to develop a facile model system of host pathogen interactions to identify basic evolutionarily conserved pathways associated with microbial pathogenesis. The model involves the killing of Caenorhabditis elegans by a variety of human pathogens. Several virulence-related genes in a variety of pathogens previously shown to be involved in mammalian infection have also been shown to play a role in Caenorhabditis elegans killing. Screening of large numbers of microbial mutants for attenuation in a mammalian model would require thousands of mice, rats or rabbits. In contrast, the Caenorhabditis elegans model allows rapid identification of mutants in microbial genes associated with pathogenesis and then these phenotypes can be confirmed in a relevant mammalian model. Expert Rev. Anti-infect. Ther. 1(1), (2003) The outcome of the interaction between a pathogen and a host depends upon the interplay between virulence factors of the micro-organism and responses of the host to infection. Some microbial virulence factors are only induced in the host and may require specialized techniques to be identified based upon detection of the expression of the corresponding virulence genes in vivo or the failure of specific mutants to survive within the host environment. Recent studies have shown that nonvertebrate hosts (such as the plant Arabidopsis thaliana, the nematode Caenorhabditis elegans, and the insects Drosophila melanogaster and Galleria mellonella), can be used to identify novel virulence determinants in a variety of microbial animal pathogens [1 4]. The most widely used of these alternative models is the extensively studied nematode C. elegans. Researchers have shown that a variety of bacterial and some fungal pathogens of humans also kill C. elegans, that this killing is dependent on genes involved in mammalian infection and that the C. elegans model can be used to identify new virulence factors [5 28]. The free-living soil nematode C. elegans has been a popular subject of study since Sidney Brenner first promoted its use in the 1960s: C. elegans has a short reproductive cycle and produces many progeny, accelerating experiments As C. elegans is a hermaphrodite and capable of self-fertilization, it is possible to easily create genetically identical populations by simply allowing a single C. elegans animal to produce progeny The entire cell lineage (the division and differentiation of each cell) has been described in exact detail and is identical in every C. elegans adult The worm is small (~1 mm) and transparent and thus easily viewed with a dissecting microscope or by using Normarski or confocal microsocopy The entire genome of the worm has been sequenced Future Drugs Ltd. All rights reserved. ISSN

2 Mylonakis, Ausubel, Tang & Calderwood C. elegans chromosomal RNA interference (RNAi) feeding library is available [101]. RNAi refers to the introduction of homologous double-stranded RNA (dsrna) to specifically inhibit expression of the corresponding gene, resulting in null or hypomorphic phenotypes [29]. In comparison to C. elegans, the study of pathogenesis in mammalian models is substantially more complicated due to difficulty of handling, long reproductive cycles, small brood sizes and the much higher complexity of mammalian hosts. Therefore, if killing of C. elegans by pathogens mimics key features of mammalian pathogenesis, then it should be possible to use C. elegans as a facile and inexpensive host to model a variety of human microbial diseases. Methodology for killing assays C. elegans feed on unicellular microbes. In the laboratory, C. elegans is usually propagated on petri plates containing nematode growth (NG) medium (a minimal medium containing NaCl, agar, peptone, cholesterol, CaCl 2, MgSO 4 and potassium phosphate), spread with a lawn of a slow-growing strain of Escherichia coli K-12 (OP50). The most commonly used wild type strain of C. elegans is Bristol N2. The pathogen killing assays described in this review simply involve transferring C. elegans animals (usually at the L4 developmental stage) from a lawn of Escherichia coli strain OP50 to a lawn of the pathogenic bacterium or yeast to be tested. For example, in the C. elegans/cryptococcus neoformans killing assays, C. neoformans strains are inoculated into 2 ml of yeast peptone dextrose (YPD) and grown at 28 C. Of the culture, 10 µl is spread on 35-mm tissue-culture plates (Falcon) containing 4-ml brain heart infusion (BHI) agar (Difco). To spread 10 µl of C. neoformans culture on the surface of an agar plate, a sterile pipette tip is held on a 20 µl automatic pipettor and bent at an acute angle. The rounded nose thus resulting is used to spread the culture uniformly over roughly two-thirds of the plate area; care is taken not to break the surface of the agar. Plates are then incubated at 28 C overnight. Ampicillin (100 µg/ml) or gentamicin (25 50 µg/ml) is added to the agar to selectively prevent growth of E. coli OP50 carried over on transfer of worms to the yeast-containing plates. Nematodes are transferred from a lawn of E. coli strain OP50 to the lawn of C. neoformans, utilizing a flatedged platinum wire pick; transfer must be rapid to prevent drying-out of the worms. Plates are examined for nematode viability at intervals using a dissecting microscope Worms are considered dead when they do not respond to touch with a platinum wire pick [5,6]. Plotting of killing curves, calculation of time for half of the worms to die (LT50) and estimation of differences in survival on different pathogenic strains are performed by using statistical software, such as STATA 6 (Stata, College Station, TX, USA) [5,6]. Screening of pathogen mutant libraries to identify novel virulence traits is usually done using a three-stage screen with an increasing number of nematodes at each stage [5]. Some pathogens both kill nematodes as well as prevent production of progeny. For other pathogens (as well as nonpathogens), however, progeny are produced. In these latter cases, such as for the yeast Cryptococcus laurentii or the bacterium Salmonella typhimurium, nematodes must be transferred every 48 h to new plates that contain the microorganism being studied. This allows determination of survival of the original worms before they produce progeny, a necessary procedure to prevent the original worms from being lost among the rapidly maturing progeny. Pseudomonas aeruginosa The important human pathogen Pseudomonas aeruginosa was the first bacterium whose pathogenic interaction with C. elegans was studied in-depth [13,16,20,26 28]. Two mechanistically different modes of C. elegans killing are observed with P. aeruginosa depending on the media on which the P. aeruginosa lawn is grown. When P. aeruginosa strain PA14 is grown on minimal (NG) agar, killing occurs over the course of 2 3 days and this killing correlates with the accumulation of large numbers of P. aeruginosa cells in the C. elegans intestine; normally, when feeding on E. coli, the intestine is bacteria-free. In contrast, when P. aeruginosa strain PA14 is grown on highosmolarity peptone-glucose-sorbitol (PGS) medium, prior to being used as a food source for C. elegans, killing of C. elegans occurs over a matter of several hours. It appears that these two types of killing are mechanistically distinct. In most cases, PA14 mutants that are attenuated in fast killing on PGS medium exhibit wild type levels of slow killing on NG medium and vice versa. Moreover, heat killed bacteria are capable of fast but not slow killing. Fast but not slow killing, can be effected by PA14 bacterial supernatants, with death of C. elegans occurring similarly as with bacteria. Fast killing correlates with the production of phenazines, a class of tricyclic pigments that includes pyocyanin. In the case of slow killing, a screen of TnphoA transposon insertion mutants of P. aeruginosa strain PA14 demonstrated that the lasr-lasi quorum-sensing system, the lema and gaca two-component regulators and toxa (encoding exotoxin A), were involved in PA14 lethality. In all, 19 of 23 genes identified in a screen as being involved in C. elegans fast or slow killing were also shown to be required for full virulence in a mouse thermal injury and infection model [20,26 28], showing the close correlation of genes required for virulence across evolutionarily disparate hosts. In addition to the fast and slow killing mechanisms described above, a third, distinct mechanism of C. elegans killling by P. aeruginosa is caused by the growth of strain PAO1 on brain heart infusion medium (BHI); this killing functions through rapid (<4 h) neuromuscular paralysis [13]. Recently, this rapid paralysis was shown to be mediated by the production of hydrogen cyanide [16]. It is not likely that the fast killing phenotype exhibited by P. aeruginosa strain PA14 is also due to hydrogen cyanide toxicity. C. elegans age-1 mutants, which are more resistant to oxidative stress, are also more 90 Expert Rev. Anti-infect. Ther. 1(1), (2003)

3 Caenorhabditis elegans model of pathogenesis resistant to PA14 killing but not PAO1 killing. Conversely, C. elegans egl-9 mutants are more resistant to PAO1 killing by an unknown mechanism but are not more resistant to PA14 killing. The EGL-9 protein, which is strongly expressed in the nematode body wall and pharyngeal muscles, has homologs in a wide range of organisms, including mammals and Drosophila. In C. elegans, EGL-9 is necessary for normal muscle function but its inactivation results in resistance against paralysis induced by the Pseudomonas aeruginosa toxin. Salmonella enterica Aballay and Ausubel, and Labrousse and colleauges demonstrated that C. elegans is also an attractive model host for a variety of Salmonella enterica serovars, including typhimurium, enteridis and dublin [9,10,19]. Unlike P. aeruginosa, however, Salmonella does not appear to kill by means of low molecular weight toxins. Like P. aeruginosa, S. typhimurium accumulates in the C. elegans intestine but unlike Pseudomonas, S. typhimurium establishes a persistent infection that cannot be cured by transferring the worms to a lawn of E. coli. As in the case of P. aeruginosa, mutations in known Salmonella virulence factors, such as phop/phoq, which encode a global regulatory system, fur-1 and ompr, which confer acid resistance and rpos, which encodes an acid-tolerance sigma factor, attenuate nematode killing, validating the model. Serratia marcescens Like P. aeruginosa, killing of C. elegans by S. marcescens involves two separate mechanisms, one toxin-based and one through a systemic infection that begins with proliferation in the intestine. Recently, Kurz and colleagues used C. elegans to screen a bank of ca transposon-induced S. marcescens mutants and isolated 23 clones with attenuated virulence [18]. Three of these clones exhibited decreased virulence in an insect model and reduced cytotoxicity in vitro and one of them was also markedly attenuated in virulence in a murine model [18]. Burkholderia pseudomallei Workers have reported that several Burkholderia species kill C. elegans. B. thailandensis is the most virulent, followed by B. pseudomallei, B. cepacia and B. mallei [15,22,25]. Killing occurs by rapid loss of locomotor function, foraging ability and pharyngeal pumping [22]. After the 8th hour, recovery by transfer to E. coli is not possible. Feeding cessation does not appear to be a defensive choice as worms do not prefer nontoxic food sources over Burkholderia. The genes responsible for aminoglycoside and macrolide efflux pumping, lipopolysaccharide (LPS) O-antigen biosynthesis, the general protein secretory machinery, flagellar biosynthesis and the biosynthesis of antihelminthic macrolides do not appear to be involved in B. pseudomallei virulence. Bacterial supernatants of B. pseudomallei and thailandensis do not kill C. elegans, although γ-irradiated lawns do. B. pseudomallei and P. aeruginosa share many phenotypic features (B. pseudomallei was once classified as a pseudomonad). Gan and colleagues tested B. pseudomallei using assays similar to those used by Tan and coworkers for P. aeruginosa [15]. C. elegans killing on PGS agar was indeed found to more rapid than on NG agar and bacterial supernatants were capable of killing; however, it is not clear, as it was in the case of P. aeruginosa, whether or not separate mechanisms of action are involved in the slow and fast killing conditions for B. pseudomallei. Gan and colleagues screened 3400 transposon mutants of B. pseudomallei and identified 39 mutants that exhibited reduced killing of C. elegans. To eliminate mutants in which the reduced killing phenotype was caused by generalized debilitation of the bacterium, Gan and colleagues compared the growth rates of the 39 mutants with the wild type strain. This secondary screen allowed them to focus on five mutants that exhibited growth rates comparable with the parent strain. These five B. pseudomallei transposon mutants, which attenuated virulence in the nematode, were also shown to be similarly attenuated in the mouse [15]. Enterococcus faecalis & other Gram-positive bacteria Garsin and colleagues and Jansen and colleagues showed that Gram-positive pathogens, such as Enterococcus faecalis, Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenesand kill C. elegans [5,8]. Like S. typhimurium, Enterococcus faecalis establishes a persistent infection in the nematode intestine and proliferates. E. faecium also accumulates in the C. elegans intestine but unlike E. faecalis, neither kills the nematodes nor is able to permanently colonize the intestinal lumen. Disruption of the E. faecalis cyl gene, a known virulence factor involved in disruption of host membranes, shows attenuation of virulence in both C. elegans and the mouse [5]. Moreover, disruption of the quorum-sensing regulatory genes fsra, fsrb and fsrc, which control the known virulence factor gele, a gelatinase, also attenuate C. elegans virulence [5,7,24]. The fsrb mutant is also attenuated in the mouse model of infection. Using a 3- stage screen of ca random E. faecalis mutants, Garsin and colleagues identified a novel gene with homology to scrb of Streptococcus sobrinus, whose disruption reduces virulence in both C. elegans and a murine model [5]. Both S. pyogenes and S. pneumoniae kill C. elegans rapidly via production of hydrogen peroxide, a small diffusible toxin that is also a virulence factor in humans [8]. Other bacteria A large number of bacteria are pathogenic to L4 C. elegans when grown on BHI [12]. Among these are the plant pathogens Erwinia chrysanthemi, Agrobacterium tumefaciens and Erwinia carotovora pv. carotovora; the enterobacteria Shewanella frigidimarina and Shewanella massilia; the fish pathogen Aeromonas hydrophila; and the insect pathogens Photorhabdus luminescens and Xenorhabdus nematophila. Of these, A. tumefaciens, E. carotovora pv. carotovora, A. hydrophila and P. luminescens require 91

4 Mylonakis, Ausubel, Tang & Calderwood live bacteria for lethality, whereas X. nematophila does not. Bacillus megaterium is nonpathogenic and inconclusive results have been obtained for Brucella spp., Mycobacterium fortuitum and Mycobacterium marinum. Cryptococcus neoformans Interactions between fungi and free-living Rhabditid nematodes have been suggested by prior ecological studies but only in the case of the endoparasitic fungus Drechmeria coniospora has a model of infection of C. elegans been established. Infection of C. elegans by D. coniospora starts by the adhesion of fungal spores to the head of the worm, followed by extension of hyphal processes into the nematode [30]. More focus has been placed important human fungal pathogen, C. neoformans. The significant numbers of serious infections with C. neoformans, the paucity of new antifungal agents and the likelihood of the emergence of drug resistance in fungi, suggested a pressing need for new model systems to study the mechanisms of fungal virulence and to identify new targets for antifungal therapy [31 40]. C. neoformans most often affects the CNS, lungs or the skin. In a statewide study performed in Alabama from 1992 to 1994, the annual incidence of cryptococcosis among HIV-infected patients was found to be per 100,000 individuals [41]. Rates of cryptococcosis in patients without HIV infection in the US are comparable to the incidence rates described for meningococcal meningitis [39]. Although the incidence of cryptococcal meningitis in developed countries probably decreased with the advent of combination antiretroviral therapy, there has been an explosion in the incidence of cryptococcosis in Africa, Thailand and India [39,41]. Currently, cryptococcal meningitis is the leading cause of culture-positive meningitis in Zimbabwe, constituting 45% of all cases and easily outnumbering the cases of pyogenic (16%) or tuberculous (12%) meningitis [39]. Among children in areas of Africa, mortality from cryptococcal infection during initial hospitalization can exceed 40% [40,42]. Study of the pathogenic mechanisms of C. neoformans has been enhanced substantially by genetic techniques, including the development of transformation protocols and homologous recombination for genetic manipulations and reproducible animal models [38,43,44]. Although it was not known whether C. elegans could feed on yeasts, Mylonakis and colleagues recently reported that C. elegans can use various nonpathogenic yeasts, including Cryptococcus laurentii and Cryptococcus kuetzingii, as a sole source of food, producing similar brood sizes compared with growth on E. coli OP50. C. elegans grown on these yeasts has a lifespan similar to (C. laurentii) or longer than (C. kuetzingii) worms fed on E. coli [6]. However, the human pathogenic yeast C. neoformans kills C. elegans and the C. neoformans polysaccharide capsule, as well as several C. neoformans genes previously shown to be involved in mammalian virulence, also play a role in C. elegans killing [6]. These include genes associated with signal transduction pathways (GPA1, PKA1, PKR1 and RAS1), laccase production (LAC1) and the α-mating type. Yeast accumulate in the intestine (FIGURE 1) but do not permanently colonize, while acapsular mutants kill without accumulation in the intestine, suggesting that neither accumulation nor capsule are strictly necessary for killing. As in bacterial pathogens, the exact mechanism of killing of C. elegans by C. neoformans is not yet clear. A green fluorescent protein (GFP) reporter gene fused to the mating pheromone MF1α is expressed only in yeast cells within the nematode intestine, analogous to expression of this promoter specifically in the CSF in a rabbit model of cryptococcal meningitis [6]. This suggests that expression of a gene associated with virulence is temporally related to C. elegans killing [6]. Host response Nematoda and Arthropoda are sister phyla and it was reasonable to hypothesize that a C. elegans defense response would also share some of the features of the defense response pathways found in insects and homologous to those in mammals [23]. Virtual searches of the C. elegans genome sequence Figure 1. Cryptococcus neoformans accumulates in the gastrointestinal tract of Caenorhabditis elegans. Intact yeast cells are present in the distended gastrointestinal tract after feeding for 24 h on C. neoformans strain H99. The round structure is the pharyngeal grinder organ of the worm, which functions to disrupt ingested organisms. Arrows point to the pharyngeal grinder organ and the intestinal lumen. 92 Expert Rev. Anti-infect. Ther. 1(1), (2003)

5 Caenorhabditis elegans model of pathogenesis revealed the presence of genes encoding presumptive homologs of a Toll receptor (tol-1), a Traf linker protein (trf- 1), a Pelle (IRAK) kinase (pik-1) and a Cactus-(I-κB)-like inhibitory protein (ikb-1) but Rel-domain transcription factors, such as Drosophila Dif, Dorsal, or Relish or mammalian NF-κB do not appear to be encoded in the C. elegans genome. However, recent experiments using C. elegans-bacterial pathogenesis models have failed to demonstrate a role for Toll signaling components in the activation of a C. elegans innate immune response, although interestingly, tol-1 did appear to play a role in a pathogen recognition/avoidance mechanism [23]. Although a C. elegans Toll signaling cascade does not appear to play a role in an immune response to bacterial pathogens, recently published work has shown that exposure of C. elegans to pathogenic bacteria does activate the transcription of genes known to be involved in defense responses in other species [4]. Mallo and colleagues. have shown using microarray analysis that the lysozyme genes lys- 1, lys-7 and lys-8 are upregulated following infection with Serratia marcescens [14]. Bacterial pathogens also induce a programmed cell death (PCD) response in C. elegans gonadal cells that may play a protective role in defense against bacterial infection [9]. Salmonella typhimurium infection causes an increased level of gonadal PCD, which is distinct from and controlled independently of a well-described basal level of gonadal PCD. PCD does not similarly occur in the soma. While the mechanism of gonadal PCD is not fully understood, these observations may provide insight into the mechanisms of nematode self-defense following bacterial infection. C. elegans mutants in ced-3 and ced-4, which are defective in the cell death pathway, are more susceptible to S. typhimurium killing [9]. The same is true for a gain-of-function mutant in ced-9, which negatively regulates the cell death pathway [9]. The failure of S. typhimurium to reach the gonad suggests the action of an indirect signal to trigger gonadal PCD. Recently, Kim and coworkers have taken a forward genetic approach to identify components of a presumptive C. elegans innate immune response pathway upstream of induced defense responses [17]. This led to the discovery that a C. elegans homolog of the mammalian p38 mitogen activated protein kinase (MAPK), is an important component of an apparent C. elegans defense response to bacterial pathogens [45]. The p38 MAPK cascade appears to be upstream of the pathogen-induced PCD response in the gonad [11]. Five-year view Further screens of a variety of pathogens in the next five years using the C. elegans model is expected to identify additional genes associated with bacterial and fungal pathogenesis. These genes may be appropriate targets for the development of new antibacterial and antifungal agents. A better understanding of the C. elegans innate immune response to pathogens will further facilitate study of host/pathogen interactions. Expert opinion The studies outlined above describe how the nematode C. elegans can be used as a facile model host to identify and characterize genes required for microbial pathogenesis. It is anticipated that the future use of the C. elegans model will enhance our understanding of the pathogenesis of significant human bacterial and fungal pathogens, provide new targets for the development of antibacterial and antifungal agents and contribute new insights on basic, evolutionarily preserved mechanisms by which bacteria and yeasts interact with hosts during the pathogenesis process. The differences in killing of C. elegans by bacteria grown on different media and the precise mechanisms of nematode killing are just two of the areas needing further exploration, as researchers expand use of this system to study the interactions between pathogens and C. elegans. Acknowledgments This work was supported by a grant from Aventis, SA to FMA. and SBC and by a postdoctoral fellowship from the Howard Hughes Medical Institute to EM. Key issues The nematode worm Caenorhabditis elegans has been used to develop a facile model system of host pathogen interactions to identify basic evolutionarily conserved pathways associated with microbial pathogenesis. The model involves the killing of C. elegans by a variety of human pathogens. Several genes previously shown to be involved in pathogenesis of several organisms in mammalian systems, have also been shown to play a role in C. elegans killing. The important human pathogen Pseudomonas aeruginosa was the first bacterium whose pathogenic interaction with C. elegans was studied in-depth. Mechanistically different modes of C. elegans killing are observed with P. aeruginosa, depending on the media on which P. aeruginosa is grown. Recently, the C. elegans model was expanded to include fungal pathogens, particularly the model human pathogen Cryptococcus neoformans. Bacterial pathogens induce a programmed cell death response in gonadal cells of C. elegans, which may play a protective role in defense against microbial infection. A forward genetic approach has been used to identify components of a presumptive C. elegans innate immune response upstream of induced defense responses. 93

6 Mylonakis, Ausubel, Tang & Calderwood References Papers of special note have been highlighted as: of interest of considerable interest 1 Mahajan-Miklos S, Rahme LG, Ausubel FM. Elucidating the molecular mechanisms of bacterial virulence using nonmammalian hosts. Mol Microbiol. 37(5), (2000). Interesting review of studies on the extensive conservation in the virulence mechanisms used by P. aeruginosa to infect evolutionarily diverged hosts. 2 Rahme LG, Ausubel FM, Cao H et al. Plants and animals share functionally common bacterial virulence factors. Proc. Natl Acad. Sci. USA 97(16), (2000). Overview of model systems that use plants and nematodes as adjuncts to mammalian models to help elucidate the molecular basis of P. aeruginosa pathogenesis. 3 Aballay A, Ausubel FM. Caenorhabditis elegans as a host for the study of hostpathogen interactions. Curr. Opin. Microbiol. 5(1), (2002). 4 Kurz CL, Ewbank JJ. Caenorhabditis elegans for the study of host-pathogen interactions. Trends Microbiol. 8(3), (2000). Excellent review on the topic. 5 Garsin DA, Sifri CD, Mylonakis E et al. A simple model host for identifying Grampositive virulence factors. Proc. Natl Acad. Sci. USA 98(19), (2001). Studies investigating the parallels between Gram-positive infection in simple and more complex organisms with a focus on E. faecalis. 6 Mylonakis E, Ausubel FM, Perfect JR, Heitman J, Calderwood SB. Killing of Caenorhabditis elegans by Cryptococcus neoformans as a model of yeast pathogenesis. Proc. Natl Acad. Sci. USA 99(24), (2002). C. elegans can be used as a simple model host in which C. neoformans pathogenesis can be readily studied. 7 Mylonakis E, Engelbert M, Qin X et al. The Enterococcus faecalis fsrb gene, a key component of the fsr quorum-sensing system, is associated with virulence in the rabbit endophthalmitis model. Infect. Immun. 70(8), (2002). 8 Jansen WT, Bolm M, Balling R, Chhatwal GS, Schnabel R. Hydrogen peroxidemediated killing of Caenorhabditis elegans by Streptococcus pyogenes. Infect. Immun. 70(9), (2002). 9 Aballay A, Ausubel FM. Programmed cell death mediated by ced-3 and ced-4 protects Caenorhabditis elegans from Salmonella typhimurium-mediated killing. Proc. Natl Acad. Sci. USA 98(5), (2001). 10 Aballay A, Yorgey P, Ausubel FM. Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans. Curr. Biol. 10(23), (2000). 11 Aballay A, Drenkard E, Hilbun LR, Ausubel FM. Caenorhabditis elegans innate immune response triggered by Salmonella enterica requires intact LPS and is mediated by a MAPK signaling pathway. Curr. Biol. 13(1), (2003) 12 Couillault C, Ewbank JJ. Diverse bacteria are pathogens of Caenorhabditis elegans. Infect. Immun. 70(8), (2002). 13 Darby C, Cosma CL, Thomas JH, Manoil C. Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 96(26), (1999). 14 Mallo GV, Kurz CL, Couillault C et al. Inducible antibacterial defense system in C. elegans. Curr. Biol. 12(14), (2002). The first demonstration of inducible antibacterial defenses in C. elegans. 15 Gan YH, Chua KL, Chua HH et al. Characterization of Burkholderia pseudomallei infection and identification of novel virulence factors using a Caenorhabditis elegans host system. Mol. Microbiol. 44(5), (2002). 16 Gallagher LA, Manoil C. Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning. J. Bacteriol. 183(21), (2001). 17 Kim DH, Feinbaum R, Alloing G et al. A conserved p38 MAPK pathway in Caenorhabditis elegans innate immunity. Science 297(5581), (2002). A genetic screen for C. elegans mutants led to the identification of two genes required for pathogen resistance: one that encodes a mitogen-activated protein (MAP) kinase kinase, and another that encodes a MAP kinase kinase kinase. 18 Kurz CL, Chauvet S, Andres E et al. Virulence factors of the human opportunistic pathogen Serratia marcescens identified by in vivo screening. EMBO J. 22(7), , (2003). Use of the C. elegans model to screen a bank of transposon-induced S.marcescens mutants lead to the isolation of clones with an attenuated virulence. 19 Labrousse A, Chauvet S, Couillault C, Kurz CL, Ewbank JJ. Caenorhabditis elegans is a model host for Salmonella typhimurium. Curr. Biol. 10(23), (2000). 20 Mahajan-Miklos S, Tan MW, Rahme LG, Ausubel FM. Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa Caenorhabditis elegans pathogenesis model. Cell 96(1), (1999). Use of systematic mutagenesis to identify mutants that fail to kill C. elegans, identified phenazines, as one of the mediators of killing and demonstrated that phenazines are also required for pathogenesis in plants and mice. 21 Nicholas HR, Hodgkin J. Innate immunity: the worm fights back. Curr. Biol. 12(21), R731 R732 (2002). 22 O Quinn AL, Wiegand EM, Jeddeloh JA. Burkholderia pseudomallei kills the nematode Caenorhabditis elegans using an endotoxin-mediated paralysis. Cell Microbiol. 3(6), (2001). 23 Pujol N, Link EM, Liu LX et al. A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans. Curr. Biol. 11(11), (2001). Deletion mutants for four genes supposed to function in a nematode Toll signaling pathway demonstrated that in C. elegans, tol-1 is important for development and pathogen recognition. 24 Sifri CD, Mylonakis E, Singh KV et al. Virulence effect of Enterococcus faecalis protease genes and the quorum-sensing locus fsr in Caenorhabditis elegans and mice. Infect. Immun. 70(10), (2002). 25 Smith MP, Laws TR, Atkins TP, Oyston PC, de Pomerai DI, Titball RW. A liquidbased method for the assessment of bacterial pathogenicity using the nematode Caenorhabditis elegans. FEMS Microbiol. Lett. 210(2), (2002) 26 Tan MW, Ausubel FM. Caenorhabditis elegans: a model genetic host to study Pseudomonas aeruginosa pathogenesis. Curr. Opin. Microbiol. 3(1), (2000). 27 Tan MW, Mahajan-Miklos S, Ausubel FM. Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proc. Natl Acad. Sci. USA 96(2), (1999). 28 Tan MW, Rahme LG, Sternberg JA, Tompkins RG, Ausubel FM. Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. Proc. Natl Acad. Sci. USA 96(5), (1999). 29 Timmons L, Fire A. Specific interference by ingested dsrna. Nature 395(6705), 854 (1998). 30 Jansson HB. Adhesion of conidia of 94 Expert Rev. Anti-infect. Ther. 1(1), (2003)

7 Caenorhabditis elegans model of pathogenesis Drechmeria coniospora to Caenorhabditis elegans wild type and mutants. J. Nematol. 26(4), (1994). 31 Mylonakis E, Flanigan T. Antifungal prophylaxis with weekly fluconazole for patients with AIDS. Clin. Infect. Dis. 27(6), (1998). 32 Mylonakis E, Barlam TF, Flanigan T, Rich J. Pulmonary aspergillosis in the acquired immunodeficiency syndrome: review of 342 cases. Chest 114(1), (1998). 33 Marty F, Mylonakis E. Use of antifungals in HIV infection. Expert Opin. Pharmacother. 3(2), (2002). 34 Mylonakis E, Merriman NA, Rich JD, Walters BC, Tashima K, Mileno MD et al. Use of cerebrospinal fluid shunt for the management of elevated intracranial pressure in a patient with active AIDSrelated cryptococcal meningitis. Diagn. Microb. Infect. Dis. 34(2), (1999). 35 Mylonakis E, Rich J, Kwakwa H et al. Muscle abscess due to Aspergillus fumigatus in an AIDS patient. Clin. Infect. Dis. 23(6), (1996). 36 Mylonakis E, Rich J, Skolnik P, De Orchis D, Flanigan T. Invasive Aspergillus sinusitis in patients with human immunodeficiency virus infection. Medicine (Baltimore) 76(4), (1997). 37 Mylonakis E, Paliou M, Sax PE, Skolnik PR, Baron MJ, Rich JD. Central nervous system aspergillosis in patients with human immunodeficiency virus infection. Medicine (Baltimore) 79(4), (2000). 38 Casadevall A, Perfect JR. In: Cryptococcus neoformans. Am. Soc. Microbiol. Washington, DC, USA, (1998). 39 Perfect J, Casadevall A. Cryptococcosis. Infect. Dis. Clin. North Am. 16(4), (2002). 40 Gumbo T, Kadzirange G, Mielke J, Gangaidzo IT, Hakim JG. Cryptococcus neoformans meningoencephalitis in African children with acquired immunodeficiency syndrome. Pediatr. Infect. Dis. J. 21(1), (2002). 41 Thomas CJ, Lee JY, Conn LA et al. Surveillance of cryptococcosis in Alabama, Ann. Epidemiol. 8(4), (1998). 42 French N, Gray K, Watera C et al. Cryptococcal infection in a cohort of HIV- 1-infected Ugandan adults. AIDS 16(7), (2002). 43 Steenbergen JN, Shuman HA, Casadevall A. Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages. Proc. Natl Acad. Sci. USA 98(26), (2001). Interesting nonmammalian model. Microscopy, fungal and ameae killing assays, and phagocytosis assays revealed that C. neoformans is phagocytosed by and replicates in Acanthamoeba castellanii, which leads to death of amoebae. 44 Levitz SM. Does amoeboid reasoning explain the evolution and maintenance of virulence factors in Cryptococcus neoformans? Proc. Natl Acad. Sci. USA 98(26), (2001). 45 Rincon M. MAP-kinase signaling pathways in T-cells. Curr. Opin. Immunol. 13(3), (2001). Websites reagents/products/descriptions/ Celegans.shtml Nov22_2002/microbiology.html Affiliations Eleftherios Mylonakis, MD, Instructor in Medicine Harvard Medical School, Tel.: emylonakis@partners.org Frederick M Ausubel, PhD, Professor of Genetics Harvard Medical School Wellman 10, Department of Molecular Biology, Robin Jian Tang, Student, Stephen B Calderwood, MD Professor of Medicine (Microbiology and Molecular Genetics), Harvard Medical School, Chief, 55 Fruit Street, Gray 5, GRJ-504, 95