Cellular Microbiology (2003)

5

(7) 435ndash444

copy 2003 Blackwell Publishing Ltd

Blackwell Science LtdOxford UKCMICellular Microbiology 1462-5822Blackwell Publishing Ltd 20035

7435444

Review Article

Celegans hostndashpathogen modelR A Alegado et al

Received 9 December 2002 revised 27 February 2003 accepted27 February 2003 For correspondence E-mail mwtanstanfordedu Tel (

+

1) 650 736 1688 Fax (

+

1) 650 725 1534

Macroreview

Characterization of mediators of microbial virulence and innate immunity using the

Caenorhabditis elegans

hostndashpathogen model

Rosanna A Alegado

1

Marianne C Campbell

1

Will C Chen

2

Sandra S Slutz

2

and Man-Wah Tan

12

1

Department of Microbiology and Immunology Stanford University School of Medicine Stanford CA 94305 USA

2

Department of Genetics Stanford University School of Medicine Stanford CA 94305 USA

Summary

The soil-borne nematode

Caenorhabditis elegans

isemerging as a versatile model in which to study hostndashpathogen interactions The worm model has shown tobe particularly effective in elucidating both microbialand animal genes involved in toxin-mediated killingIn addition recent work on worm infection by a varietyof bacterial pathogens has shown that a number ofvirulence regulatory genes mediate worm susceptibil-ity Many of these regulatory genes including thePhoPQ two-component regulators in

Salmonella

andLasR in

Pseudomonas aeruginosa

have also beenimplicated in mammalian models suggesting thatfindings in the worm model will be relevant to othersystems In keeping with this concept experimentsaimed at identifying host innate immunity genes havealso implicated pathways that have been suggestedto play a role in plants and animals such as the p38MAP kinase pathway Despite rapid forward progressusing this model much work remains to be doneincluding the design of more sensitive methods tofind effector molecules and further characterization ofthe exact interaction between invading pathogens and

C elegansrsquo

cellular components

Introduction

Hostndashpathogen interactions are a battle between twoforces On one side lies the pathogen with a stockpile of

virulence factors and in opposition is the host with defencepathways and effector molecules Determining theresources each side possesses and when they are usedis crucial to understanding pathogenesis Several animalmodels have been used over the years to elucidate themolecular mechanisms underlying hostndashpathogen inter-actions Among these mice and

Drosophila

have beenparticularly useful and rich in discoveries (reviewed inLengeling

et al

2001 Tzou

et al

2002) Recently thesoil nematode

Caenorhabditis elegans

has been used asanother host model complementary to those already inexistence Several reviews discussing the use of

C ele-gans

to address questions of hostndashpathogen interactionshave recently been published (Kurz and Ewbank 2000Mahajan-Miklos

et al

2000 Tan and Ausubel 2000Aballay and Ausubel 2002 Ewbank 2002 Tan 2002aTan and Ausubel 2002) In this review we briefly discussthe utility of

C elegans

as a hostndashpathogen model andtechniques being used to analyse

C elegans

ndashpathogeninteractions Finally we summarize published data eluci-dating bacterial virulence factors and the

C elegans

innate immune system

C elegans

as a hostndashpathogen model

In nature

C elegans

dwells in the soil and feeds onbacteria Constant contact with soil-borne microbes sug-gests that these metazoans must have evolved protectiveresponses against pathogens This evolutionary relation-ship between

C elegans

and microorganisms makes theworm an attractive hostndashpathogen model Physicaldefences such as the wormrsquos cuticle and grinder protectagainst microbial entry However these physical barriersare imperfect A small fraction of bacteria can passthrough the grinder intact and enter the intestine Once inthe gut some pathogenic bacteria are capable of prolifer-ating and killing

C elegans

by an infectious processOther bacteria have been demonstrated to kill

C elegans

through the use of toxins (Kurz and Ewbank 2000Mahajan-Miklos

et al

2000 Tan and Ausubel 2000Aballay and Ausubel 2002 Ewbank 2002 Tan 2002b

436

R A Alegado

et al

copy 2003 Blackwell Publishing Ltd

Cellular Microbiology

5

435ndash444

Tan and Ausubel 2002) Recently microarray experi-ments profiling

C elegans

genes that are transcriptionallyregulated in the presence of a bacterial pathogen identi-fied several candidate genes that may play a role in innateimmune response (Mallo

et al

2002) Moreover

C ele-gans

mutants that are more susceptible or resistant toeither bacterial toxin- or infection-mediated death havebeen characterized (see below) Together these suggestthat

C elegans

like other animal hosts has defences thatcan be genetically dissected

Since Sydney Brennerrsquos seminal paper introducing

Celegans

in 1974 the worm has been used extensively tostudy a variety of biological questions (Brenner 1974)

Caenorhabditis elegansrsquo

usefulness as a model organismis a result of its genetic tractability rapid generation timeease of propagation a well-defined cell lineage map anda fully sequenced genome that contains a large numberof vertebrate orthologues A network of publicly available

C elegans

resources also increases research efficiencyThe

Caenorhabditis

Genetics Center (httpbiosciumneduCGCCGChomepagehtm) cataloguesmaintains and distributes existing

C elegans

strains freeof charge to non-commercial laboratories In addition the

C elegans

Gene Knockout Consortium (httpelegansbcgscbccaknockoutshtml) is in the process ofgenerating publicly available deletion mutations for allknown

C elegans

genes For genes where a geneticlesion is not currently available knockdown of gene func-tion by double-stranded RNA-mediated interference(RNAi) can be performed It is now possible to feed

E coli

expressing double-stranded RNA corresponding to a par-ticular

C elegans

gene to wild-type

C elegans

todecrease activity by RNAi of that gene (Kamath

et al

2001 Timmons

et al

2001) Thus to determine the pos-sible role of a particular gene in innate immunity animalsin which that gene has been subjected to RNAi can betested for susceptibility to pathogens This innovationinstantaneously links phenotype to molecular identitybecause the DNA sequence of the

C elegans

gene ineach

E coli

strain is recorded in Wormbase (httpwwwwormbase org) a central data repository for infor-mation about

C elegans

and related nematodes (Harris

et al

2003) RNAi vectors for nearly 90 of predictedgenes on chromosome I are currently available throughthe UK Human Genome Mapping Project Resource Cen-ter (httpwwwhgmpmrcacuk) and vectors for the restof the genome should be publicly available in 2003 (JAhringer pers comm) Thus the powerful genetics of

Celegans

coupled with bioinformatics and functionalgenomics tools can be brought to bear in the study ofhostndashpathogen interactions

There are limitations to

C elegansrsquo

functionality as ahostndashpathogen model Because

C elegans

has onlyinnate immune defences questions targeted at under-

standing the acquired immune system will need to beaddressed in a vertebrate model

Caenorhabditis elegans

also lacks a mobile scavenging phagocytic cell type likethe mammalian macrophage or

Drosophila

haemocyte Inaddition not all vertebrate innate immunity genes havenematode orthologues For example the

C elegans

genome appears to lack RelNF-

k

B family members (Tanand Ausubel 2000 Pujol

et al

2001) This family of pro-teins has been shown to play a critical role in

Drosophila

and vertebrate immunity (reviewed in Caamano andHunter 2002) These limitations underscore the necessityof a variety of complementary host-pathogen model sys-tems in order to thoroughly understand the full complexityof virulencendashdefence interactions

Methods for analysing

C elegans

ndashpathogen interactions

In order to address questions of hostndashpathogen interac-tion a variety of techniques have been created andormodified Below we briefly discuss these techniques andthe questions they help answer Further technical detailsare provided in the primary literature and in reviews byTan (2002a) and Tan and Ausubel (2002)

Evaluation of pathogenicity to

C elegans

Virulence of a pathogen is determined by both genetic andenvironmental factors One common method of determin-ing whether a microorganism is pathogenic to

C elegans

is by performing host mortality assays in which virulenceof a pathogen is measured by the time required for themicroorganism to kill a predetermined proportion of itshost (Mahajan-Miklos

et al

1999 Tan

et al

1999aGarsin

et al

2001 Couillault and Ewbank 2002 Gan

et al

2002) Typically the time to death for 50 of thehost (TD50) is used In brief individual strains of microbesare spread on a defined media in Petri plates and

Celegans

are added at a specified developmental stageand monitored for time to death (Tan and Ausubel 2002)A pathogen harbouring a mutation that renders it lessvirulent would result in a longer TD50 for the host or alarger proportion of surviving animals at a specified periodpostinfection relative to wild-type animals (Tan andAusubel 2002)

To determine whether the difference in observed mor-tality rate is mediated by diffusible toxins or by infectionspecialized host mortality assays can be performed Inthese assays cultures of bacteria are placed on nitrocel-lulose filters overlying agar plates incubated andremoved before seeding with nematodes (Mahajan-Miklos

et al

1999) Infectious versus toxin-mediated mecha-nisms of nematode death can also be distinguished byheat-killing antibiotic treatment or ultravioletgamma irra-diation of the bacteria (Tan

et al

1999b Garsin

et al

C elegans

hostndashpathogen model

437

copy 2003 Blackwell Publishing Ltd

Cellular Microbiology

5

435ndash444

2001 OrsquoQuinn

et al

2001 Gan

et al

2002) In all casesdescribed above rapid death of the worms on plateslacking dividing bacteria is indicative of a toxin componentto the pathogenic interaction

If death is mediated by infection virulence of the patho-gen can be measured by calculating the number of colonyforming units (CFUs) recovered from the host In additiona competitive index can be ascertained when worms arefed mixtures of bacteria and CFUs are determined(Aballay

et al

2000 Garsin

et al

2001) In parallel bac-terial proliferation and persistence can be confirmed visu-ally by monitoring the presence of GFP-labelled bacteriain the worm over time

In addition to genetic factors the composition of themedia on which the pathogen is grown (the environment)

has been shown for five pathogens to influence the hostmortality rate For example

Escherichia coli

OP50 whichis not pathogenic to

C elegans

when grown on nematodegrowth media (NGM) is almost as pathogenic as

Entero-coccus faecalis

when it is grown on brain heart infusion(BHI) agar (Garsin

et al

2001 see Table 1 for otherexamples)

Identifying and confirming the roles of defence-related genes

Genetic screens have been used to identify

C elegans

mutants that are either more susceptible or resistant topathogens (Marroquin

et al

2000 Kim

et al

2002) Inaddition functional genomics approaches such as high

Table 1

Effect of media on

C elegans

exposed to pathogens

Species Media Phenotype Source

Gram-positive

Bacillus subtilis

NGM non-pathogenic 9

Bacillus thuringiensis

NGM toxic 10

Enterococcus faecium

BHI non-pathogenic 9

Enterococcus faecalis

BHI infectious 9

Staphylococcus aureus

BHI infectious 9

Streptococcus pneumoniae

BHI infectious 9

Streptococcus pyogenes

a

BHI non-pathogenic 9

Streptococcus pyogenes

a

Todd-Hewitt toxic 11

Agrobacterium tumefaciens

NGM infectious 6Gram-negative

Escherichia coli OP50

b

NGM non-pathogenic 2

Escherichia coli OP50

b

BHI infectious 9

Burkholderia cepacia

MYOB toxic 15 16

Burkholderia cocovenenans

MYOB non-pathogenic 15

Burkholderia mallei

c

NGM PGS non-pathogenic 16

Burkholderia mallei

c

MYOB toxic 15

Burkholderia multivorans

d

MYOB toxic 15

Burkholderia multivorans

d

NGM PGS non-pathogenic 16

Burkholderia pseudomallei

MYOB toxic 7 15

Burkholderia thailandensis

MYOB toxic 7 15

Burkholderia vietnamiensis

MYOB toxic 15 16

Pseudomonas aeruginosa

PA14

e

SKA infectious 14 16

Pseudomonas aeruginosa

PA14e PGS toxic 14 16Pseudomonas aeruginosa PA01 BHI toxic 3 8Pseudomonas fluorescens BHI PGS infectious 4 16Pseudomonas syringae SKA PGS non-pathogenic 15 16Salmonella enterica ser Paratyphi NGM non-pathogenic 1Salmonella enterica ser Typhi NGM non-pathogenic 1Salmonella enterica ser Dublin NGM infectious 1Salmonella enterica ser Enteritidis NGM infectious 1Salmonella enterica ser Typhimurium NGM infectious 1 13Yersinia pseudotuberculosis NGM biofilm 5Serratia marscescens NGM infectious 12Shewanella massalia NGM infectious 3Shewanella frigidimarina NGM infectious 3Aeromonas hydrophila NGM infectious 6Erwinia carotova NGM infectious 6Erwinia chyrsanthemi NGM infectious 6

a S pyogenes is virulent when grown in Todd-Hewitt media but not BHIb E coli OP50 is virulent when grown on BHI but not NGMc B mallei is virulent when grown on MYOB but not NGM or PGSd B multivorans is virulent when grown on MYOB but not NGM or PGSe P aeruginosa PA14 kills by infection when grown on SKA but by toxin when grown on PGSNGM = nematode growth medium BHI = brain heart infusion SKA = slow killing agar MYOB = Modified Youngrenrsquos Only Bactopeptone agar(1) Aballay et al (2000) (2) Brenner (1974) (3) Couillault and Ewbank (2002) (4) Darby et al (1999) (5) Darby et al (2002) (6) Ewbank (2002)(7) Gan et al (2002) (8) Gallagher and Manoil (2001) (9) Garsin et al (2001) (10) Griffitts et al (2001) (11) Jansen et al (2002) (12) Kurz andEwbank (2000) (13) Labrousse et al (2000) (14) Mahajan-Miklos et al (1999) (15) OrsquoQuinn et al (2001) (16) Tan and Ausubel (2000)

438 R A Alegado et al

copy 2003 Blackwell Publishing Ltd Cellular Microbiology 5 435ndash444

density cDNA microarrays have been used to identify Celegans genes that are differentially regulated in responseto pathogen (Mallo et al 2002) To determine the functionof these genes in C elegans innate immunity C elegansstrains carrying lesions in these genes can be assayed fortheir susceptibility to pathogen using the techniquesdescribed above In cases where a genetic lesion is notavailable transient knockdown of that gene can beachieved by using RNAi followed by pathogen exposure

Pathogenesis against C elegans is measured mostoften by death an absolute endpoint Thus for wormmutants that show increased susceptibility to pathogensit is essential to distinguish between immunity-defectivemutants and weak worms Lifespan assays have beenused to determine the fitness of mutant worms Suchassays have traditionally been conducted on live non-pathogenic bacteria or on non-dividing bacteria HoweverGarigan et al (2002) showed that E coli OP50 accumu-lates in the pharynx and gut of aged worms suggestingthat bacterial proliferation is linked to a shorter lifespanIndeed worms fed on dead bacteria have a 30ndash40extension of lifespan Given these findings it is possiblethat an immunocompromised worm may have a shorterlifespan even on live bacteria classified as lsquonon-patho-genicrsquo To circumvent this issue lifespan assays can beconducted on heat-killed or antibiotic-killed bacteriallawns In addition to having a normal life-span other sec-ondary defects in processes such as developmental ratefeeding egg-laying and defecation should be ruled outbefore a strain that shows enhanced susceptibility topathogen can be considered a strong candidate for car-rying innate immunity defects (Tan 2002a) Once a geneis cloned the possibility that enhanced susceptibility topathogen is a consequence of developmental defects thatis not directly relevant to defence response could betested by generating transgenic animals carrying extrach-romosomal arrays containing a rescuing fragment corre-sponding to the gene of interest under the control of aninducible promoter The ability to rescue the susceptibilityphenotype by the rescuing fragment only when the geneis induced in adult animals (when development has beencompleted) prior to exposure to pathogen would indicatethat the defect is not due to non-specific developmentalabnormalities

Microbial Interactions with C elegans

Within a defined environmental condition the ability of anorganism to cause disease is a balance between theinherent virulence determinants of the pathogen and theeffectiveness of a hostrsquos defence mechanisms In the fol-lowing two sections we discuss the virulence determi-nants that have been identified for a variety of pathogens

using C elegans as a host and the known componentsof the C elegans innate immune system

Virulence determinants of C elegans pathogens

Bacterial pathogenesis in C elegans is manifested in twoprocesses toxin-mediated and infection-mediated Micro-bial genes found to be involved in virulence are noted inTable 2

Toxin-mediated

Pseudomonas aeruginosa PA14 kills C elegans by twomechanistically distinct processes that utilize different vir-ulence determinants fast- and slow-killing (Mahajan-Mikloset al 1999 Tan et al 1999a b and Table 2) Fast-killingis mediated by diffusible toxins whereas slow-killing is aconsequence of infection (discussed below) To date genesinvolved in both phenazine biosynthesis and the Mex mul-tidrug transport system have been shown to be requiredfor fast-killing (Sanchez et al 2002 Mahajan-Miklos et al1999) These results suggest that phenazine is one of thetoxins facilitating fast-killing and that it is likely to be trans-ported out of the bacterial cell via the Mex efflux pump

Unlike strain PA14 P aeruginosa PA01 grown on brainheart infusion (BHI) agar causes a rapid onset of irrevers-ible paralysis Cyanide production appears to be the pri-mary toxic component causing paralysis and is regulatedby the quorum-sensing activators LasR and RhlR and atwo-component regulator gene gacS (Darby et al 1999Gallagher and Manoil 2001) Surprisingly although PA01and PA14 grown on BHI produce comparable amounts ofcyanide nematodes exposed to PA14 on BHI do notbecome paralysed (M Campbell and M-W Tan unpublobs) More experiments will be needed to resolve thisapparent discrepancy

Several strains of Streptococcus pyogenes are capableof killing C elegans Although S pyogenes-mediated kill-ing requires live pathogen direct contact between thepathogen and its host is not necessary suggesting thatdeath is caused by diffusible toxin(s) However the twomajor exotoxins that are involved in mammalian pathogen-esis SPE-B and SLO do not appear to be involvedInstead killing of C elegans by S pyogenes is mediatedby hydrogen peroxide and this effect can be abrogated bythe addition of catalase to the media (Jansen et al 2002)The role that hydrogen peroxide plays in S pyogenespathogenesis of mammalian cells in vivo remains to beascertained Together these findings raise the question ofthe utility of C elegans to model S pyogenes pathogen-esis in mammals

To identify the host targets of bacterial toxins severalgenetic approaches can be used If the toxin is known andcan be purified it can be used to determine host targets

C elegans hostndashpathogen model 439

copy 2003 Blackwell Publishing Ltd Cellular Microbiology 5 435ndash444

in the absence of the pathogen For example to identifyhost targets of the Bacillus thuringiensis toxin Cry5B ascreen for C elegans mutants that are Bacillus-toxin resis-tant (bre) was conducted The screen was successful inidentifying mutants resistant to Cry5B (Marroquin et al2000) Of these bre-5 has been shown to encode a puta-tive carbohydrate modifying enzyme (Griffitts et al 2001)For a toxin that cannot be purified C elegans mutantsthat show altered susceptibility can be isolated For exam-ple a genetic screen for C elegans mutants that areresistant to P aeruginosa-PA01-induced paralysis identi-fied two alleles of the egl-9 gene (Darby et al 1999)

Since egl-9 mutants are also resistant to cyanide-medi-ated killing in the absence of bacteria EGL-9 whichencodes a dioxygenase is likely to participate in the phys-iological response to cyanide (Epstein et al 2001 Gal-lagher and Manoil 2001) Constitutive activation of thehypoxic response in the absence of EGL-9 is theorized toprovide resistance to cytochrome oxidase inhibition bycyanide Alternatively reactive oxygen species producedin response to cyanide might activate an EGL-9-depen-dent stress pathway (Gallagher and Manoil 2001) If mul-tiple toxins are involved the cellular target(s) of each toxincan be elucidated by generating transgenic C elegans

Table 2 Bacterial genes important in C elegans-pathogenicity model

Pathogen Gene product function Gene identity Source

Infection Enterococcus faecalis gelatinase gelE 10

lytic factor for eukaryotic and prokaryotic cells Cyl 5quorum-sensing regulator fsrB 5serine protease sprE 10

Pseudomonas aeruginosa PA14 integral membrane protein aefA 11quorum-sensing regulator lasR 11transcriptional regulator of multidrug transporter mtrR 11transcriptional regulator of RpoN-dependent operons ptsP 11two-component regulator gacA 11two-component regulator lemA 11

Salmonella entericaTyphimurium

iron uptake acid tolerance fur-1 7Virulence regulation acid tolerance ompR 7O-antigen ligase rfaL 2Phosphopeptose isomerase gmhA 2regulator of genes activated in macrophages phoPQ 1

Toxin Pseudomonas aeruginosa ML508 multidrug transporter nalB 9transcriptional regulator of multidrug transporter nfxB 9

Pseudomonas aeruginosa PA01 2-keto-3-deoxy-6-phosphogluconate aldolase eda 4alginate biosynthesis algC 4fatty acid and phospholipid metabolism prpB 4fatty acid and phospholipid metabolism prpC 4fatty acid and phospholipid metabolism gpdA 4fatty acid degradation PA0745 4filamentous haemagglutinin (Bordatella FhaB) PA0041 4glucose-6-phosphate dehydrogenase zwf 4hydrogen cyanide synthase hcnC 4permease of ABC zinc transporter znuB 4proline biosynthesis purM 4proline biosynthesis proC 4purine biosynthesis purL 4putative amino acid permease fused to a sensor

histidine kinasePA4725 4

putative transcriptional regulator LasR signature PA1003 4quinolone signal synthesis PA2587 4quorum-sensing regulator lasR 4quorum-sensing regulator rhlR 4sarcosine oxidase soxA 4two-component regulator PA3946 4two-component regulator gacS 4type 4 pili pilW 4

Pseudomonas aeruginosa PA14 histidine kinase motif 1G2 unknown 8multidrug transporter mexA 8phenazine biosynthesis phzB 8plant bacterial virulence factor hrpM 8

Toxin and Infection Pseudomonas aeruginosa PA14 alternative sigma factor s54 rpoN 6periplasmic serine protease mucD 12

Other Yersinia pseudotuberculosis regulator of biofilm production hmsT 3

(1) Aballay et al (2000) (2) Aballay et al (2003) (3) Darby et al (2002) (4) Gallagher and Manoil (2001) (5) Garsin et al (2001) (6) Hendricksonet al (2001) (7) Labrousse et al (2000) (8) Mahajan-Miklos et al (1999) (9) Sanchez et al (2002) (10) Sifri et al (2002) (11) Tan et al (1999a)(12) Yorgey et al (1999)

440 R A Alegado et al

copy 2003 Blackwell Publishing Ltd Cellular Microbiology 5 435ndash444

strains bearing a pathogen-derived toxin gene under aninducible promoter and screening for mutations that conferresistance to the effect of the toxin

Caenorhabditis elegans has also been used as a modelto confirm the conservation of toxin targets between mam-malian and C elegans cells For example Darby andFalkow (2001) showed that the pertussis toxin (PTX) inac-tivates heterotrimeric G proteins in both mammals and Celegans Using transgenic nematodes that express PTXunder the control of a C elegans heat shock promoterthey showed that heat-shocked animals phenocopy the Celegans strain that carries a loss of function mutation in aheterotrimeric G gene

Infectious mechanisms

An infection process in C elegans typically involves directcontact between host cells and live bacteria and anincrease in bacterial load Under the slow-killing condi-tion P aeruginosa PA14 accumulates and increases itsload within the intestinal lumen of C elegans ultimatelyleading to death of the infected animals (Tan et al1999a) The gacA-gacS and lasR mutants of PA14 thatare less pathogenic in mammalian models fail to accu-mulate in the gut of C elegans and are significantly atten-uated in pathogenicity suggesting that a regulatorycascade involving the GacS-GacA two-component andquorum sensing systems is essential for nematode andmammalian pathogenesis Although many other virulencedeterminants have been shown to be essential for fullvirulence (Table 2) the exact role that each of these geneproducts plays in nematode pathogenesis remains to beelucidated

Several serovars of Salmonella enterica have beenshown to have pathogenic effects on the health of Celegans (Aballay et al 2000 Labrousse et al 2000) Senterica serovar Typhimurium causes a persistent infec-tion it accumulates in and causes distention of the gutlumen of C elegans In addition infections by Salmonellaalso result in apoptosis of germline cells (Aballay andAusubel 2001) The PhoPQ two-component regulatorswhich are required for Salmonella survival in macroph-ages are also central to C elegans killing Salmonellapathogenicity island-2 (SPI-2) whose genes are regu-lated by PhoPQ is critical for long-term survival andsystemic spread in other animal models (Miller et al1989) However SPI-2 effectors do not appear to be nec-essary for killing of C elegans by Salmonella as a mutantin the SPI-2 type III secretion apparatus (ssaV) is notattenuated (Labrousse et al 2000) The identity of PhoPQ-regulated genes that act during C elegans pathogen-esis remains to be determined It is also not knownwhether Salmonella pathogenicity island 1 (SPI-1) whichis required during adherence and invasion by the bacteria

of mammalian intestinal epithelial cells is required forcolonization of C elegans

Caenorhabditis elegans can also be killed by severalGram-positive human pathogens such as E faecalis Sta-phylococcus aureus and S pneumoniae (Garsin et al2001) In contrast Bacillus subtilis S pyogenes and Efaecium do not cause worm mortality Yet both E faecalisand E faecium can effectively colonize the C elegansintestine This result implies that bacterial colonizationmay not always be associated with lethality Among thevirulence factors required for E faecalis pathogenesis ofC elegans is Cyl a protein known to lyse eukaryotic cellsand the products of the fsr loci (Garsin et al 2001 Sifriet al 2002 and Table 2)

The majority of the virulence factors identified using Celegans as the animal host are also required for mamma-lian pathogenesis However most of these factors areregulators of virulence (Table 2) This may be due to thefact that the screens used to identify these factors werebased on identifying bacterial mutants that have an atten-uated ability to cause death Screens designed to identifybacterial mutants defective in specific interactions with thehost such as colonization of the worm intestine shouldlead to the determination of a wider variety of virulencefactors An advantage to using C elegans is that largenumbers of hosts can be used which would result ingreater statistical power to resolve even small differencesin pathogen virulence Although this approach has beenused to confirm the virulence of bacterial mutants isolatedfrom screens this power of resolution has not beenapplied to a random mutant screen Thus to criticallyevaluate the effectiveness of C elegans in discoveringnovel virulence effector genes more sensitive and specificscreens will have to be devised and tested If these factorsare also necessary for mammalian pathogenesis it ispossible that they exert their effect by targeting conservedcomponents within these hosts Conversely virulence fac-tors that are C elegans-specific are likely to target hostfactors that are unique to the species analyses of whichwould shed light into the question of species specificity inhostndashpathogen interactions

The C elegans innate immune system

Recent investigations in the fledgling field of C elegansinnate immunity have provided evidence that C eleganscan protect itself from pathogens in many ways includingthe use of physical barriers and the expression of signal-ling and effector molecules

Physical mechanisms

The cuticle is C elegansrsquo first defence against any patho-gen it encounters Access beyond the cuticle can be

C elegans hostndashpathogen model 441

copy 2003 Blackwell Publishing Ltd Cellular Microbiology 5 435ndash444

gained through the mouth anus vulva or sensory open-ings Microbes entering through the mouth immediatelyencounter the grinder made up of three pairs of simulta-neously contracting muscle cells which break open bac-teria as they pass back to the intestine (Riddle 1997) Thegrinderrsquos importance is highlighted by the fact that grinderdefective mutants have been found to be more susceptibleto P aeruginosa (Kim et al 2002 Smith et al 2002 Tan2002a)

Bacteria that manage to pass through the grinder intactface expulsion from the intestine by means of defecationFor example visualization of nematodes exposed to Ecoli DH5-a marked with GFP showed that worms do notaccumulate the bacteria in their lumen but instead expelthe bacteria though the anus (Aballay et al 2000)

Signalling pathways

Recent studies have identified several signalling pathwaysand candidate molecules that are involved in C elegansinnate immunity (Kim et al 2002 Mallo et al 2002Aballay et al 2003)

(a) The p38 MAP kinase pathway In order to geneti-cally dissect the C elegans innate immune system Kimand colleagues took advantage of C elegansrsquo hermaph-roditic nature and its ability to self-fertilize to isolate wormstrains that had enhanced susceptibility to P aeruginosaslow killing They selected mutagenized worms that diedearlier than wild type worms and recovered each strain byallowing the clonal progeny to hatch from the mutantrsquoscorpse This screen identified two p38 mitogen-activatedprotein kinase (MAPK) pathway genes nsy-1 and sek-1(Kim et al 2002) NSY-1 and SEK-1 encode a MAPKkinase kinase and a MAPK kinase respectively and anin vitro assay showed that they interact with one another(Sagasti et al 2001Tanaka-Hino et al 2002) Inactiva-tion of pmk-1 a p38 MAPK by RNAi also resulted innematodes showing increased susceptibility to pathogensuggesting that NSY-1 SEK-1 and PMK-1 form a modulethat transduces a defence signal in C elegans (Kim et al2002) The function of this pathway in immunity appearsto be conserved across phylogeny mammalian p38MAPK signalling is also important in the cellular stressand immune responses (reviewed in Kyriakis and Avruch2001) In concert with recent evidence for the role ofMAPK signalling in plant defence (Asai et al 2002) thiswork suggests that the MAPK signalling module is a cen-tral component of the defence of multicellular organismsagainst pathogen attack However except for its possiblerole in regulating Salmonella-induced programmed celldeath (PCD see below) the immune responses that liedownstream of p38 MAPK in C elegans remain to becharacterized Similarly the upstream signal(s) areunknown In C elegans the nsy-1 and sek-1 genes are

also involved in AWC neuronal symmetry and they aredownstream of the unc-43 gene product (Sagasti et al2001 Tanaka-Hino et al 2002) However the unc-43mutant does not have increased susceptibility to pathogen(Kim et al 2002) This suggests that the input of defencesignal to NSY-1 and SEK-1 is mediated by a yet to beidentified molecule

(b) The programmed cell death (PCD) pathway Expo-sure of C elegans to S enterica serovar Typhimuriumleads to an increased level of apoptosis of germ cells inthe gonad that is dependent on the cell-death (CED)machinery (Aballay and Ausubel 2001) The loss-of-func-tion ced-3 ced-4 and egl-1 and gain-of-function ced-9mutants do not undergo developmentally regulated PCDNor do they have increased germ cell apoptosis uponexposure to Salmonella However these mutants arehypersensitive to Typhimurium-mediated killing leadingAballay and Ausubel to hypothesize that PCD may func-tion to eliminate excess germ cells that could be detrimen-tal to the worm and that this response may also beinvolved in C elegansrsquo defence against environmentalinsults including infections In a recent work Aballay et al(2003) showed that the nsy-1 and sek-1 mutants as wellas worms subjected to pmk-1 (p38) RNAi are deficient inSalmonella-induced PCD Epistasis analysis revealed thatthe CED PCD pathway lies downstream of PMK-1 andthat elicitation of this p38CED PCD (as well as persis-tence of the Salmonella infection in the C elegans intes-tine) requires intact Salmonella lipopolysaccharide (LPS)Together these data establish that pathogen-elicited PCDin C elegans lies downstream of LPS signalling and p38MAP kinase

The germ cell PCD response appears to be specific toSalmonella infection since germ cells of a nematodeinfected by P aeruginosa do not have an increased levelof apoptosis and ced mutants are not hypersusceptible toP aeruginosa (Aballay and Ausubel 2001) Two interest-ing questions arise from this observation First what arethe downstream components of the PMK-1 signalling cas-cade that mediate C elegans defence against P aerugi-nosa Second since S enterica serovar Typhimurium hasbeen observed only within the intestinal lumen how is theapoptotic signal transmitted from the intestinal cells to thegerm line cells

(c) The TGF-b-like signalling pathway A transforminggrowth factor-b (TGF-b)-related pathway also appears toplay a role in C elegansrsquo defence against bacteria (Tan2001 Mallo et al 2002) In C elegans there are severalorphaned ligands and at least two well characterized TGF-b-like signalling pathways the dauer larva formation (Daf)and small (Sma) pathways (Patterson and Padgett 2000)Only the Sma pathway is required to defend wormsagainst P aeruginosa infection Worms with a geneticlesion in the ligand dbl-1 the Type I receptor sma-6 or

442 R A Alegado et al

copy 2003 Blackwell Publishing Ltd Cellular Microbiology 5 435ndash444

any of the SMADs (sma-2 3 or 4) are more susceptibleto P aeruginosa-mediated slow-killing In contrast sus-ceptibility of daf-7 and daf-5 animals is indistinguishablefrom wild-type animals (Tan 2001) Mallo and colleaguesshowed that a dbl-1 mutant is also more susceptible toSerratia marcescens when compared to wild-type C ele-gans Additionally they showed that lys-8 and F46F23which were more highly expressed in C elegans exposedto S marcescens are likely to be regulated by the Smapathway (Mallo et al 2002 Mochii et al 1999)

(d) The Toll pathway The Toll pathway plays a centralrole in mediating inducible innate defence in Drosophilaand mammals (reviewed in Kimbrell and Beutler 2001)Although several of the Toll pathway orthologues arepresent in the C elegans genome these genes do notappear to play an essential role in mediating worm innateimmunity (Pujol et al 2001) Susceptibilities of wormswith a mutation in tol-1 pik-1 ikb-1 or trf-1 do not differsignificantly from the wild-type animals in response tovarious pathogens However the tol-1 mutant displays abehavioural defect in that it fails to avoid a pathogenic Smarcescens strain after extended contact Thus althoughthe Toll signalling in C elegans functions differently thanin Drosophila it may play a role in keeping C elegansaway from potentially harmful pathogens (Pujol et al2001)

An important question that needs to be addressed ishow the nematode host recognizes the invading patho-gen The C elegans genome contains over 100 C-typelectins proteins with C-type carbohydrate recognitiondomains (Drickamer and Dodd 1999) Two C-type lectinsY54G2 A6 and W04E128 were up-regulated upon infec-tion by S marcescens as shown by a cDNA microarray(Mallo et al 2002) Investigations into the role C-typelectins play in C elegans innate immunity may prove tobe fruitful More work will also be required to determinehow the p38 MAPK TGF-b and PCD pathways interactwhen the C elegans host is engaged in an antagonisticinteraction with its pathogens Finally it will be importantto identify effector molecules that are regulated by thesepathways

Effector molecules

In an inducible defence response the activation of a sig-nalling pathway typically leads to the production of effectormolecules that directly destroy or inhibit the growth of theinvading pathogen To date inducible effector moleculesthat play a direct role in limiting bacterial growth in vivohave yet to be identified in C elegans The C elegansgenome encodes a number of molecules that possessantimicrobial activities and they may play a role indefence Two defensin-like molecules homologous toASABF (Ascaris suum antibacterial factor) ABF-1 and

ABF-2 have been identified (Kato and Komatsu 1996Kato et al 2002) ABF-2 is expressed in the pharynx inlarvae and adults where it would be likely to encounterbacteria In vitro expression of ABF-2 confirmed that itpossesses antimicrobial activity against yeast and bacte-ria However whether ABF-2 plays an important role indefending C elegans from pathogen remains to be dem-onstrated

Lysozymes may also contribute to C elegans defenceThree lysozyme genes lys-1 lys-7 and lys-8 were upreg-ulated in response to S marcescens infection (Mochiiet al 1999 Mallo et al 2002) As worms feed on bacte-ria it can be argued that the upregulation of lysozymesmay simply be the wormrsquos digestive response to foodrather than an immune response It is also possible thatsome digestive enzymes may actually function as defencemolecules For example some lsquodigestiversquo enzymes foundin bacteria-feeding amoeba are homologous to moleculesthat function to kill bacteria in mammalian macrophagesbut in the latter case they are classified as defence mol-ecules Semantics aside it will be important to distinguishif lysozymes (or other hydrolytic enzymes) play a role inimmunity or digestion lys-1 overexpression in wormsincreased resistance to S marcescens strain Db1140 butRNAi of lys-1 had little effect on the survival of worms inthe presence of this pathogen (Mallo et al 2002) How-ever given that there are multiple lysozymes present inthe worm genome the loss of one protein may not besufficient to cause a significant effect on worm survivalTo circumvent this functional redundancy future experi-ments testing the role of lysozymes in defence will haveto utilize methods such as combining genetic lesions andRNAi to inactivate multiple genes simultaneously

Concluding remarks

Since 1999 when the first papers describing the use ofC elegans as a model to study P aeruginosa pathogen-esis were published many pathogens have been shownto be able to infect andor kill C elegans To date severalscreens to identify bacterial mutants that are attenuatedin C elegans pathogenicity in the infection or toxin modelshave led to the identification and characterization of manyvirulence determinants The toxin model has been moresuccessful in identifying the bacterial genes that aredirectly involved in pathogenesis In regards to the infec-tion models the majority of the factors identified havebeen regulators of virulence A future challenge will be todesign more sensitive and specific screens that will iden-tify virulence factors that directly interact with the hostwhich in turn should help elucidate the molecular mecha-nism of C elegans infection

Several pathways have been implicated in C elegansinnate immunity Although important questions such as

C elegans hostndashpathogen model 443

copy 2003 Blackwell Publishing Ltd Cellular Microbiology 5 435ndash444

how a pathogen is recognized which signalling pathwayis triggered upon pathogen recognition and what effectormolecules are induced remain to be addressed this workhas laid the foundation for future studies of innate immu-nity in C elegans In addition the use of C elegans hasgreat potential in advancing the field of microbial patho-genesis in that it allows the application and integration oftraditional genetics approaches with recent functionalgenomics innovations to identify and characterize compo-nents that are involved in hostndashpathogen interactionssome of which are likely to be conserved across phylog-eny Thus the use of C elegans as a model host has greatpotential for offering further insights into the conservedmechanisms of innate immunity

Acknowledgements

We are grateful to two anonymous reviewers for their constructivecriticisms on the manuscript Work in our laboratory is supportedby grants from the National Institutes of Health the Donald E andDelia B Baxter Foundation the V Foundation and March ofDimes to MWT We are also grateful for predoctoral fellowshipssupport from the Ford Foundation (RAA) the National ScienceFoundation (MCC) and the Howard Hughes Medical Institute(SSS and WCC)

References

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Aballay A and Ausubel FM (2002) Caenorhabditis ele-gans as a host for the study of hostndashpathogen interactionsCurr Opin Microbiol 5 97ndash101

Aballay A Yorgey P and Ausubel FM (2000) Salmonellatyphimurium proliferates and establishes a persistent infec-tion in the intestine of Caenorhabditis elegans Curr Biol10 1539ndash1542

Aballay A Drenkard E Hilbun LR and Ausubel FM(2003) Caenorhabditis elegans innate immune responsetriggered by Salmonella enterica requires intact LPS andis mediated by a MAPK signaling pathway Curr Biol 1347ndash52

Asai T Tena G Plotnikova J Willmann MR Chiu WLGomez-Gomez L et al (2002) MAP kinase signalling cas-cade in Arabidopsis innate immunity Nature 415 977ndash983

Brenner S (1974) The genetics of Caenorhabditis elegansGenetics 77 71ndash94

Caamano J and Hunter CA (2002) NF-kappaB family oftranscription factors central regulators of innate and adap-tive immune functions Clin Microbiol Rev 15 414ndash429

Couillault C and Ewbank JJ (2002) Diverse bacteria arepathogens of Caenorhabditis elegans Infect Immun 704705ndash4707

Darby C and Falkow S (2001) Mimicry of a G proteinmutation by pertussis toxin expression in transgenic Cae-norhabditis elegans Infect Immun 69 6271ndash6275

Darby C Cosma CL Thomas JH and Manoil C (1999)Lethal paralysis of Caenorhabditis elegans by Pseudomo-

nas aeruginosa Proc Natl Acad Sci USA 96 15202ndash15207

Darby C Hsu JW Ghori N and Falkow S (2002) Plaguebacteria biofilm blocks food intake Nature 417 243ndash244

Drickamer K and Dodd RB (1999) C-Type lectin-likedomains in Caenorhabditis elegans predictions from thecomplete genome sequence Glycobiology 9 1357ndash1369

Epstein AC Gleadle JM McNeill LA Hewitson KSOrsquoRourke J Mole DR et al (2001) C elegans EGL-9and mammalian homologs define a family of dioxygenasesthat regulate HIF by prolyl hydroxylation Cell 107 43ndash54

Ewbank JJ (2002) Tackling both sides of the host-pathogenequation with Caenorhabditis elegans Microbes Infect 4247ndash256

Gallagher LA and Manoil C (2001) Pseudomonas aerug-inosa PAO1 kills Caenorhabditis elegans by cyanide poi-soning J Bacteriol 183 6207ndash6214

Gan YH Chua KL Chua HH Liu BP Hii CSChong HL and Tan P (2002) Characterization ofBurkholderia pseudomallei infection and identification ofnovel virulence factors using a Caenorhabditis eleganshost system Mol Microbiol 44 1185ndash1197

Garigan D Hsu AL Fraser AG Kamath RS AhringerJ and Kenyon C (2002) Genetic Analysis of Tissue Agingin Caenorhabditis elegans A role for heat-shock factor andbacterial proliferation Genetics 161 1101ndash1112

Garsin DA Sifri CD Mylonakis E Qin X Singh KVMurray BE et al (2001) A simple model host for identify-ing Gram-positive virulence factors Proc Natl Acad SciUSA 98 10892ndash10897

Griffitts JS Whitacre JL Stevens DE and Aroian RV(2001) Bt toxin resistance from loss of a putative carbohy-drate- modifying enzyme Science 293 860ndash864

Harris TW Lee R Schwarz E Bradnam K Lawson DChen W et al (2003) WormBase a cross-species data-base for comparative genomics Nucleic Acids Res 31133ndash137

Hendrickson EL Plotnikova J Mahajan-Miklos SRahme LG and Ausubel FM (2001) Differential rolesof the Pseudomonas aeruginosa PA14 rpoN gene in patho-genicity in plants nematodes insects and mice J Bacte-riol 183 7126ndash7134

Jansen WTM Bolm M Balling R Chhatwal GS andSchnabel R (2002) Hydrogen peroxide-mediated killing ofCaenorhabditis elegans by Streptococcus pyogenes InfectImmun 70 5202ndash5207

Kamath RS Martinez-Campos M Zipperlen P FraserAG and Ahringer J (2001) Effectiveness of specificRNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans Genome Bio 2research0002

Kato Y and Komatsu S (1996) ASABF a novel cysteine-rich antibacterial peptide isolated from the nematodeAscaris suum Purification primary structure and molecu-lar cloning of cDNA J Biol Chem 271 30493ndash30498

Kato Y Aizawa T Hoshino H Kawano K Nitta K andZhang H (2002) abf-1 and abf-2 ASABF-type antimicro-bial peptide genes in Caenorhabditis elegans Biochem J361 221ndash230

Kim DH Feinbaum R Alloing G Emerson FE GarsinDA Inoue H et al (2002) A conserved p38 MAP kinasepathway in Caenorhabditis elegans innate immunity Sci-ence 297 623ndash626

444 R A Alegado et al

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Kimbrell DA and Beutler B (2001) The evolution andgenetics of innate immunity Nat Rev Genet 2 256ndash267

Kurz CL and Ewbank JJ (2000) Caenorhabditis elegansfor the study of hostndashpathogen interactions Trends Micro-biol 8 142ndash144

Kyriakis JM and Avruch J (2001) Mammalian mitogen-activated protein kinase signal transduction pathways acti-vated by stress and inflammation Physiol Rev 81 807ndash869

Labrousse A Chauvet S Couillault C Kurz CL andEwbank JJ (2000) Caenorhabditis elegans is a modelhost for Salmonella typhimurium Curr Biol 10 1543ndash1545

Lengeling A Pfeffer K and Balling R (2001) The battleof two genomes genetics of bacterial hostpathogen inter-actions in mice Mamm Genome 12 261ndash271

Mahajan-Miklos S Tan M-W Rahme LG and AusubelFM (1999) Molecular mechanisms of bacterial virulenceelucidated using a Pseudomonas aeruginosa Caenorhab-ditis elegans pathogenesis model Cell 96 47ndash56

Mahajan-Miklos S Rahme LG and Ausubel FM (2000)Elucidating the molecular mechanisms of bacterial viru-lence using non-mammalian hosts Mol Microbiol 37 981ndash988

Mallo GV Kurz CL Couillault C Pujol N GranjeaudS Kohara Y and Ewbank JJ (2002) Inducible antibac-terial defense system in C elegans Curr Biol 12 1209ndash1214

Marroquin LD Elyassnia D Griffitts JS Feitelson JSand Aroian RV (2000) Bacillus thuringiensis (Bt) toxinsusceptibility and isolation of resistance mutants in thenematode Caenorhabditis elegans Genetics 155 1693ndash1699

Miller SI Kukral AM and Mekalanos JJ (1989) A two-component regulatory system (phoP phoQ) controls Sal-monella typhimurium virulence Proc Natl Acad Sci USA86 5054ndash5058

Mochii M Yoshida S Morita K Kohara Y and Ueno N(1999) Identification of transforming growth factor-beta ndashregulated genes in Caenorhabditis elegans by differentialhybridization of arrayed cDNAs Proc Natl Acad Sci USA96 15020ndash15025

OrsquoQuinn AL Wiegand EM and Jeddeloh JA (2001)Burkholderia pseudomallei kills the nematode Caenorhab-ditis elegans using an endotoxin-mediated paralysis CellMicrobiol 3 381ndash393

Patterson GI and Padgett RW (2000) TGF beta-relatedpathways Roles in Caenorhabditis elegans developmentTrends Genet 16 27ndash33

Pujol N Link EM Liu LX Kurz CL Alloing G TanM-W et al (2001) A reverse genetic analysis of compo-nents of the Toll signaling pathway in Caenorhabditis ele-gans Curr Biol 11 809ndash821

Riddle DL (1997) C Elegans II Plainview New York ColdSpring Harbor Laboratory Press

Sagasti A Hisamoto N Hyodo J Tanaka-Hino M Mat-sumoto K and Bargmann CI (2001) The CaMKII UNC-43 activates the MAPKKK NSY-1 to execute a lateral sig-

naling decision required for asymmetric olfactory neuronfates Cell 105 221ndash232

Sanchez P Linares JF Ruiz-Diez B Campanario ENavas A Baquero F and Martinez JL (2002) Fitnessof in vitro selected Pseudomonas aeruginosa nalB andnfxB multidrug resistant mutants J Antimicrob Chemother50 657ndash664

Sifri CD Mylonakis E Singh KV Qin X Garsin DAMurray BE et al (2002) Virulence effect of Enterococcusfaecalis protease genes and the quorum-sensing locus fsrCaenorhabditis elegans and mice Infect Immun 70 5647ndash5650

Smith MP Laws TR Atkins TP Oyston PCF dePomerai DI and Titball RW (2002) A liquid-basedmethod for the assessment of bacterial pathogenicity usingthe nematode Caenorhabditis elegans FEMS MicrobiolLett 210 181ndash185

Tan M-W (2001) Genetic and genomic dissection ofhostndashpathogen interactions using a P aeruginosa-C ele-gans pathogenesis model Pediatric Pulmonol 32 (S22)96ndash97

Tan M-W (2002a) Identification of host and pathogen fac-tors involved in virulence using Caenorhabditis elegansMethods Enzymol 358 13ndash28

Tan M-W (2002b) Cross-species infections and their anal-ysis Annu Rev Microbiol 56 539ndash565

Tan M-W and Ausubel FM (2000) Caenorhabditis ele-gans a model genetic host to study Pseudomonas aerug-inosa pathogenesis Curr Opin Microbiol 3 29ndash34

Tan M-W and Ausubel FM (2002) Alternative models inmicrobial pathogens Method Microbiol 31 461ndash475

Tan M-W Rahme LG Sternberg JA Tompkins RGand Ausubel FM (1999a) Pseudomonas aeruginosa kill-ing of Caenorhabditis elegans used to identify P aerugi-nosa virulence factors Proc Natl Acad Sci USA 96 2408ndash2413

Tan M-W Mahajan-Miklos S and Ausubel FM (1999b)Killing of Caenorhabditis elegans by Pseudomonas aerug-inosa used to model mammalian bacterial pathogenesisProc Natl Acad Sci USA 96 715ndash720

Tanaka-Hino M Sagasti A Hisamoto N Kawasaki MNakano S Ninomiya-Tsuji J et al (2002) SEK-1 MAPKKmediates Ca2+ signaling to determine neuronal asymmetricdevelopment in Caenorhabditis elegans EMBO Report 356ndash62

Timmons L Court DL and Fire A (2001) Ingestion ofbacterially expressed dsRNAs can produce specific andpotent genetic interference in Caenorhabditis elegansGene 263 103ndash112

Tzou P De Gregorio E and Lemaitre B (2002) HowDrosophila combats microbial infection a model to studyinnate immunity and hostndashpathogen interactions Curr OpinMicrobiol 5 102ndash110

Yorgey P Rahme LG Tan M-W and Ausubel FM(2001) The roles of mucD and alginate in the virulence ofPseudomonas aeruginosa plants nematodes and miceMol Microbiol 41 1063ndash1076