1 London School of Hygiene and Tropical Medicine, Dept Infectious and Tropical Diseases, Keppel St, London WC1E 7HT, UK
2 DSTL, Porton Down, Salisbury SP4 0JQ, UK
Correspondence
B. W. Wren
Brendan.wren{at}lshtm.ac.uk
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ABSTRACT |
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INTRODUCTION |
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In the wild, C. elegans is a free-living, soil-dwelling nematode feeding on micro-organisms. In the laboratory it is maintained on a uracil auxotrophic strain (OP50) of Escherichia coli, which has limited growth on NGM plates (Lewis & Fleming, 1995). A number of bacteria infect C. elegans, with various infection phenotypes. These include a variety of plant and animal pathogens such as Erwinia chrysanthemi, Agrobacterium tumefaciens, Shewanella frigidimarina, Photorhabdus luminescens, Xenorhabdus nematophilus (Couillault & Ewbank, 2002
) and Microbacterium nematophilum (Hodgkin et al., 2000
). They also include bacteria pathogenic to humans, including the Gram-negative bacteria Pseudomonas aeruginosa, which kills the worm by one of three mechanisms fast, slow or neurotoxin-mediated (Tan et al., 1999
; Darby et al., 1999
), Burkholderia pseudomallei, which kills via a neuromuscular endotoxin (O'Quinn et al., 2001
), and Serratia marcescens, which also kills via a (different) toxin (Kurz & Ewbank, 2000
); and the Gram-positive bacteria Enterococcus faecalis, Streptococcus pneumoniae and Staphylococcus aureus (Garsin et al., 2001
).
We have investigated the effect of Yersinia spp. on C. elegans. The pathogenic yersiniae are an important cause of human disease, Yersinia pestis causing bubonic and pneumonic plague (Perry & Fetherston, 1997), and Yersinia enterocolitica and Yersinia pseudotuberculosis causing food-borne enteric disease. Y. enterocolitica causes gastrointestinal syndromes ranging from an acute enteritis to mesenteric lymphadenitis and Y. pseudotuberculosis causes mesenteric lymphadenitis and septicaemia (Naktin & Beavis, 1999
). During bubonic plague Y. pestis is transmitted via an insect (flea) vector; in contrast, the enteropathogenic yersiniae are transmitted by the faecal/oral route and survive in water and soil. The yersiniae are model organisms to study bacterial pathogenesis. There are well-developed systems for genetic manipulation (e.g. transposon mutagenesis) and animal models of infection (e.g. the murine yersiniosis model). The full genome sequences of strains belonging to Y. pestis biovars Orientalis and Medievalis have been completed (Parkhill et al., 2001
; Deng et al., 2002
), and the genome sequences of Y. pseudotuberculosis and Y. enterocolitica O8 are near completion (http://bbrp.llnl.gov/bbrp/html/microbe.html and http://www.sanger.ac.uk/Projects/Y_enterocolitica). Moreover, Y. pseudotuberculosis and Y. pestis share near-identical sequence similarity but cause remarkably different diseases and have different routes of transmission, whereas Y. enterocolitica shares only 70 % sequence similarity with Y. pseudotuberculosis and Y. pestis but exhibits a disease phenotype similar to that of Y. pseudotuberculosis.
All of the human-pathogenic yersiniae possess plasmid pYV and most of the pathogenicity studies on yersiniae have largely focused on determinants found on this plasmid, which encodes the type III secretion system essential for virulence. Y. pestis possesses two additional plasmids (pMT1 and pPCP1) which also encode key virulence determinants. In addition Y. pestis possesses a number of inactivated genes, which may no longer be required for its lifestyle (Parkhill et al., 2001). Achtman et al. (1999)
have suggested that Y. pestis is a recently emerged clone of Y. pseudotuberculosis, evolving between an estimated 1500 and 20 000 years ago. Studies on the pathogenic yersiniae may, therefore, shed light on the evolutionary relationships between these three species and the mechanisms by which they survive in the environment, occupy new niches and cause disease.
In a recent report Darby et al. (2002) observed that both Y. pestis and Y. pseudotuberculosis infected C. elegans, colonizing the head of the worm and blocking feeding. It was suggested that this was due to the formation of a biofilm on the head/mouth of the worm as a defence against predation by invertebrates. Biofilms are matrix-encased bacterial communities that are specialized for surface persistence. They are more resistant to antibiotics, chlorine and components of the host innate immune system (Drenkard & Ausubel, 2002
; Singh et al., 2002
; Yildiz & Schoolnik, 1999
). Additionally, biofilm formation has been shown to be important in bacterialfungal interactions, where virulence factors studied in the context of human infection may also have a role during interactions in the bacterialfungal ecological niche (Hogan & Kolter, 2002
; Mendoza De Gives et al., 1999
).
In this study we sought to characterize the C. elegans infection caused by the three human-pathogenic Yersinia species and to identify genetic features of both the pathogen and the host which contribute to the establishment of disease in C. elegans.
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METHODS |
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Microscopy.
For EM (Fig. 2A, B, C), C. elegans were taken from plates and bisected under fixative (3 % glutaraldehyde/0·075 M cacodylate buffer; pH 7·4). Specimens were post-fixed in 1 % osmium tetroxide, dehydrated through an alcohol series and embedded in TAAB resin (TAAB Laboratories). Specimens in Fig. 2(D, E)
were fixed in 2 % paraformaldehyde/1 % glutaraldehyde and cryosectioned. For light microscopy (Fig. 2F, G
), C. elegans were fixed in 4 % paraformaldehyde, stained with 0·1 % crystal violet for 3 min and viewed with epi-illumination on 5 % agarose pads (Sulston & Horvitz, 1977
).
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RESULTS |
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Three strains of Y. pestis (virulent biovar Orientalis strains GB and CO92, and the biovar Orientalis strain Java-9, which lacks plasmid pMT1) caused a severe infection. In contrast, the hms- Y. pestis F361-66 strain (which has lost the 102 kb hms/yersinabactin locus and is attenuated in the mouse) did not infect C. elegans (Table 1). All 15 of the Y. enterocolitica strains tested (see Methods) had no effect on C. elegans.
Bacterial virulence factors
Karlyshev et al. (2001) recently tested 603 signature-tagged Y. pseudotuberculosis YPIII pIB1 transposon mutants in the murine yersiniosis model and identified 39 attenuated mutants. Of the 39 strains which were attenuated in the mouse, six showed altered pathogenicity in C. elegans, with two showing complete attenuation and four showing a change from a severe to a weak infection (see Table 2
). Additionally, a defined phoP-negative Y. pseudotuberculosis YPIII pIB1 strain was tested; it caused a severe infection phenotype, suggesting that the PhoP/Q regulatory locus (Groisman, 2001
) is not important in biofilm formation (data not shown). Two hundred randomly selected Y. pseudotuberculosis YPIII pIB1 transposon mutants were also tested in C. elegans, and a single mutant was identified which showed complete attenuation. Sequencing the DNA flanking the transposon of this mutant (Karlyshev et al., 2000
) showed that the hmsF gene had been interrupted.
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DISCUSSION |
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Bacterial biofilms are aggregates of bacteria in a hydrated polymeric matrix of their own synthesis (Costerton et al., 1999). They may form on abiotic or biotic surfaces and such biofilms may influence the properties of pathogens. This is exemplified by P. aeruginosa, as the biofilm mode of growth confers antibiotic resistance within the lungs of patients with cystic fibrosis (Costerton et al., 1995
). A frequently used assay for biofilm formation on abiotic surfaces is to grow the bacteria on polystyrene and then measure the absorbance after staining with crystal violet (Nesper et al., 2001
; Solano et al., 2002
); this assay is not appropriate for measurement of biofilm formation on a biotic surface such as C. elegans. We have shown the presence of an extracellular matrix containing bacteria on C. elegans infected with Y. pseudotuberculosis by EM (Fig. 2D, E
) and stained the bacterial mass with crystal violet (Fig. 2F, G
), and periodic acidSchiff (data not shown). These findings strongly suggest the formation of a biofilm around C. elegans infected with Y. pestis or Y. pseudotuberculosis.
Neither Y. pestis nor Y. pseudotuberculosis has previously been reported to form biofilms on abiotic surfaces; we found that in repeated experiments in triplicate, a subset of Y. pseudotuberculosis strains are able to form a biofilm to varying degrees on polystyrene (Table 1). It is noteworthy that there is no relationship between strains forming a biofilm on polystyrene and on C. elegans; the formation of the biofilm on C. elegans appears to be an interactive process involving both bacterial and host recognition molecules. We have identified a number of bacterial genes involved in the colonization of C. elegans, and a number of C. elegans mutants refractory to infection. However, although the srf genes conferring the resistance phenotype have been mapped, they have not yet been assigned to a sequence or cloned. We are currently investigating the identity of those genes.
The C. elegans srf-2, srf-3 and srf-5 mutants, which were resistant to infection, that is in which biofilm formation did not occur, show ectopic binding of the lectins wheat germ agglutinin (WGA) and soybean agglutinin (SBA) (Link et al., 1992; Politz et al., 1990
) and their role in the formation of the biofilm remains to be determined. These mutants are also resistant to infection by M. nematophilum, in which the normal infection is a localized raised lesion (Hodgkin et al., 2000
); it does not form a biofilm. It is likely that Y. pestis, Y. pseudotuberculosis and M. nematophilum attach via related molecular targets on the nematode surface. It is noteworthy that srf-2, srf-3 and srf-5 are otherwise wild-type, whereas srf-4, srf-8 and srf-9, which are not resistant to infection by either Y. pseudotuberculosis or M. nematophilum (Hodgkin et al., 2000
) have other defects including defective copulatory bursae development, uncoordinated movement and abnormal egg laying. daf-1, mutants of which were also resistant to Y. pseudotuberculosis infection, encodes a receptor tyrosine kinase forming part of the dauer-formation pathway which includes the srf-6 gene (Grenache et al., 1996
).
In several bacterial species, biofilm formation has been shown to be under genetic control. A microarray analysis of free-living and sessile P. aeruginosa showed 34 genes activated and 39 genes repressed in biofilm populations (Whiteley et al., 2001). Pseudomonas fluorescens biofilm formation was abrogated in strains with mutations in the clpP gene, which encodes a subunit of the cytoplasmic Clp protease (O'Toole & Kolter, 1998
). In Salmonella enteritidis, mutation of the bcsABZC or bcsEFG gene clusters, which are required for the synthesis of an exopolysaccharide, prevents biofilm formation. In Y. pseudotuberculosis, mutation of the hmsT gene similarly prevents biofilm formation on C. elegans (Darby et al., 2002
). It is known that HmsT is a positive regulator of the hmsHFRS operon in Y. pestis, and homologues of HmsF and HmsR are required for the synthesis of polysaccharide components of biofilms of Staphylococcus epidermidis (Gerke et al., 1998
). We have also identified a Y. pseudotuberculosis YPIII pIB1 hms gene (hmsF; Table 2
) as necessary for biofilm formation. Biofilm formation was also absent in a Y. pseudotuberculosis IP32637 defined hms-negative mutant. In addition, we have shown that a pigmentation-negative strain of Y. pestis (F361-66), which lacks the hms locus, is unable to cause biofilm formation in C. elegans. It may be significant that the blockage of fleas by Y. pestis is also dependent on the hms locus.
The murine lethal toxin (phospholipase D) encoded on the 100 kb plasmid pMT1 in Y. pestis also plays a role in the survival of Y. pestis in the midgut of fleas (Hinnebusch et al., 2002). However, pMT1 does not appear to be important for infection of C. elegans since the Y. pestis Java-9 strain that lacks this plasmid (confirmed by DNA microarray analysis; S. J. Hinchliffe & B. W. Wren, unpublished data) readily caused biofilms. By contrast, Y. pestis F361-66, which lacks the hms locus, failed to block C. elegans and would, presumably, be unable to block fleas.
We have identified six further genes in which disruption prevents or reduces biofilm formation (Table 2); two of these relate to lipopolysaccharide (LPS) biosynthesis, although the mutant 4H9 has reduced viability and lack of biofilm formation may not be solely due to reduced LPS biosynthesis. Mutation of galU and galE in Vibrio cholerae causes production of an altered LPS core oligosaccharide; mutants are unable to synthesize exopolysaccharide (VPS) or to form a biofilm (Nesper et al., 2001
). Clearly bacterial polysaccharides are intrinsic to the formation of the biofilm in the Y. pseudotuberculosis/C. elegans infection model, as they are in biofilms formed by other bacterial species.
We do not know, as yet, the relevance of the sensory transduction gene (1G6-1) in biofilm formation in Y. pseudotuberculosis YPIII pIB1, nor of the two genes (2H10 and 1C9) with no similarity at the protein level to published sequences.
The C. elegans/Y. pseudotuberculosis and C. elegans/Y. pestis infection models may be useful in the investigation of two areas of nematode/bacterial biology. As discussed above, Y. pseudotuberculosis and Y. pestis share 97 % sequence similarity yet cause markedly different disease in humans. Although the virulence of Y. pestis is conferred largely by the pYV, pPCP1 and pMT1 plasmids, the mechanism by which Y. pestis causes blockage in the flea following formation of a biofilm while Y. pseudotuberculosis can form a biofilm (on C. elegans), but cannot block the flea remains to be determined. It may relate to the second area of interest: host interaction. Acylhomoserine lactones (AHLs), which mediate quorum sensing in Gram-negative bacteria, are constituitively produced by Y. pseudotuberculosis (Atkinson et al., 1999; S. Atkinson, personal communication) but biofilms are not normally formed. It is a tenable hypothesis that quorum-sensing processes followed by biofilm formation come into operation when bacteria are co-localized by receptor-mediated binding on the worm surface. As such, the identities of srf-2, srf-3, srf-5 and daf-1 are of interest. Human orthologues may be relevant to clinical disease where bioflms form, e.g. P. aeruginosa in cystic fibrosis patients; and flea orthologues may be important in flea blockage. The C. elegans/Yersina spp. model therefore represents a useful experimental system to investigate the genetic and biochemical basis of biofilm formation by bacteria on a biotic surface, and the evolution of Y. pestis from Y. pseudotuberculosis.
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ACKNOWLEDGEMENTS |
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Received 9 May 2003;
revised 22 July 2003;
accepted 28 July 2003.