Title: Virulence factors of the human opportunistic pathogen Serratia marcescens identified by in vivo screening
Abstract: Article1 April 2003free access Virulence factors of the human opportunistic pathogen Serratia marcescens identified by in vivo screening C.Léopold Kurz C.Léopold Kurz Centre d'Immunologie de Marseille Luminy, INSERM/CNRS/Université de la Méditerranée, Case 906, 13288 Marseille, cedex 9, France Search for more papers by this author Sophie Chauvet Sophie Chauvet Present address: INSERM U382, IBDM, Campus de Luminy, Case 907, 13288 Marseille, cedex 9, France Search for more papers by this author Emmanuel Andrès Emmanuel Andrès Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du CNRS, 15 rue R.Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Marianne Aurouze Marianne Aurouze LISM/IBSM, 31 Ch.J.Aiguier, 13402 Marseille, cedex 20, France Search for more papers by this author Isabelle Vallet Isabelle Vallet LISM/IBSM, 31 Ch.J.Aiguier, 13402 Marseille, cedex 20, France Search for more papers by this author Gérard P.F. Michel Gérard P.F. Michel LISM/IBSM, 31 Ch.J.Aiguier, 13402 Marseille, cedex 20, France Search for more papers by this author Mitch Uh Mitch Uh Biotechnology Laboratory, University of British Columbia, 237-6174 University Boulevard, Vancouver, BC, V6T 1Z3 Canada Search for more papers by this author Jean Celli Jean Celli Biotechnology Laboratory, University of British Columbia, 237-6174 University Boulevard, Vancouver, BC, V6T 1Z3 Canada Present address: Centre d'Immunologie de Marseille Luminy, 13288 Marseille, cedex 9, France Search for more papers by this author Alain Filloux Alain Filloux LISM/IBSM, 31 Ch.J.Aiguier, 13402 Marseille, cedex 20, France Search for more papers by this author Sophie de Bentzmann Sophie de Bentzmann LISM/IBSM, 31 Ch.J.Aiguier, 13402 Marseille, cedex 20, France Search for more papers by this author Ivo Steinmetz Ivo Steinmetz Institute of Medical Microbiology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany Search for more papers by this author Jules A. Hoffmann Jules A. Hoffmann Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du CNRS, 15 rue R.Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author B.Brett Finlay B.Brett Finlay Biotechnology Laboratory, University of British Columbia, 237-6174 University Boulevard, Vancouver, BC, V6T 1Z3 Canada Search for more papers by this author Jean-Pierre Gorvel Jean-Pierre Gorvel Centre d'Immunologie de Marseille Luminy, INSERM/CNRS/Université de la Méditerranée, Case 906, 13288 Marseille, cedex 9, France Search for more papers by this author Dominique Ferrandon Dominique Ferrandon Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du CNRS, 15 rue R.Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Jonathan J. Ewbank Corresponding Author Jonathan J. Ewbank Centre d'Immunologie de Marseille Luminy, INSERM/CNRS/Université de la Méditerranée, Case 906, 13288 Marseille, cedex 9, France Search for more papers by this author C.Léopold Kurz C.Léopold Kurz Centre d'Immunologie de Marseille Luminy, INSERM/CNRS/Université de la Méditerranée, Case 906, 13288 Marseille, cedex 9, France Search for more papers by this author Sophie Chauvet Sophie Chauvet Present address: INSERM U382, IBDM, Campus de Luminy, Case 907, 13288 Marseille, cedex 9, France Search for more papers by this author Emmanuel Andrès Emmanuel Andrès Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du CNRS, 15 rue R.Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Marianne Aurouze Marianne Aurouze LISM/IBSM, 31 Ch.J.Aiguier, 13402 Marseille, cedex 20, France Search for more papers by this author Isabelle Vallet Isabelle Vallet LISM/IBSM, 31 Ch.J.Aiguier, 13402 Marseille, cedex 20, France Search for more papers by this author Gérard P.F. Michel Gérard P.F. Michel LISM/IBSM, 31 Ch.J.Aiguier, 13402 Marseille, cedex 20, France Search for more papers by this author Mitch Uh Mitch Uh Biotechnology Laboratory, University of British Columbia, 237-6174 University Boulevard, Vancouver, BC, V6T 1Z3 Canada Search for more papers by this author Jean Celli Jean Celli Biotechnology Laboratory, University of British Columbia, 237-6174 University Boulevard, Vancouver, BC, V6T 1Z3 Canada Present address: Centre d'Immunologie de Marseille Luminy, 13288 Marseille, cedex 9, France Search for more papers by this author Alain Filloux Alain Filloux LISM/IBSM, 31 Ch.J.Aiguier, 13402 Marseille, cedex 20, France Search for more papers by this author Sophie de Bentzmann Sophie de Bentzmann LISM/IBSM, 31 Ch.J.Aiguier, 13402 Marseille, cedex 20, France Search for more papers by this author Ivo Steinmetz Ivo Steinmetz Institute of Medical Microbiology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany Search for more papers by this author Jules A. Hoffmann Jules A. Hoffmann Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du CNRS, 15 rue R.Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author B.Brett Finlay B.Brett Finlay Biotechnology Laboratory, University of British Columbia, 237-6174 University Boulevard, Vancouver, BC, V6T 1Z3 Canada Search for more papers by this author Jean-Pierre Gorvel Jean-Pierre Gorvel Centre d'Immunologie de Marseille Luminy, INSERM/CNRS/Université de la Méditerranée, Case 906, 13288 Marseille, cedex 9, France Search for more papers by this author Dominique Ferrandon Dominique Ferrandon Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du CNRS, 15 rue R.Descartes, 67084 Strasbourg, cedex, France Search for more papers by this author Jonathan J. Ewbank Corresponding Author Jonathan J. Ewbank Centre d'Immunologie de Marseille Luminy, INSERM/CNRS/Université de la Méditerranée, Case 906, 13288 Marseille, cedex 9, France Search for more papers by this author Author Information C.Léopold Kurz1, Sophie Chauvet2, Emmanuel Andrès3, Marianne Aurouze4, Isabelle Vallet4, Gérard P.F. Michel4, Mitch Uh5, Jean Celli5,6, Alain Filloux4, Sophie de Bentzmann4, Ivo Steinmetz7, Jules A. Hoffmann3, B.Brett Finlay5, Jean-Pierre Gorvel1, Dominique Ferrandon3 and Jonathan J. Ewbank 1 1Centre d'Immunologie de Marseille Luminy, INSERM/CNRS/Université de la Méditerranée, Case 906, 13288 Marseille, cedex 9, France 2Present address: INSERM U382, IBDM, Campus de Luminy, Case 907, 13288 Marseille, cedex 9, France 3Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du CNRS, 15 rue R.Descartes, 67084 Strasbourg, cedex, France 4LISM/IBSM, 31 Ch.J.Aiguier, 13402 Marseille, cedex 20, France 5Biotechnology Laboratory, University of British Columbia, 237-6174 University Boulevard, Vancouver, BC, V6T 1Z3 Canada 6Present address: Centre d'Immunologie de Marseille Luminy, 13288 Marseille, cedex 9, France 7Institute of Medical Microbiology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:1451-1460https://doi.org/10.1093/emboj/cdg159 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The human opportunistic pathogen Serratia marcescens is a bacterium with a broad host range, and represents a growing problem for public health. Serratia marcescens kills Caenorhabditis elegans after colonizing the nematode's intestine. We used C.elegans to screen a bank of transposon-induced S.marcescens mutants and isolated 23 clones with an attenuated virulence. Nine of the selected bacterial clones also showed a reduced virulence in an insect model of infection. Of these, three exhibited a reduced cytotoxicity in vitro, and among them one was also markedly attenuated in its virulence in a murine lung infection model. For 21 of the 23 mutants, the transposon insertion site was identified. This revealed that among the genes necessary for full in vivo virulence are those that function in lipopolysaccharide (LPS) biosynthesis, iron uptake and hemolysin produc tion. Using this system we also identified novel conserved virulence factors required for Pseudomonas aeruginosa pathogenicity. This study extends the utility of C.elegans as an in vivo model for the study of bacterial virulence and advances the molecular understanding of S.marcescens pathogenicity. Introduction There is a continuing need to characterize the strategies used by bacterial pathogens during infection. This has been highlighted in recent times by the increase in resistance to multiple antibiotics of certain bacteria, and by the use of pathogens as biowarfare agents. Since certain virulence factors are necessary for full pathogenicity regardless of the host, one approach to the study of the interaction between pathogen and host is the use of simple model systems. It has been clearly demonstrated that the nematode Caenorhabditis elegans is an appropriate model host for the study of the virulence mechanisms deployed by the human opportunistic pathogen Pseudomonas aeruginosa (reviewed in Tan and Ausubel, 2000). The use of C.elegans for such studies has now been extended to a range of Gram-positive and Gram-negative bacteria (reviewed in Aballay and Ausubel, 2002; Ewbank, 2002), including the potential biowarfare agents Burkholderia pseudomallei (O'Quinn et al., 2001; Gan et al., 2002) and Yersinia pestis (Darby et al., 2002). Serratia marcescens is a Gram-negative bacterium that causes disease in plants and in a wide range of both invertebrate and vertebrate hosts (Grimont and Grimont, 1978). It is an opportunistic human pathogen and in the last three decades there has been a steady increase in nosocomial S.marcescens infections that can be life-threatening (Haddy et al., 1996; Hejazi and Falkiner, 1997; and references cited therein). While environmental S.marcescens strains are often red, due to the production of prodigiosin (Thomson et al., 2000), the strains associated with hospital outbreaks are mostly non-pigmented (Hejazi and Falkiner, 1997; Carbonell et al., 2000). As many S.marcescens strains are also resistant to multiple antibiotics (Hejazi and Falkiner, 1997; Alexandrakis et al., 2000), it represents a growing problem for public health. However, relatively little is known about the factors that contribute to S.marcescens pathogenesis within its host (Hejazi and Falkiner, 1997). Serratia marcescens is capable of killing C.elegans following the establishment of an intestinal infection (Mallo et al., 2002). We have characterized this interaction and used C.elegans to screen a bank of S.marcescens mutants. We isolated bacterial clones with a reduced virulence against the nematode. Some of them were also attenuated in their pathogenicity in two other infection models. Three candidates were tested in a murine lung infection model, and one of them displayed a very marked reduction in its pathogenicity. To our knowledge, this represents the first systematic study of S.marcescens virulence in vivo and has permitted the identification of previously uncharacterized virulence factors. Further, using this S.marcescens–C.elegans model we have identified conserved virulence factors also required for the full virulence of P.aeruginosa. Results Characterization of the infection of C.elegans by S.marcescens Several pigmented and non-pigmented strains of S.marcescens are capable of infecting and killing C.elegans (Table I). We chose the non-pigmented strain Db11 for further study, thereby eliminating any potential contribution of the pigment prodigiosin to the infectious process. This strain was first described as a pathogen of Drosophila melanogaster (Flyg et al., 1980). More recently it has been used as a model pathogen to investigate innate immunity in C.elegans (Pujol et al., 2001; Mallo et al., 2002). The steps in the infection of worms by Db11 have been outlined (Mallo et al., 2002) and its repulsive effect on worms has previously been described (Pujol et al., 2001). When wild-type (N2) worms were transferred as L4 larvae from the standard Escherichia coli strain OP50 to lawns of Db11 they became visibly sick after 2 days. They did not show any sign of starvation, but started to die 1 day later. All worms were dead after 7 days when grown on Db11 (Figure 1A). Figure 1.Characterization of the infection of C.elegans by S.marcescens. (A) The killing of C.elegans by S.marcescens requires live bacteria. Kinetics of killing of worms exposed to S.marcescens Db11 (closed squares), E.coli OP50 (open squares), heat-killed Db11 (open triangles) and heat-killed Db11 supplemented with culture supernatant (open circles). (B) A short contact with S.marcescens is sufficient to infect C.elegans. Kinetics of killing of C.elegans by S.marcescens after different periods of contact with Db11. Worms were exposed to S.marcescens Db11 permanently (closed squares), for 4 h (open diamonds), for 8 h (open triangles) or for 18 h (open squares) and were then surface-sterilized and deposited on OP50. (C) The time course of the infection of C.elegans by Db11 is age dependent. Worms were transferred from OP50 to Db11 lawns at the L1 stage (open squares), at the L4 stage (closed squares), as 1-day-old adults (open triangles), as 2-day-old adults (open diamonds) or as 3-day-old adults (open circles) and their post-transfer survival was scored. In all cases, worms were grown on NGM plates at 25°C, and between 40 and 50 N2 hermaphrodites were used in each test. The curves are representative of at least two independent trials. Download figure Download PowerPoint Table 1. Pathogenicity of different strains of S.marcescens during C.elegans infection Strain Pigmentation Mean lifespana Db11 − 4.2 ± 0.3 (145) Db1140 − 6.2 ± 0.7 (132) Sma 3 − 4.9 ± 0.1 (95) Sma 12 − 5.0 ± 0.2 (96) Sma 13 − 5.1 ± 0.2 (98) ATCC 274 + 2.6 ± 0.1 (100) Sm 365 + 5.3 ± 0.7 (91) Sm 2170 + 1.7 ± 0.3 (99) aMean survival time ± SD in days for N2 worms infected at the L4 stage at 25°C. The total number of worms used in two independent tests is given in parentheses. Under these conditions, the mean post-L4 survival time for worms cultivated on E.coli OP50 is 12.6 ± 1 (n = 88). Certain bacteria kill worms via toxin-mediated mechanisms (Aballay and Ausubel, 2002; Couillault and Ewbank, 2002; Ewbank, 2002). To address this possibility, L4 worms were transferred to heat-killed Db11 in the presence or absence of supernatants from saturated Db11 cultures. In these cases, the worms did not appear sick and their survival was at least as long as that of worms fed on OP50 (Figure 1A). This suggests that live bacteria are needed for the infection and that a stable toxin does not mediate the killing of the worm. To establish whether a permanent contact was necessary for bacterially mediated killing, worms were transferred to Db11 at the L4 stage, allowed to feed for a fixed time and then surface-sterilized and returned to OP50. A contact of 30 h was sufficient to give survival curves that were indistinguishable from those obtained when worms were in constant contact with Db11 (data not shown). With shorter periods of contact, worms died much faster than control worms that were kept on E.coli, but the time course of the infection was longer than that for worms kept permanently in contact with Db11 (Figure 1B). Older worms were more susceptible to infection and the latency period before the first observed deaths was diminished as a function of the age of the worms, presumably reflecting a decrease in the antibacterial capabilities of older worms. Conversely, the early larval stages of C.elegans were resistant to Db11 (Figure 1C and see below). To follow the fate of the bacteria upon ingestion by the worm, we used strains of E.coli OP50 and S.marcescens Db11 that express the green fluorescent protein (GFP). Db11-GFP is as virulent as Db11 with regard to the killing of C.elegans (data not shown). When L4 worms are placed on OP50-GFP, intact bacteria are not found in the intestine, as they are broken down by the grinder located in the terminal bulb of the pharynx (Figure 2A and B) (Labrousse et al., 2000). On the other hand, when worms were transferred to Db11-GFP at the L4 stage, after as little as 2 h of contact, intact bacteria were seen to accumulate in the lumen of the intestine (Figure 2C and D). If, however, L4 worms were fed briefly on Db11 before being transferred to OP50-GFP, intact fluorescent bacteria were able to pass the grinder (Figure 2E and F). This indicates that Db11 is capable of disrupting the function of the grinder. In contrast with L4 worms, no intact bacteria were observed in the intestines of earlier larval stages fed with Db11-GFP even for periods of several hours (data not shown). This suggests that the resistance of pre-L4 larvae to Db11 (Figure 1C) is most likely due to the incapacity of the bacteria to enter the intestinal lumen. After 24 h of contact with Db11-GFP, the intestinal lumen appeared distended and full of fluorescent bacteria that remained extracellular during the infection. They were restricted to the intestinal lumen, except on rare occasions when bacteria were observed in the uterus between eggs. The increase in volume of the lumen was concurrent with exponential bacterial growth (see Supplementary data, available at http://www.ciml.univ-mrs.fr/EWBANK_jonathan/SuppMat/Screen/Kurz.html). Apart from this progressive distension of the intestinal lumen, outwardly, worms showed relatively little sign of infection for the first 24 h. The muscular contractions usually associated with feeding and defecation continued and their rate of egg-laying was normal (Mallo et al., 2002). In clear contrast with worms grown on OP50 (Figure 3A), there was then a progressive vacuolation of the intestinal cells (Figure 3C) accompanied by a decrease in the volume of the worm's intestinal epithelium. After infection with Db11 for 3 days, there was also an accumulation within the lumen of autofluorescent vesicles that appeared to be derived from the intestinal epithelium (Figure 3B). Similar vesicles were observed in worms after 5 days of contact with Salmonella typhimurium (C.L.Kurz, unpublished observations). The size and number of these vesicles, which moved in the lumen as the intestine contracted, increased to such a degree that defecation became impaired. The other tissues, including the germ-line, were also gradually destroyed (Figure 3B) before the worms died. Figure 2.Early entry of intact S.marcescens into the intestinal lumen of C.elegans. Fluorescence (A, C, E) and Nomarski (B, D, F) photomicrographs of L4 N2 hermaphrodite worms fed with OP50-GFP for 2 h (A and B), with Db11-GFP for 2 h (C and D) and with Db11 for 2 h followed by brief washing and feeding with OP50-GFP for 5 min (E and F). In (A), intact bacteria (indicated by white arrows) can only be seen in the pharyngeal isthmus anterior to the terminal bulb and the grinder (indicated by the asterisk). In (C), intact Db11-GFP are in the intestinal lumen. In (E), intact OP50-GFP can freely pass the grinder after the short contact with Db11 and are found intact in the intestinal lumen. In all cases the head of the worm is to the left; scale bar, 10 μm. Download figure Download PowerPoint Figure 3.Symptoms of the infection of C.elegans by S.marcescens. Nomarski photomicrographs of N2 worms fed for 5 days post-L4 stage with OP50 (A) or Db11 for 5 days post-L4 stage (B, C). (A) The intestine is healthy with large cells (arrows), the intestinal lumen is of normal size (arrowheads) and the germ-line is clearly visible (dotted lines). (B) The intestine of the Db11-infected worm is distended (arrowheads) and full of intact bacteria. The intestinal cells are partially lysed (arrows) and the germ-line is no longer apparent. A large round vesicle can also be observed in the intestinal lumen. (C) The vacuolation of one posterior intestinal cell is highlighted (dotted lines). In all cases the head of the worm is to the left; scale bar, 10 μm. Download figure Download PowerPoint Isolation of Db11 mutants with a reduced virulence against C.elegans Having characterized the infection of C.elegans by Db11, we designed a screen to identify bacterial mutants with a reduced virulence. Given that wild-type worms infected by Db11 are able to lay eggs and these eggs hatch to give larvae that are resistant to Db11, we decided to use the mutant worm strain fer-15 that is conditionally sterile at 25°C to avoid the potential confusion between generations. The time course of survival of fer-15 on Db11 is essentially identical with that of wild-type worms (see Supplementary data). Therefore we individually screened clones from a S.marcescens Db11 mini-Tn5Cm insertion library for mutants that supported the growth and survival of fer-15 worms beyond the time observed for Db11 (see Supplementary data). From 2300 bacterial clones tested, 23 attenuated mutants were selected for further study. Each contained a single transposon insertion (see Supplementary data). To determine whether the observed attenuation in virulence was due to a problem of general metabolism and/or growth, or was the result of the disruption of the function of a virulence gene, we followed the growth of each clone in Luria broth (LB) at 25°C and 37°C. No major difference in replication rate between mutant and parental strains was observed during exponential growth. However, two clones, 8E11 and 18D4, grew more slowly on LB agar plates (data not shown). The different mutants were then individually tested for their pathogenicity during the infection of N2 worms and were classed into three categories: weakly attenuated, attenuated and strongly attenuated (Figure 4; Table II). With the exception of 10E5, all mutants supported the growth and survival of N2 worms beyond 7 days, something that was never seen with Db11 (n > 5000). Worms grown on the most strongly attenuated clone, 20C2, lived roughly twice as long as worms on Db11 (Figure 4). In the case of 10E5, a statistically significant reduction of virulence was only observed during the infection of the fer-15 strain (see Supplementary data). Figure 4.The isolated S.marcescens mutants are less virulent during their infection of C.elegans. Kinetics of killing of C.elegans infected by Db11 (closed squares), and representative weakly attenuated mutant 3H5 (open circles), attenuated mutant 7F1 (open triangles) and strongly attenuated mutant 20C2 (open diamonds). The dotted line with open squares shows the survival curve for worms fed on E.coli OP50. In all cases, worms were grown on NGM plates at 25°C and 40–50 N2 hermaphrodites were used in each test. The curves are representative of at least two independent trials. Download figure Download PowerPoint Table 2. Characterization of attenuated mutants Clone Model Within/upstream of gene encoding conserved protein/domain (gene name) Species (DDBJ/EMBL/GenBank accession No.) E valueb Function Worma Fly Cells 3H5 – wt wt No na na na 8G1 – wt wt baeS homologue X.axonopodis (AAM37649) 8e-07 Two-component system sensor 8H1 – wt wt mgtB homologue Y.pestis (NP_405238) 9e-24 Magnesium transport 10E5 – wt wt Ferrisiderophore receptor (y3343) Y.pestis (AAM86893) 4e-82 Iron transport 7D1 – – wt wt Yes (STM0278; see 22D9) S.typhimurium (NP_459276) 2e-05 Unknown 7E7 – – wt wt galR homologue Y.pestis (NP_670482) 2e-56 Galactose operon repressor 7F1 – – wt wt Amino oxidase domain (PA3713) P.aeruginosa (A83182) 5e-57 Unknown 8C7 – – wt wt No na na na 8E2 – – wt wt Yes (YPO2856) Y.pestis (NP_406362) 5e-06 Unknown 23C11 – – wt wt Not cloned na na na 18F3 – – – wt wt wbeiT homologue E.ictaluri (AAL25633) 1e-39 O-antigen biosynthesis 22D4 – – – wt wt ibpB homologue Y.pestis (NP_671393) 5e-36 Stress resistance 22D9 – – – wt wt Yes (STM0278; see 7D1) S.typhimurium (NP_459276) 3e-07 Unknown 23E6 – – – wt wt yjcE homologue Y.pestis (NP_407085) 3e-23 Unknown 10F7 – – wt No na na na 18D4 – – wt No na na na 10H4 – – – wt Yes (yfdR) E.coli (P76514) 5e-29 Unknown 8E11 – – – wt Not cloned na na na 7A8 – – – – wt DJ-1/PfpI domain (CC2959) C.crescentus (NP_421753) 2e-11 Unknown 21C1 – – – – – wt ATPase domain E.coli (S28007) 0.008 Unknown 10H1 – – – – – vibC homologue P.fluorescens (CAA70528) 2e-17 Iron transport 21C4 – – – – – – – – shlB S.marcescens (AAA50322) na Hemolysin production 20C2 – – – – – – – – wzm homologue E.coli (BAA28324) 1e-115 LPS biosynthesis wt, wild type; –, weakly attenuated; – –, attenuated; – – –, strongly attenuated; na, not applicable. a N2 worms except for 10E5, for which fer-15 worms were used. b Expected value of BLASTX; see Supplementary data for details. Certain mutants exhibit reduced virulence in other infection models To determine whether the bacterial genes necessary for full virulence during the natural infection of C.elegans were also necessary for pathogenesis in other hosts, the selected mutants were individually tested in a D.melanogaster infection model. At 20°C, almost all flies died within 24 h following injection of 50–100 Db11 bacteria into the thorax (Figure 5A). Nine of the 23 mutants showed a clear and reproducible reduction of their virulence in this insect model. This shows that certain factors are necessary for the full virulence of Db11 during infection of both the nematode and the fly, despite the different modes of infection (ingestion versus injection). Of the nine mutants, 20C2, 21C1 and 21C4 showed the strongest attenuation of virulence (Figure 5A; Supplementary data). Figure 5.The selected S.marcescens mutants are less pathogenic in other models. (A) Kinetics of killing of D.melanogaster injected in the thorax with LB medium (open squares), Db11 (closed squares), or mutants 10F7 (open diamonds), 10H4 (open triangles) or 21C1 (open circles). (B) Cytotoxic effect of Db11 and derived mutants against a polarized human epithelial cell line. The release of lactate dehydrogenase from the epithelial cells was measured after 2 h contact with the bacteria and a cytotoxicity index calculated. An index of 1 corresponds to 100% lysis. The results represent the mean and standard deviation obtained from four independent trials. (C) Kinetics of killing of 8- to 10-week-old mice (n = 10) infected intranasally with Db11 (closed squares) or the mutant 21C4 (open squares). Their survival was plotted using the Kaplan–Meier method. Download figure Download PowerPoint Serratia marcescens has previously been shown to possess a strong cytotoxic effect in vitro against several cell types (Poole et al., 1988; Carbonell et al., 1997; Hertle et al., 1999) and is also associated with nosocomial lung infections (Haddy et al., 1996). Therefore we tested the cytotoxicity of Db11 and of the 23 mutants against a human polarized pulmonary epithelial cell-line (16HBE14o−). In this test, the bacteria are in direct contact with the target cells. Three of the mutants, 10H1, 20C2 and 21C4, showed a strong attenuation of their cytotoxic effect in vitro compared with Db11 (Figure 5B). These three mutants, which also showed a reduced virulence in D.melanogaster, were then tested in vivo in a murine lung infection model. The mutant 21C4 showed a marked reduction in its pathogenicity (p < 0.0013) (Figure 5C), while 10H1 and 20C2 were at least as virulent as Db11 (data not shown). Molecular characterization of the attenuated mutants For 21 of the 23 mutants, the inserted transposon and part of the flanking genomic region was cloned; the remaining two regions have so far been refractory. The respective transposon insertion sites were identified (see Supplementary data), and analyses revealed that for 19 mutants the sites were unrelated, indicating that the screen is far from being saturated. The mutants 7D1 and 22D9 contained insertions separated by less than 250 bp (see below), confirming the role of this particular locus in virulence. Only in the case of the clone 21C4 was the transposon inserted in a region previously characterized in S.marcescens, being inserted within the hemolysin shl operon. For four mutants (3H5, 8C7, 10F7 and 18D4), no conserved open reading frame was identified in the vicinity of the transposon insertion site. On the other hand, for 16 mutants, the transposon was inserted within, or just upstream of, a gene potentially encoding a protein with a homologue in at least one other bacterial species (see Supplementary data). Based on the site of transposon insertion, the three mutants 10H1, 20C2 and 21C4 were predicted to be affected in iron uptake, lipopolysaccharide (LPS) biosynthesis or hemolysin production, respectively. For 10H1, the gene downstream of the transposon insertion site potentially encodes a VibC/EntC homologue. These proteins are involved in the biosynthesis of the siderophores vibriobactin and enterobactin necessary for iron uptake in Vibrio cholerae (Wyckoff et al., 2001) and E.coli (Nahlik et al., 1987), respectively. As judged by chrome azurol S tests (Schwyn and Neilands, 1987), the mutant 10H1 produced less siderophore compared with Db11 (E.Pradel, personal communication). Increasing the iron concentration in the culture medium had no effect on either the survival of worms grown on OP50 or the virulence of Db11 (data not shown), but increased the rate of killing of C.elegans by 10H1. At 25°C, the difference was marginal (data not shown). We have observed that at 20°C the time course of infection of C.elegans with S.marcescens is slower than at 25°C, and that this can help accentua