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.
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Table 1. Pathogenicity of different strains of S.marcescens during C.elegans infection
|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.
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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.
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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.
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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|
|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|
|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|
|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|
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.
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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 accentuate differences in survival between different worm strains on the same bacterial strain or between the same worm strain on different bacteria (C.L.Kurz, unpublished results). Therefore the effect of increasing the iron concentration was also assayed at 20°C. At this temperature, the augmentation of the virulence of 10H1 was significant (p < 0.014) (Figure 6). Taken together, these results confirm that the mutant is defective in its capacity to capture iron. The mutant 20C2, the most attenuated in the nematode, fly and cell models (Figures 4 and 5B; Supplementary data), contains an insertion in a homologue of wzm that codes for the membrane component of an ABC-2 transporter specialized in the translocation of LPS O-antigen (Sugiyama et al., 1998). Consistent with this, analysis of the 20C2 LPS by Tricine–SDS–PAGE revealed the absence of O-antigen. A derivative of Db11, Db1140 (Flyg and Xanthopoulos, 1983), also lacked O-antigen, as previously predicted (Pujol et al., 2001), in contrast with the other mutants (Figure 7; data not shown). In 21C4, the transposon is inserted 61 bp upstream of the initiation codon of shlB, the first gene of the shlBA operon. ShlA is a well characterized virulence factor of S.marcescens involved in cytotoxicity (e.g. Poole and Braun, 1988a,b; Hertle, 2002).
Figure 6. Addition of iron increases the virulence of the mutant 10H1. Kinetics of killing at 20°C of fer-15 hermaphrodites transferred as L4 worms from OP50 to 10H1 on standard NGM medium (closed squares) or on NGM medium supplemented with 0.1 mM iron (open squares).
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Figure 7. The mutant 20C2 is deficient in O-antigen. Silver-stained Tricine–SDS–PAGE gel of LPS prepared from different S.marcescens strains. Db1140 and 20C2 had no visible O-antigen ladder (upper broad band). The lower band corresponds to the LPS core.
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Regarding the other mutants, for 8E2, 10H4 and 7D1/22D9 absolutely no information is available regarding the function of the corresponding homologous proteins. For 23E6, the gene downstream of the insertion site is a homologue of the E.coli yjcE gene which encodes a presumed Na+−H+ exchanger (but see Verkhovskaya et al., 2001). For 7A8, the disrupted gene potentially encodes a DJ-1/PfpI domain-containing protein. This domain is common to a variety of protein families, including proteases and transcriptional regulators. For 7F1, the corresponding protein contains an amino oxidase domain. In the case of 21C1, the homologous protein contains an ATPase domain. For the remainder, a putative function could be assigned based on sequence comparisons (Table II; Supplementary data).
In the clone 8G1, a gene that encodes a homologue of the two-component system sensor BaeS of Xanthomonas axonopodis is disrupted. Two-component signal transduction systems regulate the expression of multiple genes in response to the environment and are known virulence factors in many pathogenic bacteria (Hoch, 2000). 8H1 contains an insertion upstream of a homologue of mgtB that encodes a highly conserved putative Mg2+ transport ATPase (see below). In 10E5, the transposon insertion is in a gene coding for a homologue of a TonB-dependent ferrisiderophore receptor and would be predicted to have a defect in iron uptake, as for 10H1. However, the difference in survival of worms on Db11 and 10E5 was small and precluded statistically significant tests of the rescuing capacity of exogenous iron.
Clone 22D4 contains a transposon inserted within a homologue of the chaperone gene ibpB. In E.coli, IbpB has been shown to protect enzymes from inactivation by heat and oxidants (Kitagawa et al., 2002). In Db11, it could serve to protect the bacteria during their growth in the intestine. 18F3 has an insertion in a homologue of the gene wbeiT of Edwardsiella ictaluri that encodes a UDP- glucose-4-epimerase and is within an O-antigen biosynthesis gene cluster (see DDBJ/EMBL/GenBank accession No. 16648662). No difference in LPS structure was observed in Tricine–SDS–PAGE analysis between 18F3 and Db11 (Figure 7), but, as this method can only detect major changes in LPS structure, we cannot exclude the possibility that 18F3 has a defect in its LPS. Lastly, 7E7 has an insertion in a clear homologue of galR of Y.pestis encoding the repressor of the gal operon, but there is no obvious link between this insertion and the observed reduced virulence of the mutant.
Interspecies conservation of virulence genes
Seven of the 12 S.marcescens disrupted loci conserved in other bacterial species have homologues in P.aeruginosa. The infection of C.elegans by P.aeruginosa is currently the best characterized nematode infection model (Tan and Ausubel, 2000; Aballay and Ausubel, 2002). We inactivated two of these homologous genes in the P.aeruginosa strain PA14 by targeted gene disruption to determine whether they encoded virulence factors. We chose the gene PA3713 of unknown function, corresponding to 7F1, and PA0151, corresponding to 10E5 and thus encoding a ferrisiderophore receptor, as mentioned above. During infection of worms, a significant reduction in virulence was observed at 20 and 25°C for both mutants compared with PA14 (Figure 8; data not shown). Neither gene had previously been identified as being required for full virulence in PA14.
Figure 8. Mutants of P.aeruginosa for the homologues of the genes disrupted in the Db11-derived mutants 10E5 and 7F1 (PA14-M151 and PA14-M3713, respectively) are attenuated in C.elegans. Kinetics of killing of C.elegans infected by P.aeruginosa strain PA14 (closed squares), PA14 mutant M151 (open circles) and PA14 mutant M3713 (open triangles). Worms were grown on NGM plates at 20°C, and 40–50 N2 hermaphrodites were used in each test. The curves are representative of at least two independent trials. The differences between PA14 and M151 or M3713 are statistically significant (p < 0.0004 and p < 0.023, respectively).
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