A conserved Toll-like receptor is required for Caenorhabditis elegans innate immunity - PubMed (original) (raw)

A conserved Toll-like receptor is required for Caenorhabditis elegans innate immunity

Jennifer L Tenor et al. EMBO Rep. 2008 Jan.

Abstract

Pathogen recognition through Toll-like receptors (TLRs) is crucial in order to mount an appropriate immune response against microorganisms. On the basis of a lack of evidence indicating that Caenorhabditis elegans uses TLRs to elicit an immune response and on the absence of genes encoding Rel-like transcription factors in its genome, it is believed that TLR-mediated immunity arose after coelomates split from pseudocoelomates and acoelomates. Here, we show that C. elegans tol-1(nr2033) mutants are killed by the human pathogen Salmonella enterica, which causes a significant pharyngeal invasion in the absence of TOL-1-mediated immunity. We also show that TOL-1 is required for the correct expression of ABF-2, which is a defensin-like molecule expressed in the pharynx, and heat-shock protein 16.41, which is also expressed in the pharynx and is part of a HSP family of proteins required for C. elegans immunity. The results indicate that TOL-1 has a direct role in defence response to certain Gram-negative bacteria and indicate that part of the TLR-mediated immunity might be evolutionarily conserved.

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Figures

Figure 1

Figure 1

TOL-1-mediated immunity is required for survival in the presence of live Gram-negative bacteria. (A) Wild-type N2 (WT), tol-1(nr2033) (P<0.0001), pmk-1(km25) (P<0.0001) and dbl-1(nk3) (P<0.0001) were exposed to Salmonella enterica. The graphs represent combined results of more than four independent experiments, each of which used 36–50 1-day-old adult hermaphrodites. (B,C) Wild-type N2, tol-1(nr2033), pmk-1(km25) and dbl-1(nk3) were exposed to live (Caenorhabditis elegans killing assay) or heat-killed Escherichia coli (lifespan assay). Although tol-1(nr2033) nematodes died faster than wild type on live E. coli (_P_=0.0024), their lifespan was similar to that of wild type (_P_=0.3966) when exposed to heat-killed E. coli. (D) tol-1(nr2033) lived longer on E. faecalis compared with wild-type (P<0.0001), whereas pmk-1(km25) (P<0.0001) and dbl-1(nk3) (P<0.0001) nematodes were highly susceptible. (E–H) The time for 50% of the nematodes to die (TD50) was determined for each nematode strain exposed to different bacteria. The graphs are representative of at least three independent experiments using 36–50 1-day-old adult hermaphrodites, unless otherwise indicated; bars correspond to mean±s.d.

Figure 2

Figure 2

Salmonella enterica invades the pharynx of tol-1(nr2033). (A–D) Confocal images show the pharynxes of wild-type N2 (WT), pmk-1(km25), dbl-1(nk3) and tol-1(nr2033) nematodes exposed for 48 h to S. enterica expressing green fluorescent protein (GFP). (E,F) Confocal images show the pharynxes of wild-type and tol-1(nr2033) nematodes exposed for 48 h to Pseudomonas aeruginosa expressing GFP. (G) The percentage of nematodes with infected pharynxes was determined for wild type, tol-1(nr2033), pmk-1(km25), dbl-1(nk3) and tol-1(nr2033)Ex[CO7F11;pRF4]; bars correspond to mean±s.d. The Mann–Whitney test indicates that differences among the groups are significantly different; _n_=6–11. (H) Linear regression was used to analyse the relationship between pharyngeal infection and death for wild-type, pmk-1(km25), dbl-1(nk3) and tol-1(nr2033) nematodes exposed to S. enterica expressing GFP. (I) Linear regression was used to analyse the relationship between pharyngeal infection and death for tol-1(nr2033) nematodes exposed to P. aeruginosa or S. enterica expressing GFP. Individual nematodes were monitored daily for pharyngeal infection (24 h before death) and mortality. Images and graphics are representative of 3–11 independent experiments. For each experiment, 20–50 1-day-old adult hermaphrodites were used.

Figure 3

Figure 3

Analysis of the role of candidate downstream components of the TOL-1 pathway in immunity to Salmonella enterica. (A) Wild-type N2 (WT), trf-1(nr2014) (_P_=0.0001) and ikb-1(nr2027) (_P_=0.0001) were exposed to S. enterica. The graphs represent combined results of more than four independent experiments, each of which used 36–50 1-day-old adult hermaphrodites. (B) The lifespan of the ikb-1(nr2027) is similar to that of wild type (_P_=0.3236), whereas the lifespan of trf-1(nr2014) is slightly longer than wild type (_P_=0.0197). (C) trf-1(nr2014) are more resistant to _Enterococcus faecalis-_mediated killing than wild type (P<0.0064), whereas ikb-1(nr2027) (P<0.6730) shows a susceptibility similar to that of wild type. (D–F) The time for 50% of the nematodes to die (TD50) was determined for each nematode strain exposed to different bacteria. The graphs are representative of at least three independent experiments, each of which used 36–50 1-day-old adult hermaphrodites; bars correspond to mean±s.d., unless otherwise indicated.

Figure 4

Figure 4

Characterization of downstream components of the TOL-1 pathway. (A,B) Confocal images show the pharynxes of trf-1(nr2014) and ikb-1(nr2027) nematodes exposed for 48 h to Salmonella enterica expressing green fluorescent protein (GFP). GFP-expressing bacteria were detected in the pharynx and intestinal lumen of both mutants. (C) The percentage of nematodes with infected pharynxes was determined for wild type (WT), trf-1(nr2014), ikb-1(nr2027), tol-1(nr2033), tol-1(nr2033); trf-1(nr2014), trf-1(nr2014) Ex[CO7F11;pRF4] and ikb-1(nr2027)Ex[CO7F11;pRF4]. For each experiment, 20–50 1-day-old adult hermaphrodites were used. The Mann–Whitney test indicates that differences among the groups are significantly different; n_=3–11. (D,E) Wild-type N2, tol-1(nr2033) (P<0.0001), trf-1(nr2014) (P_=0.0013), tol-1(nr2033);trf-1(nr2014) (P<0.0001), wild-type Ex[CO7F11;pRF4] (P<0.0024), trf-1(nr2014)Ex[CO7F11;pRF4] (P<0.0015) and ikb-1(nr2027)Ex[CO7F11;pRF4] (P<0.002) were exposed to S. enterica. The graphs represent combined results of more than four independent experiments, each of which used 36–50 1-day-old adult hermaphrodites. (F) Quantitative reverse transcription–PCR analysis of act-1, abf-2 and hsp-16.41 expression in tol-1(nr2033) relative to wild-type nematodes grown on S. enterica. Data were analysed by relative quantitation using the comparative cycle threshold method and normalization to act-1. Student's exact _t_-test indicates that differences among the groups are significantly different; _n_=3; bars correspond to mean±s.d.

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