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Long-Lived C. elegans daf-2 Mutants Are Resistant to Bacterial Pathogens

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Science  20 Jun 2003:
Vol. 300, Issue 5627, pp. 1921
DOI: 10.1126/science.1080147

Our laboratories have studied the mechanisms of aging (1, 2) and immune function (3) in Caenorhabditis elegans. Herein we show that the mechanisms that govern these two processes may be interrelated.

The human Gram-negative bacterial pathogens Pseudomonas aeruginosa and Salmonella enterica and the Gram-positive pathogens Enterococcus faecalis and Staphylococcus aureus kill C. elegans by an infection-like process with remarkable overlap between the bacterial factors required for virulence in mammals and killing in nematodes (4, 5). Additionally, a p38 MAPK (mitogen-activated protein kinase) signaling cascade is a key component of the C. elegans innate immune response, as it is in mammals (3). These experiments establish C. elegans as a useful model for studying bacterial pathogenicity and host immunity. Here we show that certain long-lived C. elegans mutants are highly resistant to killing by bacterial pathogens.

To investigate the general relation between longevity and pathogen resistance, we tested whether C. elegans daf-2 and age-1 mutants exhibit enhanced resistance to E. faecalis, S. aureus, and P. aeruginosa. daf-2 encodes an insulin-like receptor that functions upstream of the phosphatidylinositol 3-kinase (PI 3–kinase) encoded by age-1, and partial loss of function mutations in daf-2 or age-1 result in a long-lived phenotype (1). Both daf-2 and age-1 mutants were resistant to killing by E. faecalis, S. aureus, and P. aeruginosa (Fig. 1A; table S1). Most dramatic was the five- and sixfold increased survival of the daf-2(e1370) mutants relative to wild-type C. elegans when exposed to the Gram-positive pathogens E. faecalis and S. aureus, respectively (Fig. 1A; table S1).

Fig. 1.

Pathogen resistance of the C. elegans daf-2(e1370) mutant. (A) Survival of N2, daf-2(e1370), daf-16 (mgDf47),and daf-2;daf-16 C. elegans feeding on E. faecalis strain OG1RF. (B) Adult life-span of N2 and daf-2(e1370) C. elegans when feeding on B. subtilis (PY79) or E. coli (OP50) grown on NG (nematode growth) medium with FUDR (5-Fluoro-2-deoxyuridine). Assays were carried out as previously described (2, 5). STATA 6 statistical software (Stata, College Station, TX) was used to plot survival by the Kaplan-Meier method.

Life-span extension through the DAF-2 insulin-signaling pathway in C. elegans occurs by de-repression of the forkhead transcription factor DAF-16, which is normally under negative regulation by DAF-2. Therefore, strong loss-of-function alleles of daf-16 such as mgDf47 suppress the long-lived phenotype of daf-2 mutants (2). daf-16(mgDf47) also suppressed the pathogen-resistant phenotype of daf-2(e1370) (Fig. 1A; table S1). Interestingly, the daf-16 mutant exhibited a comparable degree of susceptibility to pathogen-mediated killing as wild-type worms (Fig. 1A; table S1) under the experimental conditions assayed. In these experiments, the daf-2(e1370) allele was more resistant to bacterial pathogens than the daf-2(1368) allele and the age-1(hx546) allele (Fig. 1A; table S1). This is most likely a consequence of differences in allele strengths of the daf-2 and age-1 mutants, all of which are partial loss-of-function mutants.

Because C. elegans daf-2 and age-1 mutants were identified in screens using Escherichia coli strain OP50 as the food source and because E. coli may also be pathogenic to C. elegans (57), the enhanced longevity phenotype of daf-2 and age-1 could reflect acquired resistance to E. coli– mediated killing. Indeed, nematodes lived considerably longer when feeding on the Gram-positive bacterium Bacillus subtilis, a common soil bacterium that C. elegans is likely to feed on in the wild (Fig. 1B) (8). It is not likely that B. subtilis is simply more nutritious or more readily digestible than E. coli because the rate of growth, egg to egg generation time, and number of eggs laid were the same whether feeding on B. subtilis or E. coli (fig. S1).

The daf-2 mutant retained extended longevity relative to wild-type mutants when propagated on B. subtilis (Fig. 1B), but the fractional extension in life-span was modest (76 ± 9%) compared with the fractional extension observed on pathogenic bacteria [E. faecalis, 325 ± 57%; S. aureus, 514 ± 189%; P. aeruginosa, 118% ± 14%; and E. coli, 110 ± 11% (9)]. The age-1 mutant also lived longer on B. subtilis (table S1). The extended survival of the daf-2 and age-1 mutants on the relatively innocuous B. subtilis suggests that additional factors beyond pathogen resistance are likely involved in life-span regulation. However, when feeding on pathogenic bacteria, especially Gram-positive ones, mechanisms underlying pathogen resistance appear to be the dominant contributor to the overall longevity of daf-2 and age-1 mutants.

Our data suggest that the insulin signaling pathway modulates both inherent longevity and pathogen resistance to affect overall survival in a manner dependent on the pathogenicity of the bacteria on which C. elegans is feeding. The linkage of longevity and pathogen resistance to the same signaling pathway may have general relevance to the observation that most organisms become more susceptible to infection as they age.

Supporting Online Material

www.sciencemag.org/cgi/content/full/300/5627/1921/DC1

Fig. S1

Table S1

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