Figure 3. Resistance of daf-2 pathway mutants is associated with reduced bacterial colonization, enhanced bacterial clearance, and increased immunity gene expression.

(A) akt-1(ok525), akt-2(ok393), and daf-2(e1370) animals accumulated significantly less PA14-GFP in their intestine than N2 following a 24 h exposure (pairwise comparison Chi-square, p < 0.0001). No significant difference in bacterial accumulation was observed between sgk-1(ok538) and N2 populations (Chi-square, p = 0.86). For each strain, population proportion by degree of bacterial accumulation in the intestine was categorized as fully colonized, partially colonized, or undetectable colonization corresponding to averages of 4.22 × 10⁴, 2.45 × 10⁴, and 3.48 × 10³ colony forming units of PA14-GFP, respectively. The pooled sample sizes are N=336 N2, 343 akt-1(ok525), 328 akt-2(ok393), 126 sgk-1(ok538), and 28 daf-2(e1370). (B) Pathogen clearance was enhanced in akt-1(ok525) and akt-2(ok393) compared to N2. The proportion of colonized worms before and after shift from PA14-GFP to E. coli OP50 was quantified, and the ratio of colonization after/before was plotted. The extent of clearance was significantly greater in akt-1(ok525) and akt-2(ok393) compared to N2 (log-linear analysis, p < 0.05) but not significantly different in sgk-1(ok538). (C) Quantification of live bacteria from whole worm lysates of daf-2(e1370) and N2 following exposure to PA14 for 24 h. The CFU counts were significantly less in daf-2 than N2 (t-test, p = 0.009). (D) Pathogen clearance was enhanced in daf-2(e1370) compared to N2. The initial PA14 CFUs obtained from daf-2 and N2 worms exposed to PA14 for 24 h and 18 h, respectively, were compared to PA14 CFUs from worms obtained 24 h after the shift to OP50-1. While the CFUs from initial and shifted N2 populations were not significantly different (t-test, p = 0.2), the CFUs were reduced more than 1000 fold in daf-2 worms after shift (t-test, p = 0.01). Use of equal initial exposure time produced comparable results after shift (data not shown). (E–F) Mean normalized expression level of immunity genes in akt-1(ok525), akt-2(ok393), sgk-1(ok538), and daf-2(e1370) exposed to (E) OP50 or (F) PA14, plotted as fold relative to expression level in N2 exposed to PA14 on a log₂ scale. Transcript levels were measured by quantitative RT-PCR and normalized to the average of 3 control genes. Mean and standard error of the mean from 2 to 4 independent experiments is shown. Pairwise comparisons to N2 were performed. (E) Antimicrobial gene expression is elevated in akt-1(ok525), akt-2(ok393), and daf-2(e1370) but not sgk-1(ok538). (F) Each antimicrobial gene was expressed at significantly higher levels in daf-2(e1370) than N2 under infected conditions. Taken as a whole, the set of antimicrobials was expressed at higher levels in daf-2(e1370), akt-1(ok525), and akt-2(ok393) than N2 (binomial test, p = 0.03). Antimicrobial gene expression was mixed in sgk-1(ok538) mutants (binomial test, p = 0.3) with significantly lower levels of abf-2 and lys-7 but higher levels of spp-1. Error bars indicate standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001