Salmonella enterica serovar Paratyphi A-induced immune response in Caenorhabditis elegans depends on MAPK pathways and DAF-16

Abstract

Salmonella enterica serovar Paratyphi A (S. Paratyphi A) is a pathogen that can cause enteric fever. According to the recent epidemic trends of typhoid fever, S. Paratyphi A has been the major important causative factor in paratyphoid fever. An effective vaccine for S. Paratyphi A has not been developed, which made it a tricky public health concern. Until now, how S. Paratyphi A interacts with organisms remain unknown. Here using lifespan assay, we found that S. Paratyphi A could infect Caenorhabditis elegans (C. elegans) at 25°C, and attenuate thermotolerance. The immune response of C. elegans was mediated by tir-1, nsy-1, sek-1, pmk-1, mpk-1, skn-1, daf-2 and daf-16, suggesting that S. Paratyphi A could regulate the MAPK and insulin pathways. Furthermore, we observed several phenotypical changes when C. elegans were fed S. Paratyphi A, including an accelerated decline in body movement, reduced the reproductive capacity, shortened spawning cycle, strong preference for OP50, arrested pharyngeal pumping and colonization of the intestinal lumen. The virulence of S. Paratyphi A requires living bacteria and is not mediated by secreting toxin. Using hydrogen peroxide analysis and quantitative RT-PCR, we discovered that S. Paratyphi A could increase oxidative stress and regulate the immune response in C. elegans. Our results sheds light on the infection mechanisms of S. Paratyphi A and lays a foundation for drugs and vaccine development.

Keywords: Caenorhabditis elegans; MAPK; Salmonella enterica serovar Paratyphi A; immune; oxidative stress.

Figure 1

S. Paratyphi A reduced the lifespan and thermotolerance, shortened the period of fast body movement and reduced the reproductive capacity of C elegans. (A) Representative survival curves of N2 of three independent trials and graphs of mean lifespan of at least 3 independent experiments at 25°C, and 35°C (B) on NGM plates containing either OP50 or S. Paratyphi A. Kaplan–Meier analysis was used to analyze the results of lifespan, and p values were calculated based on the log-rank test. Mean lifespan are presented as the mean ± standard deviation. ** p < 0.01, Student’s t-test. (C) Representative curves of three independent trials of age-related movements of worms treated with 5x10⁹ and 5x10¹⁰ bacteria/mL for OP50 group and 5x10⁹ bacteria/mL for S. Paratyphi A group (p <0.0001, log-rank test) and graphs of mean fast movement period of at least 3 independent experiments (mean ± SD, ** p < 0.01, *** p < 0.001, NS represents not significant, Student’s t-test). The movements of nematodes were classified as fast and not fast movement. (D) Number of eggs laid (mean ± SD, *** p < 0.001, Student’s t-test) and spawning period (E) of worms cultured with OP50 and S. Paratyphi A (mean ± SD, *** p < 0.001, Student’s t-test).

Figure 2

S. Paratyphi A-trained worms showed a bias toward OP50, a decreased rate of pharyngeal pumping, and increased colonization in the intestinal tract. (A) The chemotaxis index of wild-type N2 worms trained on OP50 or S. Paratyphi A (mean ± SD, ** p <0.01, Student’s t-test). (B) After 12 h, 24 h and 48 h of S. Paratyphi A infection, pharyngeal pumping assay were performed (mean ± SD, *** p < 0.001, Student’s t-test). (C) The number of OP50 or S. Paratyphi A colonies/worm present in the intestine over 12 h (mean ± SD, *** p <0.001), 24h. (mean ± SD, *** p <0.001) and 48 h (mean ± SD, *** p < 0.001) treatment (n=10 in triplicates Student’s t-test). (D) Fluorescence images of representative worms raised on E coli::eGFP or S. Paratyphi A::eGFP (both carry the same eGFP expression plasmid). i Wild-type N2 worms fed E coli::eGFP for 24 h. ii N2 worms fasted for 24 hours after feeding E coli::eGFP. iii Wild-type N2 worms fed S. Paratyphi A::eGFP for 24h. iv N2 worms fasted for 24 hours after feeding S. Paratyphi A::eGFP. (E) Representative survival curves (p value by log-rank test) and graphs of mean lifespan of at least 3 independent experiments (mean ± SD, ** p < 0.01 and *** p < 0.001, NS represents not significant, Student’s t-test) of wild-type N2 worms on NGM plates containing untreated bacteria and heat-killed OP50 or S. Paratyphi A. (F) Representative survival curves (p value by log-rank test) and graphs of mean lifespan of at least 3 independent experiments (mean ± SD, * p < 0.05 and ** p < 0.01, NS represents not significant, Student’s t-test) of wild-type N2 worms on NGM plates containing untreated bacteria and OP50 or S. Paratyphi A secretions.

Figure 3

S. Paratyphi A-induced immune response in C. elegans requires activation of the MAPK pathway. Representative survival curves (p value by log-rank test) and graphs of mean lifespan of at least 3 independent experiments (mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, NS represents not significant, Student’s t-test) of wild-type N2 worms and mutants of tir-1(qd4) III (A) , nsy-1(ag3) II (B) , sek-1(km4) X (C) , pmk-1(km25) IV (D) , mpk-1(sbj10) III (E), skn-1(zu67) IV (F) on NGM plates containing OP50 or S. Paratyphi A. (G) Relative mRNA levels of tir-1, nsy-1, sek-1, pmk-1, mpk-1, and skn-1 were detected by quantitative RT-PCR. Statistical analyses were calculated using two-tailed Student’s t test; * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001. (H) Western blot analyses and quantify the level of P-p38 MAPK/PMK-1 in N2 worms treated OP50 or S. Paratyphi A (KU25 pmk-1(km25) IV was analyzed to verify mutant). Actin was displayed as the loading control (* p < 0.05, two-tailed Student’s t-test).

Figure 4

Defense against S. Paratyphi A may require the participation of DAF-2 and DAF-16. Representative survival curves (p value by log-rank test) and graphs of mean lifespan of at least 3 independent experiments (mean ± SD, ** p < 0.01 and *** p < 0.001, Student’s t-test) of wild-type N2 worms and mutants of daf-2(e1370) III (A) and daf-16(mu86)I (B). (C) Fluorescence images of TJ356 zIs356 [daf-16p::daf-16a/b::GFP +rol-6(su1006)] grown on OP50 or S. Paratyphi A for 30 min. (D) Relative mRNA levels of daf-16, sod-3, hsp-12.6 and dod-19 were analyzed by quantitative RT–PCR (* p < 0.05 and ** p < 0.01, two-tailed Student’s t-test).

Figure 5

Stress response changes in C. elegans exposed to S. Paratyphi A. (A) Hydrogen peroxide assay after treatment with OP50 or S. Paratyphi A for 12, 24 or 48 hours (mean ± SD, * p < 0.05 and ** p < 0.01, Student’s t-test). (B) Relative mRNA levels of stress response-related genes lys-7, clec-174, clec-218, clec-258, clec-85 and (C) ctl-1, ctl-3, asp-12, abf-2, abf-3, clec-186, dbl-1, clec-86, C32H11.4, ugt-63, spp-1, hsp-70 and cyp-35A2 were analyzed by quantitative RT–PCR (* p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed Student’s t-test). (D) Model of the interaction between S. Paratyphi A and C. elegans.