Probiotic-mediated p38 MAPK immune signaling prolongs the survival of Caenorhabditis elegans exposed to pathogenic bacteria

Abstract

The host-microbiota cross-talk represents an important factor contributing to innate immune response and host resistance during infection. It has been shown that probiotic lactobacilli exhibit the ability to modulate innate immunity and enhance pathogen elimination. Here we showed that heat-inactivated probiotic strain Lactobacillus curvatus BGMK2-41 stimulates immune response and resistance of the Caenorhabditis elegans against Staphylococcus aureus and Pseudomonas aeruginosa. By employing qRT-PCR and western blot analysis we showed that heat-inactivated BGMK2-41 activated PMK-1/p38 MAPK immunity pathway which prolongs the survival of C. elegans exposed to pathogenic bacteria in nematode killing assays. The C. elegans pmk-1 mutant was used to demonstrate a mechanistic basis for the antimicrobial potential of BGMK2-41, showing that BGMK2-41 upregulated PMK-1/p38 MAPK dependent transcription of C-type lectins, lysozymes and tight junction protein CLC-1. Overall, this study suggests that PMK-1/p38 MAPK-dependent immune regulation by BGMK2-41 is essential for probiotic-mediated C. elegans protection against gram-positive and gram-negative bacteria and could be further explored for development of probiotics with the potential to increase resistance of the host towards pathogens.

Figure 1

Heat-inactivated Lactobacillus curvatus BGMK2-41 prolongs the survival of C. elegans exposed to pathogenic bacteria. (A) An overview of experimental approach for evaluation of BGMK2-41 effects on infected worms’ lifespan and gene/protein. Survival curve of WT animals exposed to (B) S. aureus ATCC 25923 and (C) P. aeruginosa PA14 after overnight feeding of L4 stage worms with heat-inactivated control (E. coli OP50) or BGMK2-41 bacteria (n = 25–30 worms per group, results are representative of 2 independent assays). CFUs level of (D) S. aureus ATCC 25923 and (E) P. aeruginosa PA14 present in the BGMK2-41 pre-treated worms after 1 day of exposure to pathogens (n = 10 worms per group, results are representative of 2 independent assays). All values are presented as mean ± SD and the student’s t-test was used to compare the treated group relative to control. The log-rank (Mantel-Cox) test was used to assess the p-value in nematode killing assays (***P < 0.001). The statistical analysis and graphs were done in GraphPad Prism version 8.0.0 for Mac, GraphPad Software, www.graphpad.com. (A) was designed by using free trial of Adobe Illustrator, www.adobe.com/products/illustrator.

Figure 2

Heat-inactivated Lactobacillus curvatus BGMK2-41 stimulates a canonical p38MAPK immune pathway in C. elegans. (A) Expression of immune-related genes measured by qRT-PCR in WT animals after 24 h of treatment with heat-inactivated BGMK2-41 relative to heat-inactivated E. coli OP50 control. (B) Representative western blot with densitometric analysis showing the levels of phospho-p38 MAPK isolated from WT animals after overnight BGMK2-41 treatment. HSC-70 was used as a loading control. (C) Levels of tol-1 mRNA measured in day 1 old WT worms treated with BGMK2-41. All results were obtained from three independent experiments and the data are presented as mean ± SD and student’s t-test was used to compare the treated group relative to control (*P < 0.05, **P < 0.01). The statistical analysis and graphs were done in GraphPad Prism version 8.0.0 for Mac, GraphPad Software, www.graphpad.com.

Figure 3

The p38 MAPK deficiency abrogates protective effects of Lactobacillus curvatus BGMK2-41. Survival curve of pmk-1 loss-of-function animals exposed to (A) S. aureus ATCC 25923 and (B) P. aeruginosa PA14 after overnight feeding of L4 stage worms with heat-inactivated control (E. coli OP50) or BGMK2-41 bacteria (n = 25–30 worms per group, results are representative of 2 independent assays). (C) Expression of immune-related genes analyzed by qRT-PCR in the L4 stage of the pmk-1 mutant after overnight treatment with heat-inactivated BGMK2-41 (n = 3, three independent experiments). CFUs values of (D) S. aureus ATCC 25923 and (E) P. aeruginosa PA14 present in the pmk-1 deficient BGMK2-41 treated worms after 1 day of exposure to pathogens (n = 10 worms per group, results are representative of 2 independent assays). (F) Tight junction barrier assessment by determination of clc-1 mRNA levels in day 1 old WT and pmk-1 worms treated with heat-inactivated BGMK2-41 (results from three independent experiments). All values are presented as mean ± SD. Student’s t-test was used to compare the treated group relative to control. The log-rank (Mantel-Cox) test was used to assess the p-value in nematode killing assays (*P < 0.05, **P < 0.01, ***P < 0.001). The statistical analysis and graphs were done in GraphPad Prism version 8.0.0 for Mac, GraphPad Software, www.graphpad.com.

Figure 4

Heat-inactivated Lactobacillus curvatus BGMK2-41 controls the expression of antimicrobial proteins via p38 MAPK. Expression of (A) antimicrobial peptides, (B) C-type lectins and (C) lysozymes measured by qRT-PCR in day 1 old WT and pmk-1 worms treated with heat-inactivated BGMK2-41 (results from three independent experiments). All values are presented as mean ± SD. Student’s t-test was used to compare the treated group relative to control (*P < 0.05, **P < 0.01, ***P < 0.001). The statistical analysis and graphs were done in GraphPad Prism version 8.0.0 for Mac, GraphPad Software, www.graphpad.com.