BMP signaling to pharyngeal muscle in the C. elegans response to a bacterial pathogen regulates anti-microbial peptide expression and pharyngeal pumping

Abstract

Host response to pathogens recruits multiple tissues in part through conserved cell signaling pathways. In Caenorhabditis elegans, the bone morphogenetic protein (BMP) like DBL-1 signaling pathway has a role in the response to infection in addition to other roles in development and postdevelopmental functions. In the regulation of body size, the DBL-1 pathway acts through cell autonomous signal activation in the epidermis (hypodermis). We have now elucidated the tissues that respond to DBL-1 signaling upon exposure to two bacterial pathogens. The receptors and Smad signal transducers for DBL-1 are expressed in pharyngeal muscle, intestine, and epidermis. We demonstrate that expression of receptor-regulated Smad (R-Smad) gene sma-3 in the pharynx is sufficient to improve the impaired survival phenotype of sma-3 mutants and that expression of sma-3 in the intestine has no effect when exposing worms to bacterial infection of the intestine. We also show that two antimicrobial peptide genes - abf-2 and cnc-2 - are regulated by DBL-1 signaling through R-Smad SMA-3 activity in the pharynx. Finally, we show that pharyngeal pumping activity is reduced in sma-3 mutants and that other pharynx-defective mutants also have reduced survival on a bacterial pathogen. Our results identify the pharynx as a tissue that responds to BMP signaling to coordinate a systemic response to bacterial pathogens.

FIGURE 1:

DBL-1 Pathway R-Smad SMA-3 is required for survival on bacterial pathogen. (A) BMP signaling in C. elegans through the DBL-1 pathway. (B) Survival of sma-3(wk30) on S. marcescens. n values: Control (99), sma-3 (55). (C) Survival of sma-3(wk30) on P. luminescens. n values: Control (67), sma-3 (63). Survival analyses were repeated and results were consistent across trials. Statistical analysis was done using Log-rank (Mantel-Cox) Test. *** P ≤ 0.001; **** P < 0.0001.

FIGURE 2:

Expression of sma-3(+) in pharyngeal muscle rescues survival of sma-3 mutants. (A) Survival analysis of tissue-specific sma-3 expressing strains on S. marcescens. Tissue-specific strains have sma-3 expressed under the regulation of tissue-specific promotors in a sma-3(wk30) mutant background: vha-6p for intestinal expression and myo-2p for pharyngeal expression. n values: Control (47), sma-3 (45) Intestine (58), Pharynx (41). (B) Survival of tissue-specific sma-3 expressing strains on P. luminescens. n values: Control (102), sma-3 (82), Intestine (134), Pharynx (131). Survival analyses were repeated and results were consistent across trials. Statistical analysis was done using Log-rank (Mantel-Cox) Test. ns P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P < 0.0001. Black asterisks show significance relative to control; magenta shows significance relative to sma-3 mutant.

FIGURE 3:

DBL-1 regulates expression of AMPs abf-2 and cnc-2 through SMA-3. (A) qRT-PCR results for abf-2 and cnc-2 expression in dbl-1(wk70) animals on control bacteria and P. luminescens. (B) qRT-PCR results for abf-2 and cnc-2 in sma-3(wk30) animals on control bacteria and P. luminescens. (C) Survival analysis of cnc-2(ok3226) animals on P. luminescens pathogenic bacteria. n values: Control (91), cnc-2 (74). Survival analysis was repeated and results were consistent across trials. Data for qRT-PCR represents repeated analyses from two biological replicates. Statistical analysis for qRT-PCR was done using One-way ANOVA with Šídák’s multiple comparison test using GraphPad Prism 9. Error bars represent SD. Statistical analysis for survival was done using Log-rank (Mantel-Cox) Test. ns P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; **** P < 0.0001.

FIGURE 4:

Pharyngeal expression of SMA-3 mediates regulation of AMP expression. (A) qRT-PCR results for abf-2 in transgenic sma-3 expressing animals on control bacteria and P. luminescens. (B) qRT-PCR results for cnc-2 in transgenic sma-3 expressing animals on control bacteria and P. luminescens. Data for qRT-PCR represents repeated analyses from two biological replicates. Statistical analysis for qRT-PCR was done using One-way ANOVA with Šídák’s multiple comparison test, using GraphPad Prism 9. Error bars represent SD. ns P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P < 0.001.

FIGURE 5:

SMA-3 controls pharyngeal pumping rate cell autonomously. (A.) Pumping rate in pumps/20 Seconds of sma-3(wk30) mutants and pharyngeal sma-3 rescue animals on P. luminescens bacteria. (B.) Pumping rate in pumps/20 s of sma-3(wk30) mutants and on E. coli bacteria. Ten worms per strain were counted and experiments were repeated on two to three separate biological replicates. Results were consistent across all trials. Statistical analysis was performed using One way ANOVA with Šídák’s multiple comparison test, using GraphPad Prism 9. ns P > 0.05; **** P ≤ 0.0001. C–F. L4 animals of the designated genotypes were exposed to fluorescent OP50-GFP E. coli bacteria for 24 h and 10 or more animals imaged as Day 1 adults. The experiment was repeated in triplicate. All scale bars show 100 µm.

FIGURE 6:

Mutants with pharyngeal defects have reduced survival on P. luminescens bacteria. (A–B) Pharyngeal pumping rate in pumps/20 s for pharyngeal defect mutants phm-2(ad597) and eat-2(ad1116) on P. luminescens bacteria. Ten worms per strain were counted and experiments were repeated on two separate biological replicates. Results were consistent across all trials. Statistical analysis was performed using One way ANOVA with Šídák’s multiple comparison test, using GraphPad Prism 9. (C–D) Survival analysis of phm-2(ad597) and eat-2(ad1116) on P. luminescens bacteria. N values: Control (81), phm-2 (99), Control (96), eat-2 (58). Statistical analysis was done using Log-rank (Mantel-Cox) Test. **** P ≤ 0.0001.