p38-MAPK recruits the proteolytic pathways in Caenorhabditis elegans during bacterial infection

In eukaryotic organisms, cell-signalling completely relies on Post Translational Modifications (PTMs) that can function as regulatory switches. Phosphorylation is a fundamental and frequently occurring PTM in almost all eukaryotes. Herein, we have studied the importance of protein phosphorylation using classical proteomic techniques in C. elegans upon bacterial infection. The differentially regulated proteins during bacterial infection were excised from SDS-PAGE (One-Dimensional) gel (TiO2 column elutes) and subjected to MALDI-TOF-MS which ended up in identifying 220 proteins kinetically. KEGG pathway analysis of those proteins suggested that MAPK pathway was part of the innate immunity. Thus, we have characterized p38-MAPK (one of the crucial downstream MAPKs) using immunoblotting, subcellular fractionation, coimmunoprecipitation, LC-MS/MS, bioinformatics studies and qPCR. Meanwhile, KU25 strain (pmk-1 mutant) exhibited an earlier mortality during infection suggesting the crucial role of p38-MAPK during host-pathogen interaction. Interestingly, Reactome pathway analysis of p38 interactors (CoIP coupled to LC-MS/MS) revealed the involvement of various proteolytic pathway players (ubiquitination, SUMOylation and Neddylation) during bacterial infection. Further, the regulation of SUMOylation and Neddylation was identified and validated using immunoblotting and qPCR analyses, respectively. Concisely, our study indicated that bacterial infection triggers the MAPK cascade to elicit innate immunity which in turn recruits proteolytic pathways to counteract the invading pathogen.

Automated summary

In eukaryotic organisms, cell signalling relies on post-translational modifications, and phosphorylation is a common and important modification. In this study, the importance of protein phosphorylation was investigated in C. elegans during bacterial infection. Proteomic techniques were used to identify differentially regulated proteins, and pathway analysis suggested the involvement of the MAPK pathway in innate immunity. The p38-MAPK protein, a crucial downstream component of the MAPK pathway, was characterized using various techniques. The study also revealed the involvement of proteolytic pathways in response to bacterial infection. Overall, the findings suggest that bacterial infection triggers the MAPK cascade to activate innate immunity and recruit proteolytic pathways.