Src-dependent tyrosine-phosphorylation of NM2A has a protective role against bacterial pore-forming toxins

Pore-forming toxins (PFTs) are key bacterial virulence factors that disrupt host plasma membrane (PM) integrity, triggering cellular stress and initiating repair mechanisms. The cytolysin Listeriolysin O (LLO), secreted by Listeria monocytogenes, has well established roles in infection, yet the host signaling responses to LLO-induced damage remain poorly understood. Here, we identify a previously unrecognized protective pathway in which LLO triggers rapid activation of the tyrosine kinase Src, leading to phosphorylation of the non-muscle myosin II heavy chain NMHC2A at tyrosine 158. While Src activation and NMHC2A tyrosine phosphorylation have been observed during Listeria infection, we demonstrate here that both responses are directly driven by LLO. This phosphorylation event does not alter NMHC2A motor activity in vitro but is required for cytoskeletal reorganization and efficient responses to PM damage. Using Caenorhabditis elegans, we further show that phosphorylation of the NMHC2A homolog NMY-2 at the conserved tyrosine 163 is required for survival under PFT-induced stress and heat shock, revealing an evolutionarily conserved defense mechanism. Together, our findings establish Src-mediated NMHC2A phosphorylation as a critical link between PFT-induced PM damage sensing and actomyosin remodeling, advancing our understanding of host defense against bacterial toxins. Author SummaryPathogenic bacteria often produce toxins that create pores in the membranes of host cells, compromising cell survival and helping the infection spread. To counter this, host cells must quickly detect and repair the damage. We studied how cells respond to Listeriolysin O (LLO), produced by the human pathogenic bacterium Listeria monocytogenes. We found that LLO activates the host Src kinase, which in turn modifies a key structural protein, non-muscle myosin II (NMII). Although this modification does not affect NMIIs normal motor function, it is crucial for organizing the cellular skeleton and protecting the cells membrane after toxin damage. Importantly, we found that this protective mechanism is conserved in the worm Caenorhabditis elegans, where it also defends against a similar bacterial toxin and even heat stress. Our findings reveal a new way that cells use existing structural proteins to rapidly respond to external threats, providing insight into how organisms protect themselves from harmful microbes and environmental stress.