Dynamic regulation of extracellular adenosine triphosphate in Serratia marcescens

TUTTOBENE, MARISEL ROMINA; SCHACHTER, JULIETA; ALVAREZ, CORA L.; VESCOVI, ELEONORA GARCÍA; SCHWARZBAUM, PABLO JULIO

Los Cocos

Congreso; SAMIGE 2022; 2022

Serratia marcescens is a highly ubiquitous Gram-negative enteric bacterium that can be isolated from most abiotic environmental sources, as well as from plants, insects, and nematodes. In the clinical setting, S. marcescens is the cause of urinary tract, respiratory, wound, ocular, cardiac, bloodstream, and surgical infections, mostly affecting intensive care unit patients. In 2017, the World Health Organization declared S. marcescens, along with other Enterobacteriaceae, a priority research target to develop alternative antimicrobial strategies given the high frequency of clinical isolates resistant to carbapenems.In our previous work, we have demonstrated that S. marcescens is able to be internalized by nonphagocytic cells. We showed that, once inside the cell, Serratia is able to inhabit and proliferate inside large membrane-bound compartments. These vesicles exhibit autophagic-like features, as they acquire markers typically recruited throughout the progression of autophagosome biogenesis in the antibacterial process. Serratia maneuvers the normal progression of host cell traffic, and this contribute to explaining the potential for Serratia to establish infection and persist in the host. In addition, ShlA, a pore-forming toxin, is responsible for inducing autophagy in nonphagocytic CHO epithelial cells, previous to the internalization process. In this study we seek to analyze the role of extracellular ATP (eATP), on Serratia dependent autophagy of CHO cells, a mammalian cell model. The CHO cell possesses two families of nucleotide receptors, metabotropic (P2Y) and ionotropic (P2X), with various subtypes displaying high affinity for eATP. Preincubation of CHO cells with an excess of apyrase (an ATP diphosphohydrolase, to remove eATP) or with the P receptor blocker suramin inhibited by ∼ 50% the autophagic response induced by ShlA. P2X blockers did not have any effect, indicating that a potential effect of eATP might be P2Y mediated.To estimate ATP release from CHO cells, the kinetics of eATP accumulation was quantified by real time luminometry. CHO cells were exposed to wild-type S. marcescens, to the shlA mutant strain, and to a E. coli overexpressing the toxin. Exposure of CHO cells to wild-type S. marcescens, as well as to E. coli-ShlA, promoted a 3-fold increments of [eATP], while no ATP release was detected in the shlA mutant strain. Hydrolysis of eATP by nucleotidases of CHO cells and those of S. marcescens and E. coli were very low and therefore did not affect the experimental eATP kinetics. Results indicate that trigger of the autophagy response depends on Serratia ShlA, which promotes ATP release from CHO cells, and the resulting eATP acting on P2Y receptors of the target cell.