EXOTOXINS HLYA AND SHLA CONTROL CYTOTOXIC PHENOTYPES OF TARGET EUKARYOTIC CELLS THROUGH PURINERGIC SIGNALING

TUTTOBENE, M.R.; DENIS, MARÍA FLORENCIA LEAL; ALVAREZ CORA LILIA; SCHACHTER JULIETA; SAFFIOTI, NICOLÁS; LAURI NATALIA; OSTUNI MARIANO ANIBAL; HERLAX, VANESA; GARCÍA-VÉSCOVI, ELEONORA; SCHWARZBAUM, PABLO JULIO

Mar del Plata

Congreso; REUNIÓN CONJUNTA SAIC SAB AAFE AACYTAL; 2023

SAIC, SAB, AAFE, AACYTAL

Aim: we characterized the effects of exotoxins from gram(-) bacteria,i.e., HlyA from uropathogenic strains of Escherichia coli and ShlAfrom Serratia marcescens, on eukaryotic cell models. Focus wasgiven to the capacity of these toxins to induce intracellular ATP re-lease from target cells, and consequent purinergic signaling. Humanerythrocytes (RBCs) were exposed to HlyA, while CHO (chinesehamster ovary) cells were exposed to ShlA. Methods: luciferin-lu-ciferase luminometry was used to quantify extracellular ATP (eATP).Electrical impedance was used to quantify cell volume of RBCs.Autophagy in CHO cells was revealed by an EGFP-LC3 green fluo-rescent punctate pattern. Significance level at P<0.05. Results: bothexposure of RBCs to HlyA, and of CHO cells to ShlA, induced a sig-nificant increase of [eATP] (3-40 fold over 40 min exposure), indic-ative of intracellular ATP release from target cells. This release waspartially inhibited (40-70%) by carbenoxolone and mefloquine, twoinhibitors of the hemichannel pannexin 1. Toxin dependent eATP ac-cumulation was partially opposed by nucleotide hydrolysis of endog-enous ectonucleotidases from target cells. In HlyA treated RBCs,eATP activated P2X receptors leading to swelling (1.5-fold), whileosmotically blocking this swelling reduced ATP release by 80%. InShlA treated CHO cells, eATP activated a P2Y2 receptor, that in turntransactivated α5β1, leading to autophagy. Conclusions: exposureof exotoxins to target cells led to intracellular ATP release from tar-get cells, partially mediated by pannexin 1. The resulting eATP, inspite of eATP hydrolysis by ectonucleotidases, was capable of acti-vating purinergic signaling, so as to: 1 (for HlyA)- induce an osmoticimbalance leading to swelling in RBCs, a prerequisite for hemolysis;2 (for ShlA)- induce P2Y2-α5β1 integrin activation, a prerequisitefor autophagy in CHO cells. In the absence of purinergic signaling,the cytotoxic phenotypes induced by the toxins are highly reduced.