Microvesicles released from erythrocytes treated with alpha hemolysin of E.coli

KAREN STRACK; VANESA HERLAX; SABINA MATÉ

Workshop; Imaging Techniques for Biotechnological and Biomedical Applications Workshop; 2016

Uropathogenic strains of E. coli deliver the toxin alpha-hemolysin (HlyA) to optimize the host environment for the spread of infection. It is synthesized as a protoxin (ProHlyA) and needs to be activated in bacterial cytosol to the active form by acylation at two internal lysine residues. Recently, we have demonstrated that at sublytic concentration HlyA induces the shape transition; discocyte-echinocyte-spherocyte1 and finally erytosis2 or lysis. Although ProHlyA is an inactive protein it also induces early morphologic shape transitions in erythrocytes1. This sequence of morphologic changes observed for HlyA is the same one occurring with amphiphiles that induce exovesicle formation. Within this context, we studied the release of microvesicles from human erythrocytes treated with sublytic concentrations of HlyA.HlyA and ProHlyA-treated erythrocytes were observed by Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). Images show that HlyA-treated erythrocyte present some vesicles around the cell, which has an echinocyte shape, while ProHlyA does not. Then microvesicles were purified by ultracentrifugation from the supernatant of HlyA-treated erythrocytes at two toxin concentrations. TEM images show that the sizes of the purified microvesicles are different depending on the toxin concentration. At 35 nM the population of microvesicles is heterogenous, showing a diameter ranging from 170 to 250 nm, instead at 70 nM this population is more homogenous presenting a diameter of 200-220 nm. Results indicate that human erythrocytes treated with sublytic concentration of HlyA induce the release of microvesicles. Actually we are studying if HlyA is delivered in these microvesicles as a mechanism of spread of the toxin in circulation.Referencias:1-Vázquez, R.F., et al. Biochim Biophys Acta 1858 (8): 1944-53, 2016.2-Carrizo Velasquez, F. et. al. Biochim Biophys Acta 1848: 2779?2788, 2015.