INIBIOLP   05426
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE LA PLATA "PROF. DR. RODOLFO R. BRENNER"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Imaging the interaction of Escherichia coli alpha-hemolysin with model membranes
Autor/es:
SABINA M. MATÉ; FELIPPE PAVINATTO; MARÍA E. VELA; MARÍA A. DAZA MILLONE; OSVALDO N. OLIVEIRA JR; ROMINA F. VAZQUEZ; VANESA S. HERLAX
Lugar:
La Plata
Reunión:
Workshop; Imaging Techniques for Biotechnology and Biomedical Applications; 2016
Resumen:
Uropathogenic strains of Escherichiacoli produce different virulence factors that enable the bacteria tocolonize, break the host defense barriers, invade and disseminate, causingsevere infections [1]. One of the main virulence factors produced is the 110 kDa proteintoxin alpha-hemolysin (HlyA) which is considered as the prototype member of theRTX toxin family of Gram negative bacteria. HlyA is synthetized as a protoxin(ProHlyA) that is transformed into the active form in the bacterial cytosolprior to its secretion. This post-translational modification consists in theamide-linkage of fatty-acyl moieties to the ε-amino groups of two internallysine residues. Many mammalian cell types?including red blood cells, myeloid andlymphoid cells, renal epithelial cells, among others?are attacked by HlyA. Theprecise mechanism by which this acylated protein exerts its toxic effects stillremains unclear. Notwithstanding, cell membranes constitute the primary targetsof the toxin and, hence, protein-membrane interactions play a critical role inthis process. The lytic activity of HlyA proceeds through a complexmechanism, and pore formation has been proposed to participate in the toxin's mechanismof cytolysis. Three stages seem to be involved that ultimately lead to celllysis: binding, insertion, and oligomerization of the toxin in the membrane.The mechanism of HlyA insertion in lipid bilayers is not fully known; but as wehave previously reported, the insertion is highly dependent on the physicalproperties of the bilayer [2]. As a firststep in the study of HlyA?membrane interaction we examined the interaction of HlyAwith lipid bilayers by AFM, which is a powerful tool for high-resolutionimaging with nanometer resolution under physiological conditions. We providethe first direct visualization of the interaction of HlyA with supported lipidbilayers that mimic the composition of red blood cell membranes. Real-time AFMimaging demonstrated the occurrence of a preferential accumulation of the toxininto lipid-packing defects arising at the interfaces between coexisting lipid phases[3].As the nextstep we examined the interaction of HlyA and ProHlyA with lipid monolayersusing BAM visualization and Polarization-modulation infrared reflectionabsorption spectroscopy (PM-IRRAS). These measurements revealed that theadsorption of HlyA and ProHlyA to the lipid monolayers produced an increase inthe lipid acyl chains disorder. When pure proteins films were analyzed byPM-IRRAS, different secondary structure elements were found exposed at theair-water interface, being the entire HlyA polypeptide chain more extended thanits unacylated counterpart. These differences in protein arrangement at ahydrophobic-hydrophilic interface could be an important factor for biologicalactivity. References [1]. Wiles, T.J.,R.R. Kulesus, and M.A. Mulvey, 2008, Exp Mol Pathol, 85(1): p. 11-9.[2]. Bakas, L., H. Ostolaza,W.L. Vaz, and F.M. Goni, 1996, Biophys J, 71(4):p. 1869-76.[3]. Maté, S.M., R.F.Vazquez, V.S. Herlax, M.A. Daza Millone, M.L. Fanani, B. Maggio, M.E. Vela, andL.S. Bakas, 2014, Biochim Biophys Acta, 1838(7):p. 1832-41.