Imaging the interaction of Escherichia coli alpha-hemolysin with model membranes

SABINA M. MATÉ; ROMINA F. VAZQUEZ; MARÍA A. DAZA MILLONE; FELIPPE PAVINATTO; VANESA S. HERLAX; OSVALDO N. OLIVEIRA JR; MARÍA E. VELA

La Plata

Workshop; Imaging Techniques for Biotechnology and Biomedical Applications; 2016

Uropathogenic strains of Escherichia coli produce different virulence factors that enable the bacteria to colonize, break the host defense barriers, invade and disseminate, causing severe infections. One of the main virulence factors produced is the 110 kDa protein toxin alpha-hemolysin (HlyA) which is considered as the prototype member of the RTX toxin family of Gram negative bacteria. HlyA is synthetized as a protoxin (ProHlyA) that is transformed into the active form in the bacterial cytosol prior to its secretion. This post-translational modification consists in the amide-linkage of fatty-acyl moieties to the epsilon-amino groups of two internal lysine residues. Many mammalian cell types—including red blood cells, myeloid and lymphoid cells, renal epithelial cells, among others—are attacked by HlyA. The precise mechanism by which this acylated protein exerts its toxic effects still remains unclear. Notwithstanding, cell membranes constitute the primary targets of the toxin and, hence, protein-membrane interactions play a critical role in this process. The lytic activity of HlyA proceeds through a complex mechanism, and pore formation has been proposed to participate in the toxin's mechanism of cytolysis. Three stages seem to be involved that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin in the membrane. The mechanism of HlyA insertion in lipid bilayers is not fully known; but as we have previously reported, the insertion is highly dependent on the physical properties of the bilayer. As a first step in the study of HlyA–membrane interaction we examined the interaction of HlyA with lipid bilayers by AFM, which is a powerful tool for high-resolution imaging with nanometer resolution under physiological conditions. We provide the first direct visualization of the interaction of HlyA with supported lipid bilayers that mimic the composition of red blood cell membranes. Real-time AFM imaging demonstrated the occurrence of a preferential accumulation of the toxin into lipid-packing defects arising at the interfaces between coexisting lipid phases.As the next step we examined the interaction of HlyA and ProHlyA with lipid monolayers using BAM visualization and Polarization-modulation infrared reflection absorption spectroscopy (PM-IRRAS). These measurements revealed that the adsorption of HlyA and ProHlyA to the lipid monolayers produced an increase in the lipid acyl chains disorder. When pure proteins films were analyzed by PM-IRRAS, different secondary structure elements were found exposed at the air-water interface, being the entire HlyA polypeptide chain more extended than its unacylated counterpart. These differences in protein arrangement at a hydrophobic-hydrophilic interface could be an important factor for biological activity.