Relevance of Fatty Acid Covalently Bound to Escherichia coli α-Hemolysin and Membrane Microdomains in the Oligomerization Process

Abstract

␣-Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of Escherichia coli that causes lysis of several mammalian cells, including erythrocytes of different species. HlyA is synthesized as a protoxin, pro-HlyA, which is activated by acylation at two internal lysines Lys-563 and Lys-689. It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes, but these experiments have yielded conflicting results about the structure of the pore. In this study, HlyA cysteine replacement mutant proteins of amino acids have been labeled with Alexa-488 and Alexa-546. Fluorescence resonance energy transfer measurements, employing labeled toxin bound to sheep ghost erythrocytes, have demonstrated that HlyA oligomerizes on erythrocyte membranes. As the cytotoxic activity is absolutely dependent on acylation, we have studied the role of acylation in the oligomerization, demonstrating that fatty acids are essential in this process. On the other hand, fluorescence resonance energy transfer and the hemolytic activity decrease when the erythrocyte ghosts are cholesterol-depleted, hence indicating the role of membrane microdomains in the clustering of HlyA. Simultaneously, HlyA was found in detergent-resistant membranes. Pro-HlyA has also been found in detergent-resistant membranes, thus demonstrating that the importance of acyl chains in toxin oligomerization is the promotion of protein-protein interaction. These results change the concept of the main role assigned to acyl chain in the targeting of proteins to membrane microdomains.

E. coli Alpha Hemolysin and Properties

“…This circumstance obtains for HlyA, where an oligomer was found at lytic concentrations in sheep-erythrocyte ghosts. In contrast, no oligomeric structure was found for proHlyA (Herlax et al, 2009). …”

“…To carry out this study, two populations of HlyA K344C mutant proteins, one labelled with donor (Alexa-488) and the other with acceptor fluorophores (Alexa-546), were bound to sheep-erythrocyte ghosts. Our report showed that an oligomer was involved in the hemolytic mechanism of HlyA (Herlax et al, 2009). FRET can be used to study the distribution of molecules in membranes because the average spacing between molecules of interest will depend primarily on their lateral arrangement.…”

“…Interestingly, such proteins are linked to saturated acyl chains, which species partition well into those domains (Pike, 2003). Although covalently bound fatty acids had not been implicated in the targeting of HlyA to membranes, their involvement in the targeting to membrane microdomains was studied (Herlax et al, 2009). For this purpose-and taking into account that these microdomains are enriched in cholesterol and sphingolipids-the hemolytic activity of the toxin on sheep erythrocytes was compared with the activity on cholesterol-depleted erythrocytes.…”

“…We previously found that the fatty acids covalently bound to the toxin induce conformational changes that expose intrinsically disordered regions so as to promote protein-protein interactions. Thus, the oligomerization process of the toxin is facilitated by microdomains within the membrane (Herlax & Bakas, 2007), (Herlax et al, 2009). …”

“…Fatty acids expose specific regions that induce protein-protein interaction in the oligomerization process that takes place within the membrane microdomains of erythrocytes. The irreversibility of the toxin's membrane binding promoted by fatty acids might result from the formation of the HlyA oligomeric structure (Herlax & Bakas, 2003, (Herlax et al, 2009). …”