CONICET | Buscador de Institutos y Recursos Humanos

Microbial toxins that act on eukaryotic cells would have some kind of interaction with membranes containing cholesterol. The effects that cholesterol might have on toxin activity can be: either a specific interaction with cholesterol or a modulation of toxin activity. HlyA is one of the key virulence factors released by invading E. coli strains. It causes lysis of various mammalian cells, including erythrocytes of different animal species. Several previous results suggested a role of cholesterol in the mechanism of action of HlyA, such as: HlyA oligomerizases on membrane microdomains which are enriched in sphingomyelin and cholesterol; the leakage of fluorescent probes from DOPC/Cho liposomes is higher compared to liposomes composed of DOPC only. Also, oligomerization and hemolytic activity decreases in ghost erythrocytes cholesterol depleted by methyl- β-cyclodextrin. The aim of our work was to study the role of cholesterol in the lytic mechanism of α-hemolysin of E. coli. Dot blot experiments demonstrated that HlyA interacts specifically with cholesterol and does not interact with sphingomyelin nor phosphatidylcholine. Using the monolayer technique we demonstrated that HlyA induces a bigger change in the surface pressure of a DOPC:Cho:SM monolayer than in one composed of only DOPC. This indicates that HlyA largely inserts in a monolayer containing cholesterol than in the one that is devoid of it. On the other hand, an inactive single mutant protein W578C, which binds in a lesser extent to Cho than to the wild type toxin, does not insert in the monolayer composed of DOPC:Cho:SM, but it inserts in a major extent than HlyA in a DOPC one. All together these results suggested that cholesterol allows the stabilization of the monomer in order to favor a proper conformation to form an active oligomer (pore). W 578 might be involved in this interaction.