New insights into the mechanism of action of E. coli alpha hemolysin: Cholesterol as a modulator of toxin activity?

ROMINA F. VAZQUEZ; SABINA M. MATÉ; LAURA S. BAKÁS; MARISA M. FERNÁNDEZ; EMILIO L. MALCHIODI; VANESA S. HERLAX

Guarujá, San Pablo

Congreso; XI Congress of the Pan- American Section of the International Society on Toxinology and XII Congress of the Brazilian Society of Toxinology; 2013

International Society on Toxinology

Introduction: Alpha hemolysin (HlyA) is a 110 kDa protein toxin considered as the prototype of the RTX toxin family and represents an important virulence factor of many uropathogenic E. coli strains. It is synthesized as a protoxin and needs to be activated in bacterial cytosol to the active form by acylation at two internal lysine residues. HlyA is then secreted as a soluble protein across both membranes by the type I secretion system. HlyA has a wide range of host cells, acting on a variety of cell types from several species ?e. g., red blood cells, fibroblasts, granulocytes, lymphocytes, and macrophages? and also binds to and disrupts protein-free liposomes. HlyA forms highly dynamic pores of a proteolipidic nature since conductance and membrane lifetime are dependent on membrane composition. Many previous results suggest a possible role of cholesterol (Cho) in the mechanism of action of this toxin. Given that different steps are involved in the assembly of HlyA pores, in this work we have analyzed to what extent Cho can modulate the changes associated with HlyA binding, insertion, oligomerization and pore formation. Methods and results: Leakage experiments using large unilamellar vesicles (LUVs) of different lipid composition show that although HlyA can provoke the leakage of liposomes content composed solely of phophatidylcholine (PC), the leakage percentage is increased in Cho-containing ones. Lysis inhibition assays demonstrate that pretreatment with Cho inhibits hemolytic activity of HlyA. In this way, HlyA-lipid interactions were explored by a lipid dot-blot assay, indicating that HlyA interacts preferentially with Cho. This interaction was further confirmed by Surface Plasmon Resonance (SPR) measurements. Despite this, pretreatment with Cho seems not to affect HlyA binding to erythrocyte ghosts. Regarding the insertion process, studies using the monolayer technique indicate that HlyA inserts in a bigger extent in Cho-containing monolayers. Conclusion: We can conclude from these results that HlyA interacts specifically with Cho, facilitating the insertion of the toxin into membranes. The interaction with Cho, an essential component of all animal cell membranes, may favor a conformational state of the protein that allows the correct insertion and further oligomerization that leads to pore formation, enhancing the hemolytic activity of HlyA.