The key role of W578 in the hemolytic process of E.coli alpha hemolysin

ROMINA VAZQUEZ; SABINA MATÉ; LAURA BAKÁS; VANESA HERLAX

San Javier, Tucumán

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica (SAB); 2012

Sociedad Argentina de Biofísica

Alpha-hemolysin is a major virulence factor of E.coli strains that causes 50% of extraintestinal infections in humans. It is a member of the RTX toxin family, which shares several functional and genetic features. HlyA contains four tryptophans (W) in its sequence at positions: 431, 479, 578 and 913. Previous results demonstrated that oxidation of HlyA´s Ws by N-bromosuccinimide (NBS), leads to inactivation of the toxin. This result suggests that Ws may play a critical role in the lytic mechanism of HlyA. In order to study this thoroughly we analyzed the hemolytic behavior of four mutant proteins where one of the four Ws was mutated by cysteine (C) (W431C, W479C, W578C and W913C). Only W578C mutant protein is hemolytically inactive. Three stages seem to be involved that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin in the membrane. Experiments presented in this work pointed out the study of each steps, in order to investigate the reason of the inactivity of W578C. The binding percentage of proteins to membranes was not affected by the absence of W578. Instead irreversibility of the binding was affected, since W578C binds reversibly to membranes as ProHlyA, the unacylated and inactive toxin; while the other mutant proteins bind irreversibly like HlyA. The oligomerization process was studied by FRET, demonstrating that W578C oligomerizases on membrane as Wt. The insertion step was studied by the monolayer technique. Our results demonstrated that in a DOPC monolayer W578C inserts more than the Wt toxin. Instead, in monolayer containing cholesterol this mutant protein practically does not insert into it, while the HlyA insertion is increased. Recently, we have found that HlyA interacts specifically with cholesterol (Cho). In this context we performed a lipid dot blot assay with a concentration range of Cho for HlyA and W578C. Results showed that W578C binds in a lesser extent to Cho than the wild type toxin. Taking all results into account, we can conclude that W578 might be involved in the interaction with cholesterol. This interaction might facilitate the adequate conformational change of the protein that induces the correct insertion of the toxin into membranes, finally forming the oligomer that leads to the lysis of the cell.