Why erythrocytes of different species present different susceptibility towards E.coli alpha-hemolysin ?

R. VAZQUEZ; V. HERLAX; S.MATÉ; C. WOLF; L.BAKÁS

Bilbao

Conferencia; 51st International conference on the bioscience of lipids; 2010

-Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of E. coli that causes lyses of several mammalian cells, including erythrocytes of different species which present different susceptibility toward the toxin. In this context, the aim of this work was to study the factors that determine the differences in this susceptibility. In this work, we evaluated the susceptibility of rabbit, horse and sheep erythrocytes. The hemolytic activity demonstrated that rabbit erythrocytes were the most susceptible to HlyA, followed by horse and sheep. Taking into account that oligomerization is a requirement for the hemolytic process, we studied the binding and the oligomerization of the toxin on ghost erythrocytes by fluorescence. The membrane fluidity was determined by Laurdan generalized polarization (GP) images taken in a two-photon fluorescence microscope and the lipid composition by HPTLC and LCMS. The results showed that the binding percentage of the toxin to erythrocytes of the three species is similar (approx. 30%). Instead, HlyA oligomerizes on all the erythrocytes, but surprisingly it oligomerizes more on rabbit erythrocytes, which are the only membranes that increase the GP value due to the interaction with the toxin. On the other hand, the lipid composition of the three species is very different. The main differences are in the content of phosphatidylcholine and sphingomyelin and their molecular species composition. In conclusion, the composition of the membrane determines the degree of oligomerization of the toxin resulting in different susceptibilities toward HlyA, despite the presence or not of a receptor. -Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of E. coli that causes lyses of several mammalian cells, including erythrocytes of different species which present different susceptibility toward the toxin. In this context, the aim of this work was to study the factors that determine the differences in this susceptibility. In this work, we evaluated the susceptibility of rabbit, horse and sheep erythrocytes. The hemolytic activity demonstrated that rabbit erythrocytes were the most susceptible to HlyA, followed by horse and sheep. Taking into account that oligomerization is a requirement for the hemolytic process, we studied the binding and the oligomerization of the toxin on ghost erythrocytes by fluorescence. The membrane fluidity was determined by Laurdan generalized polarization (GP) images taken in a two-photon fluorescence microscope and the lipid composition by HPTLC and LCMS. The results showed that the binding percentage of the toxin to erythrocytes of the three species is similar (approx. 30%). Instead, HlyA oligomerizes on all the erythrocytes, but surprisingly it oligomerizes more on rabbit erythrocytes, which are the only membranes that increase the GP value due to the interaction with the toxin. On the other hand, the lipid composition of the three species is very different. The main differences are in the content of phosphatidylcholine and sphingomyelin and their molecular species composition. In conclusion, the composition of the membrane determines the degree of oligomerization of the toxin resulting in different susceptibilities toward HlyA, despite the presence or not of a receptor.