Transfer of Escherichia coli alpha- Hemolysin from outer membrane vesicles to erythrocytes

VANESA HERLAX; M. FLORENCIA HENNING; A.M BERNASCONI; LAURA BAKÁS

Montevideo, Uruguay

Congreso; 6th Internacional Conference of Biological Physics, 5 th Southern Cone Biophysical Congress and 34 th Annual Meeting of the Argentinean Biophysical Society; 2007

ICBP y SAB

Alpha-Hemolysin (HlyA) is an extracellular protein toxin (110 kDa) secreted by E. coli, targeting plasma membranes of eukaryotic cells. Although the secretion mechanism of HlyA mediated by the type I secretion system  has been thoroughly analyzed, recently it was found that this toxin is also released to external media associated with bacterial outer membrane derived vesicles (OMVs), but the lytic action mechanism of the toxin in this form has not been studied yet. OMVs purified from the culture filtrate were negatively stained and then examined by electron microscopy. Many bilayered spherical vesicles with diameter ranging from 50 to 200 nm were observed. The presence of HlyA in OMVs produced by E. coli WAM 1824 was confirmed by Western blot analysis employing polyclonal antibodies directed against HlyA. Our results confirm that HlyA is the only protein present on OMVs responsible of the lysis of red blood cells. The specific hemolytic activity was approximately 100 fold higher than purified free HlyA showing the same relative lysis efficiency and specificity for red blood cells from different species (rabbit and horse) In order to determine if the mechanism of hemolytic action of OMVs associated- HlyA implies a membrane fusion we analyzed the lipid mixing between OMVs and LUV (POPC/PE/ CHOL) labeled with NBD-PE (donor) and Rh-PE (acceptor). Our results show that fusion event is not involved in the lytic mechanism of OMVs-HlyA as previously found regarding free-HlyA. Instead a transfer of HlyA from OMV´s to red blood cells occurs demonstrated by western blot. In conclusion, OMVs could be an important auxiliary way of secretion, acting mainly as a concentration mechanism of HlyA near the target cells lysing them by  toxin transfer  from OMVs to target cell membranes.