INIBIOLP   05426
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE LA PLATA "PROF. DR. RODOLFO R. BRENNER"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Study on lytic action mechanism of Escherichia coli a-hemolysin associated to outer membrane vesicles
Autor/es:
VANESA HERLAX; M. FLORENCIA HENNING; LAURA BAKAS
Lugar:
Madrid, España
Reunión:
Congreso; Congreso Iberoamericano; 2006
Resumen:
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. In conclusions, OMVs could be an important auxiliary way of secretion, acting mainly as a concentration mechanism of  HlyA near the target cells  lysing them  by transference of  the toxin from OMVs to target cell membranes . Because OMVs associated-HlyA represents a very high percentage of the toxin present in the culture supernatants, a revision on the results published up to date, mainly at  sublytic concentration where small amounts of LPS can stimulate the release of inflammatory mediators by mononuclear phagocytes  should be done .