Interaction of acylated and unacylated forms of E. coli alpha-hemolysin with lipid monolayers: a PM-IRRAS study.

Uropathogenic strains of Escherichia coli produce virulence factors, such as the protein toxin alpha-hemolysin (HlyA), that enable the bacteria to colonize the host and establish an infection. HlyA is synthetized as a protoxin (ProHlyA) that is transformed into the active form in the bacterial cytosol by the covalent linkage of two fatty-acyl moieties to the polypeptide chain before the secretion of HlyA into the extracellular medium. The aim of this work was to investigate the effect of the fatty acylation of HlyA on protein conformation and protein-membrane interactions. Polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) experiments were performed at the air-water interface, and lipid monolayers mimicking the outer leaflet of red-blood-cell membranes were used as model systems for the study of protein-membrane interaction. According to surface-pressure measurements, incorporation of the acylated protein into the lipid films was faster than that of the nonacylated form. PM-IRRAS measurements revealed that the adsorption of the proteins to the lipid monolayers induced disorder in the lipid acyl chains and also changed the elastic properties of the films independently of protein acylation. No significant difference was observed between HlyA and ProHlyA in the interaction with the model lipid monolayers; but when these proteins became adsorbed on a bare air-water interface, they adopted different secondary structures. The assumption of the correct protein conformation at a hydrophobic-hydrophilic interface could constitute a critical condition for biologic activity.

Culture supernatants from V. cholerae O1 ElTor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity / Cepas de V. cholerae O1 biotipo ElTor aisladas de diferente origen geográfico inducen vacuolización celular y citotoxicidad

OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

OBJETIVO: Analizar el efecto vacuolizante de cepas de V. cholerae O1 ElTor aisladas de diferente origen geográfico, incluyendo México. MATERIAL Y MÉTODOS: Se realizaron pruebas de hemolisis, vacuolización y citotoxicidad en células Vero, así como PCR, análisis por RFLP y clonación molecular. RESULTADOS: Todas las cepas indujeron el efecto vacuolizante. Las cepas del ribotipo 2, aisladas de las costas del Golfo en Estados Unidos, presentaron títulos altos de vacuolización. El gen hlyA fue amplificado en las nueve cepas mediante PCR, aunque sólo ocho fueron hemolíticas. Se clonó el gen hlyA de una cepa toxigénica (2514-88, ctxAB+) y de una cepa no toxigénica aislada en México (CM 91-3, ctxAB-). El sobrenadante de las clonas recombinantes indujo hemólisis, efecto vacuolizante y citotoxicidad. El RFLP mostró alta similitud del gen hlyA de las cepas estudiadas. CONCLUSIÓN: El efecto vacuolizante es un fenotipo ampliamente distribuido en cepas epidémicas de V. cholerae O1 biotipo ElTor.