RESUMEN
Vibrio vulnificus hemolysin (VVH) is thought to be a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins. To date, the structure-function relationships of CDCs produced by Gram-negative bacteria remain largely unknown. We show here that the aromatic ring of phenylalanine residue conserved in Vibrionaceae hemolysins is essential for oligomerization of VVH. We generated the VVH mutants; substituted Phe 334 for Ile (F334I), Ala (F334A), Tyr (F334Y), or Trp (F334W); and tested their binding and oligomerizing activity on Chinese hamster ovary cells. Binding in all mutants fell by approximately 50% compared with that in the wild type. Oligomerizing activities were completely eliminated in F334I and F334A mutants, whereas this ability was partially retained in F334Y and F334W mutants. These findings indicate that both hydrophobicity and an aromatic ring residue at the 334th position were needed for full binding activity and that the oligomerizing activity of this toxin was dependent on the existence of an aromatic ring residue at the 334th position. Our findings might help further understanding of the structure-and-function relationships in Vibrionaceae hemolysins.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Fenilalanina/química , Vibrio vulnificus/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Células CHO/efectos de los fármacos , Cricetinae , Cricetulus , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacología , L-Lactato Deshidrogenasa/metabolismo , Mutagénesis , Mutagénesis Sitio-Dirigida , Mutación , Fenilalanina/genética , Multimerización de Proteína , Relación Estructura-Actividad , Vibrio vulnificus/genéticaRESUMEN
Vibrio vulnificus hemolysin (VVH), a pore forming toxin, is thought to be a virulence factor of this bacterium. It is well known that VVH induces apoptosis as well as cell lysis in susceptible target cells. Although pore formation is an essential step in cell lysis, it is unknown whether this step is necessary for VVH-induced apoptosis. In this study, Chinese hamster ovary (CHO) cells were exposed to non-oligomerized mutant F334I, in which phenylalanine 334 was replaced by isoleucine. The rate of apoptosis caused by the wild type VVH (VVH wt) was 41.5 +/- 6.4 %, whereas that caused by F334I was 0.4 +/- 0.8% at the same concentration. Our results clearly showed that oligomerization is essential for the cell lytic activity as well as apoptotic activity of this toxin.