Measurement of Haemolysin Activities in Vibrio vulnificus

Abstract

VvhA produced by Vibrio vulnificus exhibits cytolytic activity to human cells including erythrocytes. Since haemolysis by VvhA may provide iron for bacterial growth and pathogenicity, we investigated the expression of VvhA to elucidate the regulatory roles of Fur, a major transcription factor controlling iron-homeostasis. Fur repressed the transcription of vvhBA operon via binding to the promoter region. However, haemolysin content and haemolytic activity were lowered in cell-free supernatant of fur mutant. This discrepancy between the levels of vvhA transcript and VvhA protein in fur mutant was caused by exoproteolytic activities of the elastase VvpE and another metalloprotease VvpM, which were also regulated by Fur. vvpE gene expression was repressed by Fur via binding to the Fur-box homologous region. Regulation of VvpM expression by Fur did not occur at the level of vvpM transcription. In vitro proteolysis assays showed that both proteases efficiently degraded VvhA. In addition, the extracellular levels of VvhA were higher in culture supernatants of vvpE or vvpM mutants than in the wild type. Thus this study demonstrates that Fur regulates haemolysin production at the transcription level of the vvhBA operon and at the post-translation level by regulating the expressions of two VvhA-degrading exoproteases, VvpE and VvpM. This protocol can be applied to other Vibrio strains with haemolysin activities, such as Vibrio parahaemolyticus (V. parahaemolyticus) or other human pathogen strains with similar heamolysin activities.