Identification and Regulatory Characteristics of gbpA Encoding a N-acetylglucosamine Binding Protein in Vibrio vulnificus

Abstract

Attachment of pathogenic bacteria to host cell is crucial for the initial stage of pathogenesis. Previous study showed that VVMO6_03494 encoding putative adhesin is up-regulated by transcriptional regulator IscR which is responsible for the survival and virulence of V. vulnificus. VVMO6_03494 is a homologue of Vibrio cholerae gbpA encoding N-acetylglucosamine binding protein. In this study, the role of V. vulnificus GbpA and its regulatory characteristics were investigated. Amino acid sequence analysis revealed that V. vulnificus GbpA is predicted to be a secreted chitin binding protein. The gbpA mutant showed significantly reduced ability to bind for chitin compared to its parental wild type. The purified GbpA protein was capable of binding to the chitin and N-acetylglucosamine, the monomer of chitin, in vitro. Also, GbpA was not secreted in the mutant impaired in type II secretion system. These indicated that GbpA is an N-acetylglucosamine-sensitive chitin binding protein and secreted via type II secretion system. Furthermore, the disruption of gbpA resulted in a substantial decrease in adherence to mucin containing N-acetylglucosamine as a component of carbohydrate moiety in vitro and the lower adherence was restored to the level of wild type by exogenous addition of purified GbpA. In addition, the gbpA mutant was significantly diminished in adherence to HT-29 MTX cells, mucin-secreting human intestinal epithelial cells and exhibited reduced the virulence in the infection of mice. Combined results suggested that GbpA contributes to the pathogenesis of V. vulnificus by facilitating the adherence to the mucus layer of human intestinal epithelium. It was observed that the cellular level of gbpA transcripts is decreased in the growth-dependent manner, implying that the expression level of gbpA could be controlled by quorum sensing. The qRT-PCR analysis and western blot analyses revealed that the expression of gbpA is up-regulated by IscR and down-regulated by SmcR, quorum-sensing master regulator, at both the transcription and translation level. The EMSA showed that both IscR and SmcR bind to the promoter region of gbpA. In summary, V. vulnificus GbpA is a tightly regulated virulence factor responsible for attachment to chitin and mucus layer.