Functional Analysis of HlyU, a Transcriptional Regulator of Vibrio vulnificus

Abstract

Vibrio vulnificus is an opportunistic pathogen capable of causing severe food-borne diseases to susceptible individuals with hematological disorders or immunocompromised states. This bacterium possesses several virulence factors including capsular polysaccharide, lipopolysaccharide, toxins, and proteins involved in iron acquisition, motility, attachment and adhesion. RtxA toxin is one of the major virulence factors that plays an important role in the pathogenesis of V. vulnificus by triggering cellular damages after host cell contact, which results in necrotic and apoptotic cell death. Expression of rtxA is up-regulated by HlyU, a transcriptional regulator that directly binds to the promoter region of the rtxHCA operon. To further examine the function of HlyU, the hlyU mutant was constructed and its virulence was evaluated. Compared with the wild type, the hlyU mutant showed significantly reduced virulence in mice and tissue culture, indicating HlyU is essential for the virulence of V. vulnificus. In a growth phase-dependent manner, HlyU appeared to up-regulate the expression of rtxA. To further identify the genes regulated by HlyU, transcriptomic profiles of the wild type and hlyU mutant were analyzed by RNA-sequencing. In addition to rtxA, genes encoding virulence factors such as hemolysin and phospholipase were down-regulated in the hlyU mutant, suggesting that HlyU contributes to the V. vulnificus pathogenesis by regulating various virulence genes. This study also revealed that the induction of rtxA was mediated by HlyU upon exposure to host cells or under anaerobic conditions, and it might be attributed to altered activity rather than amounts of HlyU. A mutagenesis study proposed that Cysteine 30 is a critical residue of HlyU for rtxA activation. Taken together, HlyU might act as a regulator for the various virulence genes and induce rtxA under host environment by altering its activity.