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Molecular Characterization of HlyU, a Global Regulator of Vibrio vulnificus Virulence Genes
Author(s) -
Choi Sang Ho,
Lee ZeeWon,
Bang YeJi,
Jang Kyung Ku
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.756.15
Subject(s) - biology , vibrio vulnificus , virulence , mutant , hemolysin , microbiology and biotechnology , wild type , gene , operon , bacteria , genetics
An opportunistic pathogen Vibrio vulnificus produces a large pore‐forming toxin, RtxA, which triggers necrotic and apoptotic cell death. Previous studies reported that a transcriptional regulator HlyU upregulates rtxA expression by direct binding to the promoter region of the rtxHCA operon. To examine the function of HlyU, the hlyU mutant was constructed and its virulence was evaluated. Compared with the wild type, the hlyU mutant exhibited low cytotoxicity toward mucin‐secreting HT‐29 MTX cells and reduced mortality in mice, suggesting that HlyU is essential for the virulence of V. vulnfiicus . To further identify the genes regulated by HlyU, transcriptomic profiles of the wild type and the hlyU mutant were analyzed by RNA‐seq. In addition to rtxA , genes encoding virulence factors such as hemolysin and phospholipase were down‐regulated in the hlyU mutant, implying that HlyU contributes to the V. vulnificus pathogenesis by regulating various virulence genes. To elucidate the mechanism of HlyU in rtxA regulation, the levels of rtxA and hlyU transcripts in V. vulnificus cells grown in different conditions were determined. Induction of the rtxA expression occurred in the wild‐type cells either exposed to the INT‐407 human epithelial cells or grown under anaerobic conditions, but not in the hlyU mutant, indicating that HlyU mediated the induction. Since the levels of hlyU transcript in the wild‐type cells were not significantly changed in those conditions, the rtxA activation might be attributed to altered activity rather than amounts of HlyU. To better understand this activity alteration of HlyU, C30, C96, and M87, which were predicted as essential amino acids from structural analysis, were mutated by site‐directed mutagenesis. Among the mutants, V. vulnificus expressing HlyU‐C30S showed a decreased rtxA level and cytotoxicity toward the HT‐29 MTX cells than the wild type, suggesting that C30 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.