IscR Is a Global Regulator Essential for Pathogenesis of Vibrio vulnificus and Induced by Host Cells

Abstract

A mutant that exhibited less cytotoxic activity toward INT-407 human intestinal epithelial cells than the wild type was screened from a random transposon mutant library of Vibrio vulnificus, and an open reading frame encoding an Fe-S cluster regulator, IscR, was identified using a transposon-tagging method. A mutational analysis demonstrated that IscR contributes to mouse mortality as well as cytotoxicity toward the INT-407 cells, indicating that IscR is essential for the pathogenesis of V. vulnificus. A whole-genome microarray analysis revealed that IscR influenced the expression of 67 genes, of which 52 were upregulated and 15 were downregulated. Among these, 12 genes most likely involved in motility and adhesion to host cells, hemolytic activity, and survival under oxidative stress of the pathogen during infection were selected and experimentally verified to be upregulated by IscR. Accordingly, the disruption of iscR resulted in a significant reduction in motility and adhesion to INT-407 cells, in hemolytic activity, and in resistance to reactive oxygen species (ROS) such as H 2 O 2 and tert-butyl hydroperoxide (t-BOOH). Furthermore, the present study demonstrated that iscR expression was induced by exposure of V. vulnificus to the INT-407 cells, and the induction appeared to be mediated by ROS generated by the host cells during infection. Consequently, the combined results indicated that IscR is a global regulator that contributes to the overall success in the pathogenesis of V. vulnificus by regulating the expression of various virulence and survival genes in addition to Fe-S cluster genes. Most of virulence factors of pathogenic bacteria act cooperatively to obtain maximum effectiveness during pathogenesis while their expression is coordinately regulated by common global regulators in response to environmental conditions, and this coordinated regulation facilitates the cooperation of virulence factors and is crucial to the overall success of the pathogens during infection (1, 2). Vibrio vulnificus is an opportunistic Gram-negative pathogen that frequently contaminates oysters. It has been proposed that numerous virulence factors account for the fulminating and destructive nature of V. vulnificus infections and contribute to not only disease development but also the survival and multiplication on or within the host (for a recent review, see reference 3). However, studies about global regulators involved in the regulation of V. vulnificus virulence factors are still very limited.Iron-sulfur proteins containing the Fe-S cluster as a cofactor carry out multiple important cellular processes such as electron transfer, metabolic reactions, and gene regulation and are widely distributed (for recent reviews, see references 4 and 5). A highly conserved isc operon, iscRSUA-hscBA-fdx, was discovered to encode all of the proteins required for the biogenesis of the majority, if not all, of the Fe-S cluster proteins in Escherichia coli (for recent reviews, see references 5 and 6). Expression of the isc operon is autoregu...

Characterization of the Vibrio vulnificus 1-Cys Peroxiredoxin Prx3 and Regulation of Its Expression by the Fe-S Cluster Regulator IscR in Response to Oxidative Stress and Iron Starvation

“…Proteins (10 g) were resolved on SDS-PAGE and immunoblotted as described previously (30). For immunoblotting of IscR, the anti-IscR polyclonal antibody prepared previously was used (30). When necessary, the wild type grown aerobically to an A 600 of 0.3 was exposed to various concentrations of an iron chelator, 2,2Ј-dipyridyl (DP) (Sigma) for 10 min and then harvested.…”

“…His-tagged proteins were expressed in E. coli BL21(DE3) and purified by affinity chromatography according to the manufacturer's procedure (Qiagen, Valencia, CA). Similarly, the expression and purification of the His-tagged thioredoxin A (TrxA), thioredoxin reductase (TrR), and IscR were carried out using pBANG1104, pOH053, and pJK0928 (Table 1), respectively, as described previously (17,30).…”

“…The V. vulnificus strains grown aerobically or anaerobically to an A 600 of 0.3 were harvested to isolate total proteins. Proteins (10 g) were resolved on SDS-PAGE and immunoblotted as described previously (30). For immunoblotting of IscR, the anti-IscR polyclonal antibody prepared previously was used (30).…”

“…Among the genes up-regulated by IscR, a gene was predicted to encode a putative Prx (here called Prx3) (30). However, the catalytic mechanism of Prx3 and IscR regulation of its expression have not yet been experimentally verified.…”

“…A transcriptome analysis previously predicted that V. vulnificus IscR is a global regulator that controls the expression of many genes involved in important cellular processes in addition to Fe-S cluster biogenesis (30). Among the genes up-regulated by IscR, a gene was predicted to encode a putative Prx (here called Prx3) (30).…”