Functional and Regulatory Characteristics of IscR, a Global Regulator of Vibrio vulnificus

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농업생명과학대학 농생명공학부
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서울대학교 대학원
Vibrio vulnificusFe-S clusterIscRAphAPeroxiredoxin
학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2014. 2. 최상호.
Pathogenic bacteria have evolved global regulatory mechanisms to facilitate cooperation of the numerous virulence factors during pathogenesis. In the present study, a homologue of IscR, an Fe-S cluster-containing transcriptional regulator was identified from Vibrio vulnificus, a causative agent of food-borne diseases, and its role and regulatory characteristics were assessed. A mutant that exhibited less cytotoxic activity toward INT-407 human intestinal epithelial cells 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, 52 of which were up-regulated and 15 down-regulated. Among these, twelve 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 up-regulated by IscR. Accordingly, the disruption of iscR resulted in a significant reduction in motility and adhesion to the INT-407 cells, hemolytic activity, and resistance to reactive oxygen species (ROS) such as H2O2 and t-BOOH. Furthermore, the present study demonstrated that the 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 contributing 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.

Furthermore, the regulatory mechanisms for the iscR expression of V. vulnificus were evaluated. The expression of iscR was found to be upregulated by a transcriptional regulator AphA, a homologue of the low cell density regulator AphA of the Vibrio species, in the exponential phase of growth. The promoter activity of iscR appeared to be activated and repressed by AphA and IscR, respectively. EMSA and DNase I protection assay showed that both AphA and IscR bind to the iscR regulatory region and the binding site for AphA overlapped with part of the binding site for IscR. Mutational analysis suggested that AphA upregulates the iscR expression only in the presence of functional IscR. An examination of the roles of AphA and the binding sites revealed that the binding of AphA would hinder the IscR-mediated repression of the iscR transcription. The combined results show that V. vulnificus AphA upregulates iscR expression by antagonizing its negative autoregulation. Furthermore, the disruption of aphA resulted in significantly reduced virulence in tissue cultures and in mice. Accordingly, AphA contributes the pathogenesis of V. vulnificus possibly by promoting the production of IscR, which activates the genes required for survival and virulence.

The transcriptome analysis revealed that Vibrio vulnificus IscR upregulates a gene encoding a putative antioxidant, homologous to human peroxiredoxin 5. This gene was further identified as a peroxiredoxin-encoding gene of V. vulnificus and named as prx3. The prx3 mutant was hypersusceptable to killing by hydrogen peroxide and peroxynitrite, indicating that V. vulnificus Prx3 is required for survival under oxidative and nitrosative stress. In addition, mouse mortality test suggested that Prx3 is essential for the virulence of V. vulnificus. The expression of prx3 was increased upon iron starvation in IscR-dependent manner, implying that IscR-dependent sensing of the cellular Fe-S cluster status involves the regulation of prx3. Escherichia coli dual plasmid system assay showed that IscR3CA mutant (apo-form of IscR) also activates the prx3 expression, suggesting that Fe-S cluster of IscR is dispensible for the activation of prx3. qRT-PCR and primer extension analyses showed that the expression of prx3 in the iscR3CA mutant was more increased than that in the wild type. These results might be contributed to the increased level of IscR3CA in the iscR3CA mutant. A direct interaction between IscR3CA and the promoter region of prx3 was demonstrated by an EMSA, and a IscR3CA binding site, centered at 44 bp upstream of the transcription start site, was identified by a DNase I protection assay. The binding site for IscR3CA on the prx3 promoter matched the type 2 binding motif of Escherichia coli IscR, reinforcing that apo-IscR also activates the prx3 expression. Taken together, the expression of V. vulnificus Prx3, essential for the survival under conditions of oxidative and nitrosative stress and virulence in mice, is regulated by IscR.
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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