Functional and regulatory characteristics of IscR, a global regulator of Vibrio vulnificus

다국어 초록 (Multilingual Abstract)

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.

목차 (Table of Contents)

• Abstract I
• Contents V
• List of Figures X
• List of Tables XII
• Chapter I. Background 1
• I-1. Vibrio vulnificus 2
• I-2. Disease caused by V. vulnificus 4
• I-3. Virulence factors and molecular pathogenesis of V. vulnificus 6
• I-4. Objective of this study 15
• Chapter II. IscR is a global regulator essential for the pathogenesis of Vibrio vulnificus and induced by host cells 17
• II-1. Introduction 18
• II-2. Materials and Methods 21
• II-2-1. Bacterial strains, plasmids and culture conditions 21
• II-2-2. Identification of V. vulnificus iscR and generation of iscR mutant 29
• II-2-3. Complementation of the iscR mutant 30
• II-2-4. Cytotoxicity and mouse mortality 30
• II-2-5. Transcriptome analysis 31
• II-2-6. Quantitative real-time PCR (qRT-PCR) 32
• II-2-7. Purification of V. vulnificus iscR and electrophoretic mobility shift assay 37
• II-2-8. Motility and adhesion assays 38
• II-2-9. Hemolysis assay and survival under oxidative stress 38
• II-2-10. Western blot analysis 39
• II-2-11. Microarray data accession number 40
• II-3. Results 41
• II-3-1. Identification of V. vulnificus IscR 41
• II-3-2. IscR is important for virulence 41
• II-3-3. IscR-regulated genes involved in pathogenesis 45
• II-3-4. Effects of the iscR mutation on the virulence-related phenotypes of V. vulnificus 57
• II-3-5. Effects of host cells on IscR expression 64
• II-4. Discussion 69
• Chapter III. Low cell density regulator AphA upregulates the expression of Vibrio vulnficus iscR gene encoding the Fe-S cluster regulator IscR 73
• III-1. Introduction 74
• III-2. Materials and Methods 77
• III-2-1. Bacterial strains, plasmids, and culture conditions 77
• III-2-2. Generation of aphA and aphA iscR mutants 77
• III-2-3. RNA purification and transcript analyses 78
• III-2-4. Overexpression and purification of V. vulnificus AphA and IscR 79
• III-2-5. Electrophoretic mobility shift assay (EMSA) and DNase I protection assay 79
• III-2-6. E. coli dual plasmid system 80
• III-2-7. Cytotoxicity assay 81
• III-2-8. LD50 determination 82
• III-3. Results 83
• III-3-1. Effects of the cell growth and aphA mutation on the iscR expression 83
• III-3-2. Effects of iscR or aphA mutation on activity of iscR promoter 86
• III-3-3. IscR and AphA bind specifically to the iscR regulatory region 89
• III-3-4. Identification of binding sites for IscR and AphA using DNase I protection analysis 91
• III-3-5. AphA upregulates the iscR expression only in the presence of functional IscR 95
• III-3-6. Examination of the roles of AphA and binding sites in the control of PiscR activity 97
• III-3-7. AphA is important for virulence 100
• III-4. Discussion 103
• Chapter IV. Evidence that a Vibrio vulnificus peroxiredoxin gene, required for survival under oxidative and nitrosative stress and virulence, is regulated by Fe-S cluster regulator IscR 107
• IV-1. Introduction 108
• IV-2. Materials and Methods 111
• IV-2-1. Bacterial strains, plasmids, and culture conditions 111
• IV-2-2. Generation of prx3 mutant 111
• IV-2-3. Site-specific mutagenesis of IscR 112
• IV-2-4. Growth of V. vulnificus under oxidative and nitrosative stress 112
• IV-2-5. Mouse mortality test 113
• IV-2-6. Construction of a prx3-luxCDABE transcription fusion and measurement of cellular luminescence 113
• IV-2-7. E. coli dual plasmid system 114
• IV-2-8. RNA purification and analysis of prx3 transcripts 115
• IV-2-9. Western blot analysis 116
• IV-2-10. Overexpression and purification of V. vulnificus IscR3CA 116
• IV-2-11. Electrophoretic mobility shift assay (EMSA) and DNase I protection assay 116
• IV-3. Results 118
• IV-3-1. Identification of V. vulnificus prx3 gene 118
• IV-3-2. Effect of prx3 mutation on the growth of V. vulnificus under oxidative and nitrosative stress 120
• IV-3-3. Effect of prx3 mutation on virulence of V. vulnificus in mice 123
• IV-3-4. Transcription of prx3 is controlled by IscR and iron 125
• IV-3-5. Transcription of prx3 in the presence of functional IscR or IscR3CA 127
• IV-3-6. Effect of iscR or iscR3CA mutation on the expression of prx3 130
• IV-3-7. IscR3CA binds specifically to the prx3 regulatory region 134
• IV-3-8. Identification of the IscR3CA binding site using DNase I protection analysis 136
• IV-4. Discussion 139
• Chapter V. Conclusion 142
• References 147
• 국문초록 167