S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Agricultural Biotechnology (농생명공학부) Theses (Ph.D. / Sc.D._농생명공학부)
Characterization of two peroxiredoxins and an MFS transporter Induced by oxidative stress in vibrio vulnificus
패혈증 비브리오균에서 산화 스트레스에 의해 유도되는 두 Peroxiredoxin과 MFS 수송단백질의 특성 규명
- 농업생명과학대학 농생명공학부
- Issue Date
- 서울대학교 대학원
- Vibrio vulnificus; Oxidative stress; Transcriptome analysis; Peroxiredoxin (Prx); Major facilitator superfamily transporter (Mfs); SoxR
- 학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2014. 8. 최상호.
- Vibrio vulnificus is an opportunistic human pathogen whose infection can cause diseases such as gastroenteritis and life-threatening septicemia. Oxidative stress resulting from toxic reactive oxygen species (ROS) is imposed to bacteria by a wide range of factors, including aerobic metabolism, host immune response, redox-active metals and drugs. Therefore, coping with oxidative stress is crucial for successful pathogenesis of V. vulnificus as well as the survival in various conditions. In this study, to better understand the bacteriums strategies to survive oxidative stress, I have analyzed transcriptome changes of V. vulnificus in response to oxidative stress using a strand-specific RNA-sequencing. By analyzing RPKM (reads per kilobase of transcript per million reads) fold changes of each gene, I identified 108 up-regulated genes and 42 down-regulated genes by oxidative stress (P-value≦0.05, 1.5 fold threshold). The genes most highly up-regulated included those encoding antioxidant proteins, such as peroxiredoxin 1, peroxiredoxin 2, thiol peroxidase Bcp, iron-binding ferritin-like antioxidant protein Dps, and cytochrome c peroxidase, some of which are newly identified in V. vulnificus. Also, the genes encoding a wide variety of transporters were significantly up-regulated, implying their potential roles in relieving oxidative stress by transporting their substrates. Meanwhile, the most down-regulated genes were associated with biosynthesis of amino acids and aerobic metabolism, suggesting the repression of the cellular processes that are not necessary for the survival at the moment and/or the processes that produce ROS.
Among the genes whose expression was up-regulated by oxidative stress, genes encoding two peroxiredoxins (Prxs), prx1 and prx2, were identified. Prxs are ubiquitous antioxidant enzymes reducing toxic peroxides. Both Prxs, Prx1 and Prx2, have two conserved catalytic cysteines, CP and CR, and thus classified into 2-Cys Prxs. In contrast to Prx1, which is highly homologous in amino acid sequences to Prxs from other bacteria, Prx2 is more homologous to eukaryotic Prx than to Prx1. Prx2 utilized thioredoxin A (TrxA) as a reductant, while Prx1 required alkyl hydroperoxide reductase F (AhpF). Prx2 contained GGIG and FL motifs similar to the motifs conserved in sensitive Prxs and exhibited sensitivity to overoxidation. MS analysis and CP-SO3H specific immunoblotting demonstrated overoxidation of CP to CP-SO2H (or CP-SO3H) in vitro and in vivo, respectively. In contrast, Prx1 was robust and CP was not overoxidized. Discrete expression of the Prxs implied that Prx2 is induced by trace amounts of H2O2 and thereby residential in cells grown aerobically. In contrast, Prx1 was occasionally expressed only in cells exposed to high levels of H2O2. A mutagenesis study indicated that lack of Prx2 accumulated sufficient H2O2 to induce Prx1. Kinetic properties indicated that Prx2 effectively scavenges low levels of peroxides because of its high affinity to H2O2, while Prx1 quickly degrades higher levels of peroxides because of its high turnover rate and more efficient reactivation. The combined results revealed that the two Prxs are differentially optimized for detoxifying distinct ranges of H2O2, and proposed that Prx2 is a residential scavenger of peroxides endogenously generated, while Prx1 is an occasional scavenger of peroxides exogenously encountered. Further, genome sequence database search predicted widespread coexistence of the two Prxs among bacteria.
A mfs gene encoding a novel major facilitator superfamily transporter, Mfs, was firstly identified as one of the most significantly up-regulated genes by oxidative stress in V. vulnificus. Amino acid sequence analysis predicted that Mfs contains 12 transmembrane helices and is located in cytoplasmic membrane of the bacterium. Bioinformatic analyses revealed that Mfs homologues are present only in a limited number of bacteria including Vibrio species. Functions of mfs were assessed by comparing the growth of wild type and mfs mutant under various conditions. The growth of mfs mutant was comparable to that of wild type under normal and oxidative stress conditions. However, mfs mutant was not able to grow to the substantial level when exposed to a ROS-generating antibiotic, bleomycin, revealing that the gene product of mfs contributes to resistance of V. vulnificus to bleomycin. Consistently, overexpression of mfs enhanced the bacterial resistance to bleomycin. The expression of mfs was under the positive control of SoxR, a central regulator of oxidative stress response. SoxR directly bound to the mfs promoter in vitro and activated mfs expression in response to oxidative stress. Induction of mfs was observed when the cell was exposed to bleomycin, which was also under the control of SoxR. The combined results suggested that Mfs is a novel SoxR-regulated transporter conferring resistance to bleomycin, which is considered as a part of SoxR-mediated oxidative stress defense mechanisms involved in the removal of the source of ROS.