Isolation and Quantitative Cell Count of Vibrio vulnificus and Vibrio cholerae using MPN-PCR Method

이재화 부경대학교 대학원 2020 국내석사

Genomic, transcriptomic, and metagenomic analyses of Vibrio species and microbial community composition of crabs harvested at different seasons and locations

김수연 Seoul National University 2016 국내박사

Vibrio vulnificus and Vibrio parahaemolyticus are gram-negative, motile, nonspore-forming opportunistic pathogens that causes foodborne illness associated with the consumption of contaminated seafood. Although many cases of foodborne outbreaks caused by V. vulnificus and V. parahaemolyticus have been reported, the genomes of only few of them have been completely sequenced and analyzed using bioinformatics. In order to characterize overall virulence factors and pathogenesis of V. vulinificus and V. parahaemolyticus associated with foodborne outbreak in South Korea, new strains V. vulnificus FORC_016 and V. parahaemolyticus FORC_008 were isolated from blood of food-poisoning patient or flounder fish and their genome was completely sequenced. The genomic analysis of FORC_016 revealed that the genome consists of two circular DNA chromosomes, and contains 4,461 predicted open reading frames (ORFs), 129 tRNAs, and 34 rRNA genes. V. parahaemolyticus FORC_008 have two circular DNA chromosomes containing 4,494 predicted ORFs, 129 tRNAs, and 31 rRNA genes. The genomic analysis revealed that the V. vulnificus FORC_016 has major virulence genes such as RTX, cytolysin, and metalloprotases. Furthermore, comparative genome analysis identified unique virulence genes of FORC_016 strain, suggesting that this pathogen have unique pathogenesis mechanism which different from other V. vulnificus. While the strain FORC_008 does not have genes encoding thermo-stable direct hemolysin (TDH) and TDH-related hemolysin (TRH), its genome encodes many other virulence factors including hemolysins, pathogenesis-associated secretion systems, and iron acquisition systems, suggesting that it may be a potential pathogen. Subsequent cytotoxicity test of the strain FORC_008 revealed its high cytotoxicity activity, substantiating this. This report provides an extended understanding on V. vulnificus and V. parahaemolyticus in genomic level and would be helpful for rapid detection, epidemiological investigation, and prevention of foodborne outbreak in South Korea. Because the foodborne illness occurs via consumption of contaminated food, it is important not only understanding of the virulence factors but also the transcriptome alteration of pathogens caused by contacting with foods. To identify differentially expressed genes of pathogen under contact with foods, V. vulnificus FORC_016, an opportunistic marine pathogen, was selected for the transcriptome analysis. Swimming crab, a common niche of V. vulnificus, was selected for the model foods. The transcriptomic profiles of V. vulnificus exposed or unexporsed to crab in 1 or 4h were analyzed using a strand-specific RNA-sequencing. By analyzing RPKM (reads per kilobase of transcript per million reads) fold changes of each gene, I identified that 922 and 648 genes were differentially expressed under exposure to crab for 1h and 4h (P value < 0.05, 2 fold threshold). Regardless of incubation time, the genes related with energy production, cell growth, oligopeptide transport, and glucose metabolism were up-regulated, while genes associated with amino acid biosynthesis, nitrogen metabolism, and other sugar metabolism were down-regulated. These result suggested that V. vulnificus could metabolize the component of crab. Also, the genes encoding thermolabile hemolysin was up-regulated, suggesting this virulence gene might be have crucial role for pathogenesis of V. vulnificus FORC_016 when consumed the V. vulnificus FORC_016 contaminated crab. The swimming crab, Portunus trituberculatus, is the most consumed edible crab in South Korea, and their production and consumption have been increased. Although the foodborne illness caused by consuming of swimming crab have been reported each year, the bacterial community in swimming crab has not been fully understood yet. In order to identify the bacterial members in swimming crab depending on seasons and locations, the microbiota in 65 crabs which were collected from different locations in spring and autumn was analyzed by pyrosequencing. The bacterial communities in autumn crab were more diverse in than those in spring. Psychrobacter, Vagococcus, and Carnobacterium were the most abundant genera in spring, whereas Roseovarius was predominant in autumn, but their proportions were influenced by the pathogenic bacterial proportion. These results indicated that the microbiota in swimming crab significantly influenced by seasonal temperature change. The proportion analysis on Vibrio species indicated that intake of crab could cause the foodborne illness. This study provides the extended understanding on composition of bacterial community in swimming crab and the factors influencing crab microbiome.

Functional analysis of vibrio vulnificus SmcR, a quorum-sensing master regulator, as a novel control target

김승민 서울대학교 대학원 2012 국내박사

Quorum sensing has been implicated as an important signal transduction system regulating the expression of numerous virulence genes in bacterial pathogens. Vibrio vulnificus is a model pathogen for studying many other foodborne pathogens because it causes life-threatening septicemia and gastroenteritis with various potential virulence factors controlled by quorum sensing. Recently, SmcR, a homologue of Vibrio harveyi LuxR, has been identified from V. vulnificus, and proposed as a quorum-sensing master regulator. In the present study, the roles of SmcR during an infectious process were examined in a series of experiments using biofilm cells, and comparing virulence of the smcR mutant with that of the parental wild type. When compared to the smcR mutant, the wild type showed a significant 2-fold increase in biofilm detachment rate in the extended time-course. To determine the genes involved in the biofilm detachment among SmcR-regulated genes, transcriptional profiles of the wild-type and smcR mutant biofilms were analyzed. The differentially expressed genes in the smcR mutant include genes that are known to stimulate biofilm dispersal in other bacteria. Interestingly, the smcR expression was induced upon exposure to intestinal epithelial cells. Thus, the ability of the wild type to detach cells from the biofilms was enhanced as biofilms were exposed to INT-407 human intestinal epithelial cells. On the other hand, the smcR mutant biofilms were not detached even upon exposure to INT-407 cells. In this regard, the defects in biofilm detachment in the smcR mutant resulted in a decrease in both intestinal colonization to new infection sites and histopathological damage in jejunum tissues from the mouse intestine after the intragastric administration of biofilm cells. Furthermore, the LD50 in ICR mice (specific-pathogen free) after intragastric infections of the smcR mutant was approximately 102 times higher than that of the parental wild type, suggesting that the smcR mutant biofilms were impaired in its ability to disperse and colonize new sites in the intestine in vivo. These differences between the wild-type and smcR mutant strain were not observed when planktonic cells were used. Therefore, these results indicate that upon entry into the host intestine, SmcR enables V. vulnificus biofilms to detach to find appropriate sites of infection and initiate a new infection cycle, demonstrating the importance of SmcR in V. vulnificus pathogenesis. After the detachment of cells from the biofilms, flagellum-mediated motility is essential for dispersal of detached cells into the new colonization site. In the present study, the functions of FlhF and regulatory characteristics of the flhF expression of V. vulnificus were investigated. A deletion mutation of FlhF abolished motility, flagella formation, and flagellin synthesis, and introduction of flhF in trans complemented the defects. The flhF mutant revealed decreased expression of the class III and IV flagella genes, indicating that FlhF is a key regulator for the flagella biogenesis of V. vulnificus. The influence of global regulatory proteins on the expression of flhF was examined, and SmcR was found to downregulate the flhF expression at the transcriptional level. SmcR represses the flhF expression only in the stationary phase of growth and exerts its effects by directly binding to the flhF promoter region. Finally, an SmcR binding site, centered at 22.5-bp upstream of the transcription start site, was identified by a DNase I protection assay. The combined results demonstrate that a quorum sensing master regulator SmcR influences the motility and flagella biogenesis of V. vulnificus through modulating the expression of FlhF in a growth phase-dependent manner. During the initial stage of infection immediately after biofilm detachment, smcR expression is repressed because of low cell density, and expression of flhF is allowed, leading to flagella synthesis. The flagella prime V. vulnificus for initial colonization of host intestinal tissue, which is an important step required for the onset of its infectious cycle. In contrast, upon establishing preferred colonization niches with the increase in population density, the necessity of motility is superfluous, even detrimental, for a successful infection of hosts by the bacteria. In fact, flagellins of many enteropathogens have been well characterized as a major inducer as well as a target of host innate immune responses. Therefore, flagellar synthesis needs to be sophisticatedly regulated by quorum sensing regulatory pathways for optimal colonization and disease progression. These previous results led me to confirm that V. vulnificus quorum sensing is essential for the survival and pathogenesis of V. vulnificus. So identification and characterization of small molecules that inhibit quorum sensing are required for delineating novel strategies to control foodborne pathogens with low incidence of bacterial resistance. Therefore, a high throughput screening of small molecule libraries was performed to identify inhibitors of the V. vulnificus quorum sensing. Using a reporter strain PVVMO6_03194::luxAB whose activity entirely depends on SmcR, I identified a small molecule named U-262, 6-(phenylsulfonyl)nicotinonitrile. U-262 suppresses the activities of exoprotease and elastase without inhibiting bacterial growth itself. Attenuated cytotoxic activity, prolonged survival period and alleviated illness in mice were observed after treating V. vulnificus with the chemical. U-262 also decreased the luminescence of V. harveyi and the total protease activities of V. anguillarum which are regulated by quorum sensing, suggesting that it inhibits other quorum sensing of Vibrio spp.. Western blot analysis demonstrated that the chemical decreases the cellular level of SmcR in a dose dependent manner, indicating that the upper quorum-sensing signaling cascade is inhibited by the chemical. Meanwhile, E. coli dual plasmid system and EMSA showed that specific interactions between U-262 and SmcR resulted in the reduced DNA binding activity of SmcR, implying that U-262 affects both the cellular amount and the activity of SmcR. Taken together, these results suggested that U-262 is a novel anti-microbial agent inhibiting the quorum sensing of Vibrio spp. without the antibiotic resistance. It will be useful to protect food from the Vibrio spp. and help to enhance a public health.

Cloning and sequencing of the virulence regulatory genes of vibrio vulnificus

신성희 전남대학교 대학원 1997 국내박사

V. cholerae와 V. parahaemolyticus에서 toxRS 유전자는 다양한 환경으로부터 신호를 받아들이고 병원성 인자들의 발현을 조절하는 조절 유전자로써 밝혀져 있다. 그러나 치명적인 패혈증을 일으키는 V. vulnificus 병원성 인자들의 발현을 조절하는 조절유전자에 대해서는 알려진 바 없다. 이에 본 저자는 V. vulnificus에서 병원성 인자들의 발현을 조절하는 조절유전자를 밝히기 위하여 먼저 V. cholerae와 V. parahaemolyticus의 toxRS 유전자의 염기서열을 비교 분석하여 한 쌍의degenerate primer 만들어 이들 유전자와 상동성을 보이는 V. vulnificus의 핵산절편을 중합효소 연쇄반응에 의해 증폭하였다. 약 900 개의 염기를 가진 핵산절편이 증폭되었으며 이 절편을 클로닝하고 염기서열을 분석하였고 20개의 제한효소로 절단하였다. 클로닝된 핵산 절편의 염기서열은 V. cholerae와 V. parahaemolyticus의 toxRS 유전자의 염기서열과 약 60%의 상동성을 보였으며, 특징적으로 제한효소 HindIII에 의해 약 300개와 600개의 염기를 가진 두개의 작은 절편으로 절단되었다. 그래서 본 저자는 이 클로닝된 핵산 절편들을 V. vulnificus의 완전한 toxRS 유전자를 클로닝하기 위한 포식자로써 사용하였다. V. vulnificus ATCC 29307의 염색체를 분리하고 이를 여러 가지 제한효소로 절단한 다음 Southern blot 분석법에 의해 포식자와 반응하는 염색체 절편들을 검사하였다. 약 4.0kb의 크기를 가진 BglII 절편, 약 2.0kb의 크기를 가진 EcoRV 절편, 약 3.0kb의 크기를 가진 SstI 절편과 약 1.2kb와 6.1kb의 크기를 가진 HindIII 절편들이 양성반응을 보였다. 이들 양성반응을 보인 절편들 중에서 여러 가지 유전자 조작에 적절한 크기를 가진 BglII 절편을 완전한 toxRS 유전자를 클로닝하는데 사용하였다. BglII 절편을 다시 제한효소 HindIII로 절단하여 HindIII 절편과 HindIII-BglII 절편 각각을 클로닝하고 염기서열을 분석하였다. 2,694개의 염기를 가진 두 개의 절편은 완전한 toxRS 유전자를 포함하고 있었으며 V. cholerae와 V. parahaemolyticus의 toxRS 유전자와 약 60%의 상동성을 보였다. V. vulnificus ATCC 29307 외 같은 종의 다른 균주들과 같은 Vibrio 속의 다른 종들에서도 클로닝된 toxRS 유전자가 존재하는지를 알아보기 위해 DNA-colony blot 분석법을 시행하였다. 모든 V. vulnificus 균주들은 높은 stringency(68℃)에서도 강한 양성반응들 보였으나 다른 Vibrio 종들에서는 높은 stringency 에서는 양성반응을 보이지 않았으며 낮은 stringency(60℃와 55℃)에서 약한 양성반응을 보인 경우도 있었다. 이 결과는 핵산 염기서열 분석결과를 뒷받침하며 모든 V. vulnificus 균주들은 클로닝된 toxRS 유전자와 같은 유전자를 가지고 있으며 몇몇 다른 Vibrio 종들에서도 클로닝된 toxRS 유전자와 약간의 상동성을 지닌 유전자를 가지고 있는 것으로 판단되었다. ToxRS 단백의 발현과 장차의 계속적인 연구를 위하여 toxRS 유전자의 open reading frame 만을 포함한 절편을 중합 효소연쇄반응으로 증폭하여 클로닝하였으며 기타 몇 가지 subclone을 구축하였다. 결론적으로 본 연구를 통해 V. cholerae와 V. parahaemolyticus의 toxRS 유전자와 상동성을 보이는 V. vulnificus의 toxRS 유전자를 동정하고 클로닝하여 염기서열을 밝힐 수 있었다. 앞으로 계속적인 연구를 통하여 환경으로부터 신호를 받아들이고 병원성 인자들의 발현에 미치는 V. vulnificus의 toxRS 유전자의 역할은 밝혀지리라 생각한다. A number of pathogenic bacteria have been reported to have genes for transducing signals from the environment and regulating the expression of virulence genes. The toxRS regulons of V. cholerae (Vc-toxRS) and V. parahaemolyticus (Vp-toxRS) are the two well-known ones. Reports concerning the virulence regulating genes of V. vulnificus, which causes fatal septicemia in susceptible patients of underlying hepatic diseases and immunocompromised conditions, are not available yet. As an effort to dissect the virulence regulatory system of V. vulnificus, the homolog of Vc-toxRS and Vp-toxRS was cloned and sequenced. By comparing the sequences of the two previously reported toxRS genes, a set of degenerate primers targeting to relatively conserved regions was designed. A DNA fragment of 864 bp was amplified from the chromosome of V. vulnificus by PCR, and cloned into a cloning vector. The DNA sequence of the insert showed about 60% homology with the Vc-toxRS and Vp-toxRS. With enzymatic restriction and sequence analysis, a HindIII site was found. The DNA fragment was used as an authentic probe for the cloning of toxRS homolog in V. vulnificus (Vv-toxRS). The chromosomal DNA extracted from V. vulnificus ATCC 29307 was digested with various restriction enzymes and analyzed by Southern blotting. The BglII fragments of 4.0 kb and the HindIII fragments of 1.2 and 6.1 kb showing positive signals were used for the chromosomal Vv-toxRS cloning. An 1.6 kb BglII-HindIII fragment and an 1.2 kb HindIII fragment was finally cloned and sequenced. The two fragments with 2,696 bp included 2 open reading frames (ORFs), Vv-toxR and Vv-toxS, showing significant homologies to Vc-toxRS and Vp-toxRS. The intact chromosomal Vv-toxRS fragment was identified and cloned by PCR amplification. The fragments including only the ORF of toxR or toxS was also amplified with PCR and constructed into subclones for the further study. The Vv-toxR DNA sequence shared homologies of 54.7% and 62.0% with the Vc-toxR and Vp-toxR, respectively. At the amino acid level, Vv-ToxR was 55.0% and 63.0% homologous to the Vc-ToxR and Vp-ToxR, respectively. The Vv-toxR showed 64.9% and 66.0% DNA, and 65.7% and 71.5% amino acid sequence homologies with Vc-toxS and Vp-toxS respectively. The Vv-toxRS homologs in other Vibrios were screened by using the cloned Vv-toxRS DNA fragment as a probe through the DNA-colony blot hybridization. All strains of V. vulnificus showed positive signals at high stringency and some other Vibrio species showed weak positive signals at low stringencies but no positivity at high stringency. These results indicate that all V. vulnificus strains have toxRS, and the Vv-toxRS homologs might be generally distributed in other Vibrio species including V. cholerae and V. parahaemolyticus. In conclusion, the Vv-toxRS were successfully identified, cloned, and sequenced and it will be used for studying virulence regulatory mechanisms.

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

임종규 서울대학교 대학원 2014 국내박사

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.

패혈증비브리오균 유래 정족수인식 신호물질 합성 유전자의 탐색 및 조절자의 특성 조사

李美愛 韓國外國語大學校 大學院 2006 국내석사

The expression of virulence factors in Vibrio vulnificus is regulated via cell-density dependent manner. The elastase (VvpE), one of the majorvirulence factors, is regulated by autoinducer (AI)-2 synthesized by LuxS. Another putative AI, which was able to induce the vvpE gene expression, was detected from the media used to grow V. vulnificus. Since this putative AI synthesis was not influenced by deletion of the luxS gene and itsaddition to V. harveyi BB170 (AI-2 sensor) failed to induce the bioluminescence, it was not believed to be an AI-2. Instead, this non-AI-2 molecule induced V. harveyi BB886 (sensor for AI-1 [N-acylated homoserine lactone; AHL]). The effort for its chemical identification was not successful when applying the standard methods for typical AHL’s. Therefore, genetic approach was applied to characterize the nature of the non-AI-2 molecule. The lactonase-deficient mutant of V. vulnificus was constructed. This mutant showed the same degrees of AI production and vvpE expression as the wild-type. In addition, overexpressed lactonase of V. vulnificus did not degrade the non-AI-2 molecule. The BLAST search of V. vulnificus genomes revealed the orf homologous to the cqsA of V. cholerae, and thus the cqsA-null mutant of V. vulnificus was also constructed. This mutant, however, did not showed any difference in quorum-sensing-related phenotypes. Therefore, the non-AI-2 signal molecule of V. vulnificus is not a typical AHL found in many Vibrio spp., and its synthesis is independent of CqsA found in V. cholerae.

Vibrio vulnificus의 人血淸殺菌力에 對한 感受性과 Vibrio感染이 마우스의 Hematocrit値에 미치는 影響

전상남 全北大學校 1986 국내석사

Vibrio vulnificus, Vibrio parahemolyticus 및 콜레라菌의 正常 人血淸殺菌力에 對한 感受性과, 이들 Vibrios, Salmonella typhimurium 또는 大腸菌感染이 마우스의 hematocrit値에 미치는 影響에 關하여 比較實驗하여 다음과 같은 結果를 얻었다. 人血淸殺菌作用은 V. parahemolyticus에 對해서 보다 V. vulnificus 및 콜레라菌에 對하여 더 높았다. 熱處理人血淸에서의 供試菌株의 生存菌數가 熱非處理人血淸에서의 그것보다 많았는데, 이는 血淸補體가 Vibrios 殺菌에 有意하게 關與함을 强力히 示唆한다. 마우스를 10^7生存菌數로 腹腔注射하여 感染시키면, 感染 2-6時間內에 多少의 血液濃縮을 일으켰다. 이와는 反對로 V. vulnificus S. typhimurium 및 大腸菌은 感染後 4 또는 6時間後에 血液稀釋을 일으켰다. 以上의 結果로 V. vulnificus는 다른 Vibrios에 比하여 血淸殺菌作用에 對하여 더 感受性이 있고, 또한 V. vulnificus는 血液濃縮을 일으키지 않는다고 結論할 수 있다. Vibrio vulnificus, a halophilic Vibrio has gained worldwide attention as a cause of severe and frequently fatal wound infections and life-threatening septicemia. For this reason, V.vulnificus is thought to produce extreme hemoconcentration and rapid death after infection, and because V.vulnificus is thought to be less susceptible to bactericidal activity of normal human serum, the present study was undertaken to assess the susceptibility of V.vulnificus to human serum bactericidal activity with that of V.parahemolyticus and V.cholerae and to assess the effect of Vibrio species, Salmonella typhimurium and E.coli on hematocrit values in experimentally infected mice. Serum bactericidal activity against both V.vulunificus and V.cholerae was higher than against V.parahemolyticus. Survival of the test strains in heat-inactivated human serum was grester than that in heat-uninactivated serum Both V.parahemolyticus and V.cholerae produced slight hemoconcentration within 2 to 6 hr after intraperitoneal injection of 10^7 viable bacteria into mice. In contrast, V. vulunificus, S.typhimurium and E.coli produced hemodilution rather than hemoconcentration after 4 or 6 hr after infection. With these results the author can conclude that V.vulunificus is more susceptible to serum bactericidal activity than other Vibrio species, and V.vulunificus did not produce hemoconcentration.

양식장내의 Vibrio vulnificus 분포 및 억제방법 모색

宋桂珉 순천대학교 2000 국내석사

2000년 1월부터 2000년 9월까지 가두리 양식장의 비브리오 패혈증균의 분포와 이들의 억제 방법을 연구하였다. 가두리 양식장의 해수, 개펄, 어패류를 실험대상으로 삼았다. 해수에서의 종속영양세균은 1.1×10³CFU~3.9×10⁴CFU/㎖의 범위로 외해인 대조구, 양식장내의 표층, 심층에서 큰 차이를 나타내지 않았고, 어패류의 경우 1.0×10³CFU~1.1×10^6CFU/g의 범위로 종에 관계없이 시기별로도 큰 변화를 보여주지 않았다. 비브리오균군의 분포는 4월까지는 저질과 어패류에서 다소 검출되었으나 온도가 상승하는 5월부터 전 검체에서 검출되었으며 7~9월에 가장 많은 분포를 보였다. 비브리오 패혈증균은 온도가 14℃이하인 4월까지 검출되지 않다가 17℃이상인 5월부터 검출되었고 19℃인 6월부터 9월까지는 대다수 검체에서 거의 검출되었다. Chelating agent인 EDTA의 경우 500mg/ℓ에서 1시간 이상 처리할 경우 비브리오 패혈증균의 생장을 억제하였으며 800mg/ℓ에서는 균이 완전히 사멸되었다. Citric acid는 같은 농도에서 전혀 억제되지 않았다. -20℃와 4℃에서 24시간 저온 처리한 경우 균의 감소는 거의 없었다. 한편 광촉매인 TiO2에 의해서는 15분~1시간 이내에 완전히 사멸되는 효과를 나타내었다. Distribution of Vibrio vulnificus in the mariculture farm and inhibition of this bacterial pathogen was analyzed. The number of heterotrophic bacteria in oyster, mussel, and sea bream was similar as 1.0×10³CFU ~ 1.1×10^6CFU/gram dry weight from January to September, 2000. Vibrio spp. was recovered only from the sediment, shellfish, and fish until April. From May, Vibrio spp. was recovered from all of the samples, and the highest distribution rate was recorded from July to September. Distribution of V. vulnificus was temperature-dependent. From June to September whose temperature was above 19℃, V. vulnificus was recovered from almost of the samples. Treatment of 500mg/ℓ of EDTA for 1 hour inhibited the growth of the pathogen significanthy, moreover over 800mg/ℓ of EDTA, the bacteria was completely inhibited. However, citric acid was not useful for the inhibition of this bacteria. Photocatalyst, TiO2 showed potent bactericidal effect on V. vulnificus within 1 hour.

Cloning and molecular biological characterization of catalase-peroxidase katG gene Vibrio vulnificus

김춘미 전남대학교 대학원 2001 국내박사

Vibrio vulnificus는 연안해수에 서식하는 호염성 미생물로서 창상 감염과 원발성 패혈증의 형태로 사람에서 병을 일으킨다. 이 병원균은 자연환경과 감염과정에서 급격한 환경변화에 따른 스트레스에 직면하게 된다. 특히 계절변화에 따른 온도, 염도, 그리고 영양분의 변화나, host defense mechanism을 통한 다양한 스트레스에 적절히 대응하여 살아 남아야 한다. Alternative sigma factor σ^38(RpoS)는 여러 환경 스트레스에 대항하여 생존하는데 필요한 단백질군을 암호화하는 유전자들의 발현을 조절한다고 잘 알려져 있다. V. vulnificus에서 rpoS 유전자를 찾고자 하던 중에 rpoS 대신에 catalase-peroxidase 유전자를 클로닝하게 되었다. 본 논문에서는 catalase-peroxidase katG 유전자를 클로닝 하였으며, KatG 단백질의 분자생물학적인 특성과 생리학적인 역할을 조사하였다. V. vulnificus type strain인 ATCC 29307의 cosmid genomic library를 이용하여, 유해한 활성산소 species인 과산화수소를 물과 산소로 전환시키는 catalase-peroxidase katG (Vv-katG) 유전자를 클로닝 하였으며 그 염기서열을 분석하였다. Vv-katG 유전자는 총 2,172 nucleotide로 이루어져 있으며, 분자량 79,530-Da의 723개의 아미노산으로 이루어진 단백질을 암호화하고 있다. V. vulnificus의 katG 유전자는 그 DNA 염기서열에 있어 V. cholerae의 catalase-peroxidase 유전자와 78%의 상동성을 나타냈다. 그리고 Vv-KatG의 아미노산 서열은 Vibrio cholerae catalase-peroxidase (85%), Synechococcus sp. catalase-peroxidase (77%), Legionella pneumophila catalase-peroxidase KatB (75%), Agrobacterium tumefaciens catalase(72%), Synechocystis sp. catalase HPI (71%), Bacillus stearothermophilus catalase (66%), 그리고 E. coli hydroperoxidase HPI (59%) 등과 같은 여러 세균의 catalase-peroxidase와 높은 상동성을 나타냈다. V. vulnificus 에서 KatG catalase-peroxidase의 생리학적 역할을 알아보기 위해 positive selection suicide vector인 pKAS32를 사용하여 Vv-katG deletion mutant를 제조하였다. Vv-katG mutant는 H₂O₂처리에 대해 현저한 감수성을 나타냈다. 그리고 wild type V. vulnificus를 60 μM H₂O₂로 30분 동안 전처리 하여 Vv-KatG를 induction 시킬 경우 치사 농도인 4 mM H₂O₂ 에 대해서도 높은 저항성을 나타내었다. 이러한 adaptation 현상은 katG mutant에서는 관찰되지 않았다. Catalase-peroxidase 활성이 사라졌음에도 불구하고, katG 돌연변이주는 정상 균주와 비교해서 배양 24 시간까지 정상적인 배가시간을 보였다. 그러나 오랜 기간동안 교반 배양 시 돌연변이주의 viability 는 배양 24 시간 이후 정상 균주에 비해 보다 빠르게 감소하였다. 게다가, 낮은 온도와 인공바닷물의 영양 결핍 상태에서 배양 시 돌연변이주의 viability는 정상 균주에 비해 보다 신속히 감소하였다. 돌연변이주의 경우 일단 배양이 불가능한 VBNC 상태가 되면 정상 균주에서 보였던 어떠한 resuscitation도 관찰되지 않았다. Non-denaturing PAGE 분석 결과 Vv-KatG 는 V. vulnificus에 존재하는 유일한 catalase 이며, catalase-peroxidase KatG는 cytoplasmic fraction에 위치하고 있음을 알 수 있다. 60 μM H₂O₂ 전처리에 따른 Vv-KatG induction 은 Western blot analysis에 의해 증명되었다. H₂O₂ 첨가에 의해 induction된 세균은 지수생장기와 휴지기 모두에서 더 높은 수준의 Vv-KatG 발현을 보여주었다. katG 유전자 발현의 조절 단계를 알아보기 위해 chromosomal 또는 plasmid P_katG::lacZ transcriptional reporter strain을 사용하여 Vv-lacZ deletion mutant에서 katG 유전자의 전사활성을 조사하였다. 60 μM H₂O₂ 전처 리에 따른 어떠한 katG transcription도 관찰되지 않았다. 이를 토대로 Vv-KatG 발현은 전사 후 수준에서 조절되는 것으로 사료된다. 이상의 분자생물학적 및 생리적인 연구 분석 결과를 종합해 볼 때, V. vulnificus catalase-peroxidase가 자연환경에 생존할 수 있는 oxidative stress, starvation, low temperature, 그리고 hyperosmolarity 등의 다양한 스트레스에 대한 저항성에 중요한 역할을 담당할 것으로 사료된다. Vibrio vulnificus is a halophilic estuarine bacterium that causes necrotizing wound infections and fatal septicemia. The microorganism encounters a wide variety of stresses in the natural niche and during infection process. V. vulnificus experiences fluctuations of temperature, salinity, and nutrients through changing seasons. The pathogen also has to survive against the stresses imposed by host defense mechanisms. The alternative sigma factor σ^38 (RpoS) are well known to regulate expression of multiple genes coping with a wide spectrum of stresses. While trying to identify rpoS, we came to clone a catalase-peroxidase instead. In the present study, we cloned the catalase-peroxidase katG gene and investigated molecular biological and physiological roles of the KatG protein. Using a cosmid genomic library of V. vulnificus type strain ATCC29307, we have cloned and sequenced the katG gene of V. vulnificus (Vv-katG). The catalase- peroxidase is involved in the dismutation of H₂O₂, leaving O₂ and water as byproducts. Vv-katG is composed of a total of 2,172-bp nucleotides encoding 723 amino acid protein with estimated molecular mass of 79,530-Da. The DNA sequence of the Vv-katG showed 78% homology with catalase-peroxidase gene of V. cholerae. The deduced amino acid sequence of the Vv-KatG showed high homologies to multiple bacterial catalase-peroxidase such as Vibrio cholerae catalase-peroxidase (85%), Synechococcus sp. catalase-peroxidase (77%), Legionella pneumophila catalase-peroxidase KatB (75%), Agrobacterium tumefaciens catalase (72%), Synechocystis sp. catalase HPI (71%), Bacillus stearothermophilus catalase (66%), and Escherichia coli hydroperoxidase HPI (59%). To analyze the physiological role of the KatG catalase-peroxidase, a Vv-katG deletion mutant was constructed by allelic replacement using a positive selection suicide vector, pKAS32. Vv-katG mutant showed a marked sensitivity to H₂O₂ treatment. Adaptation of wild type V. vulnificus with 60 μMH₂O₂ pretreatment for 30 min resulted in an enhanced resistance to lethal dose of 4 mM H₂O₂. The adaptation phenomenon was not observed in the katG mutant. Despite the absence of catalase-peroxidase activity, the mutant showed normal doubling time until 24 hr culture when compared with the isogenic wild type. However, viability decreased more rapidly after 24 hr in the mutant, when the old cultures were vigorously agitated. In addition, viability of the mutant decreased more rapidly when the strain was rendered to low temperature and starvation in artificial seawater. No resuscitation of the mutant was observed once the cells became non-culturable. The Vv-KatG was the only catalase observed on the non-denaturing PAGE analysis and was localized only to the cytoplasmic fraction. Vv-KatG induction by 60 μM H₂O₂ pretreatment was proved by Western blot analysis. H₂O₂ induced cells showed higher level of Vv-KatG during both exponential and stationary phases. To determine whether the katG expression is controlled at the transcriptional level, transcriptional activity of katG gene was examined in the Vv-lacZ deletion mutant using a chromosomal or a plasmid P_katG::lacZ transcriptional reporter strain. No significant katG transcription was not observed by the 60 μM H₂O₂ pretreatment. Vv-KatG expression appeared to be regulated at the post-transcriptional level. By functional analysis, we propose that V. vulnificus catalase-peroxidase plays a pluripotent role in resisting to oxidative stress, carbon starvation, low temperature, and hyperosmolarity that V. vulnificus might encounter in environment.

Characterization and Attenuation of Regulatory Network of Vibrio vulnificus rtxA Encoding a MARTX Toxin

이지원 서울대학교 대학원 2020 국내박사

Bacterial pathogens have evolved the ability to survive and develop diseases in several different environments within the host. The ability requires the production of various virulence factors whose expressions are coordinately controlled by regulatory networks in response to environmental changes. The opportunistic human pathogen Vibrio vulnificus can cause food-borne diseases from gastroenteritis to life-threatening septicemia. Among a wide array of virulence factors produced by V. vulnificus, a multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin RtxA encoded by the rtxA gene plays an essential role in the virulence of the pathogen. It has been previously reported that the expression of rtxA is negatively and positively regulated by direct binding of H-NS and HlyU to the rtxA promoter, PrtxA, respectively. In the present study, I have further examined additional regulatory proteins as well as environmental signals involved in the rtxA expression and identified a small-molecule inhibitor that attenuates the virulence of V. vulnificus. As a result, a leucine-responsive regulatory protein (Lrp) was found as a positive regulator of rtxA. Electrophoretic mobility shift and DNase I protection assays revealed that Lrp activates the rtxA expression by binding directly and specifically to PrtxA. Notably, DNase I cleavage of the PrtxA regulatory region showed phased hypersensitivity, suggesting that Lrp probably induces the DNA bending in PrtxA. Lrp activates rtxA in an independent manner with H-NS and HlyU, and leucine inhibits the binding of Lrp to PrtxA and thus decreases the Lrp-mediated activation. Moreover, a cyclic AMP receptor protein (CRP) acts as a negative regulator of the rtxA transcription, and exogenous glucose relieves the CRP-mediated repression. Interestingly, biochemical and mutational analyses demonstrated that CRP binds directly and specifically to the upstream regions of PrtxA, which presumably changes the DNA conformation in PrtxA and represses rtxA. Furthermore, CRP represses the expressions of lrp and hlyU by directly binding to their upstream regions, forming coherent feedforward loops with Lrp and HlyU. Taken together, a regulatory network comprising CRP, Lrp, H-NS, and HlyU coordinates the expression of rtxA in response to changes in host environmental signals such as leucine and glucose. This collaborative regulation will contribute to the precise expression of rtxA during the pathogenesis of V. vulnificus. My next concern was about new approaches called anti-virulence strategies that target virulence of bacterial pathogens in an attempt to control the virulence of V. vulnificus. Anti-virulence strategies have the advantage of less selective pressure for inducing resistance than conventional strategies that target viability of the pathogens. Therefore, I performed a high-throughput screening of the small-molecule library containing 8,385 compounds to inhibit HlyU, a transcriptional activator essential for the expression of V. vulnificus rtxA. A small molecule [N-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)-3-phenylprop-2-ynamide] was identified as an inhibitor of the HlyU activity and named CM14. CM14 reduces HlyU-dependent virulence gene expression in V. vulnificus, but does not suppress the bacterial growth or cause host cell death. Treatment of CM14 decreases hemolysis of human erythrocytes and impedes host cell rounding and lysis caused by V. vulnificus. Remarkably, co-administration of CM14 improves the survival of mice infected with V. vulnificus by alleviating hepatic and renal dysfunction and systemic inflammation. As revealed by biochemical, mass spectrometric, and mutational analyses, CM14 covalently modifies the Cys30 residue of HlyU to prevent the protein from binding to the target DNA. Based on these results, a possible molecular mechanism is proposed for the covalent modification of HlyU by CM14. Because HlyU is a conserved transcriptional activator of virulence genes in Vibrio species, CM14 is also capable of reducing the expressions of multiple virulence genes in other Vibrio species and attenuating their virulence-related phenotypes. The combined results suggest that this small-molecule could be an anti-virulence agent against HlyU-harboring pathogenic Vibrio species with a low possibility of developing resistance. 세균성 병원균은 여러 다른 환경에서 생존 및 증식하며 숙주 내에서 질병을 일으키는 능력을 진화시켜 왔다. 이러한 능력은 환경 변화에 따라 조절 시스템에 의해 공동으로 발현이 조절되는 다양한 독성 인자의 생산을 필요로 한다. 기회감염균인 인간 병원균 패혈증 비브리오균은 식중독을 유발함으로써 위장염과 생명을 위협하는 패혈증을 야기할 수 있다. 패혈증 비브리오균이 생성하는 다양한 독성 인자들 중, rtxA 유전자에 의해 발현되는 Multifunctional-autoprocessing repeat-in-toxin (MARTX) toxin RtxA는 이 병원균의 독성에 있어 필수적인 역할을 한다. 기존 연구에 따르면, 패혈증 비브리오균의 전사 조절자 H-NS와 HlyU만이 rtxA 발현을 각각 음성적으로 또는 양성적으로 조절한다고 알려져 있다. 본 연구에서는, rtxA 발현에 관여하는 추가적인 조절 단백질과 환경 신호를 조사하고, 패혈증 비브리오균의 독성을 제어하는 소분자 저해 물질(small-molecule inhibitor)을 발굴하였다. 그 결과, Leucine-responsive regulatory protein (Lrp)이 rtxA의 양성 조절자로 발견되었다. Electrophoretic mobility shift assay (EMSA) 및 DNase I 보호 분석 (DNase I protection assay) 결과, Lrp가 PrtxA에 직접적이고 특이적으로 결합하여 rtxA 발현을 활성화시킴을 확인하였다. 특히, PrtxA 조절 영역의 DNase I cleavage는 phased hypersensitivity를 보였는데, 이는 Lrp가 아마도 PrtxA의 DNA 굽힘(DNA bending)을 유도함을 시사한다. Lrp는 H-NS, HlyU와 독립적인 방식으로 rtxA를 활성화시키며, 류신은 Lrp가 PrtxA에 결합하는 것을 저해하고 Lrp에 의해 매개되는 rtxA 발현을 감소시켰다. 또한, cAMP receptor protein (CRP)은 rtxA 발현의 음성 조절자로 작용하며, 외인성 포도당은 CRP에 의해 매개되는 rtxA 억제를 완화하였다. 흥미롭게도, 생화학 및 돌연변이 분석은 CRP가 PrtxA의 upstream region에 직접적이고 특이적으로 결합하여 PrtxA의 DNA 형태를 변화시키고 rtxA를 억제할 수 있음을 입증하였다. 더불어, CRP는 lrp와 hlyU의 upstream region에 직접 결합함으로써 Lrp와 HlyU의 발현을 억제하고, 동 단백질들과 coherent feedforward loop를 형성함으로써 rtxA 발현을 간접적으로 억제하였다. 결론적으로, CRP, Lrp, H-NS, HlyU로 구성된 조절 네트워크는 패혈증 비브리오균의 감염 동안 류신과 포도당 등의 숙주 환경 신호의 변화에 반응하여 rtxA 발현을 공동으로 조절한다. 이러한 공동 조절은 패혈증 비브리오균의 발병 동안 rtxA의 정확한 발현에 기여할 것이다. 패혈증 비브리오균의 독성을 제어하려는 시도에서, 세균성 병원균의 독성을 표적으로 하는 항독성 전략이라는 새로운 방식이 개발되어 본 연구에 적용되었다. 항독성 전략은 생존 능력(viability)을 표적으로 하는 기존의 전략보다 내성을 유도하는 선택적 압력이 낮다는 장점이 있다. 따라서, 패혈증 비브리오균의 독성에 필수적인 전사 조절자 HlyU를 저해하기 위해8,385개의 화합물을 포함하는 small molecule library의 대량 발굴 탐색(high-throughput screening)을 수행하였다. 그 결과, [N-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)-3-phenylprop-2-ynamide]의 소분자가 HlyU 활성의 저해제로서 발견되었고, CM14으로 명명되었다. CM14은 패혈증 비브리오균의 HlyU에 의해 활성화되는 독성 유전자 발현을 감소시키지만, 균의 생장을 저해하거나 숙주 세포 사멸을 일으키지 않았다. CM14의 처리는 패혈증 비브리오균이 야기하는 인간 적혈구의 용혈을 감소시키고 숙주 세포의 rounding 및 용해를 저지하였다. CM14의 투여는 패혈증 비브리오균에 감염된 쥐의 간 및 신장 기능 장애와 전신성 염증을 완화시킴으로써 쥐의 생존에 기여하였다. 생화학적, 질량 분석, 돌연변이 분석에서 밝혀진 바와 같이, CM14은 HlyU의 Cys30 잔기를 공유 변형(covalent modification)시키고 동 단백질이 표적 DNA에 결합하는 것을 막는다. 이러한 결과를 바탕으로, CM14에 의한 HlyU의 covalent modification 에 대해 가능한 분자 메커니즘이 제안되었다. HlyU는 다양한 비브리오 종 내 보존된 독성 유전자 전사 활성화 인자이기 때문에, CM14은 다른 비브리오 종의 여러 독성 유전자 발현을 감소시키고 이들의 독성을 약화시켰다. 종합적으로, 본 연구는 CM14이 저항성을 발생시킬 가능성이 낮으며, HlyU를 보유하는 다양한 독성 비브리오 종에 대한 항독성 물질이 될 수 있음을 제시한다.