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.

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.

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.

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.

(The) roles of vibrio vulnificus and its virulence factor RtxA in dendritic cell-mediated Th17 responses

이아림 Graduate School, Korea University 2019 국내박사

비브리오 불니피쿠스(Vibrio vulnificus)는 그람 음성균으로, 오염된 해산물이나 벌어진 상처를 통해 호스트의 체내로 침입, 위장염부터 패혈증까지 유발하는 위험한 균이다. 비브리오균에 감염되었을 때, 항원 제시 세포(Antigen Presenting Cell, APC)인 수지상세포(Dendritic Cell, DC)에 의해 주로 매개되는 염증성 반응 관련된 여러 가지 전염증성 사이토카인(proinflammatory cytokine)의 생성이 촉진된다고 알려져 있다. 이를 통해 다양한 T 도움 세포 반응이 유도되는데, 그 중에서도 T 도움 17 세포의 경우, 세포의 반응이 비정상적으로 과하게 일어나면 조직이 손상되어 병원균의 침투를 야기하는 반면, 적정 수준의 반응은 감염에 대한 호스트의 방어 체계에 관여한다고 알려져 있다. 본 연구에서는 비브리오균 불니피쿠스 균의 감염이 수지상세포의 성숙(maturation)과 활성(activation)을 통해 T 도움 17 세포로의 분극화를 유도하는지와 그 과정에 비브리오 불니피쿠스 균의 독성인자가 관여하는지에 관해 알아보고자 하였다. 첫째, 수지상세포는 비브리오 불니피쿠스 균에 감염되어 CD40, CD80등의 공동자극분자(co-stimulatory molecule)와 MHC Ⅱ의 발현이 증가하여 성숙과 활성화되었다. RT-PCR과 ELISA를 통해, T 도움 17 세포로 분화시키는 interleukin-1β (IL-1β)와 interleukin-6 (IL-6) 역시 mRNA와 단백질 수준에서 증가하였다. 더불어, 비브리오 불니피쿠스 균에 노출된 수지상세포를 림프노드에서 분리한 naïve T 세포와 공배양하였을 때 T 도움 17 세포군이 증가하였고, 이는 IL-6의 중화 항체 처리에 의해 다시 감소하였다. 추가로 비브리오 불니피쿠스 균에 노출된 수지상세포를 마우스 체내로 주입하였을 때에도 마우스의 림프노드 내 T 도움 17 세포군은 증가하였다. 마지막으로, 비브리오균을 경구 투여한 후 장내 대부분의 면역 세포가 존재하는 점막 고유층의 면역세포군을 분석하여 생체 내에서도 T 도움 17 세포군이 증가함을 확인하였다. 위의 결과들은 비브리오 불니피쿠스 균이 수지상세포를 통해 T 도움 17 세포 반응을 유도하고, 이는 면역병리학적 효과와 관련이 있을 것이라는 것을 보여준다. 둘째, T 도움 17 세포는 호스트의 방어 기작과 병리학적 염증 반응을 매개하는 전염증성 T 도움 세포의 한 종류이다. 우리는 이전의 연구에서 호스트의 수지상 세포가 비브리오 불니피쿠스 균에 노출되었을 때 IL-1β와 IL-6와 같은 여러 사이토카인의 생성을 통해 T 도움 17 세포 반응을 유도한다고 보고하였다. 비브리오 불니피쿠스 균은 성공적인 병리생리학을 결정짓는 주요 독성인자인 RTX 독소 (RtxA)를 생성하는데, 본 연구에선 비브리오 불니피쿠스가 수지상세포의 성숙과 활성화를 통해 T 도움 17 세포반응을 유도하는 과정에서 RtxA가 관여하는지에 관해 알아보았다. 비브리오 불니피쿠스 야생형 (WT)에 의해 증가하였던 수지상세포의 표면 표지자인 CD40의 발현은 rtxA 유전자에 돌연변이가 발생한 비브리오 불니피쿠스 균이 감염되었을 때 감소하였고, T 도움 17 세포 분화와 관련된 사이토카인의 mRNA와 단백질 생성 수준 역시 rtxA 유전자 돌연변이체에 의해 감소하였다. 야생형과 rtxA 유전자 돌연변이체 비브리오 불니피쿠스 균에 노출된 수지상세포를 순수 CD4 T 세포와 공배양한 경우에서도 역시 T 도움 17 세포 반응은 돌연변이체에서 감소하였다. 이는 비브리오균을 경구 투여한 후 장내 대부분의 면역 세포가 존재하는 점막 고유층의 면역세포군을 분석한 결과에서도 마찬가지였다. rtxA 유전자의 돌변연이로 인해 감소한 수지상세포의 성숙 정도와 T 도움 17 세포 분화 관련 사이토카인의 발현, T 도움 17 세포의 반응 등은 rtxA 유전자 복귀 돌연변이체(revertant)에 의해 다시 회복되었다. rtxA 유전자의 상위 조절자인 hlyU 유전자의 돌연변이체로 실험하였을 때 역시 rtxA 유전자 돌연변이체와 같은 결과를 얻었다. 따라서, 본 연구에서 비브리오 불니피쿠스 균은 RtxA 독소를 통해 T 도움 17 세포 반응을 유도하고, 이는 비브리오 불니피쿠스 균에 대한 호스트의 적응 면역 반응(adaptive immune response)과 연관이 있다는 것을 처음으로 규명하였다. 위의 연구는 비브리오 불니피쿠스 균의 RtxA 독소를 이용한 비브리오 불니피쿠스에 의한 질병의 예방과 치료 목적의 약물의 개발에 도움이 될 것으로 생각된다. Vibrio vulnificus (V. vulnificus) is a halophilic, gram-negative bacterium, which causes life-threatening septicemia and gastroenteritis through the consumption of contaminated seafood or wound infection. In addition, V. vulnificus infection is known to stimulate the production of several pro-inflammatory cytokines, which are associated with inflammatory responses mediated predominantly by dendritic cells (DCs), functioning as antigen-presenting cells. Here, I investigated whether V. vulnificus infection induced the maturation and activation of murine DCs, which have the ability to polarize T helper (Th) cells into Th17 cells. Dysregulated Th17 cell responses are known to cause tissue damage, promoting the penetration of pathogens; however, Th17 cells are also involved in host defense against infection. Infection with V. vulnificus significantly increased the expression of cell surface molecules, including CD40, CD80 and major histocompatibility complex class II, leading to the maturation and activation of DCs. In the present study, the analysis of the cytokine profiles of DCs upon infection with V. vulnificus revealed the preferential production of interleukin-1β (IL-1β) and IL-6, through which V. vulnificus-infected DCs induced the polarization of Th17 cells when naïve CD4+ T cells were co-incubated. The reduction of Th17 cell generation through the use of anti-IL-6 neutralizing antibodies indicated that the Th17-polarizing capacity of V. vulnificus was predominantly dependent on DC-derived IL-6. The in vivo administration of V. vulnificus-infected DCs consistently increased the Th17 cell population in the lymph nodes of mice. Finally, the oral administration of V. vulnificus in mice also increased Th17 cell responses in the lamina propria of the small intestine. These results collectively demonstrated that V. vulnificus induced inflammatory Th17 cell responses via DCs, which may be associated with the immunopathological effects caused by V. vulnificus infection. Th17 cells are a subset of pro-inflammatory T helper cells that mediate host defense and pathological inflammation. In the previous study, I have reported that host DCs infected with V. vulnificus induce Th17 responses through the production of several pro-inflammatory cytokines, including IL-1β and IL-6. V. vulnificus produces RTX toxin (RtxA), an important virulence factor that determines successful pathophysiology. In this study, I investigated the involvement of RtxA from V. vulnificus in Th17 cell induction through the activation and maturation of DCs. The increased expression of the DC surface marker CD40 caused by V. vulnificus wild-type infection was reduced by rtxA gene mutation in V. vulnificus. The mRNA and protein levels of Th17 polarization-related cytokines also decreased in V. vulnificus rtxA mutant infected DCs. In addition, the co-culture of Th cells and DCs infected with rtxA mutant V. vulnificus resulted in reduction in DC-mediated Th17 responses. Th17 cell responses in the small intestinal lamina propria decreased in mice inoculated with V. vulnificus rtxA mutant as compared to those inoculated with the wild-type strain. These decreases in DC maturation, Th17-polarizing cytokine secretion, and Th17 responses attributed to rtxA mutation were restored following infection with the rtxA revertant strain. Furthermore, the mutation in the hlyU gene encoding the activator of rtxA1 gene reproduced the results observed with rtxA mutation. Taken together, V. vulnificus, by means of RtxA, induces inflammatory Th17 responses, which may be associated with adaptive responses of the host against V. vulnificus infection. In conclusion, these findings indicate that V. vulnificus RtxA induces Th17 cell responses via the increased activation and maturation of BMDCs with IL-1β and IL-6 production, Th17-polarizing cytokines, in vitro and in vivo. Therefore, I revealed the novel virulence factor of V. vulnificus that is involved in adaptive immune responses against V. vulnificus infection.

Research on the Vibrio vulnificus Infectious Disease Biomarker and Development of Antibiotic-free Inhibitors Using Nanotechnology

이영민 대구한의대학교 일반대학원 2020 국내석사

Vibrio vulnificus (V. vulnificus) is an anaerobic halophilic bacterium that often induces gastrointestinal epithelial cells death, and gastroenteritis. VvhA, a hemolytic toxin, has been considered to be a major exoprotease that causes an apoptosis process affecting gastrointestinal epithelial cells. In this study, we carried out an investigation of the cellular mechanism of a 36 kDa glycoprotein isolated from RVS fruits (RVS glycoprotein) and curcumin nanosphere (CN) to improve the therapeutic potential of curcumin during the apoptosis of human intestinal epithelial cells induced by VvhA produced by V. vulnificus. The recombinant protein (r) VvhA stimulated the apoptosis by producing intracellular ROS, which was significantly inhibited by RVS glycoprotein and CN. We found that RVS glycoprotein and CN blocked phosphorylation of NF-κB, which are responsible for mitochondria-mediated apoptotic factors in a cell treated with rVvhA. In rVvhA-treated ileal-ligated mouse, CN blocked the level of intestinal apoptosis. These results indicate that RVS glycoprotein inhibits mitochondrial apoptosis stimulated by rVvhA through the blockage of ROS-mediated signaling pathway in HCT116 cells. Furthermore, CN is effective delivery system to improve the bioavailability of curcumin in the gut and has inhibitory effects on mitochondrial apoptosis induced by V. vulnificus. 비브리오균(Vibrio vulnificus)은 혐기성의 호염균으로서 인체로 유입되어 다양한 독소를 만들어 위장관 세포 사멸 및 위장염을 유발한다고 알려져 있다. 그 중 VvhA는 위장 상피 세포에서 세포자멸사 과정을 일으키는 용혈성 독소로 알려져 있다. 현재까지 비브리오균의 감염에 대한 안전한 항생제-프리 치료법은 밝혀진 바가 적다. 본 연구는 인간의 장 상피 세포에서 VvhA에 의해 유도된 세포자멸사에 있어서 in vitro 및 in vivo 실험을 통해 옻나무 과육에서 추출한 당단백질(RVS glycoprotein)과 curcumin (커큐민)의 생체이용률 한계를 향상시킨 curcumin nanosphere (CN)의 효과를 밝혔다. 장 상피 세포에서 재조합 단백질 (r) VvhA는 세포 내 ROS를 생성함으로써 세포자멸사를 촉진하였으며, 이는 RVS glycoprotein 및 CN에 의해 억제되었다. 특히, rVvhA에 감염된 세포에서 전사인자 NF-κB의 활성 억제를 통하여 세포자멸사에 관련된 인자들(Bax, Bcl-2, cleaved caspase-3)의 발현을 감소시켰다. 또한, in vivo 실험을 통해 rVvhA가 처리된 회장 결찰 마우스모델의 세포자멸사에 대한 CN의 효과를 밝혔다. 본 실험에서 CN은 rVvhA에 의해 유도된 마우스 장의 NF-κB 활성, Bax, Bcl-2, 그리고 cleaved caspase-3의 발현을 모두 정상화시켰다. 결론적으로, 본 연구에서 RVS glycoprotein이 장 상피 세포에서 ROS 생산에 매개한 신호 전달 인자의 억제를 통해 rVvhA에 의해 유도된 미토콘드리아 세포자멸사를 억제한다는 것을 알 수 있다. 또한 CN은 장에서 커큐민의 생체 이용률을 향상시킨 효과적인 전달 시스템이며 장 상피 세포에서 ROS 생산에 매개한 신호 전달 인자의 억제를 통해 rVvhA에 의해 유도된 미토콘드리아 세포자멸사에 대한 억제 효과를 갖는다는 것을 밝힌 최초의 연구이다.