CTXphi infection of Vibrio cholerae requires the tolQRA gene products

CTXphi is a lysogenic filamentous bacteriophage that encodes cholera toxin. Filamentous phages that infect Escherichia coli require both a pilus and the products of tolQRA in order to enter host cells. We have previously shown that toxin-coregulated pilus (TCP), a type IV pilus that is an essential Vibrio cholerae intestinal colonization factor, serves as a receptor for CTXphi. To test whether CTXphi also depends upon tol gene products to infect V. cholerae, we identified and inactivated the V. cholerae tolQRAB orthologues. The predicted amino acid sequences of V. cholerae TolQ, TolR, TolA, and TolB showed significant similarity to the corresponding E. coli sequences. V. cholerae strains with insertion mutations in tolQ, tolR, or tolA were reduced in their efficiency of CTXphi uptake by 4 orders of magnitude, whereas a strain with an insertion mutation in tolB showed no reduction in CTXphi entry. We could detect CTXphi infection of TCP(-) V. cholerae, albeit at very low frequencies. However, strains with mutations in both tcpA and either tolQ, tolR, or tolA were completely resistant to CTXphi infection. Thus, CTXphi, like the E. coli filamentous phages, uses both a pilus and TolQRA to enter its host. This suggests that the pathway for filamentous phage entry into cells is conserved between host bacterial species.     

Characterization of VPI pathogenicity island and CTXphi prophage in environmental strains of Vibrio cholerae

Environmental isolates of Vibrio cholerae of eight randomly amplified polymorphic DNA (RAPD) fingerprint types from Calcutta, India, that were unusual in containing toxin-coregulated pilus or cholera toxin genes but not O1 or O139 antigens of epidemic strains were studied by PCR and sequencing to gain insights into V. cholerae evolution. We found that each isolate contained a variant form of the VPI pathogenicity island. Distinguishing features included (i) four new alleles of tcpF (which encodes secreted virulence protein; its exact function is unknown), 20 to 70% divergent (at the protein level) from each other and canonical tcpF; (ii) a new allele of toxT (virulence regulatory gene), 36% divergent (at the protein level) in its 5' half and nearly identical in its 3' half to canonical toxT; (iii) a new tcpA (pilin) gene; and (iv) four variant forms of a regulatory sequence upstream of toxT. Also found were transpositions of an IS903-related element and function-unknown genes to sites in VPI. Cholera toxin (ctx) genes were found in isolates of two RAPD types, in each case embedded in CTXphi-like prophages. Fragments that are inferred to contain only putative repressor, replication, and integration genes were present in two other RAPD types. New possible prophage repressor and replication genes were also identified. Our results show marked genetic diversity in the virulence-associated gene clusters found in some nonepidemic V. cholerae strains, suggest that some of these genes contribute to fitness in nature, and emphasize the potential importance of interstrain gene exchange in the evolution of this species.     

The Vibrio cholerae O139 Calcutta bacteriophage CTXphi is infectious and encodes a novel repressor.

CTXphi is a lysogenic, filamentous bacteriophage. Its genome includes the genes encoding cholera toxin (ctxAB), one of the principal virulence factors of Vibrio cholerae; consequently, nonpathogenic strains of V. cholerae can be converted into toxigenic strains by CTXphi infection. O139 Calcutta strains of V. cholerae, which were linked to cholera outbreaks in Calcutta, India, in 1996, are novel pathogenic strains that carry two distinct CTX prophages integrated in tandem: CTX(ET), the prophage previously characterized within El Tor strains, and a new CTX Calcutta prophage (CTX(calc)). We found that the CTX(calc) prophage gives rise to infectious virions; thus, CTX(ET)phi is no longer the only known vector for transmission of ctxAB. The most functionally significant differences between the nucleotide sequences of CTX(calc)phi and CTX(ET)phi are located within the phages' repressor genes (rstR(calc) and rstR(ET), respectively) and their RstR operators. RstR(calc) is a novel, allele-specific repressor that regulates replication of CTX(calc)phi by inhibiting the activity of the rstA(calc) promoter. RstR(calc) has no inhibitory effect upon the classical and El Tor rstA promoters, which are instead regulated by their cognate RstRs. Consequently, production of RstR(calc) renders a CTX(calc) lysogen immune to superinfection by CTX(calc)phi but susceptible (heteroimmune) to infection by CTX(ET)phi. Analysis of the prophage arrays generated by sequentially integrated CTX phages revealed that pathogenic V. cholerae O139 Calcutta probably arose via infection of an O139 CTX(ET)phi lysogen by CTX(calc)phi.     

Statins as a newly recognized type of immunomodulator

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, or statins, are effective lipid-lowering agents, extensively used in medical practice. Statins have never been shown to be involved in the immune response, although a report has indicated a better outcome of cardiac transplantation in patients under Pravastatin therapy. Major histocompatibility complex class II (MHC-II) molecules are directly involved in the activation of T lymphocytes and in the control of the immune response. Whereas only a limited number of specialized cell types express MHC-II constitutively, numerous other cells become MHC-II positive upon induction by interferon gamma (IFN-gamma). This complex regulation is under the control of the transactivator CIITA (refs 6,7). Here we show that statins act as direct inhibitors of induction of MHC-II expression by IFN-gamma and thus as repressors of MHC-II-mediated T-cell activation. This effect of statins is due to inhibition of the inducible promoter IV of the transactivator CIITA and is observed in several cell types, including primary human endothelial cells (ECs) and monocyte-macrophages (Mstraight phi). It is of note that this inhibition is specific for inducible MHC-II expression and does not concern constitutive expression of CIITA and MHC-II. In repressing induction of MHC-II, and subsequent T-lymphocyte activation, statins therefore provide a new type of immunomodulation. This unexpected effect provides a scientific rationale for using statins as immunosuppressors, not only in organ transplantation but in numerous other pathologies as well.     

Prophage CTXphi genome variability and its role in alteration of Vibrio cholerae El Tor virulence characteristics

Comparative analysis of CTXphi prophage genome of 366 V. cholerae El Tor strains isolated from infected people and water was carried out using the polymerase chain reaction. Four groups of vibrios, which carry different combinations of ctxA, zot, and ace genes from core region of CTXphi prophage coding key (cholera enterotoxin) and accessory (Zot and Ace toxins) pathogenicity factors, were determined: ctxA(+) zot(-) ace(+), ctxA(-) zot(+) ace(+), ctxA(-) zot(+) ace(-), ctxA(-) zot(-) ace(+). Vibrios that had lost all tested genes were also revealed. Genomic rearrangements occurring in water environment in virulent V. cholerae strains, which acquired foreign pathogenicity genes necessary for their existence in human organism, were proposed as one of the mechanisms of formation of clones with an incomplete or no prophage. Infection process in model animals challenged with wild and isogenic strains of V. cholerae differing in the set of the phage genes (ctxA, zot, and ace) was comparatively analyzed. It was shown that variability of CTXphi prophage genome was an important factor of modification of cholera vibrios virulent characteristics. Obtained data point to usefulness of ctxA, zot, and ace phage genes detection in wild V. cholerae isolates as it could permit evaluation of their virulent potential determining the severity of the infection.     

Molecular analyses of a putative CTXphi precursor and evidence for independent acquisition of distinct CTX(phi)s by toxigenic Vibrio cholerae

The genes encoding cholera toxin (ctxA and ctxB) are encoded in the genome of CTXphi, a filamentous phage that infects Vibrio cholerae. To study the evolutionary history of CTXphi, we examined genome diversity in CTX(phi)s derived from a variety of epidemic and nonepidemic Vibrio sp. natural isolates. Among these were three V. cholerae strains that contained CTX prophage sequences but not the ctxA and ctxB genes. These prophages each gave rise to a plasmid form whose genomic organization was very similar to that of the CTXphi replicative form, with the exception of missing ctxAB. Sequence analysis of these three plasmids revealed that they lacked the upstream control region normally found 5' of ctxA, as well as the ctxAB promoter region and coding sequences. These findings are consistent with the hypothesis that a CTXphi precursor that lacked ctxAB simultaneously acquired the toxin genes and their regulatory sequences. To assess the evolutionary relationships among additional CTX(phi)s, two CTXphi-encoded genes, orfU and zot, were sequenced from 13 V. cholerae and 4 V. mimicus isolates. Comparative nucleotide sequence analyses revealed that the CTX(phi)s derived from classical and El Tor V. cholerae isolates comprise two distinct lineages within otherwise nearly identical chromosomal backgrounds (based on mdh sequences). These findings suggest that nontoxigenic precursors of the two V. cholerae O1 biotypes independently acquired distinct CTX(phi)s.     

pIIICTX,a predicted CTXphi minor coat protein,can expand the host range of coliphage fd to include Vibrio cholerae

CTXphi is a filamentous bacteriophage that encodes cholera toxin. CTXphi infection of its host bacterium, Vibrio cholerae, requires the toxin-coregulated pilus (TCP) and the products of the V. cholerae tolQRA genes. Here, we have explored the role of OrfU, a predicted CTXphi minor coat protein, in CTXphi infection. Prior to the discovery that it was part of a prophage, orfU was initially described as an open reading frame of unknown function that lacked similarity to known protein sequences. Based on its size and position in the CTXphi genome, we hypothesized that OrfU may function in a manner similar to that of the coliphage fd protein pIII and mediate CTXphi infection as well as playing a role in CTXphi assembly and release. Deletion of orfU from CTXphi dramatically reduced the number of CTXphi virions detected in supernatants from CTXphi-bearing cells. This defect was complemented by expression of orfU in trans, thereby confirming a role for this gene in CTXphi assembly and/or release. To evaluate the requirement for OrfU in CTXphi infection, we introduced fragments of orfU into gIII in an fd derivative to create OrfU-pIII fusions. While fd is ordinarily unable to infect V. cholerae, an fd phage displaying the N-terminal 274 amino acids of OrfU could infect V. cholerae in a TCP- and TolA-dependent fashion. Since our findings indicate that OrfU functions as the CTXphi pIII, we propose to rename OrfU as pIII(CTX). Our data also provide new evidence for a conserved pathway for filamentous phage infection.     

The Zymovars of Vibrio cholerae: multilocus enzyme electrophoresis of Vibrio cholerae

Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest. Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia. The mean genetic diversity was 0.339. It is shown that the same O antigen (both O1 and non-O1) may be present in several genetically diverse (different zymovars) strains. Conversely the same zymovar may contain more than one serogroup. It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase). Here it is shown that this rare allele is present in 1 V. mimicus and 4 non-O1 V. cholerae. Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars. The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V. cholerae.     

A variant type of Vibrio cholerae SXT element in a multidrug-resistant strain of Vibrio fluvialis

Vibrio fluvialis strain H-08942 was isolated from an infant aged 6 months who was suffering from cholera-like diarrhea in India. This strain showed the typical multidrug-resistance phenotype of an SXT element. It was resistant to sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm) and streptomycin (Sm), in addition to other antibiotics such as ampicillin (Am), furazolidone (Fz), nalidixic acid (Na), and gentamicin (Gm). The SXT element is a Vibrio cholerae-derived integrative and conjugative element (ICE) that has also been referred to as a conjugative transposon. Our goal was to find a relationship between these resistant phenotypes and the presence of the SXT element in this unique strain. By using PCR, we detected the antibiotic resistance genes, the integrase gene and the attP attachment site of SXT element. Cloning and DNA sequencing results showed that both the SXT integrase gene and its attP site of V. fluvialis were similar but not identical to those of V. cholerae. The SXT integrase gene of V. fluvialis has a 99% identity to that of V. cholerae, and the attP site of SXT of V. fluvialis is variant and shorter (641 bp) than that of V. cholerae (785 bp). It was possible for the SXT of V. fluvialis to be transferred by conjugation to a laboratory strain of Escherichia coli. Here, we report the detection of a variant SXT element in species other than V. cholerae, with molecular characterization and analysis of its integrase gene and its attP site.     

Quorum-sensing- and type VI secretion-mediated spatiotemporal cell death drives genetic diversity in Vibrio cholerae

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Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.     

Variation in the genome of CTXphi prophage of Vibrio cholerae biovar El Tor caused by Tn5-Mob transposon

A key pathogenicity factor of the cholera etiologic agent is cholera toxin (CT) whose synthesis is encoded by the ctxAB operon forming apart of the CTXphi ptophage. Alterations in the virulent properties of the cholera vibrios are based on the variability of the CTXphi prophage containing the genes for ctxAB, zot, ace, cep, orfU, and psh in its core region. At the same time, the mechanism of the porophage genome reorganization needs further and more profound analysis. The goal of this work was to demonstrate that transposon Tn5-Mob (Kmr), when introduced into the chromosome of the V. cholera model strain MAK757 El Tor biovar containing two copies of the CTXphi prophage provoked a reorganization in the CTXphi prophage consisting in the deletion of zot, ace, cep, orfU genes. The level of the CT biosynthesis in the insertion mutants MAK757 chr::Tn5-Mob still retaining only the ctxAB operon, increased more than 2000 times as compared to that of the original strain. The enhanced CT production was shown to be associated with the altered structure of the chromosomal DNA region containing one copy of the ctxAB operon encoding this protein biosynthesis. The mutation in the CTXphi genome induced by Tn5-Mob was unstable. Among 600 isolated colonies obtained after dissemination of the MAK757 chr::Tn5-Mob transposant capable of CT overproduction in the full medium with no antibiotics, 5.8% gave clones that in parallel to the loss of Kmr marker, appeared to be deprived of the ctxAB operon thus becoming non-toxinogenic. The observed formation of the V. cholerae insertion mutants both capable of CT overproduction and non-toxinogenic ones, may be indicative of an important role played in the evolution of the cholera pathogen by the CTXphi genome variability induced by Tn elements. The plasmidless V. cholerae El Tor strain characterized by type II CT hyperproduction thus obtained in our experiments could be used for the production of this protein routinely applied to construct efficient cholera diagnostic and prophylactic preparations.     

霍乱弧菌Ⅵ型分泌系统的效应蛋白对细菌细胞壁的降解机制

【背景】肽聚糖(Peptidoglycan,PG)是细菌细胞壁的重要组成部分,而霍乱弧菌Ⅵ型分泌系统(Type Ⅵ Secretion System,T6SS)可以分泌具有肽聚糖水解酶活性的效应蛋白到受体细菌中杀死细胞,这类水解酶的作用机制尚未研究清楚。【目的】通过对细菌细胞壁的PG成分进行研究,建立细胞壁PG成分分析方法,并对霍乱弧菌T6SS分泌的2个破坏细胞壁的效应蛋白TseH和VgrG3的作用机制进行解析。【方法】使用显微镜观察TseH和VgrG3异位表达对宿主细菌生长的影响;纯化大肠杆菌细胞壁,使用透射电子显微镜(Transmission Electron Microscope,TEM)观察提纯的细胞壁形态;使用纯化的TseH和VgrG3分解消化PG,利用超高效液相色谱-飞行时间质谱(Ultra-Performance LiquidChromatography-Time-of-FlightMassSpectrometry,UPLC-TOFMS)分析鉴定消化后的产物成分;通过分析结果推导结构。【结果】通过透射电子显微镜观察,发现提纯的PG呈现半透明的薄膜泡状;通过UPLC-TOFMS的分析以及逆向推导,得到了提纯的PG被VgrG3水解酶降解之后的3种主要产物,分别是二糖二肽(Disaccharide,Di)、二糖三肽(Disaccharide Tripeptide,Tri)和二糖四肽(Disaccharide Tetrapeptide,Tetra)。【结论】建立了提纯PG和UPLC-TOFMS分析PG成分的方法,揭示了效应蛋白VgrG3而非TseH可以降解PG多糖链N-乙酰葡糖胺和N-乙酰胞壁酸之间的β(1-4)糖苷键的功能。由于攻击细胞壁的效应蛋白在革兰氏阴性细菌中广泛存在,本研究不仅为鉴定这类重要效应蛋白的功能提供了有效的方法,而且对研究靶向细胞壁的新型抗生素也有重要的指导作用。