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.     

Decrease in culturability of Vibrio cholerae caused by glucose.

The culturability of Vibrio cholerae O1 serotype Inaba strain 569B was decreased by the addition of glucose to cell suspensions in starvation media. A similar effect was observed with sucrose, maltose, and fructose. We term this inhibitory effect glucose shock. It was not observed with arabinose or xylose or with carboxylates, such as acetate and pyruvate. No acidification of the medium occurred in the presence of these carbohydrates. Glucose shock was prevented by the addition of nitrogen or phosphorus sources. In the presence of phosphate, the bacterium produced formic acid from glucose. The phenomenon of glucose shock was also observed in V. cholerae O1 serotype Inaba strain RIMD 2203082 but not in strain RIMD 2203088 (O1 Inaba), IID 936 (O1 Ogawa), or RIMD 2214034 (non-O1). The culturability of Escherichia coli, Enterobacter aerogenes, and Listonella anguillarum did not decrease in starvation media with added glucose. Hence, the phenomenon should have ecological significance in determining the distribution of bacteria in marine ecosystems in situations where carbohydrates are abundant, but nitrogen and phosphorus are limiting.     

The histone-like nucleoid structuring protein (H-NS) is a repressor of Vibrio cholerae exopolysaccharide biosynthesis (vps) genes

The capacity of Vibrio cholerae to form biofilms has been shown to enhance its survival in the aquatic environment and play important roles in pathogenesis and disease transmission. In this study, we demonstrated that the histone-like nucleoid structuring protein is a repressor of exopolysaccharide (vps) biosynthesis genes and biofilm formation.     

DNA环介导恒温扩增技术快速检测霍乱弧菌

霍乱弧菌是一种重要的食源性致病菌,主要引起急性肠道传染病,其快速检测具有重要意义。根据霍乱弧菌的mdh管家基因序列,设计2对特异性检测引物,利用DNA环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP),经反应体系优化,成功建立了霍乱弧菌的LAMP快速检测方法。该方法最佳反应温度为65℃,60min完成检测,对培养菌的检测限为25CFU/mL,污染食品中霍乱弧菌的检测限为32CFU/g。对33株同种或近源细菌进行LAMP检测,仅霍乱弧菌得到阳性扩增。LAMP方法实践应用结果表明,对1057份虾、蟹、牡蛎、肉类、人腹泻物等样本进行检测,共检出85份阳性,与国际标准(ISO TS21872-1-2007)检测结果的符合率为100%。结果表明,本研究建立的霍乱弧菌LAMP检测方法特异性强、灵敏度高、操作简便,有利于霍乱弧菌疫情的监测。     

A two-nucleotide deletion renders the mannose-binding lectin 2 (MBL2) gene nonfunctional in Danish Landrace and Duroc pigs

The mannose-binding lectins (MBLs) are central components of innate immunity, facilitating phagocytosis and inducing the lectin activation pathway of the complement system. Previously, it has been found that certain single-nucleotide polymorphisms (SNPs) in porcine MBL1 and MBL2 (pMBL1, pMBL2) affect mRNA expression, serum concentration, and susceptibility to disease, but the combinatory effect of pMBL1 and pMBL2 genotypes needs further elucidation. In the present study, pMBL1 and pMBL2 alleles, combined pMBL haplotypes, and MBL-A concentration in serum were analyzed in purebred Landrace (N?=?30) and Duroc (N?=?10) pigs. Furthermore, the combined pMBL haplotypes of 89 Piètrain × (Large White × Landrace) crossbred pigs were studied, and the genotypes of 67 crossbreds challenged with Escherichia coli were compared to their individual disease records. In the purebred animals, three non-synonymous SNPs and a two-nucleotide deletion were detected in the coding sequence of pMBL2. The two-nucleotide deletion was present at a frequency of 0.88 in the Landrace pigs and 0.90 in the Duroc pigs, respectively. In the crossbreds, the T allele of the SNP G949T in pMBL1—previously shown to have profound effect on MBL-A concentration even in the heterozygote condition—was detected in 47 % of the animals. Finally, an association was found between low-producing MBL genotypes and low body weight on the day of weaning in the same animals.     

RecA-independence of the process of amplification caused by the RS-1 sequence of Vibrio cholerae

The processes of amplification and deamplification of a plasmid DNA segment flanked by direct repeats of RSI-sequence of Vibrio cholerae and carrying the structural genes of cholera toxin inside the recombinant plasmid in E. coli cells have been studied. These processes determined by RSI-sequences are shown to take place independent of the RecA-system of E. coli cells.     

Vibrio fluvialis: a new serogroup (19) possessing the Inaba factor antigen of Vibrio cholerae O1

A serogroup of Vibrio fluvialis possessing the C (Inaba) antigen but not the B (Ogawa) nor A antigen of V. cholerae O1 is described. The O-antigen of this serogroup was identical with that of bioserogroup 1875-variant of a marine Vibrio species. As the O-antigen of this serogroup was not agglutinated by any of O-antisera for the 18 serogroups of V. fluvialis already recognized, it was designated O-serogroup 19 of this species.     

Novel transmissible factors in a non-O1 Vibrio cholerae and a Vibrio sp

Transmissible factors encoding production of lacunae (L factors) were demonstrated in a non-O1 Vibrio cholerae and a Vibrio sp. of recent environmental origin. Lacunae were produced in lawns of non-O1 V. cholerae indicator strains under the same assay conditions as those where lacunae were produced by the well characterized P fertility plasmid of V. cholerae O1 and the V fertility factor found in a non-cholera vibrio strain. The origin of the lacunae produced by strains harbouring the V and L factors was examined. No vibriocin or phage activity was found in culture supernates or in lacunae produced by the strains, suggesting that, as in the case of the P plasmid, the lacunae probably represent sites of active mating. Unlike the P plasmid, neither the Vn or L factor could be detected or isolated by conventional plasmid techniques.     

Brucellosis in captive Rocky Mountain bighorn sheep (Ovis canadensis) caused by Brucella abortus biovar 4

Nine (four female, five male) captive adult Rocky Mountain bighorn sheep (Ovis canadensis) contracted brucellosis caused by Brucella abortus biovar 4 as a result of natural exposure to an aborted elk (Cervus elaphus) fetus. Clinical signs of infection were orchitis and epididymitis in males and lymphadenitis and placentitis with abortion in females. Gross pathologic findings included enlargement of the testes or epididymides, or both, and yellow caseous abscesses and pyogranulomas of the same. Brucella abortus biovar 4 was cultured in all bighorn sheep from a variety of tissues, including testes/epididymides, mammary gland, and lymph nodes. All bighorn sheep tested were positive on a variety of standard Brucella serologic tests. This is the first report of brucellosis caused by B. abortus in Rocky Mountain bighorn sheep. It also provides evidence that bighorn sheep develop many of the manifestations ascribed to this disease and that infection can occur from natural exposure to an aborted fetus from another species. Wildlife managers responsible for bighorn sheep populations sympatric with Brucella-infected elk or bison (Bison bison) should be cognizant of the possibility of this disease in bighorn sheep.     

Three-dimensional structure of the detergent-solubilized Vibrio cholerae cytolysin (VCC) heptamer by electron cryomicroscopy

Vibrio cholerae cytolysin (VCC) is a pore-forming toxin that inserts a lytic water-filled channel into susceptible host membranes. Assembly of the toxin on cell surfaces may be enhanced by two tandem lectin domains, in addition to direct interactions with lipids and cholesterol within the membrane itself. We used single-particle electron cryomicroscopy (cryoEM) to generate a low-resolution molecular structure of the detergent-solubilized VCC oligomer to 20 Å resolution. After confirming a heptameric arrangement of individual protomers, sevenfold averaging around the central pore was utilized to improve the structure. Docking of the previously determined VCC protoxin crystal structure was possible with rigid-body rearrangements between the cytolytic and lectin domains. A second cryoEM reconstruction of a truncated VCC mutant supported the topology of our model in which the carboxyl-terminal lectin domain forms “spikes” around the toxin core with the putative carbohydrate receptor-binding site accessible on the surface of the oligomer. This finding points to an assembly mechanism in which lectin domains may remain bound to receptors on the cell surface throughout assembly of the cytolytic toxin core and explains the hemagglutinating activity of purified toxin. Our model provides an insight into the structural rearrangements that accompany VCC-mediated cytolysis and may aid in the engineering of novel pore-forming toxins to attack specific cells towards therapeutic ends.     

Colonization of the gut of the blue crab (Callinectes sapidus) by Vibrio cholerae

Attachment of Vibrio cholerae to the mucosal surface of the intestine is considered to be an important virulence characteristic. Vibrio cholerae, an autochthonous member of brackish water and estuarine bacterial communities, also attaches to crustacea, a significant factor in multiplication and survival of V. cholerae in nature. The ability of V. cholerae to attach to the gut wall of the blue crab (Callinectes sapidus) was examined, and attachment was observed only in the hindgut and not the midgut of crabs, confirming a requirement for chitin in the attachment of V. cholerae to invertebrate and zooplankton surfaces. The new finding of attachment of V. cholerae to the hindgut of crabs may be correlated with the epidemiology and transmission of cholera in the aquatic environment. The crab model may also prove useful in elucidating the mechanism(s) of ion transport in crustacea.     

Novel insights into Haemagglutinin Protease (HAP) gene regulation in Vibrio cholerae

Quorum sensing is the phenomenon, whereby bacteria use signal molecules to communicate with each other. For example, to establish a successful infection, pathogenic bacteria become virulent only when they reach a certain local concentration in their host. Bassler and others have highlighted the surprising observation that quorum sensing seems to repress Vibrio cholerae virulence factor expression (e.g. cholera toxin), in contrast to what has been observed for virulence gene expression in other bacteria. Here, I present a novel insight that may clarify the way V. cholerae quorum‐sensing signals regulate its genes. Chironomids (Diptera; Chironomidae), which occur worldwide and are frequently the insect found most abundantly in fresh water bodies, are natural reservoirs of V. cholerae. Quorum‐sensing signals in V. cholerae up‐regulate the production of an extracellular enzyme, haemagglutinin protease (HAP), which degrades chironomid egg masses and prevents the eggs from hatching. HAP, therefore, is a virulence factor against chironomids. Indeed, in a survey carried out over the course of a year, V. cholerae and chironomids showed a pattern that mirrored the dynamics of predator‐prey populations. Globally, the numbers of chironomids are much larger than those of humans, so quorum‐sensing signals of V. cholerae and HAP gene regulation should be understood with regard to their role in chironomids rather than humans. Further research is needed to understand the role of cholera toxin in the environmental existence of V. cholerae.     

Dependent population dynamics between chironomids (nonbiting midges) and Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, is a natural inhabitant of the aquatic ecosystem. Chironomid (nonbiting midges) egg masses were recently found to harbour V. cholerae non-O1 and non-O139, providing a natural reservoir for the cholera bacterium. Chironomid populations and the presence of V. cholerae in chironomid egg masses were monitored. All V. cholerae isolates were able to degrade chironomid egg masses. The following virulence associated genes were detected in the bacterial isolates: hapA (100%), toxR (100%), hlyA (72%) and ompU (28%). The chironomid populations and the V. cholerae in their egg masses followed the phenological succession and interaction of host-pathogen population dynamics. A peak in the chironomid population was followed by a peak in the V. cholerae population. If such a connection is further substantiated for the pathogenic serogroups of V. cholerae in endemic areas of the disease, it may lead to a better understanding of the role of chironomids as a host for the cholera bacterium.