The Gram-negative, motile bacterium Campylobacter jejuni is a causative agent of food-borne gastroenteritis. Cytolethal distending toxin (CDT) is one of the important virulence factors for C. jejuni pathogenesis. It was not previously known how CDT is released from C. jejuni into the surrounding environment. In our study, CDT proteins were observed in the periplasmic fraction and all CDT subunits from C. jejuni were released from the bacterial cells in association with OMVs. The OMV-associated toxin caused cytolethal distending effects on tissue culture cells. Our results strongly suggest that the release of OMV-associated CDT is a route by which C. jejuni delivers all CDT toxin subunits (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.The Gram-negative, motile bacterium Vibrio cholerae is primarily known as the causal organism of the severe dehydrating diarrheal disease cholera. OMVs released from non-O1 non-O139 V. cholerae (NOVC) strain V:5/04 induced an inflammatory response in human host cells. The inflammatory potential is mediated by the nucleotide-binding domain, leucine-rich repeat containing family members NOD1 and NOD2. Physiochemical analysis in conjunction with NOD1/2 reporter assays in HEK293T cells confirmed the presence of the NOD1/2 active peptidoglycan (PGN) in OMVs. Deletion of the quorum sensing master regulator HapR specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. These findings suggest that OMVs from a NOVC strain delivered PGN to the host cells, where they elicited an immune response mediated by NOD1 and NOD2.The Gram-negative, non-motile coccobacillus Aggregatibacter actinomycetemcomitans is a natural inhabitant of the oral cavity, but the bacterium can translocate from the oral cavity into the bloodstream and thereby be transported to other regions of the body. A. actinomycetemcomitans is implicated in aggressive forms of periodontitis. The mechanism behind this aggressive periodontitis was not fully known. In addition to several virulence factors, this organism also produces CDT. We have demonstrated that OMVs released by A. actinomycetemcomitans contain several virulence factors, including CDT. We showed that OMVs delivered CDT to the host cells and that CDT was localized inside the nucleus, which led to a cytolethal distending effect on two different cell lines tested: HeLa cells and human gingival fibroblasts (HGF). These results suggest that A. actinomycetemcomitans OMVs could deliver biologically active CDT toxin into the periodontal tissue and may contribute to periodontitis.In our earlier studies, we discovered that an M6 family metalloprotease PrtV was an essential factor for V. cholerae survival from predator grazing. Pure PrtV protein effectively degraded human blood plasma components. In addition, it also showed a dose-dependent cytotoxic effect in the human intestinal HCT8 cell line. V. cholerae produces a large amount of outer membrane vesicles (OMVs) during the normal course of cell growth. OMVs are composed of periplasmic proteins, membrane lipids, lipopolysaccharides and outer membrane proteins. We showed that OMVs can transport several biologically active toxins and enzymes to the surrounding environment and ultimately into the host cells. We have initiated analysis of OMV-associated secretion of virulence factors in V. cholerae. It was observed that PrtV is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs and OMV-associated PrtV protein is biologically active and more stable than the free, soluble PrtV protease.

Vibrio spp., natural inhabitants of aquatic environments, are one of the most common causes of bacterial gastroenteritis in the world, being spread to humans via the ingestion of seafood, contaminated drinking water or exposure to seawater. The majority of Vibrio spp. are avirulent, but certain strains may sporadically be human pathogenic. Vibrio cholerae may cause cholera and fatal wound infections, Vibrio parahaemolyticus may cause gastroenteritis and Vibrio vulnificus may cause wound infections and sepsis. To expand current knowledge of the occurrence, ecological niche and persistence of potential human pathogenic Vibrio spp. in aquatic environments, occurrence and laboratory studies were performed. The seasonal variation of Vibrio spp. in clams and mussels from Mozambique and Sweden were studied, with isolated strains characterized and compared with those isolated from water samples collected in India. Results showed that the numbers of Vibrio spp. in Mozambican clams peaked during the warmer rainy season and that the dominating species was V. parahaemolyticus. Biochemical fingerprinting and virulence screened by PCR revealed a high similarity among strains from the different aquatic environments. However, isolate functional hemolytic analyses and antibiotic resistance patterns differed between strains; Swedish and Indian strains were less sensitive to the tested antibiotics and had a lower hemolytic capacity than those from Mozambique. Molecular analysis of bacterial DNA from Swedish mussels showed the presence of the three Vibrio spp. most commonly linked with human illness, as well as their associated virulence genes. The strains isolated from marine and clinical environments were equally and highly harmful to the tested eukaryotic cells. The persistence of clinical V. cholerae in aquatic environments was investigated in vivo. Strains were exposed to mussels, with bacterial uptake and elimination then examined. The mussels were able to avoid the most potent strain by complete closure of shells. The less potent strain was accumulated in mussel tissue in low levels and one marine control strain to a higher degree. Mussels eliminated the pathogenic strain less efficiently than they did the marine strain. One clinical and one marine strain were then exposed to 4°C for 21 days, with the temperature then increased to 20°C. The clinical strain was more prone to lose culturability than the marine strain at 4°C, the former performed significantly better in regaining culturability after the temperature up-shift. Subsequently, the persistence of the clinical strain in natural bottom sediment, incubating as above, was studied and results showed a similar decrease in culturable numbers in the sediment as in the water. As the clinical V. cholerae strains did not carry any of the standard set of virulence genes, the ability to change from non-culturable to culturable may be of great importance to strain pathogenicity. The results also show that natural bottom sediment may be a potential reservoir of human pathogenic Vibrio spp.

Vibrio cholerae, the causal agent of cholera typically encodes two critical virulence factors: cholera toxin (CT), which is primarily responsible for the diarrhoeal purge, and toxin-co-regulated pilus (TCP), an essential colonisation factor. Nontoxigenic strains expressing TCP can efficiently acquire the CT gene through lysogenic conversion with CTXΦ, a filamentous phage that encodes CT and uses TCP as a receptor.  V. cholerae is a Gram-negative bacterium and a natural inhabitant of estuarine and coastal waters throughout both temperate and tropical regions of the world. In the aquatic environment, V. cholerae encounters several environmental stresses, such as change in salinity, UV stress, nutrient limitation, temperature fluctuations, viral infections and protozoan predation. To fully understand the pathogenic and virulence potential of V. cholerae, knowledge is required of its interactions with, not only human, but also environmental factors. By using the nematode Caenorhabditis elegans as host model, we were able to identify a previously uncharacterised protein, the extracellular protease PrtV. PrtV was shown to be required for the killing of. elegans and also necessary for survival from grazing by the ciliate Tetrahymena pyriformis and the flagellate Cafeteria roenbergensis. The PrtV protein, which belongs to a M6 family of metallopeptidases was cloned and purified for further characterisations. The purified PrtV was cytotoxic against the human intestinal cell line HCT8. By using human blood plasma, fibrinogen, fibronectin and plasminogen were identified as candidate substrates for the PrtV protease. Outer membrane vesicles (OMVs) are released to the surroundings by most Gram-negative bacteria through “bulging and pinching” of the outer membrane.  OMVs have been shown to contain many virulence factors important in pathogenesis. Therefore, we investigated the association of PrtV with OMVs. PrtV was not associated with OMVs from the wild type O1 strain. In contrast, in an LPS mutant lacking two sugar chains in the core oligosaccharide PrtV was found to be associated with the OMVs. The OMV-associated PrtV was shown to be proteolytically and cytotoxically active. V. cholerae strains are grouped into >200 serogroups. Only the O1 and O139 serogroups have been associated with pandemic cholera, a severe diarrhoeal disease.  All other serogroups are collectively referred to as non-O1 non-O139 V. cholerae. Non-O1 non-O139 V. cholerae can cause gastroenteritis and extraintestinal infections, but unlike O1 and O139 strains of V. cholerae, little is known about the virulence gene content and their potential to become human pathogens. We analysed clinical and environmental non-O1 non-O139 isolates for their putative virulence traits. None of them carry the genes encoding CT or the TCP, but other putative virulence factors were present in these isolates. The incidence of serum resistance was found to vary considerably and was independent of encapsulation. Three strains were strongly serum-resistant, and these same strains could also kill C. elegans.

The bacterial pathogen Vibrio cholerae is responsible for ongoing cholera outbreaks in Haiti and elsewhere. Association of V. cholerae with the human host is responsible for fatal disease, but the bacteria also reside as natural inhabitants of aquatic environments, commonly attaching as biofilms to chitinous surfaces of copepods and crabs. Prior studies in V. cholerae demonstrated that competence for genetic transformation, a mechanism of horizontal gene transfer (HGT), requires the TfoX regulator protein that is triggered by chitin, and the HapR transcription factor that is made in response to quorum sensing (QS) signals produced by V. cholerae and Vibrios. To define regulatory components connecting extracellular signals to natural competence, I first demonstrated that QS molecules produced by Vibrios within multi-species chitinous biofilms are required for DNA uptake by V. cholerae, confirming the critical role of QS signals in HGT. Second, I identified by transposon-mutagenesis a new positive regulator of competence, CytR (cytidine repressor), only studied prior in E. coli as a regulator of nucleoside scavenging. Specific mutations in V. cholerae CytR impaired expression of competence genes and halted DNA uptake; and the addition of exogenous cytidine had similar affects as predicted in E. coli. V. cholerae and other competent Vibrios encode TfoX, HapR, and CytR, although none of these regulators directly controls genes coding for the DNA uptake apparatus. Thus, these results have uncovered a regulatory network, likely used by many Vibrios, that contains additional factors linking several extracellular chemical molecules (cytidine, chitin, and QS signals) to DNA uptake. My study has begun to define a molecular mechanism by which both environment and genetics contribute to genome evolution for this important marine pathogen.

Thesis (Ph.D.)--University of Wollongong, 2009. / Typescript. Includes bibliographical references: leaf 188-214.

An evanescent wave fiber-optic biosensor was used in an effort to develop an assay for the rapid detection of two biological toxins: cholera toxin and botulinum toxin. The Analyte 2000 fiber-optic biosensor utilizes a sandwich immunoassay format. Gangliosides or liposomes are directly adsorbed to the surface of the fiber-optic waveguide through hydrophobic interactions. The waveguide is exposed to a sample containing the toxin of interest, then subsequently exposed to a polyclonal detection antibody conjugated to the fluorophore cyanine 5. Excitation light from a 635nm laser diode is propagated through the waveguide and fluorescent molecules within approximately 100nm of the waveguide are excited. The emission light from the excited cyanine 5 molecules reverberates into the waveguide and is quantitated in pico Amperes and displayed on a computer. The exotoxins of Vibrio cholerae and Clostridium botulinum, cholera and botulinum toxin, respectively, were used for pote ntial assay development. Assay development utilizing the biosensor was attempted for the detection of botulinum toxin in buffer. The limit of detection remained too high to generate a positive signal for the detection of botulinum toxin. Biosensor assays were developed to detect cholera toxin in buffer, oyster homogenate, pure culture and induction media. A cholera toxin standard curve was generated with a limit of detection of 1 ng/ml. The values were normalized by setting 100 ng/ml of cholera toxin to a value of 100. Signals were detected in oyster homogenate spiked at 5 ug/ml as well as unspiked oyster homogenate. A Western blot showed that there were cross reactive proteins in the oyster matrix at molecular weights different from those of the cholera toxin. Cholera toxin production by three strains of Vibrio cholerae with values estimated to range from 100 pg -- 100 ng was detected with the biosensor. Additionally, oysters were harvested from Tampa Bay and placed in a 10 gallon tank filled with different types of induction media. The tank was inoculated with Vibrio cholerae and the oysters and induction medium were analyzed at varying times for the presence of cholera toxin. Vibrio cholerae cells were viable through 24 hours but no toxin was detectable.

Les éléments intégratifs et conjugatifs (ICEs) sont des éléments génétiques mobiles bactériens largement reconnus en tant qu'importants vecteurs de la transmission des gènes de résistance aux antibiotiques. Comme tous les éléments génétiques mobiles, ils participent grandement à la plasticité génomique de leur hôte, en permettant le transfert horizontal de gènes conférant des avantages pour l'adaptation de leur hôte à différentes conditions environnementales. Les ICEs se transfèrent horizontalement par conjugaison, par un mécanisme similaire à celui emprunté par les plasmides conjugatifs. Cependant, au contraire des plasmides, les ICEs ne possèdent pas un intermédiaire circulaire extrachromosomique réplicatif : ils s'intègrent plutôt dans le chromosome de leur hôte par recombinaison site-spécifique, à la manière d'un prophage. Une conséquence de ces deux caractéristiques est qu'ils sont également transmis verticalement lors de la division cellulaire de leur hôte. Lorsque soumis à certaines conditions, les ICEs s'excisent de leur hôte sous forme d'une molécule circulaire qui est le substrat pour leur transfert conjugatif vers un nouvel hôte. Une fois transférée, la molécule de l'ICE est recircularisée et s'intègre dans le chromosome. Les ICEs de la famille SXT/R391 sont largement distribués dans les souches de Vibrio cholerae et dans les ?-protéobactéries apparentées et ont été isolées dans une diversité d'endroits géographiques. Les membres de cette famille comportent un large squelette de gènes conservés qui codent pour leurs fonctions principales d'intégration/excision, de transfert conjugatif et de régulation. À l'intérieur de ce squelette sont retrouvées des régions.variables - dont la nature et les combinaisons diffèrent d'un élément à l'autre - qui codent pour des fonctions accessoires telles les résistances aux antibiotiques. Le patron de combinaisons des régions variables entre différents ICEs a mené à l'hypothèse qu'ils sont soumis à de fréquents échanges de matériel génétique par recombinaison. L'intégration site-spécifique de tous les membres de cette famille dans le même locus chromosomique permet la coexistence de deux ICEs semblables mais non identiques dans la même cellule, ce qui fournit un substrat à la recombinaison inter-ICE et permet la formation d'éléments hybrides. Cette thèse présente l'étude d'un nouveau système de recombinaison homologue identifié dans le squelette des ICEs SXT/R391 et sa participation dans la formation d'ICEs hybrides. Ce système de recombinaison, composé de la recombinase Bet et de l'exonucléase Exo est apparenté au système de recombinaison Red du bactériophage ? et est retrouvé chez tous les membres de la famille SXT/R391. La première portion du projet a visé l'identification des déterminants génétiques impliqués dans la formation d'hybrides. Les résultats obtenus démontrent que les ICEs hybrides peuvent être formés par trois voies différentes : i) Bet/Exo, d'une manière indépendante de RecA, ii) RecA et iii) Bet/Exo et RecA, de manière coopérative. Les résultats démontrent également que l'excision des éléments du chromosome ainsi que leur transfert conjugatif vers une nouvelle cellule ne sont pas nécessaires pour former des hybrides. Cette portion du projet met en évidence que les ICEs de la famille SXT/R391 sont capables de promouvoir leur propre diversité à l'aide du système de recombinaison homologue RecA-indépendant qu'ils codent. La deuxième partie du projet s'est portée sur la régulation de l'expression du système de recombinaison Bet/Exo. Les résultats obtenus démontrent que la transcription des gènes de recombinaison est induite en présence d'agents causant des dommages à l'ADN, par le biais des activateurs de transcription de l'ICE, SetC et SetD. Ces activateurs sont sous le contrôle du répresseur principal SetR, dont la répression est levée par les conditions qui induisent la réponse SOS de l'hôte, et sont responsables de l'expression concertée des gènes d'intégration/excision et de transfert conjugatif. Bien que la transcription des gènes de recombinaison soit fortement induite en présence d'agents induisant la réponse SOS, l'analyse de leur profil traductionnel démontre que leur traduction est fortement régulée. Des atténuateurs de traduction putatifs positionés en amont de Bet et Exo pourraient être responsables de ce niveau additionnel de régulation. L'analyse de l'organisation génétique du locus de recombinaison a mis en évidence que bet et exo font partie d'un opéron polycistronique comprenant 12 autres gènes du squelette conservé des ICEs SXT/R391, dont les fonctions ne sont pas connues. Finalement, une analyse in silico visant à identifier tous les systèmes de recombinaison apparentés au sein des génomes séquencés de bactéries, plasmides et virus a permis de démontrer qu'un nombre important de ces systèmes existent chez une diversité d'espèces bactériennes appartenant à des taxons éloignés. Bien que la majorité de ces systèmes soient codés par des bactériophages, plusieurs sont retrouvés sur des plasmides conjugatifs. L'ensemble des résultats présentés dans cette thèse permet une meilleure compréhension de l'évolution des éléments génétiques mobiles bactériens, et plus particulièrement des éléments intégratifs et conjugatifs de la famille SXT/R391.

Includes an appendix of author's previously published papers. / Bibliography: leaves 123-160. / 160, [105] leaves, [30] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1990

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-24T18:59:28Z No. of bitstreams: 1 MIchel Francisco Abanto Marin_Tese.pdf: 17075383 bytes, checksum: 82ec8815a4daba1b96b41d8a1b92b852 (MD5) / Made available in DSpace on 2013-09-24T18:59:28Z (GMT). No. of bitstreams: 1 MIchel Francisco Abanto Marin_Tese.pdf: 17075383 bytes, checksum: 82ec8815a4daba1b96b41d8a1b92b852 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Vice Direção de Ensino, Informação e Comunicação. Rio de Janeiro, RJ, Brasil. / Vibrio cholerae é o agente causal da cólera, uma infecção ancestral, epidêmica e pandêmica, que é um dos principais problemas de saúde pública no mundo. A humanidade já passou por, pelo menos, seis pandemias de cólera e, atualmente, vive no contexto da sétima pandemia. V. cholerae é classificado em mais de 200 sorogrupos e alguns biotipos. Linhagens de V. cholerae O1 do biotipo clássico e do biotipo El Tor estão relacionadas à sexta (1899–1923) e à sétima (1960 -….) pandemias de cólera, respectivamente. A sétima pandemia chegou à América Latina em 1991 sendo que, inicialmente, sua origem foi relacionada ao Sudoeste Asiático, região endêmica e epidêmica de cólera. Uma segunda hipótese sugere uma origem ambiental local. A partir do início deste século, a epidemia se extinguiu, restando apenas eventuais relatos de casos isolados. Sob a ótica da genética e genômica do V. cholerae, estudamos a linhagem da epidemia da América Latina (AL). Nossos objetivos eram inferir sua origem e identificar marcadores genômicos que possibilitassem seu monitoramento. Utilizamos informações obtidas in vitro e in silico para estabelecer relações genéticas entre isolados de V. cholerae de antes, durante e depois da epidemia que atingiu a América Latina. Analisamos regiões do genoma core e do genoma acessório. Realizamos análises de genômica comparativa com informação de 355 V. cholerae, clínicos e ambientais, de diferentes anos e regiões geográficas. Com base em três genes do genoma core, nossos resultados mostram que a linhagem da epidemia da AL pertence ao clado El Tor, e por sua vez, os genótipos dos principais determinantes de virulência ctxB e tcpA, (genoma acessório), os agrupa com isolados do início da sétima pandemia, incluindo o canônico N16961. As análises genômicas e filogenéticas revelaram a presença de um profago (WASA1) e de uma variante exclusiva da ilha genômica VSP-II. A VSP-II da linhagem da AL é caracterizada por seu gene da integrase e uma inserção de 7 Kb, que contém três genes putativos. Curiosamente, os dois marcadores, que fazem parte do genoma acessório desta linhagem, WASA1 e VSP-II, apresentam relação de similaridade com elementos genéticos identificados em Vibrio vulnificus/Vibrio parahaemolyticus e Vibrio vulnificus/V. cholerae do ambiente, respectivamente. Estes marcadores seriam, portanto, uma fração do mobiloma que evoluiu e se fixou na linhagem de V. cholerae da epidemia da América Latina. / Vibrio cholerae is the causative agent of cholera, an ancestral, epidemic and pandemic infection, which is a major public health concern worldwide. Mankind has gone through at least six cholera pandemics and currently lives in the context of the seventh pandemic. V. cholerae is classified into more than 200 serogroups and some biotypes. V. cholerae O1 classical and El Tor biotype are related to the sixth (1899 to 1923) and seventh (1960 - ....) cholera pandemics, respectively. The seventh pandemic reached Latin America (LA) in 1991 and its origin was related, immediately, to Southwest Asia, where cholera is endemic and epidemic. A second hypothesis emerged suggesting a regional environmental origin. Since the beginning of this century, the LA epidemic ended, occurring only occasional case reports. Here, we studied, from the perspective of V. cholerae genetics and genomics, the lineage of the LA cholera epidemic. Our objectives were to infer their origin and identify genomic markers that would enable its monitoring. In order to establish the genetic relationships among V. cholerae strains isolated before, during and after the epidemic in Latin America, we used in vitro and in silico approaches. Comparative analyzes, targeting the core and accessory genome of 355 clinical and environmental V. cholerae strains, from different years and geographical regions, were performed. MLSA (multi locus sequence analysis), based on three genes of the core genome, revealed that the LA epidemic lineage belongs to the El Tor clade. Additionally, the genotypes of the major virulence determinants, ctxB and tcpA (accessory genome), show that the LA lineage is related with strains from the beginning of the seventh pandemic, including the canonical N16961. Considering this large set of genomes, we confirm the prophage WASA1 as a LA lineage marker. The LA lineage is also characterized by an unique VSP-II genomic island. The VSP-II AL harbors a particular integrase gene and a 7.0 kb insert which contains three putative genes. Interestingly, the two LA markers, WASA1 and VSP-II, which are part of its accessory genome, have similarity with genetic elements identified in Vibrio vulnificus / Vibrio parahaemolyticus and V. vulnificus / environmental V. cholerae, respectively. Therefore, these two elements are a fraction of the V. cholerae mobilome that evolved and fixed in the LA lineage.