Made available in DSpace on 2014-06-12T18:03:27Z (GMT). No. of bitstreams: 2 arquivo3705_1.pdf: 1739643 bytes, checksum: d5c033b4b7bcbc4ee46e7a8e20bbe0df (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A emergência do Vibrio cholerae sorogrupo O139 como um segundo agente etiológico da cólera serviu de alerta para o surgimento de outros clones epidêmicos que possam passar despercebidos pelos métodos tradicionais de diagnóstico da cólera, geralmente baseados no uso de antissoro contra o sorogrupo O1 tradicional. Em estudos prévios, a partir da análise de 179 cepas de V. cholerae não-O1/não-O139 isoladas de casos clínicos de cólera no Brasil, foram selecionadas sete cepas de V. cholerae O26, e outra cepa de sorogrupo não tipável (17155), que possuíam genes de virulência associados ao desenvolvimento desta enfermidade. Destas, duas cepas (4756 e 17155) possuíam o gene rfb, específico do sorogrupo O1, sugerindo serem genotipicamente deste sorogrupo, e também expressaram a toxina colérica (CT) em cultura. Este trabalho buscou uma análise genética mais detalhada destas oito cepas comparando a classificação sorológica com outros marcadores moleculares. Neste sentido foi realizada a amplificação, clonagem e seqüenciamento da região espaçadora ribossomal 16S-23S (ISR) de diferentes operons de V. cholerae. A partir da análise da seqüência de cinco grupos de operons distintos (de um total de 210 clones seqüenciados), foram construídas três árvores filogenéticas em que as cepas 4756 e 17155 ficaram agrupadas no mesmo clado com cepas O1 controle, e as demais cepas O26 foram agrupadas separadamente. Conclui-se, desta forma, que as cepas 4756 e 17155 são filogeneticamente do sorogrupo O1 e a diferença nos resultados de sorologia pode ser uma conseqüência de soroconversão provocada por mudanças em genes de biossíntese do antígeno O

Vibrio cholerae y Shigella dysenteriae, entre otras bacterias enteropatógenas, causan enfermedades gastrointestinales, la segunda causa de muerte en niños menores de cinco años en el mundo. Los sucesos de gastroenteritis que desencadenan dichos microorganismos son mediados por la acción molecular de toxinas secretadas de tipo AB, CTX y STX, respectivamente para V, cholerae y S, dysenteriae. Dichas toxinas causan desbalances iónicos y lisis celular, generando deshidratación, pérdida de electrolitos y nutrientes. El correcto funcionamiento de ambas toxinas requiere una relación estricta de cinco subunidades de la proteína B por cada subunidad A, sin embargo, los mecanismos moleculares que regulan la expresión y mantienen la relación 5:1 entre las subunidades son poco entendidos. El objetivo de este estudio es analizar la regulación post transcripcional de las toxinas CTX y STX mediante aproximaciones experimentales in vitro y usando reacciones libres de células, en un entorno altamente puro y controlado. Nuestros resultados in vitro indican que CTX se expresa de manera más eficiente que STX. En el caso de CTX, la subunidad B tuvo mayor expresión que A, con una relación cercana a 5:1 como visto en estudios previos realizados in vivo. Por el contrario, STX fue sintetizada con baja eficiencia independientemente de la presencia o ausencia de la región codificantes para STXA, desviándose notablemente de una relación activa de 5 B por cada subunidad A. La adición de ppGpp, molécula implicada en la respuesta al estrés en bacterias, resultó en una ligera disminución de la expresión de ambas toxinas. En conclusión, nuestros resultados indican que la relación de 5 a 1 entre B y A de las toxinas CTX de V. cholerae es regulada durante la síntesis de proteínas. Por el contrario, la ausencia de regulación a nivel transcripcional o traduccional de STX del presente estudio in vitro, fue también observada en estudios in vivo, indicando que en la célula existen otros factores que podrían modular la estequiometría de síntesis. Si bien ambas toxinas, CTX y STX, permanecen a la misma familia de exotoxinas, los resultados del presente estudio indican que la regulación de su expresión difiere ampliamente entre ellas. Los resultados aquí reportados brindan nuevas perspectivas para el desarrollo de moléculas inhibidoras, con especial énfasis en perturbar el ribosoma de V, cholerae, para un desbalance en la síntesis de la toxina CTX y así reducir la patogenicidad de la bacteria. / Vibrio cholerae and Shigella dysenteriae, among other enteropathogenic bacteria, cause gastrointestinal diseases, the second cause of death in children under five years of age in the world. The events of gastroenteritis that trigger these microorganisms are mediated by the molecular action of secreted toxins of type AB, CTX and STX, respectively for V. cholerae and S. dysenteriae. These toxins cause ionic imbalances and cell lysis, generating dehydration, loss of electrolytes and nutrients. The correct functioning of both toxins requires a strict relationship of five subunits of protein B for each subunit A, however, the molecular mechanisms that regulate expression and maintain the 5: 1 ratio between the subunits are poorly understood. The objective of this study is to analyze the post transcriptional regulation of CTX and STX toxins by in vitro experimental approaches and using cell-free reactions, in a highly pure and controlled environment. Our in vitro results indicate that CTX is expressed more efficiently than STX. In the case of CTX, subunit B had greater expression than A, with a ratio close to 5: 1 as seen in previous studies conducted in vivo. In contrast, STX was synthesized with low efficiency regardless of the presence or absence of the region coding for STXA, deviating markedly from an active ratio of 5 B for each subunit A. The addition of ppGpp, a molecule involved in the stress response in bacteria, resulted in a slight decrease in the expression of both toxins. In conclusion, our results indicate that the ratio of 5 to 1 between B and A of CTX toxins of V. cholerae is regulated during protein synthesis. On the contrary, the absence of transcriptional or transcriptional STX regulation of the present in vitro study was also observed in in vivo studies, indicating that the cell may harbor other factors that could modulate the synthesis stoichiometry. Although both toxins, CTX and STX, belong to the same family of exotoxins, the results of the present study indicate that the regulation of their expression differs widely. The results reported here provide new perspectives for the development of inhibitory molecules, with special emphasis on disturbing the ribosome of V. cholerae, to provoke an imbalance in the synthesis of CTX toxin and thus to reduce the pathogenicity of the bacteria. / Tesis

Please read the abstract in the section, 00front, of this document / Dissertation (MSc)--University of Pretoria, 2001. / Microbiology and Plant Pathology / MSc / unrestricted

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Distributional ecology is a branch of ecology which aims to reconstruct and predict the geographic range of free-living and symbiotic organisms in terrestrial and aquatic ecosystems. More recently, distributional ecology has been used to map disease transmission risk. The implementation of distributional ecology for disease transmission has, however, been erroneous in many cases. The inaccurate representation of disease distribution is detrimental to effective control and prevention. Furthermore, ecological niche modeling experiments are generally developed and tested using data from terrestrial organisms, neglecting aquatic organisms in case studies. Both disease and aquatic systems are often data limited, and current modeling methods are often insufficient. There is, therefore, a need to develop data-driven models that perform accurately even when only limited amounts of data are available or when there is little to no knowledge of the species' natural history to be modeled. Here, I propose a data-driven ecological niche modeling method that requires presence-only data (i.e., absence, pseudoabsence, or background records are not needed for model calibration). My method is expected to reconstruct environmental conditions where data-limited aquatic organisms are more likely to be present, based on a density-based clustering algorithm as a proxy of the realized niche (i.e., abiotic, and biotic environmental conditions occupied by the organism). Supported by ecological theories and methods, my central hypothesis is that because density-based clustering machine-learning modeling prevents extrapolation and interpolation, it can robustly reconstruct the realized niche of a data-limited aquatic organism. First, I assembled a comprehensive dataset of abiotic (temperature) and biotic (phytoplankton) environmental conditions and presence reports using Vibrio cholerae, a well-understood aquatic bacterium species in coastal waters globally (Chapter 2). Second, using V. cholerae as a model system, I developed detailed parameterizations of density-based clustering models to determine the parameter values with the best capacities to reconstruct and predict the species' distribution in global seawaters (Chapter 3). Finally, I compared the performance of density-based clustering modeling against traditional, correlative machine-learning ecological niche modeling methods (Chapter 4). Density-based clustering models, when assessed based on model fit and prediction, had comparable performance to traditional 'data-hungry' machine-learning correlative methods used in modern applications of ecological niche modeling. Modeling the environmental and geographic ranges of V. cholerae, an aquatic organism of free-living and parasitic ecologies, is a novel approach itself in distributional ecology. Ecological niche modeling applications to pathogens, such as V. cholerae, provide an opportunity to further the knowledge of directly-transmitted emerging diseases for which only limited data are available. Density-based clustering ecological niche modeling is termed here as Marble, honoring a previous, experimental version of this analytical approach, and is expected to provide new opportunities to understand how an ecological niche modeling method influences estimates of the distribution of data-limited organisms of complex ecology. These are lessons applicable to novel, rare, and cryptic aquatic organisms, such as emerging diseases, endangered fishes, and elusive aquatic species. / Master of Science / Distributional ecology is a branch of ecology which aims to reconstruct and predict the geographic distribution of land and water organisms. In the case of diseases, a correct representation of their geographic distributions is key for successful management. Previous studies highlight the need to develop new models that perform accurately even when limited amounts of data are available and there is little to no knowledge of the organisms' ecology. This thesis proposes a data-driven method, originally termed Marble. Marble is expected to help reconstruct environmental conditions where data-limited aquatic organisms are more likely to be found. Supported by ecological theories and methods, my hypothesis is that because Marble prevents under- and over-fitting, this method will produce results which better fit the data. Using V. cholerae, an aquatic organism, as a model system, I compared the performance of Marble against other traditional modeling algorithms. I found that Marble, in terms of model fit, performed similarly to traditional methods used in distributional ecology. Modeling the ecology of V. cholerae is a new approach in and of itself in ecological modeling. Furthermore, modeling pathogens provides an opportunity to further the knowledge of directly transmitted diseases, and Marble is expected to provide opportunities to understand how algorithm selection can reconstruct (or not) the distribution of data-limited aquatic organisms of diverse ecologies.

Vibrio cholerae é uma bactéria autóctone em ecossistemas aquáticos, os fatores responsáveis pela virulência podem contribuir com a patogenicidade, influenciados por fatores genéticos e ambientais. Considerando a importância de conhecer e monitorar o V. cholerae, o estudo pretende caracterizar isolados da especie e padronizar uma metodologia para detecção em amostras de água. Os isolados foram avaliados por metodologias clássicas e moleculares, para confirmar espécie. Também, foi avaliada a presença de genes de virulência, susceptibilidade aos antibióticos e resposta em modelo invertebrado. Tres marcadores moleculares foram avaliados por PCR quantitativa. Observou-se que setenta dos isolados pertenciam a espécie V. cholerae e mostraram variação na prevalência dos genes de virulência e ao perfil de suscetibilidade ao antibióticos. Mostrou uma influencia da temperatura e concentração do inoculo no modelo invertebrado. Os marcadores moleculares selecionados mostraram a viabilidade da metodologia proposta neste estudo pela alta especificidade e sensibilidade. / Vibrio cholerae is an autochthonous bacterium in aquatic ecosystems, factors responsible for virulence may contribute to pathogenicity, influenced by genetic and environmental factors. Considering the importance of knowing and monitoring V. cholerae, the study pretend to characterize selected isolates and to standardize a methodology for detection in water samples. The isolates were evaluated by classical and molecular methodologies to confirm species. Also, the presence of factors associated with virulence, antibiotics susceptibility and response in invertebrate model were evaluated. Three molecular markers were evaluated by quantitative PCR. It was observed that seventy of the isolates belonged to the V. cholerae species and showed a variation in the prevalence of the virulence genes and the antibiotic susceptibility profile. Also, showed an influence of the inoculum temperature and concentration on the invertebrate model. The selected molecular markers showed the viability of the methodology proposed in this study for the high specificity and sensitivity.

Vibrio cholerae é uma bactéria autóctone em ecossistemas aquáticos, os fatores responsáveis pela virulência podem contribuir com a patogenicidade, influenciados por fatores genéticos e ambientais. Considerando a importância de conhecer e monitorar o V. cholerae, o estudo pretende caracterizar isolados da especie e padronizar uma metodologia para detecção em amostras de água. Os isolados foram avaliados por metodologias clássicas e moleculares, para confirmar espécie. Também, foi avaliada a presença de genes de virulência, susceptibilidade aos antibióticos e resposta em modelo invertebrado. Tres marcadores moleculares foram avaliados por PCR quantitativa. Observou-se que setenta dos isolados pertenciam a espécie V. cholerae e mostraram variação na prevalência dos genes de virulência e ao perfil de suscetibilidade ao antibióticos. Mostrou uma influencia da temperatura e concentração do inoculo no modelo invertebrado. Os marcadores moleculares selecionados mostraram a viabilidade da metodologia proposta neste estudo pela alta especificidade e sensibilidade. / Vibrio cholerae is an autochthonous bacterium in aquatic ecosystems, factors responsible for virulence may contribute to pathogenicity, influenced by genetic and environmental factors. Considering the importance of knowing and monitoring V. cholerae, the study pretend to characterize selected isolates and to standardize a methodology for detection in water samples. The isolates were evaluated by classical and molecular methodologies to confirm species. Also, the presence of factors associated with virulence, antibiotics susceptibility and response in invertebrate model were evaluated. Three molecular markers were evaluated by quantitative PCR. It was observed that seventy of the isolates belonged to the V. cholerae species and showed a variation in the prevalence of the virulence genes and the antibiotic susceptibility profile. Also, showed an influence of the inoculum temperature and concentration on the invertebrate model. The selected molecular markers showed the viability of the methodology proposed in this study for the high specificity and sensitivity.

Travaux de recherche effectués sous la supervision de la docteure Marylise Duperthuy (directrice) et de la docteure Catherine Paradis-Bleau (codirectrice). / L’augmentation de l’incidence des infections bactériennes par des souches résistantes, multirésistantes, voire même ultrarésistantes, aux antibiotiques, combinés à la crise de découverte de nouvelles molécules depuis les années 1960 et du sous-investissement chronique de certains états dans la recherche publique, pourrait coûter la vie à 10 millions d’êtres humains par an d’ici 2050. L’écrasante majorité des souches bactériennes résistantes aux antibiotiques sont des bactéries à Gram négatif. Ceci est notamment dû à la composition intrinsèque de leur enveloppe, leur permettant d’être insensibles à de nombreuses molécules pourtant létales pour d’autres types de bactéries. L’étude des composants et des mécanismes de biosynthèse de l’enveloppe, qui sont essentiels au maintien de l'intégrité des bactéries et peuvent être impliqués dans leur virulence, devrait permettre l’identification de nouvelles cibles thérapeutiques. Dans le premier chapitre, nous allons tout d’abord faire un tour d’horizon des éléments composant l’enveloppe des bactéries à Gram négatif, des mécanismes de résistance aux antibiotiques et du danger que représentent les bactéries à Gram négatif. Mes résultats de recherches, présentés sous forme de quatre articles aux chapitres deux à cinq, sont précédés d’une mise en contexte des recherches propres aux deux laboratoires, portant d’une part sur les biofilms et la mobilité de Vibrio cholerae et d’autre part sur l’enveloppe de Escherichia coli. Dans le premier article, nous avons déterminé par quel mécanisme la polymyxine B en concentration sous-inhibitrice affecte la formation de biofilm chez Vibrio cholerae. Nous avons observé que la polymyxine B affecte principalement le flagelle par une action mécanique. La formation de biofilm nécessitant le flagelle dans ses premières étapes de formation, nous avons conclu que l’action de la polymyxine B prévenait ce changement d’état. Dans le second article, nous avons conçu un protocole d’évolution expérimentale qui nous a permis d’identifier des mutations dans différents gènes permettant à Vibrio cholerae de se déplacer plus rapidement en présence de concentration sous-inhibitrice de polymyxine B. Nous avons alors identifié chez plusieurs souches différentes mutations ayant probablement réduit ou anéanti la fonction des protéines IhfA, DacB, VacJ (MlaA) et MlaF. En nous basant sur la littérature, nous proposons que la perte de fonction de ces 5 protéines induisant l’augmentation de la mobilité en présence de polymyxine B puisse s’expliquer selon trois mécanismes, impliquant la stabilité de l’enveloppe, la sécrétion de vésicules de membrane ou une altération de l’expression de différents gènes. Dans le troisième article, nous avons étudié l’implication du stress oxydatif dans l’arrêt de la synthèse de la paroi qui est fatal au mutant ∆elyC de Escherichia coli. Nous avons alors démontré que la lyse du mutant ∆elyC est causée par la surproduction de radicaux hydroxyles dans son enveloppe. Cette molécule cause des dommages dans l’enveloppe suffisamment importants pour provoquer l’arrêt de synthèse de la paroi. Le mécanisme provoquant cette surproduction de radicaux toxiques reste encore à déterminer. Par le biais de l’étude de la fonction du facteur ElyC, nous avons découvert une nouvelle vulnérabilité dans l’homéostasie de l’enveloppe qu’il nous tarde de pouvoir exploiter. Dans le dernier article, nous avons étudié l’importance de la voie de synthèse de l’antigène commun aux entérobactéries dans le processus létal apparaissant en l’absence du facteur ElyC. Nous avons découvert que le mutant ∆elyC accumule un élément commun aux voies de synthèse de la paroi et de l’antigène commun aux entérobactéries, l’undécaprényl pyrophosphate. Nous avons également découvert que l’augmentation du recyclage de cette dernière via la surexpression du gène codant pour la protéine PgpB permettait de prévenir la lyse du mutant. Notre hypothèse est que l’absence de ElyC induit une mauvaise répartition de l’undécaprényl phosphate entre les voies de synthèse de la paroi et de l’antigène commun aux entérobactéries. La suractivité de la voie de synthèse de l’antigène commun aux entérobactéries induirait l’accumulation d’undécaprényl pyrophosphate provoquant l’inhibition de Pbp1b, empêchant l’ajout de nouvelles sous-unités à la paroi conduisant ainsi à la lyse du mutant ∆elyC. Ainsi l’ensemble de ces travaux a permis de mieux comprendre et d’identifier des vulnérabilités de l’enveloppe de Vibrio cholerae et Escherichia coli, de préciser certains mécanismes ainsi que d’entrevoir le rôle de divers facteurs impliqués dans l’homéostasie de leur enveloppe. / The increased incidence of bacterial infections by resistant, multiresistant and even extensively drug-resistant strain, combined with the crisis of new molecules discovery since the 1960s and the chronic underinvestment of certain states in public research, could cost 10 million human lives per year by 2050. The overwhelming majority of resistant bacteria are the Gram-negative one. This is mainly due to the intrinsic composition of their envelope, allowing them to be insensitive to many molecules yet lethal for other types of bacteria. The study of envelope components and biosynthetic mechanisms, which are essential for the maintenance of bacterial integrity and could be involved in their virulence, should lead to the identification of new therapeutic targets. The first chapter gave an overview of the different elements composing Gram-negative bacteria envelope, mechanisms of resistance and the threat that Gram-negative bacteria represent. The presentation of my work, divided in four articles from chapter two to chapter five, was preceded by a contextualization of the research projects specific to both laboratories, on biofilm and motility of Vibrio cholerae on one hand and on Escherichia coli envelope from the other hand. In the first paper, we determined the mechanism by which a sub-inhibitory concentration of polymyxin B affects biofilm formation in Vibrio cholerae. We observed that polymyxin B mainly affected the flagellum by a mechanical action. Since biofilm formation requires the flagellum in the early stages of its formation, we concluded that the action of polymyxin B prevented this change in Vibrio cholerae lifestyle. In the second paper, we designed an experimental evolution protocol which allowed us to identify mutations in different genes that increase Vibrio cholerae motility in the presence of sub-inhibitory concentration of polymyxin B. We then identified that different mutations had altered ihfA, dacB, vacJ (mlaA) and mlaF genes in different mutants. These mutations have probably induced reduction or loss of function of the proteins for which they respectively code. Based on literature, we hypothesize that the loss function of these proteins inducing increase in mobility in the presence of polymyxin B could be explained by three 7 mechanisms involving envelope stability, secretion of membrane vesicles or altered expression of various genes. In the third paper, we investigated the involvement of oxidative stress in the fatal peptidoglycan synthesis arrest occurring in the ∆elyC mutant of Escherichia coli. We then demonstrated that lysis of the ∆elyC mutant is caused by the overproduction of hydroxyl radicals in its envelope. This molecule causes multiple and sufficiently important damages in the envelope to stop the synthesis of the wall. The mechanism causing this overproduction of toxic radicals has yet to be determined. By studying the function of the ElyC factor, we have discovered a new weakness in envelope homeostasis that we are eager to exploit. In the last paper, based on a previously formulated hypothesis, we investigated the importance of the enterobacterial common antigen synthesis pathway in the lethal process occurring in the absence of ElyC. We found that the ∆elyC mutant accumulates common element to the cell wall and enterobacterial common antigen synthesis pathways, undecaprenyl pyrophosphate. We also observed that the ∆elyC mutant lysis phenotype could be suppressed by increasing undecaprenyl pyrophosphate recycling via the overexpression of the gene coding for the phosphatase PgpB. We propose that the absence of ElyC induces a misallocation of undecaprenyl phosphate between the cell wall and enterobacterial common antigen synthesis pathways, in favors the latter pathway. Overactivation of the enterobacterial common antigen pathway would induce undecaprenyl phosphate accumulation, causing inhibition of Pbp1b, thus blocking addition of new subunits to the cell wall and leading to its lysis. Ultimately, all the original data generated by my research project has led to a better understanding of envelope vulnerabilities of Vibrio cholerae and Escherichia coli, to clarify certain mechanisms but also to glimpse the role of various factors involved in the homeostasis of their envelope. / L'aumento dell’incidenza delle infezioni da batteri resistenti, multi-resistenti e anche estremamente resistenti agli antibiotici, combinata con la rarefazione della scoperta di nuove molecole fra gli anni 1960 e il sotto investimento cronico di certe nazioni nella ricerca pubblica, potrebbe costare la vita a 10 milioni di essere umano all'anno nel 2050. La stragrande maggioranza delle specie resistenti agli antibiotici sono batteri Gram- negativi. Questo è dovuto principalmente alla composizione intrinseca dei loro involucri che permette loro di essere insensibili a molte molecole che sono letali per altri tipi di batteri. Lo studio delle componenti e i meccanismi di sintesi dell’incurvo, che sono essenziali nell’integrità dei batteri e che possono essere coinvolti nella loro virulenza, dovrebbe permettere l'identificazione di nuovi bersagli terapeutici. Nel primo capitolo, avremo una panoramica dei diversi elementi che compongono l'involucro dei batteri Gram-negativi, dei meccanismi di resistenza e della minaccia che i batteri Gram-negativi rappresentano. La presentazione del mio lavoro, diviso in quattro articoli dal capitolo due a cinque, sarà preceduta da una contestualizzazione dei progetti di ricerca specifici di ciascuno dei due laboratori, sul biofilm e la motilità di Vibrio cholerae da un lato e sull'involucro di Escherichia coli dall'altro. Nel primo articolo, abbiamo determinato il meccanismo con cui la polimixina B in concentrazione sub-inibitoria influenza la formazione de biofilm in Vibrio cholerae. Abbiamo osservato che la polimixina B danneggia principalmente il flagello attraverso un'azione meccanica. Poiché la formazione del biofilm richiede il flagello nelle prime fasi della sua formazione, abbiamo concluso che l'azione della polimixina B ha impedito questo cambiamento nello stile di vita di Vibrio cholerae. Nel secondo articolo, abbiamo messo a punto un protocollo di evoluzione sperimentale che ci ha permesso di identificare diversi geni che aumentano la motilità di Vibrio cholerae in presenza di una concentrazione sub-inibitoria di polimixina B. Abbiamo quindi identificato vari mutazioni in diversi varianti che hanno probabilmente provocato la riduzione o il soppresso della funzione delle proteine IhfA, DacB, VacJ (MlaA) e MlaF. Sulla base della letteratura, proponiamo che la perdita di funzione di queste proteine inducendo un aumento della 9 mobilità in presenza di polimixina B può essere spiegato attraverso tre meccanismi coinvolgendo la stabilità dell'involucro, la secrezione di vescicole di membrana o l’alterazione dell’espressione di diversi geni. Nel terzo articolo, abbiamo studiato il coinvolgimento dello stress ossidativo nell'arresto della sintesi del peptidoglicano che è fatale al mutante ∆elyC di Escherichia coli. Abbiamo poi dimostrato con numerosi esperimenti che la lisi del mutante ∆elyC è causata dalla sovrapproduzione di radicali idrossilici nel suo involucro. Questa molecola causa importanti danni nell’involucro provocando l’inibendo della sintesi del peptidoglicano. Il meccanismo che causa questa sovrapproduzione di radicali tossici deve ancora essere determinato. Studiando la funzione del fattore ElyC, abbiamo scoperto una nuova vulnerabilità nell'omeostasi dell’involucro che siamo in fretta di sfruttare. Nel nostro ultimo articolo, basandoci su un'ipotesi precedentemente formulata, abbiamo studiato l'importanza della via di sintesi dell'antigene comune agli enterobatteri nel processo letale che si svolge in assenza di ElyC. Abbiamo scoperto che il mutante ∆elyC accumula un elemento comune alle vie di sintesi del peptidoglicano e dell'antigene comune agli enterobatteri, l'undecaprenil pirofosfato. Abbiamo anche scoperto che l'aumento del riciclaggio di quest'ultimo attraverso la sovraespressione del gene pgpB previene la lisi del mutante. Proponiamo che l'assenza di ElyC induce una allocazione diffetosa del undecaprenil fosfato tra le vie di sintesi del peptidoglicano e dell'antigene comune agli enterobatteri. L'iperattivazione della via dell'antigene comune agli enterobatteri provoca l'accumulo di undecaprenil pirofosfato, causando l'inibizione di Pbp1b e dunque dell’aggiunta di nuove unità al peptidoglicano, provocandone quindi la lisi. Quindi, questo lavoro ha portato a una migliore comprensione o identificazione delle vulnerabilità dell'involucro di Vibrio cholerae ed Escherichia coli, a chiarire certi meccanismi essenziali ma anche a intravedere il ruolo di vari fattori coinvolti nell'omeostasi del loro involucro.

Cholera is a water-borne disease that continues to ravage resource poor communities around the world especially those in developing countries. The disease is caused by Vibrio cholerae microorganisms whose natural habitat is the aquatic ecosystem. It is believed that this microorganism prior to becoming the primary cause of cholera acquired virulence factors expressed by two separate genetic elements. These genetic elements are known as VPIФ and CTXФ were acquired in that order for known physiological reasons. However only V. cholerae in possession of the CTX genetic element are capable of causing cholera disease. At present only two serotypes are known to have the ability to cause cholera and these are V. cholerae serotypes O1 and O139. SODIS (Solar disinfection) is an extremely low cost refined technology that can be used for the disinfection of water especially in areas where there is a considerable amount of sunshine. Although this technology is a composite of various factors the underlying principle is the use of solar ultraviolet radiation (SUVR). The preliminary target of SUVR is the cytoplasmic membrane and this was confirmed by flow cytometric analysis. The consequences of leaky cytoplasmic membrane include cellular death to the microorganism as well as an increase in cholera toxin secretion. The main objective of this study was to investigate the effect of solar ultraviolet radiation on the growth of V. cholerae and on the secretion of cholera toxin and to provide supporting information for the use of SODIS in South Africa while observing the possible role that climate may play in the onset of cholera disease. The initial part of the study evaluated the culturability, biomass increase and cholera toxin secretion in both a nutrient poor and a nutrient rich media by two toxigenic and one non toxigenic strain of V. cholerae. A series of pH and temperature combinations were used to achieve this objective. The result revealed that the microorganisms survived in both media. An increase in biomass was observed for all the bacteria grown in the nutrient rich media whereas in the poor nutrient media the bacteria remained culturable but no increase in biomass was observed. Interestingly lower temperatures seemed to provide more optimal growth conditions while high temperature on most occasions favoured cholera toxin secretion, in both media.The second part of the study required the exposure of the microorganisms to SUVR. A SODIS approach was used with a few modifications. The V. cholerae strains were exposed to solar radiation during all the seasons of the year. Evaluation of the viability, the increase in biomass and the detection of cholera toxin secretion was determined after each exposure to solar radiation. The results seem to suggest that the effect of SUVR depended on the season of the year, the nature of the media, strain, solar conditions and in the duration of solar exposure, in no particular order. The secretion of cholera toxin was mainly dependent on the media used, the season of the year and on the serotype of the strain. This study represents the first report on the evaluation of SUVR for the disinfection of water under South African conditions (Pretoria area) during all seasons of the year with variations in solar radiation levels and temperature. Furthermore what actually happened to V. cholerae during solar exposure in terms of cell morphology, cell viability and secretion of cholera toxin is also reported and this can give an insight of the possible role that SUVR may play in the onset of cholera. The main recommendation emanating from this study is the sensitisation of communities worldwide about the capacity that, SUVR carries to lighten the burden of communicable water borne diseases especially, in resource limited areas through the implementation of SODIS. / Life and Consumer Sciences / M. Sc. (Life Science)