<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

The waterborne bacterial pathogen Vibrio cholerae uses a process of cell-to-cell communication called quorum sensing (QS) to coordinate transcription of four sRNAs (Qrr1-4; quorum regulatory RNAs) in response to changes in extracellular QS signals that accumulate with cell density. The Qrr sRNAs are predicted to negatively control translation of several mRNAs, including hapR, which encodes the master QS transcription factor that controls genes for virulence factors, biofilm formation, protease production, and DNA uptake. The Qrr sRNAs are also predicted to positively control vca0939, which encodes a GGDEF family protein that promote biofilm formation by elevating intracellular levels of the second messenger molecule c-di-GMP. Using complementary in vivo, in vitro, and bioinformatic approaches, I showed that Qrr sRNAs base-pair with and repress translation of the mRNA encoding HapR. A single nucleotide mutation in Qrr RNA abolishes hapR pairing and thus prevents cholera toxin production and biofilm formation that are important in disease, and also alters expression of competence genes required for uptake of DNA in marine settings. I also demonstrated that base-pairing of the Qrr sRNAs with vca0939 disrupts an inhibitory structure in the 5' UTR of the mRNA. Qrr-activated translation of vca0939 was sufficient to promote synthesis of c-di-GMP and early biofilm formation in a HapR-independent manner. Thus, these studies define the non-coding Qrr sRNAs as a critical component allowing V. cholerae to sense and respond to environmental cues to regulate important developmental processes such as biofilm formation.

<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

L'objectif de cette thèse est de définir la chorégraphie de ségrégation des deux chromosomes circulaires de Vibrio cholerae, c'est à dire le positionnement de l'information génétique au cours de la croissance de la cellule, ainsi que les mécanismes dirigeant ces ségrégations. Il a longtemps été supposé que les bactéries étaient trop petites pour avoir une organisation intra-cellulaire, et le manque de techniques appropriées ne permettait pas d'infirmer cette hypothèse. Or la taille des chromosomes comparée à celle de la bactérie impose une compaction et aujourd'hui, de nouvelles techniques de microscopie et d'analyse génétique permettent d'affirmer que les chromosomes bactériens étudiés jusqu'à maintenant ont tous une organisation et une chorégraphie de ségrégation précises et différentes selon les espèces. Toutes les espèces étudiées à ce jour ont un chromosome circulaire unique : la réplication du chromosome commence à une origine unique bidirectionnelle, les deux fourches de réplication se déplacent le long des deux bras de réplication (ou réplichores) et finissent la réplication au terminus, diamétralement à l'opposée de l'origine de réplication sur la carte du chromosome. Peu d'espèces ont été étudiées, et Vibrio cholerae émerge progressivement comme un nouveau modèle : son génome est réparti sur deux chromosomes, et la chorégraphie de plusieurs chromosomes dans une cellule n'a jamais été décrite. De plus, cette espèce semble être au croisement évolutif entre Caulobacter crescentus et Escherichia coli : Vibrio cholerae a d'une part une morphologie en croissant, des systèmes de partition aux origines et un positionnement de l'origine du chromosome I, semblables à C. crescentus, et d'autre part un système de compaction du terminus et un set de gènes impliqués dans la maintenance du chromosome ayant co-évolué, qu'on ne retrouve que dans peu d'espèces proches d'E. coli. Une autre caractéristique intéressante de V. cholerae est que le chromosome II semble avoir été acquis récemment et n'est donc peut être pas gouverné par les mêmes mécanismes que le chromosome I, comme en témoignent le positionnement de son origine et son terminus, inédits pour des chromosomes bactériens. Parmi les Vibrios (environ 60 espèces principalement retrouvées dans les environnements aquatiques), certaines espèces sont des pathogènes dévastateurs pour les poissons, le corail, les crustacés ou les fruits de mer. Mais la plus documentée est Vibrio cholerae, car elle provoque chez l'Humain une maladie provoquée par l'ingestion d'eau contaminée qui peut être mortelle si le patient n'est pas réhydraté à temps. Bien que facilement traitable, le choléra fait encore de nombreuses victimes dans les pays en développement où les structures de santé et les règles d'hygiène font parfois défaut. Ainsi l'étude de Vibrio cholerae présente un intérêt médical, mais également par extension aux autres Vibrios, un intérêt environnemental non négligeable.

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)

Philosophiae Doctor - PhD (Biotechnology) / Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo;s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes. / South Africa

Thesis (MPH.) -- University of Limpopo, 2015 / Cholera is an acute enteric infection caused by the ingestion of bacterium Vibrio cholerae present in faecally contaminated water or food. Primarily linked to insufficient access to safe water and proper sanitation, its impact can be even more dramatic in areas where basic environmental infrastructures are disrupted or have been destroyed. The aim of the study was to investigate the factors contributing to the prevalence of cholera and the environmental risk factors associated with cholera in the Vhembe district of Limpopo province between 2008 and 2012. The objectives of the study were to identify environmental risk factors for cholera and to determine the number of cholera cases in the Vhembe district. The study used a quantitative, retrospective and cross-sectional research method. The records of 317 patients who met the study criteria were reviewed using an audit tool. The Statistical Package for Social Sciences (SPSS) version 22 was used to analyze the data. The results revealed that lack of adequate hygiene practices, limited access to safe drinking water, lack of safe food preparation and handling, and inadequate sanitation system are risk factors associated with cholera. The study recommends prevention, control of cholera outbreak and case management. Keywords: Cholera, outbreak, Vibrio cholerae 01 and 0139, Watery diarrhea (ricewater), Prevalence, Risk factors.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-07T11:44:28Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_DiversidadeGeneticaIsolados.pdf: 1765217 bytes, checksum: e159b664332d4cd1ed8d170a0a2c6559 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-02-12T15:56:27Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_DiversidadeGeneticaIsolados.pdf: 1765217 bytes, checksum: e159b664332d4cd1ed8d170a0a2c6559 (MD5) / Made available in DSpace on 2014-02-12T15:56:27Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_DiversidadeGeneticaIsolados.pdf: 1765217 bytes, checksum: e159b664332d4cd1ed8d170a0a2c6559 (MD5) Previous issue date: 2009 / Vibrio cholerae, agente etiológico da cólera, é uma bactéria nativa de ambientes aquáticos de regiões temperadas e tropicais em todo o mundo. A cólera é endemica e epidemica em países da África, Ásia e Americas Central e do Sul. Neste trabalho o objetivo foi estudar a diversidade genética de isolados desta espécie, de ambientes aquáticos da Amazônia brasileira. Um total de 148 isolados de V.cholerae não-O1 e não-O139 (NAGs) e O1 ambientais da Amazônia, obtidos entre 1977 e 2007, foram caracterizados e comparados a linhagens clínicas de V.cholerae O1 da sexta e sétima pandemias. Utilizou-se os perfis de macrorestrição definidos em eletroforese em gel de agarose em campo pulsado (PFGE), para determinar a relação clonal entre V.cholerae non-O1 e O1 ambientais e clínicos. A presença de genes de virulência (hlyA/hem, hlyB, hlyC, rtxA, rtxC, tcp, ctx, zot, ace, stn/sto) e integrons de classe 1, 2 e 3 (intI 1, 2 e 3), foi analisada utilizando-se a reação em cadeia da polimerase. A análise dos perfis de macrorestrição revelou que os NAGs apresentaram uma grande diversidade genética comparada aos V.cholerae O1. Isolados de NAGs e O1 segregaram em distintos grupos e a maioria dos O1 ambientais apresentou relação clonal com isolados clínicos da sétima pandemia de cólera. A distribuição dos genes de virulência entre os NAGs é diferente a dos O1, os quais, em geral, foram positivos para todos os genes de virulência estudados exceto stn/sto e integrons de classe 1, 2 e 3. Alguns V.cholerae O1 ambientais pertencentes a linhagem da sétima pandemia, apresentaram uma extensiva perda de genes. Diferentes NAGs foram stn/sto+ e intI 1+. Dois alelos do gene aadA foram encontrados: aadA2 e aadA7. De modo interessante os V.cholerae O1 ambientais pertencentes à linhagem pandêmica, só foram isolados durante o período da última epidemia de cólera na região Amazônica brasileira (1991-1996). / Vibrio cholerae, the etiologic agent of cholera, is an autochtonus bacterium of aquatic environment in temperate and tropical regions of the world. Cholera is endemic and epidemic in many countries in Africa, Asia and Central and South America. In this study our goal was to detemine the genetic diversity of V.cholerae environmental isolates from aquatic ecosystems in the Amazon region of Brazil. A total of 148 environmental strains of V.cholerae non-O1 and non-O139 (NAG) and O1 serogroups, isolated from the Amazon region since 1977 to 2007, were characterized and compared with clinical strains of V.cholerae O1 from sixty and seventh cholera pandemic. PFGE (pulsed-field gel electrophoresis) was performed to determine the clonal relationships between V.cholerae non-O1, O1 environmental and clinical strains. The presence of virulence genes (hlyA/hem, hlyB, hlyC, rtxA, rtxC, tcp, ctx, zot, ace, stn/sto) and class 1, 2 and 3 integrons (intI 1, 2 e 3) was analyzed by polymerase chain reaction. Whole genome macrorestriction analysis revealed that the environmental V.cholerae NAGs were more diverse than the environmental O1 strains, both groups segregate in distinct clusters and most of environmental O1 strains show a clonal relationship with seventh cholera pandemic strains. The distribution of virulence genes in NAGs strains is largely different from that of O1 strains which, in general, were positive for all virulence genes analyzed excepting for stn/sto and class 1, 2 and 3 integrons. Some O1 environmental strains, belonging to the seventh pandemic lineage, went through an extensive gene loss. Distinct NAGs strains were stn/sto+ and intI 1+. Two alleles of aadA were found: aadA2 and aadA7. Interestingly, V.cholerae O1 environmental strains belonging to the pandemic lineage were only isolated during the period of cholera epidemic in the Amazon region of Brazil (1991-1996).

The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the MVs and released from the surface of bacteria. MV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37, thereby, enhancing bacterial survival by avoiding this innate immune defense of the host. We also studied another virulence factor of V. cholerae, the pore-forming toxin VCC, which was found to be transported by MVs. MV-associated VCC is biologically active and triggers an autophagic response in the target cells. We suggested that autophagy serves as a cellular defense mechanism against the MV-associated bacterial virulence factor of V. cholerae. Listeria monocytogenes is a Gram-positive intracellular and facultative anaerobic food-borne pathogen causing listeriosis. It causes only sporadic outbreaks in healthy individuals, however, it is dangerous for a fetus or newborn child, and for pregnant and immunocompromised people, leading to a deadly infection in one third of the cases. We have analyzed MVs produced by L. monocytogenes and their interaction with eukaryotic cells. Confocal microscopy analysis showed that MVs are internalized into HeLa and HEK293 cells and are accumulated in lysosomes. Moreover, L. monocytogenes produces MVs inside the host cells and even inside the phagosomes. We found that the major virulence factor of L. monocytogenes, the cholesterol-dependent pore-forming protein listeriolysin O (LLO), is entrapped inside the MVs and resides there in an oxidized inactive state. LLO is known to induce autophagy by making pores in the phagosomal membrane of targeted eukaryotic cells. In our studies, we have shown that MVs effectively abrogated autophagy induced by Torin1, by purified LLO or by another pore-forming toxin from V. cholerae. We also found that MVs promote bacterial intracellular survival inside mouse embryonic fibroblasts. In addition, MVs have been shown to have a strong protective activity against host cell necrosis initiated by pore-forming toxin. Taken together, these findings suggested that in vivo MVs production from L. monocytogenes might be a relevant strategy of bacteria to manipulate host responses and to promote bacterial survival inside the host cells.

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