Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 175-194).

Cholera is an acute diarrheal illness burdening several developing countries caused by toxigenic Vibrio cholerae, where endemics maintain a seasonal pattern and occur more than once a year. Cholera is endemic in certain regions of Africa and South America, and southern Asia, where outbreaks are associated with poor hygiene and sanitary conditions. Every year, 3–5 million cases of cholera are diagnosed, and it causes more than 100,000 deaths worldwide. Cholera toxin is secreted by the V. cholerae bacteria and causes extreme secretory diarrhea, most commonly in poor hygiene environment. Watery diarrhea, vomiting, and abdominal cramps characterize the illness and approximately 5–10% of patients die of severe fluid loss if left untreated. The structure and function of the cholera toxin, its subunits, receptor, and impact on hyperactivation of cyclic adenosine monophosphate (cAMP) were sufficiently described in the 1970s. These findings fit with epidemiologic observations, which determined that the cholera toxin must first enter intestinal cells by binding to monosialoganglioside (GM1) on the host’s epithelial surface. The correlation between increase risk of V. cholerae infection and individuals with a particular ABO blood group type is unclear because of the scarce information and few studies conducted. Thus, this study reviews published research articles to better understanding the association between the blood group O and susceptibility to developing severe cholera symptoms. Several large studies have recorded an association between ABO blood groups and different infectious agents). Anthropological surveys suggest that the racial and geographic distribution of human blood types reflects tendencies towards specific erythrocyte types susceptible to infectious disease, such as cholera and malaria. Experimentally, the V. cholerae toxin has been extensively used as an experimental adjuvant, and its association with ABO groups is of practical importance for the development of an oral cholera vaccination. The results of previous studies provided strong evidence that individuals with blood type O are more vulnerable than other persons to severe cholera symptoms, even though the biologic basis for this association remains unknown.

No abstract. See Front page / Dissertation (MSc (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

CRISPR-Cas defense systems are employed by their hosts to prevent parasitization by mobile genetic elements. The discovery of nuclease-deficient CRISPR-Cas systems contained within transposon ends suggested a repurposing of the contained defense system. One such Type I-F3 CRISPR-Cas system was found inside Tn6677, a Tn7-like transposon within the genome of a Vibrio cholerae strain. Tn6677 requires coordination between the contained CRISPR-Cas system and the transposition proteins for effective transposition. Isolation of this system, and reduction to its minimal components, enabled RNA-guided integration of donor DNA in Escherichia coli. Base-pairing interactions between the user-specified CRISPR RNA and the target sequence precede the integration of donor DNA approximately 49-bp downstream of the end of the target sequence. This system is specific regardless of the supplied RNA guide, and successfully integrates donors of different lengths. The donor DNA is indicated by flanking cognate transposon end sequences. While clearly functional, the mechanism by which the transposition proteins and the CRISPR-Cas proteins interact remained unclear. To this end we purified the multi-protein RNA-guided DNA binding complex (Cascade) from the transposon-encoded minimal I-F3 CRISPR-Cas system in complex with the transposition protein TniQ. De novo modeling revealed the unexpected dimerization of TniQ, and its location within the complex, bound to the Cas6-end of the transposon-encoded Type I-F3 Cascade. Additional models obtained from DNA-bound structures of the complex demonstrate initial steps in target binding alongside novel conformations of Cascade subunits. This work reveals the mechanism by which the Tn6677 components guide integration and will enable rational engineering of these systems for further experimentation and tool development.

Publicación a texto completo no autorizada por el autor / Presenta a los bacteriófagos que son biocontroladores naturales de las bacterias como una alternativa para el control de las poblaciones de Vibrio cholerae, causantes de la enfermedad de transmisión alimentaria (ETA). El objetivo de este trabajo es aislar y caracterizar bacteriófagos capaces de infectar a Vibrio cholerae. Se toman muestras de aguas residuales de Lima - Perú. Los métodos empleados son para el aislamiento de Vibrio cholerae enriqueciendo, aislando en medio TCBS y bioquímica. Para el aislamiento y purificación del bacteriófago Φ K14 se realiza las pruebas de enfrentamiento en caldo, goteo, propagación y titulación. Para la caracterización microbiológica del bacteriófago Φ K14 se realiza las pruebas de rango de hospedero, multiplicidad de infección y curva de un paso. Para la caracterización fisicoquímica son las pruebas de estabilidad a diferentes condiciones ambientales (temperatura, pH y sensibilidad al cloroformo) y se realiza la microscopía electrónica. El bacteriófago K14 de Vibrio cholerae es caracterizado microbiológica y fisicoquímicamente de un total de 3 bacteriófagos aislados de aguas residuales es caracterizado microbiológica y fisicoquímicamente. Presenta una multiplicidad de infección (MOI) óptima de 0.001. El periodo latente del fago K14 es de 10 a 15 minutos y el tamaño de explosión de 25 UFP por célula infectada. Es estable a temperaturas de 40 oC, 50 oC y 60 oC e inestable a los 70 oC y 80 oC. El bacteriófago K14 no es sensible al cloroformo. Es inestable a pH 3 y estable del pH 7 a pH 9 pero tiene mayor estabilidad a pH 8. Según la micrografía electrónica el bacteriófago Φ K14 pertenece según sus estructuras a la familia Myoviridae. / Tesis

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.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser.

Le choléra est une diarrhée hydrique sévère volontiers épidémique causée par des Vibrio cholerae O1 toxinogènes. Ses déterminants environnementaux ont donné naissance à un paradigme influent sur les stratégies de lutte, qui sont se sont avérées peu efficientes en Afrique comme en Haïti. Elles pourraient être améliorées par une meilleure compréhension de la dynamique des épidémies. La synthèse bibliographique des influences de l'environnement sur les épidémies de choléra en Afrique y montre les limites du paradigme environnemental. L'étude multidisciplinaire des origines de l'épidémie de choléra en Guinée en 2012 suggère fortement qu'elle fut importée par voie humaine depuis la Sierra Leone voisine. Une description spatio-temporelle du choléra au Mozambique démontre l'hétérogénéité de sa transmission et amène à questionner le concept d'endémicité du choléra. Depuis son importation en Haïti en octobre 2010, l'épidémie de choléra présente également une répartition spatiale et temporelle très hétérogène. Son importante rétractation en saison sèche et son absence d'enracinement significatif dans l'environnement laissent espérer la possibilité d'une élimination rapide à condition d'apporter une réponse ciblée à tous les foyers épidémiques du pays. Une stratégie d'élimination basée sur nos recommandations y est actuellement menée par le Ministère de la Santé, l'UNICEF et leurs partenaires. Après des résultats spectaculaires en 2013 et au premier semestre 2014, la situation s'est à nouveau dégradée pendant la saison des pluies. Une élimination du choléra dans saison sèche à venir demeure cependant réaliste si nous parvenons à convaincre et remobiliser les acteurs de terrain. / Cholera is an epidemic acute watery diarrhea caused by toxigenic bacteria Vibrio cholerae O1. Its environment determinants have been at the source of a popular paradigm. Many recent control strategies have shown little efficiency in Africa or in Haiti, but they could be improved by a better comprehension of the epidemics dynamic. The bibliographic synthesis of environment influences on cholera in Africa highlights the limits of the environmental paradigm on this continent. A multidisciplinary study of the origin of cholera epidemic in Guinea in 2012 strongly suggests it was humanly imported from nearby Sierra Leone. A space-time description of cholera in Mozambique demonstrates heterogeneous transmission patterns and challenges the concept of cholera endemicity. Since its importation in Haiti in October 2010, cholera transmission also exhibits a marked spatio-temporal heterogeneity. Cholera important retraction during the dry season and its absence of significant establishment in the Haitian environment suggest it may be possible to rapidly eliminate cholera in the country, provided that every outbreak focus receives a targeted response. An elimination strategy based on our recommendations is currently implemented by Haitian Ministry of Health, UNICEF and their partners. After spectacular results in 2013 and during the first half of 2014, the situation has slowly deteriorated during the rainy season. However, cholera elimination during the coming dry season remains realistic provided that we succeed in persuading and remobilizing the partners present on the field.

A poluição, alteração física do habitat e a introdução de espécies invasoras via água de lastro, representam os maiores impactos antropogênicos para os ambientes costeiros. Foram pesquisadas em amostras da região costeira de S. Paulo, regiões portuárias brasileiras e de tanques de lastro de navios, bactérias das espécies Vibrio cholerae (Vc), V. parahaemolyticus (Vp) e V. vulnificus (Vv) que são as que têm maior implicação na saúde pública. As amostras foram avaliadas levando-se em conta parâmetros físico-químicos e microbiológicos e suas relações com a presença de Vc, Vp e Vv. As relações clonais foram verificadas através das técnicas de ERIC, BOX e REP-PCR. Foram identificadas 90 cepas de Vp e 11 de Vc. Foram observadas correlações entre alguns parâmetros microbiológicos e a presença de vibrios. A análise clonal permitiu verificar a alta diversidade das cepas. Concluiu-se que Vc e Vp são autóctones do ambiente costeiro brasileiro e podem ser tornar reservatórios para determinados fatores associados à virulência, gerando cepas com potencial epidêmico. / Pollution, physical alteration of habitat and the introduction of alien species through ballast water constitute the biggest anthropogenic impacts on coastal environments. We examined samples taken from the coastal region of S. Paulo state, Brazilian ports and ship ballast tanks, for bacteria of the species Vibrio cholerae (Vc), V. parahaemolyticus (Vp) and V. vulnificus (Vv) which have the most significant implication for public health. The samples were evaluated for microbiological and physical-chemical parameters as well as the presence of Vc, Vp and Vv. Clonal relationships of bacterial isolates were determined through ERIC, BOX and REP-PCR. A total of 90 strains of Vp and 11 of Vc were identified. Correlations between some microbiological parameters and the presence of vibrios were observed. The clonal analysis revealed extensive strain diversity. We concluded that Vc and Vp are autochthonous bacteria of the Brazilian coastal environment that can become reservoirs for factors associated with virulence, and are capable of generating strains with epidemic potential.