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Keller, Ninette. "Hypoglycaemia in virulent canine babesiosis prevalence and risk factors /." Diss., University of Pretoria, 2004. http://upetd.up.ac.za/thesis/available/etd-03082005-092252/.
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Kitahara, Nao. "Study on screening of novel pathogenic factors of Candida albicans by proteome analysis and its putative virulent mechanism." Kyoto University, 2016. http://hdl.handle.net/2433/215600.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第19774号
農博第2170号
新制||農||1040(附属図書館)
学位論文||H28||N4990(農学部図書室)
32810
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 充美, 教授 栗原 達夫, 教授 矢﨑 一史
学位規則第4条第1項該当
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Yang, Guowei. "Photorhabdus virulence factors." Thesis, University of Bath, 2005. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.425889.
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Guttmann, Daniel Mark. "Bacillus thuringiensis virulence factors." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621944.
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Wright, K. "Genomics and virulence factors of Fusobacterium necrophorum." Thesis, University of Westminster, 2016. https://westminsterresearch.westminster.ac.uk/item/9ywy3/genomics-and-virulence-factors-of-fusobacterium-necrophorum.
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Fusobacterium necrophorum, a Gram negative, anaerobic bacterium, is a common cause of acute pharyngitis and tonsillitis and a rare cause of more severe infections of the head and neck. At the beginning of the project, there was no available genome sequence for F. necrophorum. The aim of this project was to sequence the F. necrophorum genome and identify and study its putative virulence factors contained using in silico and in vitro analysis. Type strains JCM 3718 and JCM 3724,F. necrophorum subspecies necrophorum (Fnn) and funduliforme (Fnf), respectively, and strain ARU 01 (Fnf), isolated from a patient with LS, were commercially sequenced by Roche 454 GS-FLX+ next generation sequencing and assembled into contigs using Roche GS Assembler. Sequence data was annotated semi-automatically, using the xBASE pipeline, BLASTp and Pfam. The F. necrophorum genome was determined to be approximately 2.1 – 2.3 Mb in size, with an estimated 1,950 ORFs and includes genes for a leukotoxin, ecotin, haemolysin, haemagglutinin, haemin receptor, adhesin and type Vb and Vc secretion systems. The prevalence of the leukotoxin gene was investigated in strains JCM 3718, JCM 3724 and ARU 01, as well as a clinical collection of 25 Fnf strains, identified using biochemical and molecular tests. The leukotoxin operon was found to be universal within the strain collection by PCR. HL-60 cells subjected to aliquots of concentrated high molecular weight culture supernatant, predicted to contain the secreted leukotoxins of strains JCM 3718, JCM 3724 and ARU 01, were killed in a dose-dependent manner. The cytotoxic effect of the leukotoxin against human donor white blood cells was also tested to validate the HL-60 assay. The differences in the results between the two assays were not statistically significant. Ecotin, a serine protease inhibitor, was found to be present in 100 % of the strain collection and had a highly conserved sequence with primary and secondary binding sites exposed on opposing sides of the protein. During enzyme inhibition studies, a purified recombinant F. necrophorum ecotin protein inhibited human neutrophil elastase, a protease that degrades bacteria at inflammation sites, and human plasma kallikrein, a component of the host clotting cascade. The recombinant ecotin also prolonged human plasma clotting times by up to 7-fold for the extrinsic pathway, and up to 40-fold for the intrinsic pathway. The genome sequence data provides important information about F. necrophorum type strains and enables comparative study between strains and subspecies. Results from the leukotoxin and ecotin assays can be used to build up an understanding of how the organism behaves during infection.
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Monteil, Vanessa. "Analyses phénotypiques et génotypiques de différentes souches de dengue : applications en épidémiologie et recherche de facteurs de virulence." Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM5038.
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De 50 à 100 millions de cas de maladie de dengue sont recensés chaque année dans le monde. Le virus de la dengue présente aujourd’hui un problème de santé publique avec son émergence en Europe et particulièrement en France. L’infection par le virus peut être asymptomatique ou être responsable de trois pathologies: l’une avec des symptômes grippaux (DF), une autre avec des hémorragies modérées (DHF) et une dernière avec des hémorragies sévères entraînant un syndrome de choc (DSS).Les facteurs de l’hôte jouent un rôle important dans le développement de formes sévères mais les facteurs viraux impliqués restent peu décrits. Le but de ce travail de thèse était de mieux comprendre ces facteurs viraux au travers de l’étude des dynamiques de circulation de souches de dengue 3 génotype III en Afrique et de la caractérisation de trois souches de dengue de sérotype I du Cambodge. Ce travail nous a permis de mettre en évidence la circulation de variants pendant les épidémies, permettant de supposer que la présence de variants permet une meilleure dissémination, ainsi que des caractéristiques génotypiques et phénotypiques particulières in vitro aux souches associées aux formes hémorragiques et aux formes avec syndrome de choc chez l’homme. Ces travaux ont été complétés par le développement d’un système original de détection du virus de la dengue et des autres virus du genre Flavivirus. Ce travail de thèse a permis d’identifier de potentiels facteurs de virulence propres au virus, ouvrant la voie à la recherche sur le rôle de certaines protéines virales dans la pathogénicitéFrom 50 to 100 million cases of dengue illness occurred every year in the world. Today, dengue virus is a public health problem with its emergence in Europe, particularly in France. DENV infection can be asymptomatic or be responsible for three distinct pathologies: one with flu-like symptoms (DF), another with moderate hemorrhage (DHF) and the last one with severe bleeding leading to shock syndrome (DSS). Host factors have an important role in the development of severe forms but implicated viral virulence factors stay not well described. The aim of this research work was to better understand these viral factors through study of dengue serotype 3 genotype III dynamics of circulation in Africa and through the characterization of three dengue serotype 1 strains in Cambodia. This work highlighted the circulation of variants during epidemics, allowing us to suppose that the presence of variants permits a better dissemination, as well as specific phenotypic and genotypic characteristics in vitro of strains associated with hemorrhagic forms or forms with shock syndrome in humans. These works were completed by the development of an original system of detection of dengue virus and other viruses of genus Flavivirus. This research work allowed identifying potential virulence factor specific to virus, opening the way for research on the role of certain viral proteins in pathogenicity
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Ranc, Anne-Gaëlle. "Phenol Soluble Modulins et lipopolysaccharide de Legionella pneumophila : rôle dans la réponse immunitaire innée." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1010/document.
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Legionella pneumophila (Lp) est une bactérie ubiquitaire dans les environnements aqueux et responsable d’une pneumopathie potentiellement sévère : la légionellose. La majorité des souches impliquées appartiennent au sérogroupe 1 (Lp1) et à un sous- groupe spécifique de souches portant un épitope particulier dites mAb3/1+. Cependant, la différence de distribution entre les souches retrouvées dans l’environnement et celles impliquées en clinique n’est pas clairement élucidée. Notre travail a porté sur la détection de deux facteurs de virulence de Lp. Nous avons voulu mettre en évidence l’existence de Phenols Soluble Modulines (PSMs), peptides uniquement décrit chez Staphylocoques et avons ainsi pu démontrer l’activité de peptides prédits par analyse in silico chez Lp capables d’activer la réponse inflammatoire par la voie du NF-?B et sont dotés d’une action cytotoxique. Notre deuxième axe d’étude a porté sur le lipopolysaccharide (LPS) de Lp. Afin de vérifier si la prédominance de certaines souches était liée à un biais diagnostique, nous avons voulu tout d’abord vérifier la sensibilité de 3 tests urinaires diagnostiques envers le LPS extrait de souches de différents sous- groupes de Lp1 et sérogroupes de Lp et avons ainsi pu montrer que ces tests sont capables de détecter tous les LPS de Lp1. La sensibilité envers le LPS des autres sérogroupes est très variable mais reste insuffisante pour permettre leur détection. Nous avons ensuite utilisé ces LPS extraits pour vérifier la réponse immunitaire innée en fonction des souches de Lp1. Ainsi les souches mAb3/1+ activent moins le système immunitaire que les souches mAb3/1-, ce qui pourrait expliquer alors une moins bonne clairance de ces souches permettant leur multiplication à l’origine d’une infection. Au final, notre travail a permis d’étudier deux facteurs de virulence potentiels au sein de Lp, pouvant expliquer partiellement la prédominance de certaines souches en pathologie humaineLegionella pneumophila (Lp) is a ubiquitous intracellular bacterium found widely in the environment and is the cause of an opportunistic infection named legionellosis. The majority of the strains involved belong to serogroup 1 (Lp1) and to a specific subgroup named mAb3/1+, linked to a specific epitope expressed at the cell membrane. However the distribution difference between the strains found in the environment and the ones involved in pathology is not fully understood. We here studied two virulence factors of Lp. We first demonstrated the existence of Phenols Soluble Modulines (PSMs), smalls peptides that only have been described for Staphylococcus and found that the peptides that were predicted for Lp by in silico analysis were able to activate the innate immune response by NF-?B pathway and were able to have a cytotoxic activity. We also studied the lipopolysaccharide (LPS) of Lp. To found out if the predominance of some strains was linked to a diagnosis biais, we first evaluated the sensitivity of 3 urinary antigens tests against extracted LPS of strains belonging to all the sous-groups of Lp1 and serogroups of Lp. We then demonstrated that those tests are able to detect all LPS of Lp1, independently of mAb3/1 character. The sensitivities of the 3 tests were very variable for the other serogroups of Lp, but were too low to be able to detect those LPS in practice. We then used these extracted LPS to evaluate the innate immune response for different strains of Lp1. We demonstrated that mAb3/1- strains needed lower dose of LPS to activate the innate immune response than mAb3/1+ strains, which could be linked to a better clearance of the bacteria from the host, which doesn’t develop an infection. This work has studied two potentially virulent factors of Lp, which could partially explain the predominance of some strains of Lp in human pathology
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Seixas, Rui Emanuel Antunes de. "Virulence of Salmonella typhimurium 1,4,[5],12:i:- : the new emergent strain." Doctoral thesis, Universidade de Lisboa, Faculdade de Medicina Veterinária, 2017. http://hdl.handle.net/10400.5/13925.
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Tese de Doutoramento em Ciências Veterinárias na Especialidade Sanidade AnimalSalmonella serovar 1,4,[5],12:i:- is presently considered one of the major serovars responsible for human salmonellosis worldwide. A multidisciplinary approach, including the fields of epidemiology, spatial statistics, clinical and applied microbiology was used to perform an extensive characterization of Salmonella 1,4,[5],12:i:- isolates obtained by the National Health Institute Dr. Ricardo Jorge, which was lacking due to the recent emergence. It was observed that cases are reported in most districts, being more frequent in the Portuguese coastland. Spatial statistical analysis showed a significant geographic clustering, pointing out for the importance of evaluating these areas to identify risk factors, in order to establish adequate prevention programs. The most relevant antimicrobial profile in this serovar is the tetra-resistance pattern (R-type ASSuT), displaying resistance to ampicillin, streptomycin, sulphonamides and tetracyclines. A high occurrence of R-type ASSuT isolates was observed in the isolates under study, with the majority harboring the resistance genes frequently associated with the European clone, namely blaTEM, sul2, straA-straB, tetB. Additionally, resistance to quinolones and 3rd generation cephalosporin was also detected. In Portugal, the rapid spread of Salmonella 1,4,[5],12:i:- R-type ASSuT might be related with the diversity of pulsotypes and also the presence of a core of virulence factors, including biofilm production. Biofilm-forming ability varied between sample locations and collection year, and can be one of the virulence features related with the rise of this serovar. Furthermore, biofilm formation was evaluated in vitro using a simulated human intestinal environment. In such conditions was observed an impairment of biofilm production, revealing that conditions mimicking the human intestinal tract can influence the biofilm-forming ability of the isolates under study. This research highlight the critical importance of close surveillance of Salmonella 1,4,[5],12:i:- in Portugal, including R-type ASSuT isolates. Information gathered may unravel Salmonella 1,4,[5],12:i:- features, prevent the dissemination to other regions and also benefit the medical community in order to rationalize salmonellosis antimicrobial therapeutics.
RESUMO - Virulência de Salmonella Typhimurium 1,4,[5],12:i:-, a nova estirpe pandémica* - Salmonella é uma bactéria Gram-negativa pertencente à família Enterobacteriaceae, sendo uma das principais responsáveis pela morbilidade e mortalidade associadas a toxinfecções alimentares. Pode manifestar-se num espectro de sintomatologia variado, incluindo a gastroenterite, a bacteriémia e a infecção focal. Este género incluí mais de 2600 serovares descritos, distribuídos por apenas duas espécies: Salmonella enterica que inclui todos os serovares patogénicos para os humanos e Salmonella bongori. Actualmente, um dos principais serovares responsáveis pela salmonelose humana em todo o mundo é o 1,4,[5],12:i:-. Este serovar é uma variante monofásica de Salmonella Typhimurium, muito semelhante a nível molecular, sendo caracterizado pela ausência da expressão do gene fljB. Devido à sua recente emergência, estudos que avaliem este serovar são escassos, particularmente em Portugal, o que definiu o âmbito desta investigação, que teve como objectivo a caracterização epidemiológica e microbiológica, tanto do ponto de vista fenotípico e genotípico, de isolados de Salmonella 1,4,[5],12:i:- obtidos em Portugal a partir de diferentes origens, incluindo amostras humanas, animais e ambientais. Numa primeira fase foi realizada uma caracterização demográfica, epidemiológica e espacial de todos os casos de Salmonelose 1,4,[5],12:i:- humana notificados em Portugal pelo Instituto Nacional de Saúde Dr. Ricardo Jorge (INSA), durante um período de 10 anos, desde 2001 a 2011. Foram recolhidos dados sobre a origem, ano e mês de amostragem, género, idade, distrito e município de residência dos pacientes. Foi realizada a análise estatística descritiva, bem como, a análise estatística espacial através do software SaTScan™, combinada com análise através de software de georeferenciação, o QGIS™, de forma a caracterizar a epidemiologia e identificar agrupamentos espaciais de risco superior de infecção por Salmonella 1,4,[5],12:i:- em Portugal. Globalmente, observou-se que em Portugal, a maioria dos distritos tem casos notificados de infecção por Salmonella 1,4,[5],12:i:-. Verificou-se também um aumento da incidência durante o intervalo de 2004 a 2011, com um maior número de casos na região litoral do país, incluindo distritos como Porto, Lisboa e Aveiro, o que pode ser explicado pela maior densidade populacional nestas áreas. A maioria das infecções ocorreu durante Maio e Outubro, e o menor número em Fevereiro, afectando principalmente indivíduos jovens.[...]*O autor escreve segundo o antigo Acordo Ortográfico
This work was supported by National Health Institute Doutor Ricardo Jorge (INSA) and funded by Centre for Interdisciplinary Research in Animal Health (CIISA)
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Scott, David Albert. "Virulence factors of oral anaerobic spirochetes." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0001/NQ30378.pdf.
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Scott, David 1964 Jan 11. "Virulence factors of oral anaerobic spirochetes." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=34446.
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The research work presented in this thesis involved the examination of several fundamental questions concerning the role of oral anaerobic spirochetes (OAS) in the etiology of periodontal disease (PD). OAS are unusual, helical, Gram-negative bacteria that are considered putative periodontopathogens due to numerical association with diseased sites and the enzymatic arsenal available to OAS that appears consistent with disease symptoms. T. denticola is the most commonly isolated OAS from periodontal pockets and as such is the focus of most investigations into the role of OAS in PD.As free iron is severely limited in humans the means by which OAS may obtain sufficient iron to prosper in the sub-gingiva was examined. The resultant model suggests T. denticola obtains iron through the $ beta$-hemolysis of erythrocytes and the binding of liberated hemin by a 47-kDa outer membrane sheath (OMS) protein. The kinetics of the ligand-receptor interaction are presented and the receptor has been purified to apparent homogeneity from T. denticola. $ sp3$H-labeled hemin is not transported into the cell. Evidence is presented to show that T. denticola produces an iron reductase, which may facilitate the transport of ferrous iron across biological membranes. It is also shown that T. denticola (Td), T. vincentii (Tv) and T. socranskii (Ts) do not produce siderophores. In growth assays, under conditions of iron-limitation, T. denticola may use inorganic iron, a source unlikely to be available in vivo.
Hyaluronidase (Hase) activity is elevated in the gingival crevice during episodes of disease. Hase, when injected into the periodontal cavity under experimental conditions has been shown to result in connective tissue degradation. It is also known that T. pallidum, the agent of syphilis, produces a Hase that is critical to pathogenesis. Evidence is presented herein to show that Td, Tv and Ts all produce a hyauronoglucosaminidase (HGase). The identity and specificity of the Td HGase is confirmed through the use of enzyme inhibitors and activators, by electron microscope observations of the enzyme using the Hase inhibitor gold sodium thiomalate and anti-Apis mellifera venom antibodies and examination of the purity of the HA substrate using other polysaccharide degrading enzymes. As the HGase of these OAS would not migrate through a substrate-SDS PAGE system, we have employed hyaluronate (HA)- and chondroitin sulfate (CS)-absorbed nitrocellulose membranes to visualize HGase activity. The 59 kDa HGase of Td has been purified to apparent homogeneity through the conjugation of HA and CS to Affigel 701 beads.
The last subject to be addressed by this thesis pertains to the ultrastructure of oral spirochetes. Using the copper-containing dye, Alcian blue, we have shown that T. denticola produces an exopolysaccharide layer, in an electron microscopy investigation. The development of a stain for dark-field microscopy has simplified the observation of this layer. The exopolysaccharide layer may have relevance to the evasion of phagocytosis, to protection against colicins, immunoglobulins and bacteriophages, to adherence and perhaps to the immunogenicity of OAS inhabiting the sub-gingiva.
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Cahill, Lori A. (Lori Anne). "Virulence factors in non-gastric Helicobacters." Thesis, Massachusetts Institute of Technology, 1998. http://hdl.handle.net/1721.1/50433.
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Zhang, Xing. "Exploring fungal virulence using C. elegans." Thesis, Aix-Marseille, 2020. http://theses.univ-amu.fr.lama.univ-amu.fr/200924_ZHANG_406xehco6dvggp718z420kj_TH.pdf.
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Parmi les candidats figuraient plusieurs entérotoxines thermolabiles, une famille de protéines qui est élargie dans le génome de D. coniospora par rapport à d'autres champignons pathogènes. Nous nous sommes concentrés sur 3 (DcEntA-C). L'expression de DcEntA et de DcEntB, mais pas de DcEntC, rendait les vers malades et plus sensibles à l'infection. Normalement, l'infection par D. coniospora provoque l'induction de l'expression de gènes codant des peptides antimicrobiens des familles nlp et cnc. Il est intéressant de noter que l'expression de la seule entérotoxine DcEntA a bloqué la transcription des gènes nlp et cnc. DcEntA a agi en inhibant la translocation nucléaire du facteur de transcription STAT STA-2, nécessaire à l'expression des gènes de défense. Nous avons démontré que cet effet était spécifique car DcEntA a induit une forte expression d'un gène inductible par une infection indépendante de STA-2. En revanche, les vers exprimant l'entérotoxine DcEntB présentaient une élévation de l'expression de nlp-29 dépendante de STA-2. DcEntB est localisé au niveau du nucléole et affecte la taille et la morphologie du nucléole. La base moléculaire de ces différences et l'importance relative de ces facteurs au cours de l'infection ont été étudiées en détail. Notre résultat révélé la complexité des stratégies de virulence fongique. Globalement, en disséquant le mode d'action des différents facteurs de virulence, cette étude nous a permis de mieux comprendre la pathogénie fongique et la course évolutive entre l'hôte et l'agent pathogèneAmong the candidates were several heat-labile enterotoxins, a protein family that is expanded in the genome of D. coniospora compared to other pathogenic fungi. We focused on 3 (DcEntA-C). Expression of DcEntA and DcEntB, but not DcEntC made worms sick and more susceptible to infection. Normally, D. coniospora infection provokes the induction of expression of antimicrobial peptide genes of the nlp and cnc families. Interestingly, expression of the single enterotoxin DcEntA blocked the transcription of both nlp and cnc genes. DcEntA acted by inhibiting the nuclear translocation of the STAT transcriptional factor STA-2, required for defence gene expression. We demonstrated that this effect was specific as DcEntA induced high expression of a STA-2-independent infection-inducible gene. In contrast, worms expressing the enterotoxin DcEntB exhibited a STA-2 dependent elevation of nlp-29 expression. DcEntB was localized to the nucleolus and affected nucleolus size and morphology. The molecular basis of these differences and the relative importance of these factors during infection was explored in detail. Our result revealed the complexity of fungal virulence strategies. Overall, by dissecting the mode of action of different virulence factors, this study allowed us to understand better fungal pathogenesis and the evolutionary arms race between host and pathogen
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Angely, Christelle. "Propriétés mécaniques et fonctionnelles des cellules épithéliales respiratoires exposées à une toxine bactérienne : l’adénylate cyclase." Thesis, Paris Est, 2018. http://www.theses.fr/2018PESC0058/document.
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La recrudescence des infections respiratoires impliquant des facteurs virulents d’origine bactérienne est devenue un problème majeur de santé publique. Mieux caractériser la réponse des cellules respiratoires dans la phase initiale d’exposition à des toxines bactériennes est important sur les plans physiopathologiques et thérapeutiques. Le but de ce travail est de décrypter les mécanismes cellulaires et moléculaires impliqués lors de l’exposition des cellules épithéliales respiratoires à l’adénylate cyclase (CyaA), une toxine produite par Bordetella pertussis, l’agent responsable de la coqueluche. CyaA a été choisie car elle dispose de multiples moyens qui lui permettent d’envahir un grand nombre de cellules eucaryotes. Elle est notamment capable de transloquer son domaine catalytique directement dans la cellule cible puis d’utiliser la calmoduline endogène pour augmenter le taux d’AMPc à des niveaux supraphysiologiques. Cependant l’effet de ces changements sur la signalisation mécano-chimique (mécanotransduction) a été très peu décrit alors qu’elle affecte les fonctions et l’intégrité cellulaires. Nous proposons donc d’évaluer les fonctions cellulaires et les propriétés mécaniques et d’adhésion des cellules épithéliales respiratoires exposées à CyaA dans le but de déceler des modifications fondamentales dans les processus de mécanotransduction.Nous avons tout d’abord mené une étude préliminaire visant à définir les concentrations physiopathologiques de CyaA utilisées dans nos expériences. Nous avons ainsi déterminé le degré de viabilité cellulaire en fonction de 3 concentrations de CyaA (0.5 ; 5 ; 10 nM), ce qui a montré que la concentration 0.5 nM n’affectait pas la viabilité cellulaire tout en induisant des niveaux supraphysiologiques d’AMPc en moins d’une heure.Nous avons ensuite cherché à évaluer les effets de CyaA sur la migration et la réparation cellulaires, le battement ciliaire et la perméabilité cellulaire de cellules épithéliales représentatives des différents niveaux de l’arbre aérien. CyaA induit une diminution de la migration et de la réparation cellulaires, ainsi qu’une augmentation de la perméabilité cellulaire traduisant un affaiblissement des jonctions latérales.Une étude en immunoflorescence a ensuite été conduite sur les structures intracellulaires et interfaciales des cellules épithéliales alvéolaires exposées aux 3 concentrations de CyaA. Cette étude a montré que CyaA est capable d’induire un remodelage du cytosquelette d’actine ainsi qu’une diminution du nombre des adhérences focales. Enfin, une analyse complète des propriétés mécaniques et des paramètres d’adhésion a été conduite sur les mêmes cellules au moyen de 2 techniques de micro/nanomanipulation revisitées pour permettre à la fois l’évaluation des liens multiples et de la rigidité cellulaire (Microscopie à Force Atomique (AFM) avec indentation et Magnétocytométrie (MTC)). Pour évaluer le rôle de l’AMPc sur les changements observés, les cellules épithéliales respiratoires ont été testées avec la forme active de CyaA et la forme enzymatiquement inactive de la toxine : CyaAE5, qui ne permet pas de synthétiser l’AMPc.Les expériences AFM ont révélé que le principal effet de CyaA est de diminuer le nombre de liens intégrine-ligand associés (une altération du clustering) alors qu’à la plus faible concentration de CyaA, nous observons une augmentation de la rigidité cellulaire, accompagnée d’un renforcement des liens individuels, évolutions confirmées par les résultats MTC. CyaAE5 ne parvient pas à produire ces mêmes effets.L’ensemble des résultats suggère que CyaA affecte de façon précoce la mécanotransduction des cellules exposées et ceci en cohérence avec les effets attendus de l’augmentation d’AMPc (remodelage du CSQ, altération des jonctions latérales, inhibition de l’expression de Rac1), ce qui apporte une nouvelle vision de la cytotoxicité induite par l’adénylate cyclaseThe increase in respiratory infections involving virulent factors of bacterial origin has become a major public health issue. A better knowledge of the cell respiratory response in the course of the initial cell invasion by bacterial toxins is important from the pathophysiological and therapeutical point of views.The purpose of this work is to decipher the cellular and molecular mechanisms involved in the exposition of respiratory epithelial cells to the adenylate cyclase toxin (CyaA) produced by Bordetella pertussis which is the whooping cough agent. We have chosen this toxin for its multiple capacities of penetrating a wide range of eukaryotic cells. Indeed, this toxin enables direct translocation of its catalytic domain across the plasma membrane of target cells using the endogen calmoduline to increase the cAMP rate at supraphysiological levels. However, the effects of these changes on mechano-chemical signaling (mechanotransduction) pathways remain largely unknown while it affects cellular functions and cell integrity. So, we perform an evaluation of cellular functions as well as mechanical and adhesion properties of respiratory epithelial cells exposed to CyaA toxin in order to detect some critical modifications in the mechanotransduction processes.In a preliminary study aiming at defining physiopathological concentrations of CyaA toxin used in our experiments, we determined the cell viability degree for 3 concentrations of CyaA toxin (0.5; 5 and 10 nM). We found that the smallest concentration (0.5 nM) did not affect cell viability whereas inducing supraphysiological cAMP levels in less than one hour.Then, we assessed the effects of CyaA toxin on cell migration and repair phenomenon, on ciliary beating and on cell permeability of epithelial cells representative of the different levels of the respiratory tract. The toxin induces a decrease in cell migration and repair, an increase in cell permeability suggesting a weakening of lateral cell-cell junctions.Immunostaining was performed on intracellular and interfacial structures of alveolar epithelial cells exposed to the 3 concentrations of CyaA toxin. Results show that CyaA toxin is able to induce cytoskeleton remodeling and a decrease in the number of focal adhesions. Finally, a refined analysis of mechanical properties and adhesion parameters was performed on the same cells by 2 techniques of micro/nanomanipulation modified to permit at the same time, an evaluation of cell adhesion and cell rigidity (Atomic Force Microscopy with indentation and force spectroscopy to characterize the number of bond during adhesion reinforcement and multiscale Magnetic Twisting Cytometry). To evaluate the role of cAMP on cellular and molecular changes, we tested the enzymatically inactive form of CyaA toxin called CyaAE5 which could not permit to increase the intracellular cAMP rate.The AFM experiments have revealed that the main effect of CyaA toxin is to decrease the number of associated integrin-ligand bounds (meaning an alteration of clustering) while, at the smallest concentration of CyaA toxin, we observe an increase in cell rigidity with an individual bound reinforcement, a result consistent with MTC results. Nevertheless, CyaE5 does not exhibit such cellular effects. On the whole, these results suggest that CyaA toxin affects the mechanotransduction pathways of cells exposed to the toxin, a result which is in agreement with the expected effects of cAMP increase (notably cytoskeleton remodeling, lateral junction alteration and inhibition of Rac1 expression) what brings a new vision of the cytotoxicity induced by the adenylate cyclase toxin
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Goundry, Amy Louise. "Leishmania virulence factors : inhibitors of serine peptidases." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6002/.
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Leishmania spp. are protozoan parasites that cause a spectrum of pathologies in humans and other vertebrates, ranging symptomatically from cutaneous ulceration to visceral dissemination. In order to survive within the host, Leishmania are able to evade and modulate the host immune responses through the actions of their virulence factors; however, few putative virulence factors have been characterised during in vivo infection with Leishmania. The Leishmania major genome has revealed the presence of three peptide inhibitors of S1A family serine peptidases (ISPs), which are orthologues of a bacterial protease inhibitor, ecotin. Serine peptidases of the S1A family are absent in Leishmania; therefore, the ISPs have been proposed to inhibit the activity of host serine peptidases, such as those expressed by cells of the innate immune response. ISP2, which is expressed in the mammalian-infective metacyclic promastigote and amastigote stages, has previously been shown to inhibit neutrophil elastase (NE), a serine peptidase expressed by neutrophils, monocytes, and macrophages. This inhibition prevents the activation of a Toll-like receptor 4 (TLR4)-NE pathway during Leishmania-macrophage interaction promoting Leishmania survival and growth in macrophages in vitro. The aims of this project were to assess whether the presence or absence of ISP2 in L. major affects parasite survival in vivo, and to investigate the effects of ISP2 on immune cell dynamics in vivo, specifically with regards to cell recruitment and activation, using the C57BL/6 mouse model. Parasite burdens were performed in mice infected with L. major wild-type (WT) parasites, a cell line deficient in ISP2/3 (Δisp2/3), and a cell line re-expressing ISP2/3 (Δisp2/3:ISP2/3). L. major Δisp2/3 parasites could not be detected at the site of inoculation by 5 wk post-infection compared with WT and Δisp2/3:ISP2/3 parasites, but parasites of all three cell lines were detected in the draining lymph nodes (dLNs) throughout the course of infection. These data were corroborated using in vivo bioluminescence imaging (BLI) of luciferase-expressing (LUC2) versions of these cell lines, in which only a low bioluminescent signal was observed at the site of inoculation with the LUC2-expressing Δisp2/3 cell line over the course of infection, compared with the LUC2-expressing L. major and Δisp2/3:ISP2/3 cell lines. These results suggest that ISP2 may be important in the establishment and persistence of Leishmania infection by conferring parasite survival, particularly at the site of infection. Serine peptidases of innate immune cells, such as NE, function in the proteolytic cleavage of cytokines, chemokines, and cell receptors, to regulate immune cell recruitment and activation. Flow cytometric analysis of innate cell populations at the site of inoculation, in response to L. major WT and Δisp2/3 parasites over 5 wk of infection, was conducted. This line of investigation revealed significantly higher numbers of monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells (moDCs) at 2 wk in Δisp2/3 infection. MoDCs have crucial functions in the induction of antigen-specific T helper 1 responses, which are considered to be important for parasite clearance. MoDCs at the site of Δisp2/3 infection showed an upregulation of the DC co-stimulatory molecule CD80 compared with those from WT infection suggesting an upregulation of DC maturation. MoDCs have also been shown to be the major producers of inducible nitric oxide synthase (iNOS) during L. major infection, which catalyses the production of nitric oxide that is responsible for the killing of Leishmania. Intracellular staining of iNOS through flow cytometric techniques showed that iNOS expression in moDCs was not affected by the presence or absence of ISP2; there was, however, an increase in iNOS expression in other innate cell types, the resident macrophages and DCs, monocytes, and monocyte-derived macrophages, at the site of Δisp2/3 infection compared with those from WT and Δisp2/3:ISP2/3 infections. At the 2 wk time-point, there was also a significant increase in the concentration of IFN-γ, a cytokine that induces iNOS expression, in response to Δisp2/3 infection compared with WT and Δisp2/3:ISP2/3 infections, as determined by ELISA. Quantitative in vivo BLI of myeloperoxidase (MPO) activity of activated phagocytes was determined over a period of 7 wk, which, also, indicated differences in phagocyte activation at the site of inoculation in L. major WT and Δisp2/3 infections. Taken together, these results indicate that the immune response is more primed towards Leishmania killing in Δisp2/3 infection compared with WT infection, which suggests that ISP2 modulates these immune responses to facilitate Leishmania survival. Infections in transgenic mice deficient in NE, Ela-/-, showed similar monocyte recruitment and moDC activation responses in Δisp2/3 infection compared with WT and Δisp2/3:ISP2/3 infections, as those observed in the C57BL/6 mice. This indicates that NE may not be the major target for ISP2 in vivo, or that there may be compensations for the loss of NE by other serine peptidases in this model. Although the exact mechanism by which ISP2 modulates the recruitment and activation of the innate immune cells in vivo remains to be determined, this study has, for the first time, shown numerous differences in the innate immune responses induced following infection with either L. major WT or a mutant deficient in a putative virulence factor using in vivo techniques, such as in vivo imaging (IVIS) and flow cytometry, to compare the infections.
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Rudkin, Justine K. "Investigating the virulence factors of Staphylococcus aureus." Thesis, University of Bath, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.601682.
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Staphylococcus aureus is an important human pathogen and is a common cause of hospital -associated infections. It has the ability to cause a wide range of diseases, facilitated by a plethora of virulence factors. Resistance to antibiotics has played an important role in the recent evolution of this bacterium, allowing it to persist in the face of antibiotic treatment. The ever- increasing levels of resistance to multiple classes of antibiotics, has earned MRSA the name "superbug". However this antibiotic resistance comes at a biological cost. The aim of this thesis were to examine the negative effect of the methicillin resistance encoding type II SCCmec element on cytotoxicity and investigate the lack of an effect caused by the type IV SCCmec element We found that; The type II SCCmec element is implicated in the reduced cytotoxicity of type Il HA-MRSA. Expression of the mecA gene from the SCCmec element is directly responsible for this effect. High levels of mecA expression are required to produce the loss of cytotoxicity . Loss of cytotoxicity is caused by a lack of Agr quorum system activity. The cell wall of highly resistant type Il HA-MRSAs prevent quorum system signalling. Reduced cytotoxin expression in type IJ HA-MRSAs leads to reduced virulence in a murine model of sepsis. The relatively low level of mecA expression from the type IV element explains the maintenance of cytotoxicity in type IV CA-MRSAs. By increasing the level of mecA expression in type IV CA-MRSA we can reduce cytotoxin production. By addition of sub-inhibitory concentrations of oxacillin to the growth environment of type IV CA-MRSAs we can reduce cytotoxicity and reduce infection related morbidity in an invertebrate model of infection.
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Haghkhah, Masoud. "Study of virulence factors of Staphylococcus aureus." Thesis, University of Glasgow, 2003. http://theses.gla.ac.uk/952/.
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In vivo expression technology (IVET) is a promoter-trap strategy deigned to identify genes whose expression in induced in a specific environment, typically that encountered in a host. Signature-tagged mutagenesis (STM) uses comparative hybridisation to isolate mutants unable to survive specified environmental conditions and has been used to identify genes critical for survival in the host. Both methods have been used to identify virulence genes in S. aureus. The main aim of this project was to find any probable new genes of S. aureus that are essential for biofilm formation and infection mouse model by STM. A library of tagged insertion mutants of S. aureus and a series of selected tags in plasmids of S. aureus strain RN6390 were used. Most of the experiments with both the library and selected tags had problems with cross-hybridisation. All the selected tags were therefore sequenced and 33 tags with less than 50% identity were chosen for future experiments. A library of 825 mutants was made with the 33 selected tags. The mutants were arrayed in 25 pools of 33 mutants. Different tests were done to determine that the new library was reliable for a cross-hybridisation free screening. The library was then used in an infection model in mouse and biofilm formation. A total of 12 mutants with significantly reduced signals were sequenced. 7 out of 12 attenuated mutants showed homology to different genes in S. aureus and other bacteria. Tetrahydrodipicolinate succinylase homolog, opp-2F, acetoin utilization AcuC protein, phosphate ABC transporter, dapD, branched-chain-amino-acid transporter, pepF, and flaR genes were identified. This work was the first STM screening in a biofilm system, and the dapD gene was identified in a biofilm for the first time. 2 out of 12 genes had also been identified in previous STM screens. 5 out of 12 attenuated mutants showed homology to some hypothetical proteins. A hypothetical protein of the same locus was identified in two mutants.
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Ambrose, Helen. "The epidemiology and virulence factors of pneumocystis." Thesis, University of Oxford, 2003. http://ora.ox.ac.uk/objects/uuid:4357cc1c-07ea-4cc4-82eb-25ae5feeef89.
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Pneumocystis is a diverse group of fungi found in the lungs of mammals, and can cause a fatal pneumonia in immunosuppressed humans. The project focussed on two main areas of Pneumocystis biology. The first part investigated the transmission of P. jiroveci between humans and comprised three studies. The first study demonstrated direct transmission between a mother and her infant who had Pneumocystis pneumonia, PCP, contemporaneously. The second investigated transmission of P. jiroveci between health care workers and patients with PCP. P. jiroveci was identified in HCW samples but the genotyping data did not demonstrate direct transmission between the HCW and the patients. The third study examined a representative distribution of P. jiroveci genotypes within a whole human lung and identified two foci of infection. The second main area of research investigated PRT1, a multigene family, encoding a protein with homology to KEX-like proteases. The first part of this study attempted to identify the original single-copy PRT1 gene that encodes the progenitor kexin-like protease. Inconclusive sequence data was obtained. The second part of this study examined regulation of PRT1 gene expression, initially using an assay that characterised the length polymorphisms within the proline rich region of the PRT1 gene. In the complex populations of rat-derived Pneumocystis used it was difficult to distinguish reliable differences between the cDNA (expressed) and genomic DNA proline rich region profiles. Subsequent investigations used simple rat-derived Pneumocystis populations that were derived from inocula of 10 Pneumocystis organisms intratracheally injected into nude rats. The catalytic domain of PRT1 was sequenced from cDNA and genomic copies from each of the five low-dose populations. Two majority sequence types were identified, one genomic and the other cDNA. Three of the populations contained a cDNA majority sequence type, implying that some form of regulation of expression was occurring. This hypothesis was further investigated using a second set of simple populations, hybridisation techniques and 5' RACE.
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Fyfe, L. "Studies on Aeromonas salmonicida extracellular virulence factors." Thesis, University of Nottingham, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376521.
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Findlay, Gordon. "Biogenesis of virulence factors in Vibrio cholerae." Thesis, University of Kent, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294636.
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Dodson, Amanda. "Host factors affecting the virulence of Campylobacter." Thesis, University of Bristol, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.557965.
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The integrity of the intestinal epithelium is maintained by intercellular junctions, such as tight junctions. These are crucial in creating a barrier against the intestinal environment. Studies have shown that pathogenic bacteria can disrupt the structure of tight junction proteins to facilitate their paracellular passage into the underlying tissues. It is known that colonisation of the intestinal epithelium by Campylobacter affects intestinal integrity by disrupting occludin, an integral tight junction protein, enhancing the paracellular passage or Campylobacter. In response to the influx of foreign antigens, the host will mount an immune response elevating the levels of proinflammatory cytokines, such as TNF-a and IFN-y. Such cytokines have been shown to affect the structure of tight junctions during intestinal inflammation disrupting barrier function and facilitating the passage of luminal antigens into the underlying tissues. Published data show that some bacteria are capable of elevating cytokine levels by manipulating the host immune response, such synergistic interactions enhanced the pathogenesis of infection observed. This study was undertaken to examine how Campylobacter jejuni interacts with host epithelial cells and to determine if synergistic interactions with the products of the host immune response and/or the microbial community enhanced the pathogenesis of infection observed. In vitro Caco-2 monolayers were pre-incubated with inhibitors of the host cytoskeleton one hour prior to C. jejuni infection. Throughout the time course transepithelial electrical resistance (TER) readings were recorded at 24 hour intervals and levels of transcytosed bacteria determined. Monolayers were fixed at 24 hour time intervals and stained for occludin and E-actin. Caco-2 mono layers were inoculated with combinations of C. jejuni 81116 and/or proinflammatory cytokines and/or E. coli isolates over a time course. Throughout the time course, TER readings were recorded at intervals and levels of translocated bacteria in the basal well determined. Monolayers were fixed and stained for occludin, cell viability was determined using a tenninal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) assay. Gnotobiotic chicks were infected in vivo with combinations of C. jejuni 81116 and/or E. coli isolate eight. Two days post infection, chicks were sacrificed and tissues harvested. Bacterial colonisation was determined by serial dilution onto selective agar. Tissue sections were stained with haematoxylin and eosin, internalised bacteria were determined using fluorescence in situ hybridisation. C. jejuni is capable of endocytosis into host eukaryotic cells but depending on the strain it has being shown to be a microtubule and/or micro filament dependent process. Thus Caco-2 cells were pre-exposed to inhibitors of the host cytoskeleton to determine which route C. jejuni 81116 utilised for endocytosis. Inhibition of either microfilaments and/or microtubules initially prevented endocytosis, but by 24 hours post infection levels of endocytsosed Campylobacter were equivalent to C. jejuni infection alone. Interestingly, when both microfilaments and microtubules were subsequently inhibited C. jejuni was endocytosed into the cells at a significantly higher level (P < 0.05) than seen with C. jejejuni infection alone, suggesting that endocytosis occurs independently of the host cytoskeleton. In response to Campylobacter infection the levels of proinflammatory cytokines would be enhanced which have being linked in previous studies to an increase in the permeability of the epithelium. Indeed, preliminary experiments conducted in this study hewed that C. jejuni 81116 in the presence of IFN-y alone or in the presence of TNF-a resulted in focal redistribution of occludin into an intracellular location and an increase in cellular damage within 24 hours, which was not seen with C. jejuni infection alone, indicating synergistic interactions. Subsequent experiments performed using inhibitors of the host cytoskeleton tentatively suggests a role for actin in the reduced association of occludin with tight junction complexes. Synergistic interactions were also indicated in vitro and in vivo when cells were exposed to C. jejuni and E. coli isolates. Indeed eo-infection in vitro caused a significant decrease in TER within 6 hours, with a focal redistribution of occludin into an intracellular location correlating with an influx of C. jejuni into the basal well. When the experiment was conducted in gnotobiotic chicks although no outward signs of disease were observed microbiological testing confirmed that both C. jejuni and E. coli could be found throughout the gastrointestinal tract, with extensive damage to all sites of the gastrointestinal tract sampled. These results suggest that the endocytosis of C. jejuni into host epithelial cells can occur independently of the host cytoskeleton. Once Campylobacter has colonised the iastrointestinal tract of a susceptible host infection the resulting intestinal inflammation in conjunction with C. jejuni infection would result in rapid loss of tight junction barrier function. Furthermore the polymicrobial environment of the gastrointestinal tract may further enhance the pathogenesis of C. jejuni infection observed. Both synergistic interactions with products of the host immune response and microbial community may play a role in the development of bloody diarrhoea and Campylobacter-mediated colitis.
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Port, Gary C. "Exploring the Listeria monocytogenes secretome : identification and functional characterization of novel virulence factors and secretion systems /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/4981.
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Souibgui, Eytham. "Rôle de la clathrine dans le processus infectieux du champignon phytopathogène Botrytis cinerea." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1076.
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Les champignons sont les principaux agents pathogènes des plantes. Leur étude est donc essentielle pour contrôler les maladies et maintenir un bon rendement de production agricole. La nutrition de ces pathogènes est basée sur l'absorption de nutriments, préalablement dégradés par un arsenal d'enzymes lytiques secrétées. La sécrétion des protéines est assurée par le trafic intracellulaire mettant en jeu de nombreuses vésicules. Chez les champignons filamenteux, ces vésicules ont été visualisées en microscopie électronique mais le processus mis en jeu pour leur biogénèse n'est toujours pas élucidé. L'identification de ce mécanisme est un donc un prérequis pour comprendre la sécrétion de facteurs de virulence. Dans ce but, un mutant non pathogène altéré au niveau de l'expression du gène codant la chaine lourde de la clathrine a été sélectionné parmi une banque de mutants générés chez le champignon nécrotrophe Botrytis cinerea. Le gène codant pour la chaine lourde de la clathrine est essentiel chez de nombreux organismes, ainsi un mutant dominant négatif de la chaine lourde de la clathrine a été généré et confirme la perte de pathogénicité. La caractérisation du mutant par une approche de protéomique a mis en évidence un défaut de sécrétion de 82 protéines incluant des facteurs de virulence connus. Un défaut de production de vésicules intracellulaires a également été constaté. Par ailleurs, le marquage de la clathrine à la GFP a permis de préciser sa localisation dans les cellules fongiques. Enfin, de façon surprenante, aucun défaut d'endocytose n'a été constaté au sein des mutants déficients en clathrine. Cette étude met en évidence pour la première fois le rôle essentiel de la clathrine dans le processus infectieux d'un champignon pathogène ainsi que son rôle dans a sécrétion de facteurs de virulenceFungi are the most important plant pathogens on agricultural and horticultural crops. Study of fungal pathogens remains essential to understand pathogenic process and control plant diseases. These organisms secrete high amount of degrading enzymes involved in plant decomposition and they feed by absorption of degraded nutriments. Secretory proteins were described to be transported form Endoplasmic Reticulum and Golgi apparatus to extracellular space through intracellular vesicles. In filamentous fungi, intracellular vesicles were observed using electron microscopy but their biogenesis process is still unknown. Therefore, elucidation of the process and the identification of proteins involved in secretory vesicles biogenesis remains a challenge to understand virulence factors delivery. A nonpathogenic mutant altered in the expression of the gene coding for clathrin heavy chain was selected in a random mutant library generated in the necrotrophic pathogen Botrytis cinerea,. This gene is essential in many organisms, thus a clathrin dominant negative mutant was generated and confirming the nonpathogenic phenotype observed on several host plant. In eukaryotic cells, clathrin heavy chain is mainly described to be involved in endocytosis, but it is also essential for high density secretory vesicles formation in yeast. Characterization of the mutants using a proteomic approach revealed a secretion defect of 82 proteins including known virulence factors, as Plant Cell Wall Degrading Enzymes and elicitors. Furthermore, the clathrin mutant revealed a strong reduction of intracellular vesicles production. Clathrin was also localized in living cells using fluorescent GFP-tag protein. Endocytosis was also studied and surprisingly, any observable defect was observed for clathrin mutants. This study demonstrated for the first time the essential role of clathrin in the infectious process of a fungal pathogen and its role in virulence factors secretion
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Bertrand, Quentin. "Caractérisation de facteurs de virulence impliquant les systèmes de sécrétion bactériens." Thesis, Université Grenoble Alpes (ComUE), 2019. http://www.theses.fr/2019GREAV058.
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Pseudomonas aeruginosa est une bactérie responsable de sévères infections nosocomiales. Ces infections sont un réel problème pour la santé publique car P. aeruginosa fait partie des pathogènes ESKAPE qui ont développé des facteurs de résistance aux antibiotiques ce qui leur permet d’échapper aux traitements classiques. P. aeruginosa, en plus d’être résistante aux antibiotiques est capable d’exprimer de nombreux facteurs de virulences. Parmi ces facteurs on retrouve les systèmes de sécrétion de type V (SST5) sur lequel j’ai pu travailler durant ma thèse.Une nouvelle souche de P. aeruginosa a été découverte à Grenoble il y a quelques années. Cette souche n’exprime pas les mêmes facteurs de virulence que les souches communément étudiées mais en demeure bien plus toxique. La toxicité de cette souche est due à l’expression de deux protéines ExlB et ExlA faisant partie de la famille du SST5b et ayant un mécanisme d’action complètement nouveau car aucun homologue n’est connu. La toxine ExlA est capable de faire des pores dans les membranes des cellules eucaryotes entrainant leur mort. Le but de ma thèse était donc de comprendre à l’échelle moléculaire le fonctionnement de ExlA.Pour identifier le mécanisme d’action de cette toxine, nous l’avons divisée en deux domaines que nous avons étudiés séparément. En utilisant des techniques de RMN, de SEC-MALLS, de flottaison différentielle, de SAXS et d’AFM, nous avons pu définir que le domaine C-terminal de cette protéine était un « molten globule» en solution et était capable de faire des trous dans les membranes lipidiques reconstituées. Ce domaine semble être le domaine responsable de l’interaction d’ExlA avec les lipides. Ceci additionné à des études menées in vivo sur la toxine ExlA complète et comportant uniquement son domaine N-terminal nous a permis de définir un possible mécanisme d’action de la toxine ExlA. La somme de nos études fait l’objet d’un article en cours d’écriturePseudomonas aeruginosa is the causative agent of nosocomial infections. Those infections are a real threat to public health, considering that P. aeruginosa is a member of the ESKAPE pathogens family. Those pathogens developed numerous antibiotic resistance mechanisms that allow them to escape the lethality of common treatments. More than just resistant, P. aeruginosa is able to make use of several virulence factors, and among them, the type V secretion system, which is the main subject of my PhD.A new virulent P. aeruginosa strain was discovered in Grenoble University Hospital several years ago. This strain lacked the virulence factors of common studied cytotoxic strains but, still displayed high toxicity. This was related to the expression of two proteins, ExlB and ExlA, that are part of the T5SSb and display a complete new mechanism of action (no protein homologs are found). The ExlA toxin is able to form pores in the eukaryotic cell membrane leading to their death. The aim of my PhD was to understand on a molecular level the mechanism of ExlA toxicity.To decipher the activity of this toxin, we divided it in two domains, studied separately. Using NMR, SEC-MALLS, liposome floating assay, SAXS and AFM techniques, we were able to prove that the C-terminal domain of ExlA was a « molten globule » in solution and was able to form holes in reconstituted lipid bilayers. This domain seems to be the key to lipid interaction by the whole protein. Additional in vivo studies of the N-terminal domain and on full-length ExlA allowed us to propose a putative model of the mechanism of this novel toxin
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Portman, Jonathan Lewis. "Virulence Factor Regulation in Listeria monocytogenes." Thesis, University of California, Berkeley, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10620349.
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Listeria monocytogenes is a Gram-positive intracellular pathogen that is readily amenable to genetic manipulation and for which there are excellent in vitro and in vivo virulence models. These attributes have allowed a thorough examination of the molecular underpinnings of L. monocytogenes pathogenesis, however, there are still a number of major unresolved questions that remain to be answered. For example, it has been known for many years that L. monocytogenes rapidly changes its transcriptional profile upon access to the host cytosol, however the host cues and bacterial components that are involved in driving this change have remained continually unanswered. One large piece of evidence came when the long-sought co-factor for the primary virulence regulator, PrfA, was discovered to be the antioxidant tripeptide, glutathione. Glutathione was demonstrated to play a crucial role in the activation of PrfA in vivo— a finding that has since led to two important discoveries that are described herein. First, the activation of PrfA in vitro requires both exogenous glutathione and a metabolic licensing step that can be recapitulated by a chemically defined synthetic media. Second, glutathione also functions as a post-translational regulator of the pore-forming virulence factor, Listeriolysin O (LLO), by reversibly binding via an S-glutathionylation reaction and preventing membrane association of the LLO monomers. These discoveries elucidate numerous regulatory roles for glutathione during infection and describe how L. monocytogenes is able to sense and respond to critical host compartments to mount a successful infection.
Upon entry to the host cell cytosol, the facultative intracellular pathogen Listeria monocytogenes coordinates the expression of numerous essential virulence factors by allosteric binding of glutathione (GSH) to the Crp-Fnr family transcriptional regulator, PrfA. Here we report that robust virulence gene expression can be recapitulated by growing bacteria in a synthetic medium (iLSM) containing GSH or other chemical reducing agents. Bacteria grown under these conditions were 45-fold more virulent in an acute murine infection model and conferred greater immunity to a subsequent lethal challenge compared to bacteria grown in conventional media. During cultivation in vitro , PrfA activation was completely dependent on intracellular levels of GSH, as a glutathione synthase mutant (ΔgshF) was activated by exogenous GSH but not reducing agents. PrfA activation was repressed in iLSM supplemented with oligopeptides, but suppression was relieved by stimulation of the stringent response. These data suggest that cytosolic L. monocytogenes interpret a combination of metabolic and redox cues as a signal to initiate robust virulence gene expression in vivo.
Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutating the cysteine residue to alanine has minor effects on overall protein function. Thus, the function of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC Listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was post-translationally modified by a S-glutathionylation at this conserved cysteine residue, and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation and retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from wild-type in vitro, yet was attenuated 4-6 fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC family proteins are post-translationally modified and regulated, and help explain an evolutionary pressure behind the highly conserved undecapeptide cysteine.
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Luo, Wenyi. "Identification and characterization of virulence factors of mycoplasmas." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2009. https://www.mhsl.uab.edu/dt/2010p/luo.pdf.
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Rashid, Rebecca Ann. "Expression of virulence factors in pathogenic Escherichia coli /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/11512.
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Forslund, Anna-Lena. "Identification of new virulence factors in Francisella tularensis." Doctoral thesis, Umeå universitet, Molekylärbiologi (Teknat- och Medfak), 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-30857.
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Francisella tularensis, the causative agent of tularemia, is a highly virulent bacterium with an infection dose of less than ten bacteria. The ability of a pathogen to cause infection relies on different virulence mechanisms, but in Francisella tularensis relatively few virulence factors are known. Two F. tularensis subspecies are virulent in humans; the highly virulent subspecies tularensis, also referred to as type A, and the less virulent subspecies holarctica, also called type B. The aim of this thesis has been to improve the knowledge regarding the ability of Francisella to cause disease, with the emphasis on surface located and membrane associated proteins and structures. In addition I have also investigated how virulence is regulated by studying the role of the small RNA chaperone, Hfq. The genome of Francisella appears to encode few regulatory genes. In my work I found that Hfq has an important role in regulation of virulence associated genes in Francisella. Similar to what has been found in other pathogens, Hfq functions in negative regulation, and this is the first time a negative regulation has been described for genes in the Francisella pathogenicity island. Another protein with a key role in virulence is a homologue to a disulphide oxidoreductase, DsbA, which was identified as an outer membrane lipoprotein in Francisella. A dsbA mutant was found to be severely attenuated for virulence and also induced protection against wild-type infections, thus making it a candidate for exploration as a new live vaccine. Additional genes with homology to known virulence determinants include a type IV pilin system. The pilin homologue, PilA, was identified to be required for full virulence in both type A and type B strains. In addition, genes involved in pili assembly and secretion, pilC and pilQ, were also found to be virulence associated in the type A strain. In summary, dsbA, hfq and type IV pili associated genes were indentified to be virulence determinants in F. tularensis. DsbA is a potential target for drug development and a dsbA mutant a candidate for a new live vaccine strain. Furthermore the identification of Hfq as a novel regulatory factor opens new insights into the virulence regulatory network in Francisella.
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Houston, Simon Andrew. "Molecular genetic analysis of Bacteroides fragilis virulence factors." Thesis, Queen's University Belfast, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491954.
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B. fragilis is the most commonly isolated Gram-negative anaerobe from human clinical infections, and is the most common cause of anaerobic bacteraemia. The aim of the current study was to analyse potential virulence factors of B. fragilis, by investigating the contribution of three genes putatively involved in capsule biosynthesis, and by examination of fibrinogen binding and degradation. A novel plasmid DNA transformation protocol exploiting the anti restrictive property of Ocr protein was developed which allowed for the transformation of wild-type and mutant B.fragilis NCTC9343 strains. The current study is the first report of successful DNA transformation in strain NCTC9343 by electroporation. B. jfragilis is capable of within-strain variable expression of micro, small, and large capsules. Data presented herein indicate that the putative transcriptional regulator, upcY, is necessary for expression of the micro capsule polysaccharide biosynthesis locus, PS C/8, the putative wzz-like capsular polysaccharide processing gene, Bf1708, is required for micro capsule biosynthesis, and the putative glycosyltransferase, Bj2782, is necessary for large capsule expression. DNA inversion of the Bj2782 promoter was shown to regulate large capsule production in strain NCTC9343. Fibrinogen is the structural protein used in fibrin abscess formation, a pathological host response to B. ji-agilis intra-abdominal infections. Abscess formation is likely to be important in bacterial containment, preventing dissemination of infection and bacteraemia. All clinical isolates of B. ji-agilis tested degraded human fibrinogen. Fibrinoge~ degradation was related to secreted and outer membrane proteases. B.ji-agilis NCTC9343 bound human fibrinogen. A putative fibrinogen binding protein, Bfl705, after gene cloning and expression, was purified from E. coli and shown to bind human fibrinogen. The fibrinogen binding protein and fibrinogenolytic proteases may be important virulence factors in B. fragilis, allowing the bacteria to slow down, or prevent abscess fom1ation, resulting in dissemination of infection.
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Wang, Qinning. "Erysipelothrix rhusiopathiae : epidemiology, virulence factors and neuraminidase studies." University of Western Australia. Microbiology Discipline Group, 2003. http://theses.library.uwa.edu.au/adt-WU2004.0043.
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Erysipelothrix rhusiopathiae, a Gram-positive bacillus, has long been an important pathogen in veterinary medicine as well as a cause of serious disease in humans. Infections caused by this organism have economic impact on animal industries, causing erysipelas in swine and morbidities in other farmed animals. Human infections are commonly erysipeloid (skin cellulitis) and occasionally septicaemia or endocarditis. Little is known of the diagnosis, epidemiology and pathogenesis of such infections in Western Australia. The aims of this thesis were to establish new diagnostic techniques for the detection and recovery of E. rhusiopathiae, to describe the epidemiology of Erysipelothrix infection in Western Australia in humans and animals, and to characterize virulence-associated characteristics, especially focusing on the neuraminidase produced by the organism. A protocol using 48 h Brain Heart Infusion enrichment followed by subculture to selective agar containing antibiotics achieved the highest recovery rate of 37% in a seafood survey. Twentyone isolates of Erysipelothrix spp., of which 19 were identified as E. rhusiopathiae, were obtained. Two published PCR assays for differentiating E. rhusiopathiae and other Erysipelothrix species were evaluated and the best PCR detection rate achieved was 67% following selective enrichment. The PCR method was 50% more sensitive than the culture method. Epidemiological surveys using the above methods showed that E. rhusiopathiae infection is present in farmed animals in Western Australia. The PCR positive frequencies (3.3-3.7%) and isolate recovery rate (2.8-3.3%) in samples from pig and sheep abattoirs and carcass washings indicate a potential threat to the economy of the farmed animal industry as well as a public health concern with the occurrence of E. rhusiopathiae in meat for consumption. Positive PCR results (1.1%) from human skin swabs of patients with cellulitis and wounds may suggest the existence of Erysipelothrix colonization in the general population. Genetic relatedness of 92 isolates of Erysipelothrix species from various sources was analyzed and a total of 64 distinct PFGE patterns identified. Isolates were further classified into 20 clonal groups based on pattern similarities, and most E. rhusiopathiae were clustered into six groups. A few patterns of other Erysipelothrix species were clustered into separate groups from E. rhusiopathiae but shared greater than 70% similarity with E. rhusiopathiae. The genetic relatedness of colonial variants was well demonstrated using this method. PFGE typing promises to be a useful tool for epidemiological and taxonomic studies of Erysipelothrix. Several virulence-associated factors were characterized in 86 isolates of Erysipelothrix spp. A rapid and sensitive peanut lectin hemagglutination assay for neuraminidase was developed and the influence of media, incubation conditions and pH on the production of the enzyme was investigated. All 61 isolates of E. rhusiopathiae produced neuraminidase in cooked meat broth with titres between 1:10 and 1:320, with no significant difference in titre among isolates from different sources. The enzyme activity was not detected in non-pathogenic Erysipelothrix spp. Capsule was produced by 78.7% of isolates of E. rhusiopathiae but not by other species, while both hyaluronidase and haemolysin were produced by non-pathogenic Erysipelothrix spp. It was concluded that neuraminidase and capsule are most likely to be virulence factors of E. rhusiopathiae. The gene encoding neuraminidase was cloned from the type strain E. rhusiopathiae ATCC 19414. The cloned fragment was a functional partial nanH gene with a mol% G+C of 39.7. The predicted amino acid sequence displayed homology with many microbial neuraminidases and contained conserved sequences found in most bacterial neuraminidases. Southern hybridization experiments demonstrated that the gene was present as a single copy on the bacterial genomic DNA. A neuraminidasenegative mutant vector was constructed by insertional inactivation using a tetM cassette. This has provided starting material for developing a neuraminidase-deficient E. rhusiopathiae mutant, which will permit the study of the role of neuraminidase in pathogenesis. Based on the cloned sequence, a sensitive neuraminidase-specific nested PCR technique was designed and optimized. The specificity was tested in 61 isolates of E. rhusiopathiae, 25 Erysipelothrix species, and 62 other species of neuraminidaseproducing and non-producing bacteria. All isolates of E. rhusiopathiae were PCR positive and all other bacteria were negative; thus this PCR is a highly specific method suitable for application in clinical investigations of Erysipelothrix infection. In conclusion, the present study has contributed new knowledge of the biology of Erysipelothrix spp. and current occurrence of Erysipelothrix infections in Western Australia, as well as to the understanding of pathogenesis of E. rhusiopathiae. Development of several new cultural and molecular approaches in combination with other established techniques will facilitate future studies of the epidemiology, taxonomy and pathogenesis of this bacterial species.
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Paterson, Gavin Kirkwood. "Identification and characterisation of novel pneumococcal virulence factors." Thesis, University of Glasgow, 2003. http://theses.gla.ac.uk/3576/.
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The identification of four sortase homologues srtA-D in the pneumococcal genome prompted their investigation as candidate pneumococcal virulence factors. Not all of these sortase genes were present in both sequenced strains and so sortase gene distribution was investigated among a collection of clinical isolates. In contrast to srtB, C and D, srtA was found in all strains examined and so was selected for further study. It was subsequently found to contribute to pneumococcal virulence in mouse models of pneumonia, bacteraemia and colonisation. This is the first demonstration of a contribution of srtA to pneumococcal virulence. To complement this examination of srtA, some of the surface proteins known or likely to be anchored by SrtA were also investigated for a role in virulence in the animal model of pneumonia. In addition, two genes, annotated in the pneumococcal genomes as a macrophage infectivity potentiator protein and an exfoliative toxin A were also investigated and found to be novel pneumococcal virulence factors. However, it appears they have been incorrectly annotated and these genes do not represent a macrophage infectivity potentiator protein and exfoliative toxin A. Instead, one of them seems to be involved in the response to oxidative stress while no function for the other can yet be ascertained. Janus mutagenesis is a novel technique for manipulation of the pneumococcal genome allowing the creation of mutations that lack a selectable marker. This provides an accessible and potentially powerful method to easily alter the genome to make informative mutations. This thesis describes the first use, to our knowledge, of Janus mutagenesis to investigate pneumococcal virulence.
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Shami, Khosrow. "Virulence factors of Helicobacter pylori : a fermentation study." Thesis, University of Westminster, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323129.
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Ridha, Ghalib Swadi Abdul. "Virulence and associated factors in porcine Escherichia coli." Thesis, University of Liverpool, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.237528.
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Allen, Richard Charles. "Secreted virulence factors : evolution, ecology and therapeutic manipulation." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25789.
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Bacterial infections are an increasing cause for concern as resistance spreads to the majority of our front line antibiotics. To counter antibiotic resistance, new treatment regimens and drug targets are being investigated, including directly targeting bacterial virulence (pathogen-induced harm to the host), and therapies which target resistance mechanisms. The outcome of successful treatment with these compounds is not always killing or halting growth of bacteria, therefore selection for resistance to these types of therapeutics is complex. This complexity is increased by the secretion of many virulence factors, meaning their effects are shared with neighbouring individuals. In addition virulence factors show high phenotypic plasticity due to regulation by processes like quorum sensing (QS), which further complicates treatments targeting virulence, or the regulatory processes themselves. Using the example of quorum sensing inhibitors this study shows the importance of understanding the function and ecology of targeted virulence factors, to predict the selection for resistance to anti-virulence drugs. Later chapters elaborate on this to show how quorum sensing control affects selection on secreted virulence factors. The use of anti-virulence drugs as adjuvants is discussed, with a study showing that the interaction between QS inhibition and translation inhibitors is dependent on the environment. The selection for resistance to combinations of antibiotics and adjuvants is investigated using co-amoxiclav as an example, showing that treatment with high doses of adjuvant are robust to the evolution of resistance.
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Prasad, Joni M. "Hemostatic Factors in Bacterial Virulence and Host Defense." University of Cincinnati / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1329495133.
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Lucas, Erynn Ainslee. "Identification and Characterization of Arcanobacterium haemolyticum Virulence Factors." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/193896.
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Arcanobacterium haemolyticum, a Gram-positive bacterium, is an under-reportedagent of disease, causing pharyngitis, wound infections and a variety of invasive diseases.This work characterized a known A. haemolyticum toxin, phospholipase D (PLD), anddetermined its possible role in bacterial virulence. In addition, a novel toxin, arcanolysin(ALN), was identified and characterized. A draft genome sequence was determined andseveral additional virulence factors that may aid in disease pathogenesis were identified.PLD was present in all strains of A. haemolyticum tested, and was expressedmaximally during logarithmic growth. Recombinant PLD caused lipid raftrearrangement on the surface of HeLa cells in a dose-dependent manner. Thisrearrangement allowed maximal bacterial adhesion to the host, with a pld knockoutadhering only 39.7% to HeLa cells as compared to wildtype. Loss of production of PLDdid not affect bacterial invasion. However, PLD expressed by intracellular bacteria wascytotoxic to host cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate(MTS/PMS) viability assays. PLD caused host cell death via necrosis as determined bytransmission electron microscopy. PLD did not induce apoptosis, as caspases 3/7 and 9were not elevated in HeLa cells infected with wildtype A. haemolyticum.A. haemolyticum also expresses a Cholesterol-Dependent Cytolysin (CDC), ALN.Like pld, aln was present in all strains tested. ALN displays a variant undecapeptide andan unusual N-terminal extension not found in most other CDCs. Recombinant ALN11shows significantly increased activity against cultured cells and erythrocytes of humanorigin, compared with intermediate activity on rabbit and hamster cells, and low to noactivity on bovine and ovine cells as measured by hemolysis, cytotoxicity and membranebinding assays. ALN was less inhibited by free cholesterol when compared with otherCDCs, indicating the possibility of alternative receptor binding.The A. haemolyticum genome was sequenced to >20X coverage, and assembled to50 contigs covering ~95% of the genome. The genome is ~1.95Mb with a mol %G+C of53.1% and contained no plasmids. pld and aln have a reduced mol %G+C of 47.2% and46.5%, respectively, indicating the possibility of gene acquisition by horizontal transfer.Initial bioinformatics analysis identified genes encoding a protease, an extracellularDNase, two neuraminidases and three fimbrial biosynthetic operons were also identifiedwithin the genome.
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Au, Candy Po Yee. "Haemolytic virulence factors in Photorhabdus luminescens strain W14." Thesis, University of Bath, 2004. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413130.
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Agena, Mahmoud B. "Neonatal exposure to pathogens : determining key virulence factors." Thesis, Nottingham Trent University, 2017. http://irep.ntu.ac.uk/id/eprint/32859/.
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The neonatal stage is the most critical period for infections, with mortality rate of up to 45% amongst children under five years. The genus Cronobacter has been involved in many outbreaks in neonatal intensive care units, with recorded meningitis, bacteraemia, and necrotizing enterocolitis (NEC). Although this genus was deeply investigated in vitro with different cell lines, until now, few researches have been focused on the use of H4 cell line, which is supposed to be more representative of neonatal response. Therefore, the present PhD study aimed to investigate the interaction of selected clinical isolates and the seven-type species of the genus Cronobacter, as well as one E. coli K1 isolate with H4 cells compared with Caco-2 cell line. Physiological analyses revealed most of the strains to be motile, and capsule and biofilm producers. A link between capsule production and serum resistance was shown by some strains. Strains were examined regarding their attachment, invasion, translocation and cytotoxicity as well as for the role of host cytoskeleton in the invasion process to both cell lines. All strains, especially C. sakazakii ST12 (696 and 703) were significantly more adhesive to the H4 than Caco-2 cells; this sequence type has previously been associated with neonatal NEC. Importantly, this study indicated that some clinical strains were more invasive to H4 than Caco-2 cells such as C. sakazakii 767 (meningitis) and 701 (NECIII). Moreover, attachment and invasion of E.coli were higher in H4 than Caco-2 cells. High attachment and invasion is potentially linked with excessive inflammatory response and NEC development in neonates. Most studied strains were able to translocate human cell lines, causing necrotic damage in the polarized monolayer. More importantly, translocation of blood or meningitic isolates such as C. sakazakii 709, C. malonaticus 1569, C.turicensis 564 was higher via H4 compared with Caco-2 cells. However, translocation of E. coli K1 strain 939 was similar in both cell lines. Most of the bacterial strains were cytotoxic to both cell lines and C. sakazakii ST3 strain 798 showed an ability to kill H4 cells up to 90-fold of blank, and about 70-fold when co-cultured with Caco-2 cells. Results suggests that intact bacterial cells that able to produce new proteins while in direct contact with the host cells is essential for the cytotoxicity, indicating the potential involvement of an active secretion system in cytotoxicity. Cytochalasin D and Colchicine mostly inhibited invasion to the H4 cells and enhanced the invasion of some strains to Caco-2 up to five-fold. Only Nocodazole significantly enhanced the invasion of some strains to H4 cell, and variably effected strains’ invasion to Caco-2. Data obtained from human cytoskeleton inhibitors experiments suggested the possible strain specific role of inhibitors on both cell lines, and strains may encode different pathways for uptake, with the possible involvement of eukaryotic receptors that recognize the invading bacteria. This study indicated that the response of the H4 cells to bacterial challenge and production of inflammatory cytokines was higher than Caco-2 cells. The H4 cells produced more IL-1-β, IL-4, IL-6, IL-8, IP-10, MCP-1, and EGF-α. Furthermore, tested strains (n=12) variably affected the expression of human Toll-like receptors (TLRs) and NF-kB subunits 1 and 2 in both cell lines, only C. sakazakii strain 709 upregulated expression of TLR1-4 in H4 cells and was the strongest inducer of receptor gene expression in both cell lines. More importantly, the upregulation of NF-kB subunits 1 is more likely related to the increased inflammatory cytokine production, while no link with subunit 2. However, TLR-1 was not expressed in Caco-2 in response to these strains, which needs further investigation. The effect of the E. coli K1 strain 939 on the TLRs expression was varied and no specific pattern was detected, but in some cases it showed similarity to the effect of C. sakazakii 767 which is also linked with neonatal meningitis. As most of the investigated bacterial strains displayed virulence factors with H4 cells closely related to their clinical pathology, the present study provides an important initial work to introduce H4 cells as a new modle for neonatal cell lines to analyse neonatal infections.
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Wawrzyniak, Ivan. "Génomique et post-génomique du parasite intestinal Blastocystis sp. sous-type 7. Evaluation de son pouvoir pathogène." Thesis, Clermont-Ferrand 2, 2012. http://www.theses.fr/2012CLF22243/document.
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Blastocystis spp. est un Straménopile parasite anaérobie fréquemment rencontré dans le tractus gastro-intestinal de l’homme et de divers animaux. Ce parasite est associé à des troubles gastro‐intestinaux aspécifiques, et semble impliqué dans des désordres fonctionnels tels que le syndrome de l’intestin irritable (IBS). Ce travail de thèse s’appuie sur le séquençage du génome de Blastocystis sp. ST7 réalisé en collaboration avec le Génoscope d’Evry, l’Université Nationale de Singapour, l’Institut Pasteur de Lille et l’Université de Provence. Ce génome est constitué d’un génome nucléaire de 18,8 Mpb pour 6020 gènes, et d’un génome mitochondrial de 29 kpb localisé dans des organites apparentés aux mitochondries. L’analyse de ce génome apporte des informations au niveau de l’évolution de ce microorganisme, de son adaptation à l’environnement intestinal et de ces facteurs de virulence potentiels. En effet, les analyses in silico de ce génome ont montré que Blastocystis sp. ST7 possède plusieurs gènes codant des protéines pouvant agir à l’interface entre l’hôte et le parasite et connues chez d’autres protozoaires pour être impliquées dans des phénomènes de pathogénie. Ce sont en particulier des PKS, des NRPS, et des hydrolases dont des protéases. D’autre part, des activités protéolytiques ont été mises en évidence expérimentalement dans les surnageants de culture du parasite. Deux protéases à cystéines (une cathepsine B et une légumaïne) pouvant être impliquées dans la physiopathologie du parasite, ont été identifiées et caractérisées dans les surnageants, confirmant ainsi nos analyses in silico. Ce travail ouvre de nombreuses pistes intéressantes à explorer pour évaluer l’impact de ce parasite en santé humaineBlastocystis spp. is a highly prevalent anaerobic Stramenopile parasite found in the intestinal tract of humans and various animals. This parasite is associated with non specific intestinal disorders, and could be involved in functional disorders such as the irritable bowel syndrome (IBS). In this work, the Blastocystis sp. ST7 genome sequencing project was carried out in collaboration with the Génoscope of Evry, the National University of Singapore, the Pasteur Institute of Lille and the University of Provence. This genome consists in a nuclear genome of 18,8 Mpb encoding 6020 genes, and a mitochondria‐like genome of 29 kpb localised in the mitochondrion‐like organelles. The analysis of this genome brings information about the evolution of this micro‐organism, its adaptation to the intestinal environment and its potential virulence factors. Blastocystis sp. ST7 was predicted to harbor several genes coding proteins that could act at the parasite‐host interface, and that are known to be involved in the pathogeny of many protozoa. They are PKS, NRPS, and hydrolases among them proteases. In addition, proteolytic activities were highlighted in the parasite culture supernatants. Two cysteine proteases (a cathepsin B and a legumain) were identified and characterized from the supernatants and could play a role in the physiopathology of the parasite, that confirm our in silico analyses. This work opens new ways to evaluate the impact of this parasite in human health
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Gunther, Erik Christian. "Molecular mechanisms of brain derived neurotrophic factor secretion and action /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/5086.
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Talagrand, Emilie. "Diversité, complexité et adaptation au comportement pathogène au sein du genre Aeromonas." Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT123/document.
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Le genre Aeromonas regroupe des bactéries ubiquitaires vivant essentiellement dans les environnements hydriques. Ces pathogènes opportunistes de l’homme et de nombreux animaux possèdent un large répertoire de facteurs associés à la virulence. Bien que des pathotypes aient été proposés et que certaines espèces semblent plus fréquemment isolées en clinique humaine et vétérinaire, leur pouvoir pathogène demeure mal compris, notamment en raison du faible nombre d’études fonctionnelles et du manque d’investigations tenant compte de la diversité génétique et de la complexité des comportements biologiques du genre Aeromonas.Nous avons émis l’hypothèse que chez un pathogène opportuniste d’origine environnementale aussi polyvalent et ubiquitaire qu’Aeromonas, la structuration en complexes d’espèces avec une remarquable diversité génétique/génomique des populations, le polymorphisme des facteurs de virulence et les interactions au sein de communautés « pathogènes » puissent être des facteurs d’adaptation au comportement pathogène. Afin de vérifier cette hypothèse, nous avons étudié i) la diversification au sein d’un complexe d’espèces, « A. media », utilisé comme modèle au moyen d’une étude de population intégrant la génétique et la phylogénie multilocus, les mécanismes d’évolution, la génomique comparative mais également les données phénotypiques, de modes de vie et d’habitats et, ii) la patho-génomique de facteurs de virulence reconnus (aérolysine, entérotoxines thermostable et thermolabile, exotoxine A, protéase à sérine, composants et effecteurs du système de sécrétion de type III, et flagelline latérale) pour une population représentative de la diversité des espèces actuellement connue dans le genre (30 espèces) et iii) le comportement pathogène in vivo (modèle Caenorhabditis elegans) et in vitro (cytotoxicité et cytoadhésion, production de biofilm, motilité) et la signalisation intercellulaire (quorum-sensing de type I) à l’échelle de populations impliquées dans les aéromonoses mixtes (5% des aéromonoses humaines) définies par l’isolement d’au moins 2 clones distincts d’Aeromonas.Le phénomène de spéciation décrit avec l’exemple du complexe A. media, agrégeant 3 espèces génomiques, démontre qu’Aeromonas possède une structure de population en complexes d’espèces dont la diversité génétique et génomique ainsi que les modes d’évolution (mutations et recombinaisons) révèlent divers potentiels adaptatifs et patho-adaptatifs associés à l’émergence de lignées. Au sein du complexe A. media, l’espèce A. rivipollensis semble plus adaptée à un mode de vie associé à des hôtes et possède des gènes spécifiques de résistance à des stress environnementaux. Aeromonas possède de nombreux facteurs de virulence présentant diverses histoires évolutives. Certains montrent une phylogénie dépendante de l’évolution du core-génome, suggérant l’implication de ces gènes dans des processus de spéciation en relation avec l’adaptation à diverses niches. L’étude des performances de PCRs de virulence a révélé des insuffisances majeures dans la sensibilité des méthodes évaluées principalement liées au polymorphisme génétique des facteurs de virulence. Nous avons également montré que des populations mixtes d’Aeromonas isolées d’échantillons cliniques pouvaient modifier le déroulement de l’infection en modèles in vivo et in vitro probablement par mécanisme de coopération ou de compétition avec mise en jeu de signaux de communication cellule-cellule.L’importante complexité d’Aeromonas retrouvée à travers la structure de population, le polymorphisme des facteurs de virulence et les comportements de multicellularité sont autant de facteurs potentiels d’adaptation au comportement infectieux qui permettent d’expliquer au moins en partie les difficultés rencontrées dans l’élucidation de pouvoir pathogène de ces bactériesAeromonas groups ubiquitous bacteria mainly living in aquatic environments. These opportunistic pathogens for human and numerous animals have a large repertoire of virulence-associated factors. Although pathotypes were proposed and despite some species are more frequently isolated in human and animal infections, their pathogenicity is still poorly understood, mostly because very few comprehensive functional studies are available and because investigations taking into account the genetic diversity and the biological complexity within the genus are lacking.We assumed that for an opportunistic bacterial pathogen of environmental origin as versatile and ubiquitous as Aeromonas, the population structure in complex of species, the outstanding genetic/genomic diversity, the polymorphism of virulence factors and the interactions within pathogenic populations can act as factors driving the adaptation to a pathogenic behaviour. To test this hypothesis, we studied i) the diversification within “A. media”, a complex of species used as a model by a population study that included multilocus genetics, phylogenetics, evolutionary features, comparative genomics, as well as phenotypics, lifestyle and habitat ii) the patho-genomics of well-known virulence factors in aeromonads (aerolysin, thermolabile and thermostable enterotoxins, exotoxin A, serine protease, components and effectors of type III secretion system, and lateral flagellin) in a population that is representative of the known taxonomic diversity in the genus (30 species) and iii) the pathogenic behaviour using an in vivo model (Caenorhabditis elegans), an in vitro model (cytotoxicity, cytoadhesion, biofilm production, motility), and intercellular signals production (type I quorum-sensing) for populations involved in mixed aeromonosis, i.e. 5% of human aeromonosis defined by the isolation of at least 2 distinct clones.The phenomenon of speciation described in the complex “A. media” that aggregates 3 genomic species demonstrates that Aeromonas harbours a population structured in complexes of closely related species whose genetic and genomic diversity, as well as evolution mode (mutations and recombinations) reveal a wide adaptative and patho-adaptative potential linked to lineage emergence. Among the complex “A. media”, the species A. rivipollensis seems to be more adapted to a host-associated lifestyle and harbours specific genes for the resistance to environmental stress. Aeromonas has a wide range of virulence-associated genes, which presented diverse evolutive history. Some of them display a phylogeny linked to the core-genome evolution. These results suggest that these genes are involved in speciation processes probably related to niches adaptation. The evaluation of performances of virulence PCRs revealed major lacks of sensitivity of tested methods mainly due to the genetic polymorphism of the virulence factors. By using in vivo models and in vitro models, we also showed that Aeromonas mixed populations recovered from clinical samples could change the course of infection, likely through a cooperative or competitive mechanism that involves cell-to-cell signalling.The high complexity of Aeromonas results from its population structure, virulence factors polymorphism and multicellular behaviours. They are all putative adaptation factors to a pathogenic behaviour that may explain at least partially the difficulties encountered to elucidate pathogenicity of these bacteria
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Georgiades, Kalliopi. "Phylogénomique des bactéries pathogènes." Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX20690/document.
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La pathogénicité des bactéries a toujours été attribuée à des facteurs de virulence et les bactéries pathogènes sont considérées comme étant mieux armées, comparé à des bactéries ne provoquant pas de maladies. Selon les premières études génomiques, le fait de supprimer un certain nombre de gènes des bactéries pathogènes, limiterait leur capacité à infecter leurs hôtes. Au contraire, des études de génomique comparatives récentes, démontrent que la spécialisation des bactéries dans les cellules eucaryotes est associée à une perte de gènes massive, en particulier pour les endosymbiontes allopatriques qui sont isolés depuis longtemps dans une niche intracellulaire. En effet, les bactéries sympatriques, extracellulaires, ont souvent des génomes plus grands et présentent une résistance et une plasticité plus importante. Ces bactéries constituent, de fait, plutôt des complexes d’espèces que de vraies espèces. Certaines bactéries spécialistes, comme les bactéries pathogènes, arrivent à s’échapper de ces complexes et à coloniser une niche, bénéficiant alors d’un nom d’espèce. Leur spécialisation leur permet de devenir allopatriques et leurs pertes de gènes favorisent une évolution réductive. Ces observations nous ont conduits à réaliser une étude afin de quantifier le taux de perte de gènes lors de l’évolution de ces bactéries extracellulaires vers celle de bactéries spécialistes intracellulaires. Notre objectif était de vérifier que ce qui caractérise l’évolution des bactéries intracellulaires est bien la réduction génomique, en prenant en compte tous les événements possibles de gains de gènes. Par ailleurs, dans une étude neutre comparant les 12 espèces pandémiques les plus dangereuses pour l’homme avec les espèces non-épidémiques les plus proches, nous avons voulu identifier des spécificités génomiques associées à la capacité virulente de bactéries pathogènes et démontrer que, à part les toxines et les modules toxine-antitoxine, ce qui caractérise ces espèces ce ne sont pas les facteurs de virulence, mais la perte des gènes de régulation. Au final, les bactéries pathogènes ont un répertoire virulent dans lequel les gènes absents sont aussi importants que les gènes présentsThe virulence of pathogenic bacteria has been attributed to virulence factors and pathogenic bacteria are considered to have more genes compared to bacteria that do not cause disease. According to the first genomic studies, removing a certain number of genes from pathogenic bacteria impairs their capacity to infect hosts. However, more recent studies have demonstrated that the specialization of bacteria in eukaryotic cells is associated with massive gene loss, especially for allopatric endosymbionts that have been isolated for a long time in an intracellular niche. Indeed, bacteria living in sympatry often have bigger genomes and exhibit greater resistance and plasticity and constitute species complexes rather than true species. Specialists, including specific pathogenic bacteria, escape these bacterial complexes and colonize a niche; thereby gaining a species name. Their specialization allows them to adopt allopatric lifestyle and experience reductive genome evolution. These observations led us to design a study to quantify the rate of gene losses during the evolution of free-living bacteria to intracellular specialists. Our objective was to verify that what characterizes the evolution of intracellular bacteria is genomic reduction, taking under consideration all possible gene gain events. Furthermore, in another neutral study comparing the 12 most dangerous pandemic bacteria to Humans to their closest non-epidemic species, we wished to identify any genomic specificities associated to the virulent capacity of pathogenic bacteria and demonstrate that, besides toxins and surprisingly, toxin-antitoxin modules, pathogenic bacteria are not characterized by more virulence factors, but rather by a loss of regulatory genes. Finally, virulent bacteria exhibit a genomic repertoire in which absent genes are as important as present ones
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Bernardi, Adilson César Abreu. "Estudo de amostras de Staphylococcus coagulase-negativa quanto a formação de biofilme /." Araraquara : [s.n.], 2005. http://hdl.handle.net/11449/103991.
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Orientador: Antonio Carlos PizzolittoBanca: Isabel Yoko Ito
Banca: Sérgio Aparecido Torres
Banca: Maria de Lourdes Ribeiro de Souza
Banca: Clarice Queico Fujimura Leite
Resumo: Os Staphylococcus coagulase-negativa, particularmente, os Staphylococcus epidermidis são a causa mais freqüente de infecções relacionadas ao cateter por sua habilidade em aderir a uma superfície e entre si (aderência intercelular) formando biofilme em multicamadas sobre superfícies de polímeros. O objetivo do presente estudo foi avaliar cepas hospitalares de Staphylococcus coagulasenegativa isoladas de cateteres intravenosos, quanto à resistência a oxacilina, produção de slime, aderência ao poliestireno, habilidade de formar biofilme sobre superfícies abióticas (cateter esterilizado) e a presença de genes icaAD. Na presente pesquisa, a presença de icaA e icaD foi determinada pelo método PCR, em uma coleção de 27 amostras Staphylococcus coagulase-negativa (10 Staphylococcus epidermidis, 4 S. haemolyticus, 2 S. hominis, 2 S. lugdunensis, 1 S. saprophyticus, 1 S. schleiferi, 2 S. xylosus e 4 S. warneri). Os genes icaAD foram detectados em dez cepas S. epidermidis... (Resumo completo, clicar acesso eletrônmico abaixo)
Abstract: Coagulase-negative Staphylococcus, particularly, Staphylococcus epidermidis are frequent cause of infections associated with catheters and is attributed to the attachment ability on a surface and each other (intercellular adhesion) forming a multilayered biofilm on polymeric surfaces. The objective of the present study was to evaluate coagulase-negative Staphylococcus strains isolated from intravenous catheters by oxacillin resistance, slime production (qualitative method) and spectrophotometric assay (quantitative method), ability to form biofilm on abiotic surfaces (steriled catheter) and the presence of icaAD genes. In the present study icaA and icaD were determined by PCR method, in a collection of 27 coagulasenegative Staphylococcus (10 Staphylococcus epidermidis, 4 S. haemolyticus, 2 S. hominis, 2 S. lugdunensis, 1 S. saprophyticus, 1 S. schleiferi, 2 S. xylosus and 4 S. warneri). The icaA genes were detected in nine S. epidermidis and icaD in ten. The slime-producing ability was determined by culture on Congo red agar plates in which slime-producing strains formed black colonies in 10 S. epidermidis, 4 S.haemolyticus, 4 S. warneri, 2 S. xylosus and 1 S. chromogenes, while nonslimeformingones develop red colonies. The quantitative assay of coagulase-negative Staphylococcus was observed in 19 strains, including: 10 S. epidermidis, 3 S.haemolyticus, 3 S. warneri, 2 S. xylosus, 1 S. chromogenes. The ability of coagulasenegative Staphylococcus to form biofilm embedded in an amorphous substance wasobserved by scanning electronic microscope on abiotic surface in 10 S. epidermidis,3 S. haemolyticus, 2 S. hominis, 2 S. lugdunensis, 1 S. saprophyticus, 1 S. schleiferi,2 S. xylosus and 3 S. warneri. The oxacillin resistance was observed in 9 strains S.epidermidis, 3 S. haemolyticus, 3 S. warneri, 1 S. xylosus and 1 S. chromogenes. All strains of staphylococci were susceptible... (Complete abstract, click eletronic address below)
Doutor
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Molinos, Abós Sònia. "Noves aproximacions als estudis d’epidemiologia molecular i al diagnòstic de les infeccions causades per Staphylococcus aureus. Aportacions al coneixement dels factors de virulència i patogenicitat." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/368219.
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En les últimes dècades Staphylococcus aureus s’ha consolidat com un dels microorganismes amb més capacitat de causar infeccions, donant lloc des de malalties lleus i localitzades, de curs benigne, fins a infeccions greus, disseminades, amb elevada mortalitat. En aquesta tesi, s’han abordat tant aspectes clínics i epidemiològics dels pacients amb infeccions documentades per S.aureus, en especial bacterièmia i infeccions de pell i parts toves, com aspectes relacionats amb el patogen. En aquest últim apartat, s’ha estudiat l’epidemiologia molecular de les soques, factors de virulència i alguns tests fenotípics que ens ajudaran a mesurar la seva expressió. Es van incloure un total de 293 bacterièmies per S.aureus. La fagotipia va permetre tipar el 37.5% de les soques SARM i el 43.3% dels SASM. La detecció del gen mecA va resultar positiva en 28 soques, en concordança amb les dades obtingudes de la sensibilitat antibiòtica per CMI. En l’anàlisi del tipus SCCmec de les soques SARM, 27 d’elles corresponien al SCCmecIVc i només una presentava el tipus SCCmec IVa i ACME negatiu. Mitjançant spa typing es va demostrar el predomini d’un grup clonal, CC002 (31%) englobant dos spa tipus predominants: t002, t067 en soques SASM i el predomini de CC067 (37.14%) incloent els spa tipus t0067 i t002 en SARM. Comparant els patrons d’electroforesi en camp polsant es van agrupar les soques SARM en les clones E7 i E8, també les més prevalents en el nostre medi. En l’estudi mitjançant microarrays de 102 d’aquestes soques es va observar un predomini del CC5 (38.2%), seguit del CC30 (21.6%). La distribució dels perfils al·lèlics del sistema regulador agr mostrava un predomini del agr II (46.1%). La mortalitat de la sèpsia per S.aureus es relaciona amb la susceptibilitat de l’hoste, característiques de la soca, característiques epidemiològiques i amb el temps transcorregut fins a l’inici d’un tractament antibiòtic adequat. Així, l’estudi de mètodes ràpids per la identificació i la detecció de sensibilitat a cloxacil·lina ( BinaxNow S.aureus / PBP 2a Test, GenomEra MRSA/SA Blood Culture, MicroPhage MRSA/MSSA Blood Culture Test) ens ha permès adequar el tractament antibiòtic en un 79.25% dels casos. L’origen de la bacterièmia en el 58’4% dels casos era nosocomial i el 45.7% d’origen en el catèter. El 80% dels malalts presentaven comorbiditats associades: 35.5% neoplàsia, 30.4% DM, 14% IRC i 13.7% tractament previ amb immunosupressor. Una quarta part van presentar bacterièmia complicada, i un 8.5% bacterièmia persistent. .
D’altra banda, les infeccions de pell i parts toves per S.aureus resistents a meticil·lina d’origen comunitari (CA-SARM) representen al voltant del 20% en el nostre medi. La tipificació molecular d’aquestes soques va permetre demostrar que la majoria pertanyien a la soca USA300-ST8-SCCmec IVc i ACME negativa, variant de la clona USA300 (USA300-ST8-SCCmec IVa). No es va trobar cap associació entre la soca productora i la nacionalitat dels pacients. Les manifestacions clíniques dels pacients eren lleus i sense presència de complicacions, tant en els SASM com SARM. L’elevada prevalença de la Leucocidina de Panton Valentine (PVL) en soques SASM o SARM i la bona evolució, tot i el tractament antibiòtic inadequat, es correlaciona amb la hipòtesi que independentment de la sensibilitat antibiòtica, el drenatge de la lesió és necessari per la resolució de la infecció i posa de manifest la difícil valoració del paper de factors de virulència com la PVL en la patogènia d’aquestes infeccions.In recent decades, Staphylococcus aureus has become an important human pathogen causing a wide variety of diseases, ranging from uncomplicated infections to life-threatening septicaemia, thus leading to significant morbidity and mortality. This thesis recorded clinical and epidemiological characteristics from patients with S.aureus infections, especially bacteremia and skin-soft tissue infections as well as microorganism related factors. The second aim was to investigate the molecular typing and to characterize the virulence factors of S. aureus isolates and some phenotypic assays for its measurement. A total of 293 patients with bacteremia were included. Phage typing allowed to type the 37.5 % of MRSA strains and 43.3 % of MSSA. Regarding molecular characterization, mecA was detected in 28 strains, in accordance with the data of antibiotic susceptibility testing by microdilution method. SCCmec typing of SARM isolates showed that 27 of these were carrying SCCmecIVc and only in one case was found SCCmec IVa and ACME negative. Spa typing revealed the prevalent clonal complex CC002 (31%), enclosing two predominant spa types: t002, t067 in MSSA while the clonal complex CC067 (37.14%) including spa types t0067 and t002 was the predominant in MRSA strains. Genotyping by pulsed-field gel electrophoresis of these SARM strains grouped into two genotypes, E7 and E8. These profiles are the predominant clones in Spain. The application of microarrays for genotyping reflected that the most frequent CC was CC5 (38.2%) followed by CC30 (21.6%). Concerning the agr types, most isolates presented agr II (46.1%). Increased frequency of bacteraemia and maintenance of mortality rates are related to the susceptibility of the host, strain-specific features, epidemiological characteristics and the time from microbiological diagnosis to the starting of appropriate antibiotic treatment. The study of three diagnostic methods: BinaxNow S.aureus /MRSA PBP2, MicroPhage MRSA/ MSSA Blood Culture Test and GenomEra MRSA /SA Blood Culture for the direct identification and susceptibility S. aureus testing directly from positive blood cultures, allowed the early administration of appropriate antibiotic treatment in 79.25% of the cases. The origin of the bacteraemia was nosocomial in 58’4% of the cases and 45.7% were catheter related. Comorbidities were present in 80% of patients, being the most frequent neoplasia (35.5%), followed by diabetes mellitus (30.4%), end stage renal disease (14%) and immunosuppressive treatment (13.7%). Development of complications was present around 25% of the cases, and 8.5% of patients developed persistent bacteraemia. On the other hand, Community-Acquired methicillin resistant Staphylococcus aureus (CA-MRSA) causes approximately 20% of skin-soft tissue infections. A specific clone of CA-SARM, USA300-ST8-SCCmec IVc and characteristically lacking the ACME was the most prevalent in our environment, similar to USA300 (USA300-ST8-SCCmec Iva). We did not find any association between strain characteristics and nationality of the patients. All our patients presented an uncomplicated infection, both when MSSA and MRSA. In our study we reported a high prevalence of Panton Valentine leukocidin (PVL) producers among MSSA and MRSA and all patients presented a favourable outcome even though inappropriate antibiotic treatment was prescribed empirically. It is considered that the treatment of choice for these infections, irrespective of antibiotic susceptibility, is incision and drainage. The real pathogenic role of PVL it is still a subject of controversy, arising from results obtained from clinical studies.
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Buchan, Blake Wade Jones Bradley D. "Examining the regulation of virulence factors in Francisella tularensis." [Iowa City, Iowa] : University of Iowa, 2009. http://ir.uiowa.edu/etd/341.
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Solomonson, Matthew Morris. "Structure, proteolysis, and evolution of secreted tuberculosis virulence factors." Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/54572.
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Mycobacterium tuberculosis uses the ESX-1 type VII secretion system to export proteins to its cell surface, which permeabilize the host macrophage phagosomal membrane, allowing the bacterium to escape and spread to new cells. The structure of the type VII membrane complex and how it mediates this function is unknown, but it is hypothesized that some of the secreted proteins form an extracellular appendage that facilitates membrane lysis or direct secretion of virulence factors into the host cytoplasm. This thesis investigates the structural relationship between one of these secreted proteins, EspB, and a protease that processes it, MycP1. The x-ray crystallographic structures of both proteins are determined and described. EspB is shown to form a multimer with heptameric stoichiometry, and an EM reconstruction of this multimer is generated and used to create a model of the oligomer using symmetric Rosetta docking. The final model is supported by mass spectrometry-based detection of chemically cross-linked peptides from adjacent subunits. We use mass spectrometry to determine how EspB is proteolytically processed during secretion and discuss the effect of this processing event on the EspB ultrastructure. Finally, the structure of one of the membrane apparatus proteins, EccB1 is determined, revealing structural homology to a phage lysin. The combination of x-ray crystallography, EM, modeling, and mass-spectrometry provides an exciting first glimpse at the structure and function of the type VII secretion system - a critical factor in the TB pathogenesis cycle.Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
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Sharp, Jacqueline. "The culture, epidemiology and virulence factors of Clostridium difficile." Thesis, University of Edinburgh, 1988. http://hdl.handle.net/1842/26927.
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Vasi, József. "Characterization of two potential virulence factors in Streptococcus dysgalactiae /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 2000. http://epsilon.slu.se/avh/2000/91-576-5758-0.pdf.
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Johansson, Linda. "Host responses and bacterial virulence factors in Neisseria infections /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-017-6/.
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Holmes, Ashleigh. "Characterising virulence factors from pathogenic bacteria using fluorescent reporters." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/3317/.
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Protein translocation systems are invaluable to pathogenic bacteria, facilitating the display of virulence factors on their surface or their release into the extracellular environment. Some protein export systems are ubiquitous and essential to cell survival whereas others are horizontally acquired on prophages or pathogenicity islands (PAI), in many cases providing the bacterium with pathogenic advantages. For the majority of the known protein export systems, their structure, function and secreted substrates have been characterised, yet some proteins have been identified that are secreted via unknown mechanisms. Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of human foodborne disease worldwide. The pathogenesis of this bacterium is mainly attributed to the secretion of toxins and the presence of a Type III Secretion System (T3SS). The T3SS can translocate bacterial proteins, known as effectors, into the host cell which mediate an effect culminating in the formation of a characteristic attaching and effacing (A/E) lesion. This system is encoded on a horizontally acquired PAI termed the locus of enterocyte effacement (LEE). The LEE not only encodes the T3SS apparatus but also several effectors secreted by the system and transcription factors which regulate its expression. However, it was recently found that T3SS not only secretes LEE encoded effectors but can also secrete proteins encoded on other prophages present in the EHEC genome. Characterisation of these non-LEE encoded effectors is ongoing and this study investigates the expression, regulation and function of non-LEE encoded effector H1 (NleH1) and H2 (NleH2). NleH1 and NleH2 are secreted by the T3SS but are encoded on different prophages. This study demonstrates that expression of NleH1 and NleH2 is induced in the same in vitro conditions which stimulate the expression of the LEE but is diminished upon initial host cell contact in vitro. Transcription of nleH1 and nleH2 is dependant upon factors specific to E. coli O157:H7 and these factors are regulated by LEE encoded regulators Ler and GrlA, as they have a positive effect on nleH transcription. NleH1 and H2 are predicted serine/threonine protein kinases and are able to autophosphorylate. Yeast two hybrid screening and 2D differential gel electrophoresis did not elucidate a eukaryotic protein binding partner of NleH1 or NleH2. Transfection assays show that they do not have a significant effect upon NF-κB activation in vitro. Determining the expression, regulation and function of non-LEE encoded effectors contributes towards further understanding of how this pathogen causes disease. Streptococcus pneumoniae, also known as the pneumococcus, is another globally important human pathogen. It is a very diverse pathogen, with over 90 capsular serotypes and is naturally competent for DNA uptake. Pneumococcal pathogenesis is facilitated by the production of a pore-forming toxin, pneumolysin. Pneumolysin’s activities in pneumococcal pathogenesis extend beyond its cytolytic function as it can also activate the complement pathway and modulate the host cytoskeleton. Pneumolysin is a member of a conserved family of toxins known as the cholesterol dependant cytolysins but differs due to the lack of a secretion signal peptide within its sequence. This indicates that it is not secreted from the bacterium however it has been reported that some strains can release pneumolysin in a cell lysis-independent manner. Additional to this, pneumolysin can also localise to the cell wall, and this localisation is not strain dependent. This study characterised codon-optimised N-terminally labelled pneumolysin constructs and applied them to assess the localisation of pneumolysin. In addition, the importance of autolysin and genes which are co-transcribed with Ply upon the localisation/secretion of pneumolysin was investigated by construction of a pneumococcal strain carrying an autolysin-pneumolysin fusion which naturally occurs in equine strains. These genes were not required for the translocation of pneumolysin or its association with the cell wall. Growth of this strain, and its isogenic parent, in vitro at a low density and low temperature resulted in the pneumolysin being detected in the broth culture. This indicates that pneumolysin can be released from the cell wall and that this action is not dependant upon the genes which were deleted in the mutant. The distribution of pneumolysin on the pneumococcal surface was assessed with immunofluorescence, and LumioTM substrate fluorescence, microscopy and found to have a general distribution. As a contribution to future pneumococcal research, codon-optimised fluorescent protein reagents were developed and can be used as reporters for gene expression and protein localisation.
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Grant, Kathleen Ann. "Genetics and biochemistry of potential virulence factors of Campylobacter." Thesis, University of Reading, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270422.
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