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Propriétés mécaniques et fonctionnelles des cellules épithéliales respiratoires exposées à une toxine bactérienne : l’adénylate cyclase / Mechanical and functionnal properties of respiratory epithelial cells exposed to a bacterial toxine : the adenylate cyclaseAngely, Christelle 29 June 2018 (has links)
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 cyclase. / The 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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Development and Visualization of Bioluminescent Virulent Aeromonas hydrophila in Live CatfishOzdemir, Eda 10 August 2018 (has links)
Virulent Aeromonas hydrophila (vAh) is an important emerging bacterial pathogen causing motile Aeromonas septicemia (MAS) in farmed catfish. Understanding the pathogenicity of the disease is essential for the development of preventive measures. In this study, we aimed to develop a bioluminescent virulent A. hydrophila (BvAh) strain to understand the pathogen-host interactions during infection. To achieve this, a new bioluminescence expression plasmid, pAKgfplux3, was constructed and mobilized to vAh. Catfish were challenged with BvAh using immersion, injection, and adipose fin clip procedures, and bioluminescence signal was tracked in live catfish during infection. We developed a novel BvAh strain for the first time, conducted imaging of BvAh in live fish, detected infection routes and attachment sites of the pathogen, and determined target organs, which provided new insights on the pathogenesis of vAh. MAS progressed better in fish when protection of skin was bypassed. Abraded skin seems to provide a potential portal of entry during vAh infection.
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Molecular Epidemiology and Pathogenicity of the Very Virulent Infectious Bursal Disease Pathotype in United States PoultryStoute, Simone Tricia 24 August 2012 (has links)
No description available.
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Early Function of a Virulent Staphylococcal PhageLatham, Jacqueline M. 05 1900 (has links)
Early function of a temperature-sensitive mutant of staphylophage 44A HJD was examined during the twenty-five-minute period following infection. Host cell and phage DNA were labeled with C and3H respectively. DNA was separated into linear and covalently closed circular (CCC) forms by density-gradient centrifugation. The host, S. aureus, shows no CCC DNA, and apparently carries no plasmid. Following infection with wild type phage, CCC DNA forms occur in tritiated and 1 C DNA fractions 10 to 15 min after infection. Infection with mutant at permissive temperature also demonstrates CCC DNA with both labels. Infection with mutant at nonpermissive temperature produced no CCC DNA during the first 25 min after infection. The impaired function in this mutant may be a linker protein.
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"Study of the modulation of innate immune responses in intestinal epithelial cells by Toxoplasma gondii and its correlation with parasite virulence" / "Etude de la modulation des réponses immunitaires innées dans les cellules épithéliales intestinales par Toxoplasma gondii, et sa corrélation avec la virulence du parasite."Morampudi, Vijay V 28 October 2010 (has links)
Early innate response of intestinal epithelial cells is the first line defense against enteric pathogens. Toxoplasma gondii infections acquired naturally via the peroral route, encounter intestinal epithelial cells early post-infection. Although the population structure of T. gondii is known to be highly clonal, clinical strains of T. gondii have been classified into three genotypes based on their virulence. In this study we investigated whether human intestinal epithelial cell immune response to T. gondii is virulence dependent. We demonstrated distinct virulence of the three T. gondii genotype strains evaluated in human intestinal epithelial cells by their capacity to replicate and induce host cell cytotoxicity. The early host innate mechanisms such as activation of signaling pathways and induction of innate effectors were likewise differentially elicited by the three T. gondii strains. Low levels of TLR dependent NF-kB activation and a failure to rapidly up-regulate innate cytokine and chemokine genes was observed after virulent Type I strain infection. In contrast, early innate response to the less virulent Type II strain was rapid, efficient and led to high levels of IL-8 and IL-6 secretion, whereas response to Type III parasites was intermediate. Early expression of b-defensin 2 gene was suppressed specifically by virulent Type I strain and its activation prior to infection in intestinal epithelial cells led to decreased parasite viability. These findings provide evidence for T. gondii strain virulence dependent down-modulation of early human intestinal epithelial cell innate responses and highlight the importance of these cells in host defense against this infection.
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Computational characterisation of DNA methylomes in mycobacterium tuberculosis Beijing hyper- and hypo-virulent strainsNaidu, Alecia Geraldine January 2014 (has links)
Philosophiae Doctor - PhD / Mycobacterium tuberculosis, the causative agent of tuberculosis, is estimated to infect approximately one-third of the world’s population and is responsible for around 2 million deaths per year. The disease is endemic in South Africa which has one of the world’s highest tuberculosis incidence and death rates. The M. tuberculosis Beijing genotype are characterised by having an enhanced virulence capability over other M. tuberculosis strains and are the predominant strain observed in the Western Cape of South Africa. DNA methylation is a largely untapped area of research in M.tuberculosis and has been poorly described in the literature especially given its connection to virulence despite it being well characterised along with its role in virulence in other pathogenic bacteria such as E.coli. The overall aim was to characterise a global DNA methylation profile for two M. tuberculosis Beijing strains, hyper-virulent and hypo-virulent, using single molecule real time sequencing data technology. Moreover, to determine if adenine methylation in promoter regions has a possible functional role. This study identified and characterised the DNA methylation profile at the single nucleotide resolution in these strains using Pacific Biosciences single molecule real time sequencing data. A computational approach was used to discern DNA methylation patterns between the hyper and hypo-virulent strains with a view of understanding virulence in the hyper-virulent strain. Methylated motifs, which belong to known Restriction Modification (RM) systems of the H37Rv referencegenome were also identified. N6-methyladenine (m6A) and N4-methlycytosine (m4C) loci were identified in both strains. m6A were idenitified in both strains occuring within the following sequence motifs CACGCAG (Type II RM system), GATNNNNRTAC/GTAYNNNNATC (Type I RM system), while the CTGGAGGA motif was found to be uniquley methylated in the hyper-virulentstrain.Interestingly, the CACGCAG motif was significantly methylated (p = 9.9 x10 -63) at a higher proportion in intergenic regions (~70%) as opposed to genic regions in both the hyper-virulent and hypo-virulent strains suggesting a role in gene regulation. There appeared to be a higher proportion of m6A occuring in intergenic regions compared to within genes for hyper-virulent (61%) and hypo-virulent (62%) strains. The genic proportion revealed that 35% of total m6A occurred uniquely within genes for the hyper-virulent strain while 27.9% for uniquely methylated genes in hypo-virulent strain.
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Potential of Great Egrets to be Vectors for the Transmission of s Virulent Strain of Aeromonas Hydrophila between Channel Catfish Culture PondJubirt, Madison McCall 11 August 2012 (has links)
Aeromonas hydrophila is a Gram-negative, rod shaped, facultative anaerobic bacterium that is ubiquitous to freshwater and slightly brackish aquatic environments and can cause infections in fish, humans, reptiles, and avian species. Recent severe outbreaks of disease in catfish aquaculture have been associated with a highly virulent Aeromonas hydrophila strain (VAH) that is genetically distinct from less virulent strains. Given that A. hydrophila is known to infect birds, we hypothesized that fish eating birds may serve as a reservoir for VAH and spread the pathogen by flying to uninfected ponds. Great Egrets were used in this transmission model because these wading birds frequently predate catfish farms. We found that Great Egrets that were fed VAH infected catfish shed VAH demonstrating their potential to spread VAH. Histologically there were changes found in selected tissue samples.
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Vaccination and immune response of channel catfish (Ictalurus punctatus) against virulent Aeromonas hydrophilaGomaa, Basant Mahmoud Ali 08 August 2023 (has links) (PDF)
Virulent Aeromonas hydrophila (vAh) is a causative agent of motile Aeromonas septicemia (MAS) in catfish. There are limitations in the current therapeutic and preventative strategies against vAh. The pathogenesis of MAS as well as the immune response of catfish to vAh infection are poorly understood. The aim of this study is to: 1) develop a dual live attenuated vaccine against MAS and enteric septicemia of catfish caused by Edwardsiella ictaluri; and 2) evaluate the vAh bacterial load and gene expression patterns in catfish tissues following vAh infection. Previously, six recombinant vAh proteins (outer membrane protein, TonB-dependent receptor, three fimbrial proteins, and an ATPase) were identified to have vaccine efficacy against MAS, and live attenuated E. ictaluri vaccine strain ESC-NDKL1 was identified as an effective vector for expressing vAh antigens. A total of 29 recombinant ESC-NDKL1 strains have been constructed with the integration of one, two, or three genes encoding vAh antigens into the ESC-NDKL1 chromosome. Vaccine efficacy of the constructed strains was evaluated in channel catfish fingerlings. Four recombinant ESC-NDKL1 strains expressing two vAh antigens (ESC-NDKL1::atpase::fimMrfG, ESC-NDKL1::fim::fimMrfG, ESC-NDKL1::tdr::fimMrfG, and ESC-NDKL1::fim::ompA) showed significant protection against MAS with relative percent of survival (RPS) values of 55.72%, 60.18%, 61.74%, and 54.81%. Four triple recombinant ESC-NDKL1 strains (ESC-NDKL1::fimMrfG::ompA::fimA, ESC-NDKL1::atpase::fimMrfG::ompA, ESC-NDKL1::fim::fimMrfG::ompA and ESC-NDKL1::atpase::tdr::fim) showed the best protection with RPS values of 77.93%, 63.18%, 67.74%, and 82.35%. To gain a better understanding of vAh pathogenesis, catfish fingerlings were intraperitoneally injected with vAh strain ML09-119. The anterior kidney, liver, and spleen were collected for determination of vAh distribution and expression of thirteen pro-inflammatory, innate, and adaptive immune-related genes using real-time PCR. Results revealed that vAh spread into catfish tissues within 2 hours and peaked at 12 hours post-infection. vAh infection initiated a strong inflammatory response in catfish tissues. Additionally, our research revealed that surviving catfish were able to develop a primary immune response and possibly generation of memory B cells against MAS. Such information will facilitate the development of vaccines and therapeutic drugs for preventing and treating MAS outbreaks in catfish aquaculture.
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Caractérisation moléculaire et fonctionnelle de la protéine Srr2 et rôle dans l’hypervirulence du clone ST-17 de Streptococcus agalactiae / Molecular and functional characterization of Srr2, an ST-17 specific surface protein of Streptococcus agalactiaeSix, Anne 25 November 2013 (has links)
Streptococcus agalactiae est la première cause d’infections invasives chez le nouveau né et, malgré la mise en place de stratégies de prévention, cette bactérie reste le principal agent étiologique des infections néonatales. Les souches de séquence type 17, dites hyper-virulentes, sont particulièrement associées avec les méningites, type d’infection ayant des conséquences lourdes en terme de mortalité et morbidité. Ce clone possède des caractéristiques uniques, telle que la fixation au fibrinogène, ainsi qu’un répertoire de protéines de surface qui lui sont spécifiques. Parmi ces protéines, Srr2 appartient à une famille de larges glycoprotéines streptococcales et staphylococcales impliquées dans la pathogénicité. Un domaine central de Srr2, le domaine BR, est responsable de la fixation spécifique du fibrinogène par le clone ST-17, ainsi qu’au plasminogène et à divers composants de la matrice extracellulaire. Cette protéine promeut ainsi l’adhésion et le franchissement des barrières cellulaires. L’interaction de Srr2 avec les systèmes fibrinolytique et de coagulation de l’hôte favorise la dissémination bactérienne par l’activation de la fibrinolyse, et la persistance de la bactérie dans l’organisme par la formation d’agrégats bactériens. La liaison de Srr2 avec le fibrinogène semble également promouvoir la persistance bactérienne en favorisant l’internalisation et la survie dans les macrophages. Ainsi, la protéine Srr2 confère un avantage pour le processus infectieux du clone ST-17 dans l’hôte, et constitue une cible vaccinale intéressante pour la prévention des infections à S. agalactiae. / Streptococcus agalactiae is the leading cause of invasive infections in neonates. Despite the implementation of prevention strategies, this bacterium remains the main etiological agent of neonatal infections. Hyper-virulent sequence-type 17 strains are particularly associated with meningitis, a type of infection with serious consequences in terms of mortality and morbidity. This clone has unique characteristics, such as fibrinogen binding, and a panel of specific surface proteins. Among these proteins, Srr2 belongs to a family of large streptococcal and staphylococcal glycoproteins involved in pathogenicity. A central domain of Srr2, BR domain, is responsible for the specific binding of fibrinogen by the ST -17 clone and also binds plasminogen and various components of the extracellular matrix. Thereby, it promotes adhesion and crossing of cellular barriers. The interaction of Srr2 with fibrinolytic and coagulation systems of the host could promote bacterial spread through the activation of fibrinolysis and the persistence of the bacteria in the host by the formation of bacterial aggregates. The interaction of Srr2 with fibrinogen also seems to promote bacterial persistence in promoting the internalization and survival of the bacteria in macrophages. Thus, Srr2 confers an advantage to the infectious process of the ST- 17 clone in the host and is an attractive vaccine candidate for the prevention of S. agalactiae infections.
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Marine aquatic environment as a source of potential human pathogens : studies on prevalence, ecology and characterisation of Aeromonas spp. and Vibrio vulnificus isolated from marine environment /Dumontet, Stefano. January 2002 (has links) (PDF)
Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.
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