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No description available.

Pseudomonas aeruginosa is a Gram-negative bacterium implicated in several clinical contexts. In its association with immunocompromised hosts including cystic fibrosis patients, P. aeruginosa is able to exploit the host immune response to acquire key factors essential to its adaptation. As such, key virulence factors including the Type III Secretion System (T3SS), initially essential in acute infection, is reduced in its significance in chronic colonization. On the contrary, other phenotypes are essential for the altered priorities in chronic colonization. The signals of the host immune response initiating the phenotypic switch from the expression of acute virulence factors to chronic virulence factors have not been well defined. Additionally, the function of the type VI secretion system (T6SS), a protein secretion apparatus, in chronic infection has been well established. Clinical isolates obtained from acute and chronic P. aeruginosa infections suggested selective regulation of the T6SS, namely up regulation of the H3-T6SS in chronic infection. We used murine models of infection to understand the in vivo transcriptional regulation of the T6SS of PAO1. Itaconate, an anti-inflammatory metabolite generated by the host, selectively upregulated transcription of a H3-T6SS-associated locus, vgrG3. Here we present evidence to show how the host immune response, namely metabolic changes in response to infection may be exploited to support the organism’s adaptation to the lung microenvironment. In the evaluation of such a phenotypic response notable in chronic infections, the Type VI Secretion System (T6SS) of P. aeruginosa is selectively regulated by a host-specific metabolic product, itaconate. While P. aeruginosa contains genetic clusters for three (H1-, H2-, and H3-T6SS) evolutionarily distinct T6SSs, we found the H3-T6SS to be up-regulated significantly (p<0.05) in the presence of this anti-inflammatory signal. Characterization of this response reveals that itaconate induces metabolic stress in P. aeruginosa. In an acute pneumoniae mouse model, deletion of the H3-T6SS locus results in increased colonization of the murine lung. Analysis of bronchoalveolar lavage fluid from wild type and H3-T6SS null-infected mice reveals alterations in metabolic pathways including purine metabolism, carbon metabolism, and arginine biosynthesis. Overall our work outlines the H3-T6SS as a phenotypic response to metabolic stress induced by the host immune response, serving to mediate pathways essential in pathogenesis. Further understanding of such phenotypes as the T6SS implicated in chronic infection is essential in treatment interventions in the clinic.

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Problem: Shigella is a gram-negative enteropathogen that, when passed through fecal particles from one host to the oral cavity of another host, causes an infectious disease known as shigellosis. One of the distinctive features of the infection by Shigella is its ability to bypass its host’s autophagic defenses. It does this through the use of a Type III secretion system, found in gram-negative pathogens like Shigella, which injects virulent proteins into the host cell. One of these proteins is IcsB; however, its exact function is not well understood. This study aims to better understand the role of this protein in the infection. Methods: A yeast two-hybrid screening test is used in this case to examine the interactions between variations of the protein IcsB, and a library of host proteins. Given IcsB’s high yeast toxicity and that resulted in the total absence of yeast colony formation, the first aim was to identify IcsB variants which expression would not prevent yeast growth. The second aim was to use the mutant with reduced cytotoxicity to perform a Y2H screen that will allow for the identification of candidate host proteins interacting with IcsB. Results: Two mutations of the IcsB protein grew in the Y2HG yeast strain, indicating a significant reduction in the protein’s toxicity. Of the cultures that reacted, high stringency and strong interaction was observed between four genes and IcsB proteins. Among the four identified clones that grew, three corresponded to the gene RNF2, while the last one corresponds to a non-coding sequence. Key control experiments revealed that the interaction of IcsB with RNF2 is likely false-positive. Thus, when screened full-length IcsB using new epithelial cells cDNAVI libraries, strong interaction was observed between three genes and our IcsB proteins. All the three genes DDX3X, FANCL, and SGT1 passed the false-positive interaction tests. It is interesting to notice that DDX3X and SGT1 interacted with catalytically active and inactive IcsB, suggesting that the interactions established between IcsB and prey proteins does not require the catalytic - C306A mutation and that IcsB most likely does not function as a protease against these two proteins. By contrast, FANCL bound catalytically inactive, but not catalytically active IcsB, suggesting it could be a substrate of IcsB. The literature provides some support for the putative role of DDX3X, FANCL, and SGT1 in regulating the vacuole escape of Shigella through IcsB action. Conclusion: The aim of this study was to determine the functional of IcsB in the vacuole escape of Shigella. This study successfully identified three candidates interacting partner proteins for IcsB. Key control experiments confirmed the interaction of IcsB with DDX3X, FANCL and SGT1. This study provides a basis for further research, with further study aimed at confirming these results during Shigella infection

Das Flagellum von Salmonella enterica ist eine komplexe molekulare Nanomaschine, die zur Fortbewegung verwendet wird. Die Synthese erfordert die Sekretion extrazellulärer Bausteine durch die Zellhülle. Der Substratexport erfolgt durch ein hochkonserviertes Typ-III-Sekretionssystem. Der Kern des fT3SS ist eine komplexe Proteinsekretionsmaschine, bestehend aus den Proteinen FliPQR und FlhBA. Ziel dieser Arbeit war die molekularen Mechanismen, die eine korrekte Funktion gewährleisten, tiefergehend zu erforschen. Im ersten Kapitel wurden die molekulare Mechanismen der Qualitätskontrolle während der Synthese des fT3SS untersucht. Es wurde kürzlich gezeigt, dass die korrekte Synthese durch das fT3SS-spezifischen Chaperon FliO gewährleistet wird. Ziel war es, den molekularen Mechanismus, wie FliO an diesem Prozess beteiligt ist, aufzuklären. Die Ergebnisse zeigten, dass mehrere Aminosäuren von FliO während der Assemblierung mit FliP interagieren. Des Weiteren wurde die Relevanz des spaltbaren Signalpeptids am N-Terminus von FliP untersucht. Diese Studie zeigt, dass die Anwesenheit des Signalpeptids und seine korrekte Spaltung entscheidend, aber nicht unerlässlich für die Funktion der Flagellen sind. Das fT3SS ist in der Lage Proteine mit einer bemerkenswerten Geschwindigkeit von mehreren tausend Aminosäuren pro Sekunde zu sekretieren. Das zweite Kapitel konzentrierte sich darauf, wie das fT3SS Proteine mit hoher Geschwindigkeit sekretiert, während das Austreten kleiner Moleküle verhindert wird. Unsere Mutationsanalysen zeigten, dass eine Methioninschleife in FliP, eine sperrige Plug-Domäne in FliR und intermolekulare Salzbrücken zwischen FliQ-Untereinheiten zusammenarbeiten, um die Integrität der Membran aufrechtzuerhalten. Diese Arbeit liefert neue Einblicke in die Synthese des fT3SS Kerns und die Regulation der Substratsekretion. Beide Prozesse werden an mehreren Stellen streng kontrolliert, um eine korrekte Funktion des Flagellums sicherzustellen. / The flagellum of Salmonella enterica is a sophisticated molecular nanomachine, which is used for locomotion. Flagella synthesis requires the translocation of extracellular subunits across the cell envelop, which is mediated by a highly conserved type-III secretion system (fT3SS). The core fT3SS is a complex protein secretion machine consisting of the proteins FliPQR and FlhBA. Productive assembly is crucial for flagella function. The molecular mechanisms which ensure correct function of the fT3SS remain poorly understood. In this thesis, we aimed to gain a profound insight into the molecular mechanisms of fT3SS core assembly and function. The first chapter investigated the molecular mechanisms underlying the quality control during the assembly of the fT3SS. It was recently shown that productive assembly of the core fT3SS relies on the flagella-specific chaperone FliO. We aimed to elucidate the molecular mechanism of how FliO facilitates this process. Our results demonstrated, that several residues of FliO are interacting with FliP during the assembly process. Furthermore, we aimed to identify the relevance of the cleavable signal peptide at the N-terminus of FliP. This study showed, that the presence of the signal peptide and its correct cleavage are crucial but not essential for flagella function. The fT3SS is able to secrete proteins with a remarkable speed of several thousand amino acids per second. The second chapter focused on how the fT3SS secretes proteins at high speed while preventing the leakage of small molecules. Our mutational analyses demonstrated that a methionine loop in FliP, a bulky plug domain in FliR and intermolecular salt bridges between FliQ subunits are acting cooperatively to maintain the membrane barrier. Overall, this work provides new insights into the assembly process of the fT3SS core and the regulation of substrate secretion. Both processes are tightly controlled at multiple stages to ensure the proper functioning of the flagellum.

While some rare and severe forms of platelet function disorders are now well characterized, many common types of platelet function disorders are not yet characterized. My hypothesis was that uncharacterized platelet function disorders that impair platelet function in aggregation and/or dense granule ATP release assays are associated with increased bleeding risk. The main goal of the thesis was to study the phenotype and bleeding risks for uncharacterized platelet function disorders, through analysis of the results from clinical laboratory tests of platelet function and for a detailed analysis of their reported bleeding symptoms. First, I assessed if lumi-aggregometry provides useful diagnostic information on platelet function and can be used to help decide if an individual has a bleeding disorder. Two cohorts of individuals were studied that had dense granule ATP release assessed in response to multiple agonists as part of a work-up for a bleeding disorder. Cohort I was comprised of individuals tested between January 2007 and June 2013 and cohort II was comprised of subjects tested at least twice by this assay prior to September 2015. Among subjects tested more than once for dense granule release defects as part of the work up for a bleeding disorder (cohort I; n=133; cohort II; n=17), normal findings with all tested agonists were often confirmed by the second test (cohort I: 83%; cohort II: 100%), but impaired release with multiple agonists was not often confirmed (cohort I: 34%; cohort II: 54%) and even if it was present, the finding was not predictive of a bleeding disorder. Consequentially, it was recommended that lumi-aggregometry should not be used to diagnose platelet function disorders. Next, I studied the bleeding risks associated with uncharacterized platelet function disorders, by evaluating subjects who had abnormal findings by validated assays, namely subjects who had defective aggregation responses to two or more agonists and/or dense granule deficiency. Bleeding history was evaluated using the International Society for Thrombosis and Haemostasis bleeding assessment tool (ISTH BAT) and the likelihood for bleeding symptoms/ problems, was estimated using odds ratios (OR) collected using the clinical history assessment tool - platelet (CHAT-P) for all affected subjects, a subgroup family with a mutation RUNX1, unaffected family v members and general population controls. Individuals with platelet function disorders (n=29) and the affected members of the family with the RUNX1 mutation (n=6) had elevated ISTH BAT scores (median: 9; range:0-18 and median: 8.5, range 4-15, respectively) and an increased risk of abnormal bruising (OR 15-65 and 11-67), nosebleeds (OR 23-40 and 19-121), menorrhagia (OR 6.5-29) and excessive bleeding after trauma or dental/surgical procedures (OR 9.5-44 and 15-77 ) and wound healing problems (OR 13 and 38) compared to general population control (n=60) and unaffected (n=12) family members. Overall, the platelet function disorders in the study present with a significantly increased risk of mild, rather than severe bleeding problems. These findings are important for individuals and healthcare providers to promote evidence-based care of common uncharacterized inherited platelet function disorders for individuals with RUNX1 mutations, dense granule deficiency and/or impaired aggregation responses. / Thesis / Master of Science (MSc) / Platelets are small blood cells that help stop bleeding. People who have platelets that do not work properly are more likely to bleed. Determining who has platelet problems can be challenging as there are limitations to diagnostic tests for these conditions. Additionally, the risks for bleeding in individuals with platelet problems are unknown. We looked at individuals with bleeding problems and found that a recommended test to assess platelet dense granule release, called lumi-aggregometry, wasn’t able to reliably identify persons with bleeding problems. Based on this, we recommend that lumi-aggregometry should not be used to diagnose platelet function disorders. We also found that individuals with uncharacterized platelet function disorders have increased risks for wound healing problems and experiencing bruising, nosebleeds, menorrhagia, and excessive bleeding after dental or surgical procedures. These risks are common among other mild bleeding disorders and will be important to differentiate bleeding risk from other platelet disorders.

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No description available.

No description available.