Browsing by Subject "Pathogenesis"

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    Enterococcus faecalis aggregation substance (Asc10) as liaison between bacterium and heart valve in endocarditis.
    (2009-08) Chuang-Smith, Olivia Newton
    Aggregation Substance proteins encoded by sex pheromone plasmids increase virulence of Enterococcus faecalis in experimental pathogenesis models, including infectious endocarditis. These large surface proteins may contain multiple functional domains involved in various interactions with other bacterial cells and with the mammalian host. Aggregation Substance Asc10, encoded by the plasmid pCF10, is induced during growth in the mammalian bloodstream, and pCF10 carriage gives E. faecalis a significant selective advantage in this environment. We employed a rabbit model to investigate the role of various functional domains of Asc10 in endocarditis. The data suggested that the bacterial load of the vegetation was the best indicator of virulence. Previously identified aggregation domains contributed to the virulence associated with the wild-type protein, and a strain expressing an Asc10 derivative where glycine residues in two RGD motifs were changed to alanines showed the greatest reduction in virulence. Remarkably this strain, and the strain carrying the pCF10 derivative with the in-frame deletion of prgB were both significantly less virulent than an isogenic plasmid-free strain. In addition, mutants carrying Tn917 insertions in the prgB gene demonstrated that secreted N-terminal Asc10 fragments possess activity promoting endocarditis virulence. The data demonstrate that multiple functional domains are important in Asc10-mediated interactions with the host during the course of experimental endocarditis, and that in the absence of a functional prgB gene, pCF10 carriage is actually disadvantageous in vivo. Since Asc10 is important as a virulence factor in E. faecalis endocarditis pathogenesis, developing immunization approaches against this surface protein will be useful in combating endocarditis disease. Use of Fab fragment antibodies against Asc10 was found to decrease vegetation size and bacterial load in the rabbit endocarditis model. In addition, microarray and histological studies revealed two routes of infection in vegetation formation; one in the absence of Asc10, characterized by a robust inflammatory response, and the second in which the presence of Asc10 dampens this response, possibly impeding the influx of immune cells into the vegetation. We also employed an ex vivo porcine heart valve adherence model to study the initial interactions between Asc10+ E. faecalis and valve tissue, and to examine formation of biofilms. We found that the aggregation domains contribute most to Asc10-mediated E. faecalis valve adherence, whereas the RGD motifs have importance in later stages of valve colonization. Again, an N-terminal Asc10 fragment expressed from a prgB Tn917 insertion mutant mediated adherence of E. faecalis cells, emphasizing the importance of the aggregation domains in valve attachment. Most of the Asc10 mutants examined showed some defects in valve adherence at 4 h, corroborating results from our rabbit endocarditis model, and implying that Asc10 contributes mainly to persistence of E. faecalis during endocarditis infection. Extracellular matrix (ECM) protein studies to determine the eukaryotic Asc10 ligand in valve tissue found that fulllength Asc10 protein did not mediate E. faecalis binding to vitronectin, fibronectin, fibrinogen, von Willebrand factor, heparan sulfate, or chondroitin sulfate. In scanning electron microscopy analysis of the infected valve tissue, we found evidence of biofilm formation, including growing aggregates of bacteria, and the increasing presence of exopolymeric matrix over time. Additionally, E. faecalis cells preferentially bound to damaged tissue, though it was difficult to determine whether the bacteria caused the damage, or if it was due to deterioration of the tissue over time. This porcine heart valve tissue colonization model will serve as a useful tool in future studies of biofilm formation.
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    The production and function of titan cells in Cryptococcus neoformans
    (2012-06) Okagaki, Laura Hana
    Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 μm. Cell enlargement is observed in vivo, producing cells up to 100 μm. These morphological changes in cell size affect pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement is stimulated by coinfection with strains of opposite mating type, and ste3aΔ pheromone receptor mutant strains have reduced cell enlargement. Analysis of DNA content in this novel cell type revealed that these enlarged cells are polyploid, uninucleate, and produced daughter cells in vivo. Two G protein-coupled receptors are important for induction of the titan cell phenotype: the Ste3a pheromone receptor (in mating type a cells) and the Gpr5 protein. Both receptors control titan cell formation though elements of the cAMP/PKA pathway. This conserved signaling pathway in turn mediates its effect on titan cells through the PKA-regulated Rim101 transcription factor. Additional downstream effectors required for titan cell formation include the G1 cyclin Pcl103; the Rho104 GTPase; and two GTPase activating proteins, Gap1 and Cnc1560. These observations support growing models in which the PKA signaling pathway coordinately regulates many virulence-associated phenotypes in diverse human pathogens. In addition, altered host-pathogen interactions during the early stages of pulmonary cryptococcosis were explored. The relationship between titan cell production and phagocytosis is non-linear, where moderate increases in titan cell production result in profound decreases in phagocytosis. Production of titan cells by the wild-type strain can also confer protection to a titan deficient strain, and size alone is sufficient to protect from phagocytosis, however size does not confer protection to normal-sized cells. These data describe titan cell formation, a novel cell morphology, in C. neoformans. Signaling pathway analysis showed that titan cell production is coregulated with the other cryptococcal virulence factors. In addition, titan cells have reduced phagocytosis and can confer protection from phagocytosis to normal-sized cells. These data suggest that titan cell production is a novel virulence factor in C. neoformans.