Browsing by Subject "Virulence"
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Item Enterococcus faecalis aggregation substance (Asc10) as liaison between bacterium and heart valve in endocarditis.(2009-08) Chuang-Smith, Olivia NewtonAggregation 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.Item The Evolution of Symbiosis in Communities(2015-11) Nelson, PaulAll organisms host a menagerie of symbionts. While harmful pathogens have historically held the attention of researchers, recent technological advances have revealed a cornucopia of benign, and even beneficial, symbionts. Observations that most organisms are party to a wide variety of harmless symbionts are at odds with theory that suggests that infections by multiple symbionts should lead to the evolution of harmful pathogens. Current theory regarding the evolution of symbionts is predicated on the assumption that symbionts receive a reproductive payoff for harming their hosts. Because harming the host, or virulence, indirectly decreases symbiont infection duration, increased symbiont reproduction comes at a cost and leads to a tradeoff. A consequence of this tradeoff is that when multiple symbionts infect the same host the most virulent symbiont receives the highest reproductive payoff while all symbionts suffer decreased infection duration. Consequently, multiple infections are predicted to select for higher virulence, a prediction that runs counter to observation of the plethora of relatively harmless symbionts observed co-infecting most organisms. The three chapters of this thesis seek to bring theory in line with observations of the commonality of co-infecting commensals. The first chapter of this thesis lays out a mathematical model that uses the virulence tradeoff hypothesis to show that multiple infections do not necessarily lead to increased virulence. The second chapter extends the model developed in the first chapter to show that symbiont defense of the host can lead to the evolution of lower virulence. Finally, the third chapter examines ¬genetic variation in virulence and inhibition between symbiont species for fungal symbionts isolated from two populations of maize. Together, this work furthers our understanding of how symbionts evolve in communities and is an important step toward resolving the paradox of ubiquitous benign symbionts.Item Genetic and ecological constraints to the evolution of virulence and reproduction in a plant pathogen(2012-12) Bruns, Emily LouiseHosts and pathogens are engaged in an ongoing evolutionary struggle. In humanmanaged systems, rapid evolution of pathogen populations can reduce the effectiveness of important control methods such as antibiotics and genetic resistance in crop species. My thesis research investigates potential constraints to pathogen evolution by examining genetic and ecological factors affecting the evolution of infection and reproduction in the plant pathogen Puccinia coronata. I first investigate genetic variation underlying three pathogen life history stages within the host and show that variation in pathogen life-history stages within the host is affected by both the pathogen and host genotype. Next, I evaluate the relationship between pathogen infection and reproduction and show evidence of a trade-off between the number of resistant host genotypes infected and two key pathogen life history traits. Finally, I quantify the variation in infection and reproduction among eight different agricultural populations of P. coronata and ask whether the genetic diversity of the host population affects the evolution of pathogen infection and reproduction. While I do not find conclusive evidence that host genetic diversity affects the evolution of these traits, I do I find significant variation among populations that is not explained by pathogen population structure, indicating that selection structures pathogen populations.Item A study of Fusarium graminearum virulence factors.(2011-05) Menke, Jon R.The plant pathogen F. graminearum (Gibberella zeae) presents a two-fold threat to farmers and consumers. Not only does this filamentous fungus cause the disease Fusarium head blight (FHB) that results in significant yield loss in infected grains, it also taints these grains with potent mycotoxins harmful to humans, animals, and plants alike. Equally alarming is the evidence that grain can appear to be physically sound while still being significantly contaminated with trichothecene mycotoxins. Tri12 encodes a predicted major facilitator superfamily transporter protein suggested to play a role in the export of trichothecene mycotoxins produced by the Fusarium species. However, the role of Tri12p in toxin sensitivity and plant pathogenicity of Fusarium graminearum was previously unknown. In this study, the correct intron positions for Tri12 in F. graminearum (FgTri12) were established using cDNA sequencing, EST data, and comparative genomics. Reverse genetics was used to establish that FgTri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta. To identify the subcellular location of FgTri12p during toxin production in culture, FgTri12p was tagged with eGFP. FgTri12p::eGFP was localized in small motile vesicles, the plasma membrane, and the lumen of vacuoles within fungal cells. Treatment of cells with latrunculin A resulted in the absence of motile vesicles labeled with FgTri12p::eGFP, suggesting their formation relies upon actin polymerization. To determine if FgTri12p co-localizes with enzymes involved in trichothecene metabolism, its cellular fate was compared with FgTri1p::eGFP, a fluorescently tagged oxygenase catalyzing a key intermediate step in trichothecene biosynthesis. While FgTri1p::eGFP initially localizes to small motile vesicles and later accumulates in the vacuole, during the period of initial trichothecene biosynthesis it is targeted to the periphery of intermediate sized vesicles, presumed to be the site of toxin synthesis. These results indicate FgTri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta. The interactions between F. graminearum and its hosts – wheat, rice, or barley – differ in disease severity and the levels of trichothecenes that accumulate in response to infection. The transcriptome of the fungus in rice and wheat was examined in order to identify genes expressed in planta. The hypothesis that fungal genes expressed in planta, but not during growth in culture, could include those that determine the plant infection phenotype was tested by reverse genetics: Four genes expressed exclusively in planta were deleted and two of these were determined to significantly alter disease phenotype. FGSG_03539, also called Tri9, is a previously uncharacterized gene in the major trichothecene biosynthetic gene cluster. A mutant with a tri9 deletion has attenuated virulence and lower trichothecene levels in wheat compared to wild type or a mutant strain complemented with the intact Tri9 gene. FGSG_11164 encodes a predicted trypsin protease and deletion of this gene also results in a small but significant reduction in pathogenicity toward wheat. The results demonstrate that a reverse genetic approach using in planta gene expression data may supplement forward genetic screens for identifying genes encoding virulence factors.