Pseudomonas aeruginosa, a Gram-negative bacillus, commonly infects immunocompromised individuals and uses a variety of virulence factors to persist in these hosts. The posttranscriptional regulator, RsmA, plays a role in the expression of many virulence factors in P. aeruginosa. RsmA up regulates virulence factors used in colonizing hosts. However, regulation of rsmA is not well elucidated. Transposon mutagenesis was performed on P. aeruginosa containing a transcriptional rsmA-lacZ fusion to answer this question. Mutants were screened via β-galactosidase assay and transposon insertions identified via arbitrary PCR. A probable MFS transporter, we named mtpX, was one significant transposon mutant identified. A ΔmtpX mutant containing the rsmA-lacZ transcriptional fusion was constructed to confirm our results. Further analysis of rsmA, looking at RNA and protein levels, revealed varying results in nonmucoid versus mucoid backgrounds. Phenotypic assays were performed to characterize this unknown transporter and develop a putative mechanism as to how MtpX affects rsmA expression.

Pseudomonas aeruginosa is a Gram-negative bacillus able to colonize a wide variety of environments. In the human host, P. aeruginosa can establish an acute infection or persist and create a chronic infection. P. aeruginosa is able to establish a niche and persist in human hosts by using a wide array of virulence factors used for: movement, killing host cells, and evading immune cells and antibiotics. Understanding virulence factors and their regulation has proved to be an important means of combating the morbidity and mortality of P. aeruginosa as well as the ever-increasing threat of drug resistance. By targeting virulence factors or their regulators with antivirulence compounds, the bacterium is rendered defenseless and more readily cleared by the immune system. In this study, we examine three different contributors to virulence factor regulation. First, we examined the role of the extracellular sigma factor AlgU and its contribution to regulating a post-transcriptional RsmA. AlgU is most commonly active in chronic infecting strains that produce copious amounts of the virulence factor, alginate. We confirmed that not only was their more RsmA in this background, but that there was a previously unidentified promoter for rsmA regulated by AlgU. In concert with this study, we followed up by studying the effects of AlgR on this unknown promoter. AlgU and AlgR are known to work together, specifically on the alginate operon, and we hypothesized based off of bioinformatics data this was the case with RsmA. Second, due to increased RsmA in this chronic infection strain, we set out to identify potential unknown virulence targets of RsmA. A previously unrevealed target, pasP, was shown to directly interact with RsmA. Third, in an acute infection model strain we identified a new regulatory loop involving the two-component system AlgZ/R. In a pilW strain deficient in the motility virulence factor type IV pili, we showed increased levels of AlgZ/R compared to wildtype, PAO1. The pilW strain produced less pyocyanin, rhamnolipid, and elastase and was attenuated in J774a.1 macrophages. Overall, these studies push the understanding of virulence factor regulation and open the door to potential therapeutic targets in treating P. aeruginosa infections.

Hepatitis C virus (HCV) or human immunodeficiency virus (HIV) infection leads to a phenomenon of inflammaging, in which chronic infection or inflammation induces an immune aged phenotype with T cell dysfunction. Thus, HCV or HIV infection has been deemed as a model to study the mechanisms of T cell infammaging and viral persistence in humans. In this dissertation, T cell homeostasis, DNA damage and repair machineries were investigated in patients with chronic HCV or HIV infection at risk for inflammaging. We found a significant depletion in CD4 T cells, which was correlated with their apoptosis in chronically HCV/HIVinfected patients, compared to age-matched healthy subjects. In addition, virus-infected patients’ CD4 T cells were prone to DNA damage that extended to chromosome ends (telomeres), leading to accelerated telomere erosion - a hallmark of senescence. Mechanistically, the DNA doublestrand break (DSB) sensor MRE11, RAD50, and NBS1 (MRN) remained intact, but the DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM) and its downstream checkpoint kinase 2 (CHK2) were significantly suppressed in T cells from HCV/HIV-infected individuals. Consistently, ATM/CHK2 activation, DNA repair, and cellular functions were also impaired in primary CD4 T cells following ATM knockdown, or exposure to the ATM inhibitor (KU60019), as well as in CD4 T cells co-cultured with HCV-infected hepatocytes, or a T cell line infected with HIV-1 in the presence of raltegravir in vitro, which recapitulates the biological effects observed in T cells in the setting of HCV/HIV infection in vivo. Importantly, ectopic expression of ATM was essential and sufficient to reduce the DNA damage, survival deficit, and cellular dysfunction in T cells from both HCV and HIV-infected individuals. These results demonstrate that failure of DSB repair due to ATM deficiency leads to unrepaired DNA damage and renders virally infected patients’ T cells prone to senescence and apoptosis, thus contributing to CD4 T cell loss or dysfunction during chronic HCV or HIV infection. This study reveals a novel mechanism by which ATM deficiency promotes telomeric DNA damage and premature T cell aging, and provides a new therapeutic target for inflammaging-induced immune dysfunction during chronic viral infection.

Acinetobacter baumannii is an increasingly multidrug-resistant pathogen contributing to hospital-acquired infections necessitating the discovery of novel treatments. A bacterial second messenger, cyclic diguanosine monophosphate (cyclic di-GMP), can regulate various persistence factors that are potentially advantageous for survival in hospital environments. Cyclic di-GMP–modulating enzymes and cyclic di-GMP–binding effectors predictively are encoded in the Acinetobacter baumannii genome. I hypothesized that cyclic di-GMP controls motility, biofilm formation, and desiccation tolerance in Acinetobacter baumannii. Disrupting cyclic di-GMP–modulating enzymes or cyclic di-GMP–binding effectors should alter the regulatory effectiveness of these phenotypes. I tested the multidrug-resistant isolate Acinetobacter baumannii strain AB5075 and identified several transposon mutants that altered twitching motility, biofilm formation, and desiccation tolerance; these results suggest that cyclic di-GMP plays a role during these three responses in Acinetobacter baumannii AB5075. Inhibiting these cyclic di-GMP signaling pathways could produce novel mechanisms to combat this pathogen in the hospital environment.

<p dir="ltr">Human babesiosis is a malaria-like, tick-borne infectious disease of major public health importance with a global distribution. Babesiosis is caused by intraerythrocytic, apicomplexan parasites of the genus Babesia. In the United States, human babesiosis is primarily caused by Babesia microti and Babesia duncani. Of these parasites, B. duncani infection is lethal to susceptible patients. Current treatment for babesiosis includes either the synergistic use of atovaquone and azithromycin or the combination of clindamycin and quinine. However, the side effects and the resistance posed by these parasites called for alternative approaches for the treatment of human babesiosis. Parasite-derived proteases play several functions in the context of parasitic lifestyle and regulate basic biological processes including cell death, cell progression and cell migration. We hypothesized that proteases are promising class of drug targets in Babesia parasites. Using the SYBR-Green assay, we screened a protease inhibitor library consists of 160 compounds against B. duncani in vitro culture at 50µM and identified 13 preliminary hits. Additionally, dose response assays of hit compounds against <i>B. duncani</i> and <i>B. microti</i> in vitro cultures identified 5 compounds as effective inhibitors against parasite growth. Of these 5 compounds, we chose ixazomib, a proteasome inhibitor as a potential drug for further studies based on its lower IC50 of 58nM as well as a higher therapeutic index as compared to other hit compounds. We demonstrated that in a mouse model infected with <i>target,</i>, the most effective inhibitor, the prodrug of ixazomib at a low dose of 2.5mg/kg lowers parasite proliferation without causing any adverse effects in animals. Thus, our studies suggest that Babesia proteasome may be an important drug target, and ixazomib may be a potential compound that may be used for the treatment of human babesiosis.</p>

Prions are the causative agents of a group of lethal, neurodegenerative conditions that include sheep scrapie, bovine spongiform encephalopathy (BSE), and human Creutzfeldt-Jakob disease (CJD). Prions are derived from the conversion of a normal, primarily alpha-helical, cellular prion protein (PrPC), to an infectious, beta sheet-rich conformer (PrPSc). Many unresolved issues surround the process of PrP conversion, and we know very little about cellular responses to these unique pathogens. Our lack of knowledge relates, in part, to the difficulty of infecting cells in vitro with prions. While expression of PrPC is an absolute requirement for prion propagation, I show here that not all cells that express PrPC are capable of propagating PrPSc. The goal of this thesis is to understand the role that host factors play in sustaining prion infection and to develop systems in which the cellular response to prion infection can be assessed. We hypothesize that cellular permissiveness to prion infectivity is co-dependent on unidentified additional cellular factors. To study the role of PrPC expression in susceptibility to prion infectivity, and identify these cofactors in cell culture, we utilized cells which fail to express endogenous PrPC, but become susceptible to prions following stable expression of PrPC. Following transfection of a species specific PrP expression construct and isolation of single cell clones, we assessed PrP expression and susceptibility to prion infectivity by measuring accumulation of protease resistant PrPSc. Differential gene expression studies suggest significant transcriptional differences between susceptible and resistant clones. Using three independent gene expression databases our analyses suggest that the resistant transcriptional profile favors cell division/cycle and chromosomal regulation pathways, while the sensitive transcriptional profile is involved in protein homeostasis and quality control. The results of these studies will not only lead to a greater understanding of PrP cell biology and the mechanisms of prion pathogenesis, but should ultimately lead to sensitive and expedient methods for detecting and characterizing prion infectivity from a wide range of sources.

Cystic fibrosis (CF) is an autosomal recessive disorder affecting 70,000 individuals worldwide. This disease is characterized by the buildup of mucus in the airways leading to chronic lung infections resulting in pulmonary failure and death in 95% of CF patients. Routine surveillance of CF pathogens using traditional microbiology culture guides management and treatment of CF patients. Molecular profiling studies have revealed emerging pathogens that may play a role in CF lung disease by either directly causing infection or upregulating the virulence factors of classic CF pathogens, such as P. aeruginosa; however, routine CF culture protocols have not been modified to detect these organisms. The goal of this study was to expand the data relevant to the use of microbiology cultures for the management and treatment of CF patients at Virginia Commonwealth University Medical Center (VCUMC) by directly selecting for emerging CF pathogens in culture. This was accomplished by developing,optimizing, and implementing an agar to select for colistin-resistant non-fermenting Gram- negative rods (NF GNRS). In addition, McKay agar and anaerobic media were utilized to recover members of the Streptococcus anginosus group (SAG) and anaerobes in CF respiratory samples. The prevalences of SAG, anaerobes, and colistin-resistant NF GNRs recovered on study media from 75 adult and pediatric CF patients at VCUMC were 17.33%, 41.33%, and 4% respectively. Approximately 62% of patients culture-positive for SAG were also infected with P. aeruginosa and 53.8% of SAG recovered in culture were from CF patients experiencing PE. These findings further support the claim that interspecies interactions among emerging and classic CF pathogens may result in periods of clinical instability or PE. Twenty-eight of the 75 patients were culture-positive for Veillonella species, with the majority of samples collected during a period of surveillance. Four colistin-resistant NF GNRs were isolated on the study media alone. The selective nature of the study media prevented the mixed respiratory flora and classic CF pathogens from overgrowing and obscuring the growth of these colistin-resistant NF GNRs. The presence and role of emerging pathogens in the CF patient population at VCUMC warrants further investigation; therefore, the routine culture protocol needs to be revised to recover and select for those organisms thought to play a role in PE and lung function decline.

This thesis is about the politics and the possibilities of aetiology. Firstly, the possibilities. Does an infectious disease have one, single pathogenic cause or many, interacting causes? In the medical microbiological sciences, there is no definitive answer, one way or another, to this question: there, the conditions of aetiological possibility exist in a curious tension. Ever since the birth of the 'germ theory of disease' and the concomitant birth of the singular aetiological object, these conditions have allowed for the co-existence of a very different, and far less well understood kind of object: the multifactorial object. That SARS was caused by one, singular viral agent, a coronavirus (CoV), is now entrenched as microbiological fact. And yet, the curious thing about SARS is that the history of the 2003 outbreak is littered with moments at which the possibility of the multifactorial object presented itself to, and was actively considered by, medical microbiologists. So how did we get here - to SARS-CoV, an infectious disease that could be understood and storied in this, the most singular of ways? And what happened along the way to deny the multifactorial aetiological object any kind of existence at all? In an attempt to grapple with these questions, the thesis seeks to recover the possibility of the multifactorial object through a deep, ethnomethodological reading of the moments at which it flared up precise/y as a possibility for medical microbiologists investigating the outbreak. What emerges from that recovery operation is a sense that the multifactorial object was never actually ruled out or disproved in any way, but rather, was vanished. Put another way, the suggestion is that various medical microbiological practices and interventions, whilst establishing singularity, were serving, at the same time, to create an illusion of multifactorality's non-existence; an illusion behind which the issue of multifactorality, its possibility, could be discarded without ever having to be resolved, one way or the other. In the closing sections of this thesis a move is made towards suggesting that SARS-Co V, the singular disease, was the product of a choice-, a choice that was made to explore one aetiological possibility at the expense of another. And that is where the politics comes in. For if politics, the realm of the political, can be taken to arise in situations where various possibilities exist but not all possibilities can be chosen, then it follows that what this thesis provides is an opportunity to foreground the politics bound up with the practical doing of aetiology. As a result, and based on the experience of attempting to recover the vanished multifactorial object from the 2003 SARS outbreak, the thesis concludes with an attempt to inhabit the present in such a way as to make it possible to think, in a little more detail, about where aetiology, as understood by medical microbiologists, might be heading in the future: might recent shifts in practical, everyday, seemingly innocuous microbiological technique, have begun to make it easier to coax the multifactorial object out into a space of visibility? Might those shifts actually herald the crossing of an epistemological threshold in the medical sciences? And might the conditions of aetiological possibility be changing, and changing in ways that would drastically alter what it meant to speak of a 'disease', an 'infection' and a 'pathogen'?

A paracoccidioidomicose (PCM) é uma micose sistêmica, endêmica na América Latina, causada pelo fungo dimórfico térmico Paracoccidioides brasiliensis (P. brasiliensis), cujo principal componente antigênico é a glicoproteína de 43 kDa (gp43). Diferentes formas clínicas podem ser desenvolvidas e estão diretamente associadas com vários graus de depressão da resposta imune celular. Considerando a importância das células dendríticas na interação dos sistemas imune inato e adaptativo, e na ativação de células T \"naive\", no presente trabalho estudamos se células dendríticas interagem com leveduras de P. brasiliensis, assim como seu principal componente antigênico (gp43). Foi demonstrado pela primeira vez que células dendríticas poderiam ser infectadas por leveduras de P. brasiliensis, e esse fungo permaneceu viável após fagocitose. Analisamos por citometria de fluxo a expressão das moléculas de superfície observando diminuição significativa da expressão das moléculas de MHC-II, CD80 e CD54 em células dendríticas quando estas foram incubadas com leveduras da cepa Pb18 ou com gp43. Esse resultado mostrou que a ação do P. brasiliensis em células dendríticas poderia ser mediada pela gp43. Ao analisarmos a síntese de IL-12, observamos diminuição significativa desta interleucina, quando células dendríticas ativadas com LPS, foram cultivadas na presença de leveduras de P. brasiliensis ou gp43. Esses resultados sugerem que a gp43 pode afetar várias funções das células hospedeiras, indicando que esta inibição pode ser usada pelo P. brasiliensis para reduzir a eficiência da resposta imune, facilitando assim o estabelecimento da infecção primária indivíduos suscetíveis. / Paracoccidioidomycosis, endemic in Latin America, is a progressive systemic mycosis caused by dimorphic fungus Paracoccidioides brasiliensis, where the major antigenic component is a glycoprotein of 43kDa (gp43). The infection can evolve to different clinical forms that are associated to various degrees of suppressed cell-mediated immunity. The role of dendritic cells (DCs) in P.brasiliensis infection has never been investigated. With the recognition that DCs are able to initiate response in naïve T cells and that they also participate in Th cell education the present study was undertaken to understand whether DCs interact with P. brasiliensis or gp43, as well as to elucidate possible mechanisms and consequences of this interaction. In the present report, it was demonstrated for the first time that DCs could be infected by P. brasiliensis and survive. Our results indicate that P. brasiliensis infection and purified gp43 lead to down-regulation of MHC-II and adhesion properties of immature DCs. The down-regulation was also observed in LPS-induced DCs maturation, where the expression of MHC-II, CD80, CD54 and CD40 were significantly inhibited in the presence of P. brasiliensis or gp43. These data show that the actions of P. brasiliensis on DCs could be mediated by gp43. In addition, an inhibition of IL-12 production by gp43 was observed in LPS-induced DC maturation. These results suggest that gp43 affects many functions of the host cells, indicating that this inhibition might be used by P. brasiliensis to reduce the effectiveness of the immune response, thus facilitating the establishment and fate of primary infection in susceptible host.

Cholangiocarcinoma (CCA) is a fatal primary malignancy that is formed in the bile ducts. Cancer-associated myofibroblasts play a crucial role in CCA proliferation and invasion. Furthermore, there is a growing interest in the role of the exosome in the interaction between the cancer-associated myofibroblasts and cholangiocarcinoma which lead to CCA growth. However how cholangiocarcinoma-derived exosome affect the cancer-associated myofibroblasts in the tumor microenvironment remain unknown. In this study, we examined whether exosome produced by cholangiocarcinoma could involve in the prompt of CCA cells growth by regulation of myofibroblast. We found that cholangiocarcinoma-derived exosome could prompt elevated α-smooth muscle actin and stromal cell-derived factor one expression that induces myofibroblast proliferation. We then demonstrated that cholangiocarcinoma-derived exosome upregulated periostin expression that plays an important role in cancer metastasis. In 3D organotypic rat CCA coculture model, TCA and S1P considerably increase the growth of CCA cell. Conclusion: cholangiocarcinoma-derived exosome trigger cancer-associated myofibroblasts proliferation in the tumor microenvironment that leads to prompt CCA growth.