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Item The interaction of heavy metals with the mammalian gut microbiome(Montana State University - Bozeman, College of Agriculture, 2022) Coe, Genevieve Lea; Chairperson, Graduate Committee: Seth Walk; This is a manuscript style paper that includes co-authored chapters.Heavy metals are for the most part, naturally occurring elements found in the environment. Some are essential, meaning they are involved in critical biochemical pathways, in all branches of life. Other heavy metals are non-essential and disrupt metabolic functions in most organisms rendering them toxic. The following research explored the interactions of the mammalian gut microbiome with an essential heavy metal, iron, and a non-essential heavy metal, methylmercury, acquired through the diet. The overarching goal was to define and characterize the influence of gut microbial interactions with heavy metals on host health. Novel experimental designs using murine models were designed to examine 1) the consequences of low iron challenge on the murine gut microbiome and whether host iron availability was affected and 2) the potential influence of the gut microbiome in methylmercury elimination rate and demethylation in conventional, germ-free, gnotobiotic, and humanized mice. Culturing in vitro and toxicity assays, 16S sequencing, deep metagenomic sequencing of human stool, bioinformatic analysis, transcriptional analysis of iron biomarkers, quantification of iron and mercury by ICP-MS and HPLC-ICP-MS methods were performed as well as the use of mouse models to examine iron and methylmercury interactions with the gut microbiome in vivo. Our results from this project indicate that the gut microbiome is significantly affected by loss of iron from the diet, and does not fully recover post-iron repletion, while the host is relatively unaffected by low-iron challenge to the gut microbiome. Methylmercury elimination and demethylation is significantly faster and higher, respectively, in mice with a gut microbiome, providing novel evidence in support of a role for the gut microbiome in methylmercury demethylation and elimination. However, exact mechanisms of microbial interactions with methylmercury in the gut have yet to be elucidated. Our data also suggests the possibility of host-mediated mechanisms of methylmercury demethylation, by yet unknown mechanisms that warrant further exploration.Item Study of diverse host immune responses to viral and bacterial pathogens(Montana State University - Bozeman, College of Agriculture, 2023) Plewa, Jack Bruno; Chairperson, Graduate Committee: Mark Jutila; This is a manuscript style paper that includes co-authored chapters.Brucella abortus is the bacterium that causes brucellosis, an infection transmitted from cattle to people, often through consumption of raw milk and contact with aborted materials. With antibiotic resistance on the rise, phage therapy for bacterial infection may become a useful approach. The direct effects of phage on mammalian cells is important to understand, yet understudied. In vivo delivery of low phage MOI to the mouse lung was more effective at diminishing Brucella burden than higher doses of phage. In an in vitro model of intracellular Brucella infection, low phage MOI was capable of minimizing human THP-1 monocyte infection, but, unexpectedly, use of higher phage MOI diminished this effect. We hypothesized that recognition of these phage preparations may induce an antiviral immune suppressive response that may counteract their anti-bacterial effects. Indeed, when the type I IFN signaling pathway was disrupted in mice, phage treatment was more effective. However, when attempting to induce type I IFN in vitro using both human monocyte and mouse macrophage cell lines, we were unable to stimulate expression of type I IFN with Brucella phage, including in response to a combination of phage and bacteria. We then examined the effect of phage treatment on macrophage cell surface markers that are indicative of activation/differentiation. Interestingly, while Brucella LPS induced expression of CD71 and CD206, the addition of phage suppressed upregulation of these markers. Our discovery of immune suppressive effects of Brucella bacteriophage is an important consideration for using phage as a treatment.Item Initiation and pathogenesis of Staphylococcus aureus Pneumonia following influenza A infection(Montana State University - Bozeman, College of Letters & Science, 2019) Borgogna, Timothy Ryan; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Adrian Sanchez-Gonzalez, Kelly Gorham and Jovanka M. Voyich were co-authors of the article, 'A precise pathogen delivery and recovery system for murine models of secondary bacterial pneumonia' in the journal 'JOVE Journal of visualized experiments' which is contained within this dissertation.; Bennett Hisey, Emily Heitman, Joshua J. Obar, Nicole Meissner and Jovanka M. Voyich were co-authors of the article, 'Secondary bacterial pneumonia by Staphylococcus aureus following influenza A infection is saeR/S dependent' in the journal 'Journal of infectious diseases' which is contained within this dissertation.; Madison M. Collins, Kyle A. Glose, Kyler B. Pallister, Tyler K. Pallister and Jovanka M. Voyich were co-authors of the article, 'Uncovering the executioner: disruption of pulmonary surfactant by influenza A triggers Staphylococcus aureus Pneumonia' which is contained within this dissertation.Infection influenza A virus (IAV) leads to increased host susceptibility to secondary bacterial pneumonia. In cases such as these, Staphylococcus aureus (S. aureus) has emerged as the dominant bacterial pathogen associated with severe infection outcomes. S. aureus is a common commensal of the anterior nares and is frequently trafficked into the lower respiratory tract through inhalations, micro-aspirations, and direct mucosal dispersion. Despite recurrent exposure to the lungs and the capacity to cause severe disease, cases of S. aureus pneumonia are rare in immunocompetent hosts. Previous efforts interrogating S. aureus secondary bacterial pneumonia have largely focused on the immunomodulation that occurs during the antecedent influenza infection and have ignored the virulence contributions of the bacterial pathogen. To that end, we developed a murine model of secondary pneumonia to investigate S. aureus pathogenesis following influenza A infection. We identify that secondary bacterial pneumonia by S. aureus is dependent on the activation of the two-component regulatory system (TCS) SaeR/S. Further, studies demonstrated that in the absence of IAV infection the healthy lung environment suppresses virulence gene expression. Characterization of the lung environment revealed that the lipid constituents of pulmonary surfactant suppress S. aureus virulence production. Our data provide a model of secondary bacterial pneumonia wherein infection with IAV significantly reduces surfactant lipid concentrations within the lungs. The reduction of pulmonary surfactant lipids leads to a loss of S. aureus virulence suppression and rapid activation of the major virulence regulator saeR/S. Taken together, these data provide a strong rational for the low incidence of primary S. aureus pneumonia and the increased severity of S. aureus pneumonia following antecedent influenza A infection. Furthermore, these data highlight possible pulmonary surfactant replacement therapies that may significantly alleviate secondary bacterial pneumonia morbidity and mortality.Item The influence of an iron deficient diet on the murine gut microbiome(Montana State University - Bozeman, College of Letters & Science, 2019) Coe, Genevieve Lea; Chairperson, Graduate Committee: Jennifer DuBoisIron is an essential nutrient for mammals 1. It is involved in multiple redox reactions that are essential for the survival of most organisms 2. There are two main types of iron that are absorbed from the diet: inorganic iron and heme 3. Dietary iron ingested by mammals is mostly absorbed in the small intestine; however, it is unclear whether the gut microbiome is involved in iron homeostasis or whether iron in the diet influences the microbiome. The goal of this project is to characterize the change in microbial composition in response to iron deficiency and iron repletion in conventional mice and define a baseline model for future studies involving the more complex human gut microbiome.Item Human gut phages in health and disease(Montana State University - Bozeman, College of Letters & Science, 2018) Manrique Ronquillo, Maria del Pilar; Chairperson, Graduate Committee: Mark J. Young; Michael S. Dills and Mark J. Young were co-authors of the article, 'The human gut phage community and its implications for health and disease' in the journal 'Viruses' which is contained within this dissertation.; Benjamin Bolduc, Seth T. Walk, John van der Oost, Willem M. de Vos and Mark J. Young were co-authors of the article, 'Healthy human gut phageome' in the journal 'Proceedings of the National Academy of Sciences of the United States of America' which is contained within this dissertation.; Mark J. Young was a co-author of the article, 'Interactions of the healthy gut phage community (HGP) with the core gut bacterial community' submitted to the journal 'PLOS computational biology' which is contained within this dissertation.; Yifan Zhu, John van der Oost, Willem M. de Vos and Mark J. Young were co-authors of the article, 'Gut bacteriophages and fecal microbial transplantation outcome in subjects with metabolic syndrome' which is contained within this dissertation.; Seth T. Walk and Mark J. Young were co-authors of the article, 'Bacteriophage-enriched filtrates: a potential tool to modify the structure of the gut-associated bacterial community' which is contained within this dissertation.The human body is colonized by a diverse microbial community known as the human microbiota. Most of these microbes, reside in the human intestinal tract. The gut microbiota has coevolved with humans and has become essential for multiple physiological functions that range from digestion, to development of the immune system, protection for pathogens, and even behavior. The gut microbial community is primarily dominated by Bacteria and their viruses- bacteriophages (or phages for short). Even though our knowledge of the contribution of the former to human health is extensive, the role of bacteriophages in human health and disease has been explored considerably less. Study of bacteriophages in other microbial environments has highlighted their importance in influencing the structure and function of their host community. Therefore, understanding the role of bacteriophages in the human gut ecosystem, and overall, in human health, has become a focus of current research. The main overarching hypothesis of this thesis is that human gut bacteriophages contribute to human health. To test this hypothesis, viral metagenomic surveys of healthy and disease individuals, together with experiments in a gnotobiotic mouse model system were performed. A group of bacteriophages shared among healthy individuals and significantly depleted in individuals with IBD was identified. Moreover, a host reservoir for these phages was identified in the core gut bacterial community of healthy subjects. Study of phage dynamics during an FMT treatment in patients with metabolic syndrome further highlighted the association of bacteriophages with human health. Patients that showed significant clinical improvement harbored a richer community, and a community more similar to healthy donors than patients that did not respond to the treatment. Moreover, a set of potential phage biomarkers associated with health and treatment outcome were identified. Lastly, experiments in gnotobiotic mice demonstrated the ability of bacteriophage-enriched filtrates to modify the microbial community structure. This result highlights the potential use of bacteriophages to manipulate the human gut microbiota, and potentially restore human health.Item Investigating arsenic-microbiome interactions in the gut using murine models(Montana State University - Bozeman, College of Letters & Science, 2019) Coryell, Michael Philip; Chairperson, Graduate Committee: Seth Walk; B. A. Roggenbeck and Seth T. Walk were co-authors of the article, 'The human gut microbiome's influence on arsenic toxicity' submitted to the journal 'Current pharmacology reports' which is contained within this thesis.; M. McAlpine, N.V. Pinkham, T.R. McDermott and Seth T. Walk were co-authors of the article, 'The gut microbiome is required for full protection against acute arsenic toxicity in mouse models' in the journal 'Nature communications' which is contained within this thesis.; M. Yoshinaga, T.R. McDermott and Seth T. Walk were co-authors of the article, 'Speciation of excreted arsenicals from germ free and conventional AS3MT knockout mice exposed to inorganic arsenate' which is contained within this thesis.Drinking water contamination with arsenic is a wide-spread public health concern, potentially affecting over 140 million people across at least 40 different countries. Current understanding of biological and behavioral factors influencing clinical outcomes is insufficient to explain the variation observed in arsenic-related disease prevalence and severity. The intestinal microbiome in humans is a dynamic and active ecosystem with demonstrated potential to mediate arsenic metabolism in vitro and distinct variability between individuals. This dissertation investigates arsenic-microbiome interactions, with a focus on determining how microbiome activity influences host-response and toxicity from arsenic exposures. Chapter 2 overviews common exposure routes, important metabolic pathways, and current evidence of arsenic-microbiome interactions in humans or experimental animal models. Chapter 3, the initial approach was to experimentally perturb the microbiome of common laboratory mice during arsenic exposure, measuring arsenic excretion in the stool and accumulation in host tissues. Arsenic sensitive gene-knockout mice were used to determine the microbiome's influence on subacute arsenic-induced mortality. Disrupting microbiome function--first by antibiotic treatment, then by deriving mice germ free--dramatically reduced survival times during severe arsenic exposures. Transplantation of human fecal communities into germ free mice effectively complemented the loss of function from microbiome disruption in these mice. Chapter 4 examines microbiome's impact on arsenic metabolism in germ free and conventional mice from this same arsenic-sensitive genetic background. These mice are deficient for the primary metabolic pathway involved in arsenic detoxification in both humans and mice, facilitating a more complete experimental isolation of microbiome and host metabolisms. This study provides evidence of microbiome-dependent changes in the elimination routes and metabolic transformation of ingested arsenic and provides a new experimental model for studying arsenic metabolism in the gut.Item Regulatory effects of the thymus on the IgE response of mice(Montana State University - Bozeman, College of Agriculture, 1974) Schneller, Rhet LucyItem Characterization of non-polar compounds from Pinus ponderosa needles causing reproductive failure in mice during early gestation(Montana State University - Bozeman, College of Letters & Science, 1979) Kubik, Yolanta MiroslawaItem Cortisol acetate-induced wasting disease in germfree and conventionally reared mice(Montana State University - Bozeman, College of Agriculture, 1966) Reed, Norman DuaneItem The immunology of spontaneous cure in the Nippostrongylus brasiliensis-mouse system(Montana State University - Bozeman, College of Agriculture, 1980) Benjamin, William Hiram