Theses and Dissertations at Montana State University (MSU)
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Item Disruption of neutrophil reactive oxygen species production by Staphylococcus aureus(Montana State University - Bozeman, College of Letters & Science, 2018) Guerra, Fermin Ernesto; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Timothy R. Borgogna, Delisha M. Patel, Eli W. Sward and Jovanka M. Voyich were co-authors of the article, 'Epic immune battles of history: neutrophils vs. Staphylococcus aureus' in the journal 'Frontiers in Cellular and Infection Microbiology' which is contained within this dissertation.; Conrad B. Addisson, Nienke W. M. de Jong, Joseph Azzolino, Kyler B. Pallister, Jos (A. G.) van Strijp and Jovanka M. Voyich were co-authors of the article, 'Staphylococcus aureus SaeR/S-regulated factors reduce human neutrophil reactive oxygen species production' in the journal 'Journal of Leukocyte Biology' which is contained within this dissertation.; Kyler B. Pallister, Tyler K. Nygaard, Mark T. Quinn, and Jovanka M. Voyich were co-authors of the article, 'Staphylococcus aureus leukocidins modulate human neutrophil reactive oxygen species production' which is contained within this dissertation.Staphylococcus aureus (S. aureus) is a bacterial pathogen that causes a wide range of human disease, from skin infections to invasive endocarditis. Neutrophils are the most abundant white blood cell in the human body, and the first line of defense following S. aureus infection. Even though neutrophils are equipped with an arsenal of bactericidal mechanisms, S. aureus survives neutrophil encounter. The mechanisms used by S. aureus to survive neutrophil killing remain unresolved. Previous studies have shown that the S. aureus SaeR/S two-component gene regulatory system is essential to survive neutrophil killing. Herein, we tested the hypothesis that S. aureus uses SaeR/S-dependent mechanisms to reduce neutrophil bactericidal mechanisms. First, we determined that S. aureus uses genes under the regulation of SaeR/S to inhibit neutrophil reactive oxygen species (ROS) production independent of previously defined mechanisms. Subsequently, we helped characterize a novel S. aureus SaeR/S-regulated virulence factor that inhibits human myeloperoxidase (MPO) activity to prevent formation of the highly bactericidal agent hypochlorous acid. Thus, S. aureus SaeR/S-regulated factors disrupt the neutrophil bactericidal mechanism with most efficacy against it, which is killing by oxidative mechanisms. We then focused on the role of S. aureus SaeR/S-regulated secreted leukocidins on neutrophil ROS production. While S. aureus leukocidins show redundancy inducing neutrophil pore formation, we determined that the surface receptors engaged by leukocidins induce distinct signaling pathways leading to ROS production. We showed that specific kinases are required for the differential production of neutrophil ROS induced by the S. aureus leukocidins LukGH and Panton-Valentine leukocidin (PVL). Importantly, the signaling pathways induced by S. aureus leukocidins through neutrophil surface receptors differ from the signals induced by physiological ligands through the same surface receptors. These results suggest S. aureus leukocidins 'shortcircuit' neutrophil signals to induce aberrant ROS production. In conclusion, S. aureus SaeR/S-regulated factors prevent proper bacterial clearance by disrupting neutrophil ROS production. These data provide us with a better understanding of the specific mechanisms used by S. aureus to survive neutrophil killing leading to pathogenesis.Item Mechanisms of CRISPR-mediated immunity in Escherichia coli(Montana State University - Bozeman, College of Letters & Science, 2019) van Erp, Paul Bertram Geert; Chairperson, Graduate Committee: Blake Wiedenheft; Gary Bloomer, Royce Wilkinson and Blake Wiedenheft were co-authors of the article, 'The history and market impact of CRISPR RNA-guided nucleases' in the journal 'Current opinion in virology' which is contained within this thesis.; Ryan N. Jackson and Joshua Carter were authors and Sarah M. Golden, Scott Bailey and Blake Wiedenheft were co-authors of the article, 'Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli' in the journal 'Nucleic acids research' which is contained within this thesis.; Angela Patterson was an author and Ravi Kant, Luke Berry, Sarah M. Golden, Brittney L. Forsman, Joshua Carter, Ryan N. Jackson, Brian Bothner, and Blake Wiedenheft were co-authors of the article, 'Conformational dynamics of DNA binding and CAS3 recruitment by the CRISPR RNA-guided cascade complex' in the journal 'ACS chemical biology' which is contained within this thesis.; Tanner Wiegand, Royce A. Wilkinson, Laina Hall, Dominick Faith and Blake Wiedenheft were co-authors of the article, 'Protein overexpression reduces specific phage infectivity in prokaryotic argonaute screen' which is contained within this thesis.; Dissertation contains three articles of which Paul Bertram Geert van Erp is not the main author.Prokaryotes are under constant threat from foreign genetic elements such as viruses and plasmids. To defend themselves against these genetic invaders prokaryotes have evolved extensive defense mechanisms. In this thesis I explore two such defense systems: prokaryotic Argonautes and CRISPR-systems. CRISPR-systems acquire short sequences derived from foreign genetic elements and store them in the CRISPR locus. In subsequent rounds of infection these stored sequences are used as guides by Cas proteins to target the invaders. Escherichia coli K-12 contains a type I-E CRISPR system, consisting of two CRISPR loci and eight cas genes. five of these cas genes, together with and 61-nucleotide CRISPR-RNA guide form the RNA-guided surveillance complex Cascade. This complex finds and binds foreign DNA targets that are complementary to its RNA guide. After target binding the helicase/nuclease Cas3 is recruited to the Cascade-DNA complex for destruction of the target. The goal of this research is to understand the molecular mechanisms that lead to target recognition and destruction in the type I-E CRISPR systems. Atomic resolution structures of the proteins involved in these CRISPR systems provide the blueprints of these proteins machines. Structure guided mutational analysis coupled with in vivo and in vitro biochemical experiments are used to investigate the underlying molecular mechanisms of this CRISPR system. Together, these results explain the rules of target recognition and Cas3 recruitment. Prokaryotic Argonautes have been hypothesized to defend against mobile genetic elements such as plasmids and viruses through guided nuclease activity. To test this hypothesis, we overexpressed 8 phylogenetically diverse prokaryotic Argonautes proteins in Escherichia coli and challenged them with seven bacteriophages. This resulted in robust protection against phage Lambda and phage P1 by four of the tested Argonautes, while little impact on phage infectivity was observed for the other phages tested. However, control experiments with a nuclease inactive Argonaute mutant and expression of an unrelated control protein showed similar protection against phage Lambda and phage P1. Collectively, our data suggest that protein overexpression in general, rather than Argonaute expression in particular, results in protection against 2 specific phages.Item Secretory immunity in Balb/c mice against Salmonella typhimurium(Montana State University - Bozeman, College of Agriculture, 1990) Shope, Steven RichardItem The in vivo reconstitution of congenitally thymusless mice(Montana State University - Bozeman, College of Agriculture, 1973) Isaak, Dale DarwinItem Studies on the immunological recovery of neonatally thymectomized mice(Montana State University - Bozeman, College of Agriculture, 1968) Hunter, James ThomasItem The chemical and immunological properties of Sarcoma I tumor specific antigen(Montana State University - Bozeman, College of Agriculture, 1972) Aden, Marilyn Jo ZangerItem Studies on chemotherapy of leukemia in conventionally-reared and germfree mice(Montana State University - Bozeman, College of Agriculture, 1970) Kouttab, Nicola MitriItem Macrophages and interferon gamma in host defense against disseminated candidiasis(Montana State University - Bozeman, College of Agriculture, 1997) Qian, QinfangItem Alteration of the graft-versus-host reaction by endotoxin(Montana State University - Bozeman, College of Agriculture, 1974) Jackson, Bynum McNeilItem Immunogenic studies of Clostridium sordellii(Montana State University - Bozeman, College of Agriculture, 1968) Bairey, Miles Huxtable