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 Insights into the reciprocal communication between Neutrophils and Staphylococcus aureus(Montana State University - Bozeman, College of Letters & Science, 2015) Zurek, Oliwia Wiktoria; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Tyler K. Nygaard, Robert L. Watkins, Kyler B. Pallister, Victor J. Torres, Alexander R. Horswill and Jovanka M. Voyich were co-authors of the article, 'The role of innate immunity in promoting SAER/S-mediated virulence in Staphylococcus aureus' in the journal 'Journal of innate immunity' which is contained within this thesis.; Kyler B. Pallister and Jovanka M. Voyich were co-authors of the article, 'Staphylococcus aureus inhibits neutrophil-derived IL-8 to promote cell death' in the journal 'Journal of Infectious Diseases' which is contained within this thesis.Staphylococcus aureus (S. aureus) is a highly adaptable pathogen that can cause endocarditis, skin abscesses, tissue necrosis, and sepsis. S. aureus success can be partially attributed to its ability to colonize and subsequently infect a wide variety of host tissues. This capacity is dependent on elaborate two-component gene-regulatory systems that control expression of virulence and immunomodulatory factors. The S. aureus exoprotein expression (SaeR/S) system is recognized as a major regulator of virulence that significantly contributes to the pathogen's ability to evade killing by the human neutrophil. However, it is unclear how this system becomes activated and how the SaeR/S system modulates neutrophil function. In this study, we elucidated how S. aureus evades neutrophil killing by studying the reciprocal communication between the host and pathogen. We demonstrated that only select SaeR/S-regulated genes (as opposed to all targets) were transcriptionally up-regulated in response to stimulation by neutrophils as well as alpha-defensin and show that the mouse skin environment (that lacks alpha-defensin) promoted transcription of specific saeR/S-targets, different from the expression profile elicited following neutrophil interaction or alpha-defensin. These results were unexpected and demonstrated differential activation of saeR/S targets was dependent on specific stimuli. Furthermore, we studied the influence of SaeR/S on neutrophil function and showed that this system promoted accelerated cell death by decreasing NF-kB activity, and in-turn IL- 8 production, to promote neutrophil lysis. These findings underscored the importance of neutrophil signaling demonstrating that neutrophil-derived production of IL-8 was necessary for this cell to kill S. aureus effectively. It follows that treatment of human neutrophils with recombinant IL-8 significantly increased neutrophil staphylocidal activity. Finally, we propose that both timing and magnitude of inflammation in neutrophils play major roles in dictating the outcome of staphylococcal disease and that alteration in the innate ability of neutrophils to produce IL-8 may increase susceptibility to S. aureus infections. Taken together, the findings define novel pathogen- and host-derived factors that play pivotal roles in the course of S. aureus infection.Item Production and purification of formyl peptide receptor : explorations of protein-protein interactions(Montana State University - Bozeman, College of Agriculture, 1997) Kohler, Maria RenataItem Interaction between human neutrophils and Pseudomonas aeruginosa biofilm : morphological and biochemical characterization(Montana State University - Bozeman, College of Agriculture, 2003) Papke, Maiko Sasaki; Chairperson, Graduate Committee: Michael FranklinItem Human neutrophil flavocytochrome b : structure and function(Montana State University - Bozeman, College of Agriculture, 2002) Foubert, Thomas RichardItem Molecular interaction of human neutrophil NADPH oxidase proteins(Montana State University - Bozeman, College of Agriculture, 1996) DeLeo, Frank RobertItem Neutrophil plasma membrane reorganization following stimulation with N-formyl-met-leu-phe in the presence of dihydrocytochalasin B(Montana State University - Bozeman, College of Agriculture, 1995) Mukherjee, GoutamItem Analysis of the small GTP binding protein Rac2(Montana State University - Bozeman, College of Letters & Science, 2005) Snodgrass, Meagan Alyssa; Chairperson, Graduate Committee: Algirdas J. JesaitisHuman neutrophils serve as the first line of immune defense against invading pathogens. In many cells, including neutrophils, Rac proteins are key regulators of many diverse cellular functions through their affects on cytoskeletal organization and in neutrophils, the NADPH oxidase complex, a critical mechanism for host defense. The primary purpose of this research is to examine the possibility that Rac2 interacts directly with the actin cytoskeleton. A different small GTP binding protein, Rap2 has been shown to interact with actin through direct binding. To test our hypothesis, GST-Rac2 and GST-Rac2 R120E protein was expressed in E. coli. Rac2 was then purified and used for phage display mapping to identify potential binding epitopes on actin. Phage display analysis, which has been used to map binding epitopes on other proteins, was conducted on GST-Rac2 and GST-Rac2 R120E affinity columns. Peptide sequences from phage display on both proteins were mapped to the amino acid sequence of human actin using the program FINDMAP. This program can identify discontinuous epitope regions by permitting large gaps in the target sequence during alignment. FINDMAP results were visualized using Swiss PDB Viewer, a publicly available protein modeling program and workbench. Several possible binding epitopes for Rac2 were identified on the surface of actin. Purified GST-Rac2 was shown to bind GTPã [35S] selectively, but GST-Rac2 R120E did not bind GTPã [35S]. Fluorescence spectroscopy results suggested that the structural conformation of the purified wild type and mutant protein were similar. Protein characterization shows that while only GST-Rac2 can bind nucleotide, both proteins are structurally similar. In addition to the purification of recombinant protein expressed in E. coli, we generated vectors to express c-myc tagged Rac2 and Rac2 R120E in mammalian cell lines. Preliminary fluorescence microscopy data shows that indeed c-myc tagged Rac2 and Rac2 R120E are expressed using an inducible transfection system. Future studies will determine the effect of Rac2 R120 E on the actin cytoskeleton in mammalian cells. Taken with previous data suggesting that Rac2 and Rac2 R120E binds actin, this research strengthens the argument that Rac2 and actin interact in a direct manner.Item Modulation of the plasma membrane domain structure of human neutrophils(Montana State University - Bozeman, College of Letters & Science, 2006) Stie, Jamal Talal; Chairperson, Graduate Committee: Algirdas J. JesaitisEukaryotic cell plasma membranes form an interface between cells and their environment and function to detect and interpret environmental cues. The work described in this dissertation examines the changes that occur in membrane structure during plasma membrane function in human neutrophils and a fungal opportunist. The body of this work examines how circulating neutrophils can remain functionally inactive in the presence of perturbing influences inherent in the blood circulation, and yet rapidly activate upon exposure to proinflammatory agents. It is hypothesized that the regulated modulation of plasma membrane domain structure determines the activation of blood-leukocytes, in vivo. Experimentation is based the isolation of blood-neutrophils in either nonactivated or activated (primed) cellular states using dextran- or gelatin-based preparative methods, respectively. Analysis of plasma membrane cortical components actin, fodrin, ezrin, CD45 and CD43 by sucrose density sedimentation, flow cytometry and indirect immunofluorescence microscopy indicated significant differences in the plasma membrane structure of both neutrophil populations. In nonactivated neutrophils, cortical actin and fodrin were cytosolic, thus indicating the absence of cortical structure in this population.Item The role of the recruited neutrophil in the innate response to Aspergillus fumigatus(Montana State University - Bozeman, College of Letters & Science, 2005) Bonnett, Colin Russell; Chairperson, Graduate Committee: James B. BurrittStrong clinical and experimental evidence links qualitative and quantitative neutrophil deficiencies to fatal infections caused by A. fumigatus. Yet the role of the neutrophil in mediating the protection observed in normal hosts remains largely unknown. Recent studies indicate neutrophils from CXCR2-/- mice are unable to migrate toward chemokine gradients of KC and MIP-2, rendering these animals susceptible to fatal aspergillosis. Mice with a mutation in the gene encoding for the gp91phox component of the NADPH oxidase lack the ability to generate the reactive oxygen metabolites used by phagocytes in killing microbial pathogens, and these mice are also susceptible to invasive pulmonary aspergillosis. In this investigation of the innate response to A. fumigatus, CXCR2-/- and gp91-/- mice were used to mimic the qualitative and quantitative neutrophil defects that are known to predispose to invasive pulmonary aspergillosis, the most lethal form of Aspergillus diseases. By comparing the nature of the predisposition of these mice with the robust immunity observed in normal and immunocompetent mice, insight was gained on the involvement of this key phagocyte in the innate response. Several important parameters of the innate response in the lung were analyzed in this investigation, including (1) leukocyte recruitment and organism engagement, (2) organism killing mechanisms, (3) cytokine levels of bronchoalveolar lavage fluid, and (4) in vitro organism killing by leukocytes. Following intratracheal challenge of A. fumigatus conidia a delay of 3 hours in neutrophil recruitment to lungs of CXCR2-/- animals was observed, allowing significant conidial germination and hyphal formation not seen in normal animals. In contrast, the gp91phox-/- mice recruited neutrophils normally but failed to inhibit conidial germination and hyphal proliferation, apparently as a result of their inability to generate the appropriate conidiacidal mediators. In normal mice inoculated conidia were rapidly sequestered within neutrophil aggregates involving a response by the phagocyte NADPH oxidase detected by formazan deposition. These results suggest a previously undescribed role for neutrophils that conduct early inflammatory events following exposure to A. fumigatus conidia, involving first sequestration of ungerminated conidia within neutrophil complexes, and subsequently oxidant generation that prevents hyphal outgrowth.