Theses and Dissertations at Montana State University (MSU)
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Item Characterization of host-pathogen interactions during early Staphylococcus aureus biofilm formation on surfaces(Montana State University - Bozeman, College of Agriculture, 2022) Pettygrove, Brian Alexander; Chairperson, Graduate Committee: Philip S. Stewart; This is a manuscript style paper that includes co-authored chapters.Implanted biomaterials such as orthopedic screws, prosthetic joints, pacemakers, and catheters are essential components of modern medicine. Unfortunately, implanted foreign bodies are susceptible to biofilm infection, leading to a persistent and difficult to treat disease state. Biofilm infections readily tolerate clearance from the immune system, however much of our understanding of the mechanisms governing persistence are formulated around the biofilm state during advanced infection. By comparison we have a poor understanding of the early stages of infection. Specifically, how contaminating organisms initially evade host immune defenses and establish a robust infection remains ill-defined. In this work, we interrogated interactions between Staphylococcus aureus (S. aureus), a frequent culprit in biomaterial infections, and early contributors to host immunity. Using in vitro time-lapse microscopy, we observed that human neutrophils readily phagocytose and kill single cells or small clusters of S. aureus cells that are attached to a surface. S. aureus cells that go undiscovered during the initial stages of neutrophil surveillance form biofilm aggregates that rapidly gain tolerance to neutrophil killing. In vivo models of implant infection demonstrated that surface adherent bacteria can evade discovery due to delayed or heterogeneous neutrophil recruitment to the surface. Biofilm aggregate formation was impaired in a strain deficient in the two-component gene regulatory system SaeR/S and the resulting cells were highly susceptible to neutrophil killing. Inhibition of aggregation was dependent on serum complement proteins C3 and factor B, suggesting that SaeR/S regulated factors actively inhibit host complement to facilitate aggregation. Taken together, these data suggest that the formation of immune-tolerant biofilm aggregates may contribute to chronic device related infections by protecting bacteria from phagocyte killing. These studies provide vital insight into the host pathogen interactions on contaminated biomaterial surfaces and highlight early events that may determine infection outcome.Item Investigating neutrophil cell fate following interactions with Staphylococcus aureus(Montana State University - Bozeman, College of Agriculture, 2022) Dankoff, Jennifer Grace; Chairperson, Graduate Committee: Jovanka Voyich-Kane; This is a manuscript style paper that includes co-authored chapters.Staphylococcus aureus is a ubiquitous pathogen with a growing list of antibiotic resistant capabilities. This gram-positive bacterium is able to cause a range of diseases, from a benign state of nasal colonization to fatal endocarditis. The ability to exist along this spectrum is largely dependent on the molecular dialog that takes place between the pathogen and the host, specifically white blood cells known as neutrophils. Neutrophils are the front line of defense against S. aureus infections. By modulating neutrophil behavior and inducing premature cell death, S. aureus has an advantage during an infectious state. In this thesis, I provide a method for studying this host and pathogen dynamic, and moreover, I investigate the mechanism by which S. aureus inhibits the neutrophil inflammatory response by repressing NF-KappaB. Here I show that S. aureus secretes a protein 30-50kDa in size, which both decreases total amount of NF-KappaB and activated NF-KappaB in neutrophils. This potent mystery protein is able to repress IL-8 production and does this all in a lysis independent manner. Additionally, the mystery protein is able to inhibit NF-KappaB activity in another cell type, the monocyte. It was previously believed that the S. aureus protein SSL3 was responsible for deactivating NF-KappaB, but herein, I show this is not the case. These findings reopen the need to examine the mechanism by which S. aureus modulates neutrophil inflammatory responses. Inhibition of the inflammatory response is likely linked the premature cell death seen during S. aureus infections. By utilizing these clues, the field is closer to understanding the intricacies of this host and pathogen dynamic, opening avenues to developing novel infection treatment methods.Item Investigating the regulation of virulence by Sae in Staphylococcus aureus(Montana State University - Bozeman, College of Agriculture, 2020) Collins, Madison Paige Martin; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Ranjan K. Behera, Kyler B. Pallister, Tyler J. Evans, Owen Burroughs, Caralyn Flack, Fermin E. Guerra, Willis Pullman, Brock Cone, Jennifer G. Dankoff, Tyler K. Nygaard, Shaun R. Brinsmade and Jovanka M. Voyich were co-authors of the article, 'The accessory gene saeP of the saeR/S two-component gene regulatory system impacts Staphylococcus aureus virulence during neutrophil interaction' in the journal 'Frontiers in microbiology' which is contained within this dissertation.; Kyler Pallister and Jovanka M. Voyich were co-authors of the article, 'Differential analysis of host/pathogen RNA expression via next generation sequencing reveals Staphylococcus aureus utilizes saeR/S-mediated factors to inhibit human neutrophil functions following phagocytosis' which is contained within this dissertation.Staphylococcus aureus (S. aureus) is a common commensal bacterium known to colonize, at minimum, 30% of the human population. It is also capable of causing a range of diseases that span from minor skin- and soft-tissue infections to life-threatening diseases. The diversity of S. aureus infections is due to the ability of the bacteria to sense and respond to environmental change. Virulence regulation in S. aureus can be attributed to the use of two-component gene regulatory systems (TCS). TCS can sense a variety of encounters including: antibiotics, heat stress, or immune cell encounter. Neutrophils are a key leukocyte involved in bacterial clearance in the human host. It follows that S. aureus has evolved mechanisms to sense and respond to neutrophils. The Sae TCS, is immediately up-regulated after neutrophil phagocytosis and has been demonstrated to be critical in the success of S. aureus both in vitro and in vivo. SaeS, the histidine kinase, and the respective response regulator, SaeR, are established components of the Sae TCS and their importance during neutrophil evasion and pathogenesis is well established. However, little is known about two accessory genes, saeP and saeQ. Results described herein using human neutrophil and murine models of infection provide evidence that SaeP modulates the Sae-mediated response of S. aureus against human neutrophils and suggest that saeQ and saeP together impact pathogenesis in vivo. To identify additional host and pathogen factors important during neutrophil interaction, we used differential analysis of host/pathogen RNA expression via Next Generation Sequencing to define the influence of SaeR/S on the host-pathogen transcriptome following neutrophil phagocytosis. Results determined that in the early stages of S. aureus infection, SaeR/S-dependent factors significantly modulate neutrophil processes involved in several pathways including autophagy, TNF-alpha signaling, and NF-kappaB signaling. These results suggest S. aureus uses SaeR/S-regulated virulence factors to hijack human neutrophil function at the transcriptional level to inhibit proper killing by neutrophils and allow for S. aureus persistence within the host.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 The Staphylococcus aureus two component system, SaeR/S, modulates monocyte production of TNF-alpha to influence neutrophil functions(Montana State University - Bozeman, College of Letters & Science, 2018) Sward, Eli Winfield; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Elizabeth M. Fones, Russel R. Spaan, Kyler B. Pallister, Brandon L. Haller, Fermin E. Guerra, Oliwia W. Zurek, Tyler K. Nygaard and Jovanka M. Voyich were co-authors of the article, 'Staphylococcus aureus SaeR/S-regulated factors decrease monocyte-derived tumor necrosis factor-alpha to reduce neutrophil bactercidal activity' in the journal 'Journal of infectious diseases' which is contained within this thesis.; Kyler B. Pallister and Jovanka M. Voyich were co-authors of the article, 'Staphylococcus aureus inhibits tumor necrosis factor-alpha in monocyte subsets to influence neutrophil functions' submitted to the journal 'Journal of infectious diseases' which is contained within this thesis.Staphylococcus aureus (S. aureus) is a commensal organism that colonizes the anterior nares of more than half the population. Although most individuals colonized with S. aureus remain asymptomatic, showing no signs of complications, colonization is associated with a predisposition to infection. S. aureus infections include skin- and softtissue infections as well as life-threatening infections, such as necrotizing fasciitis, necrotizing pneumonia, and sepsis. To date, it is not clearly understood how S. aureus transitions from a commensal organism to a deadly pathogen but evidence highlights that this capacity is largely dependent on two-component gene-regulatory systems that control expression of cytolytic and immunomodulatory virulence factors. The SaeR/S twocomponent system (SaeR/S TCS) of S. aureus is critical for the regulation of virulence factors that enables immune evasion and attenuates killing of S. aureus by human neutrophils. However, the precise SaeR/S-dependent mechanisms used by S. aureus to overcome and effective neutrophil response remains incompletely define. To advance our understanding, we studied SaeR/S-dependent immunomodulation of TNF-alpha. TNF-alpha is an important inflammatory mediator because it can recruit neutrophils to the site of infection and promote increased neutrophil killing of S. aureus. Using primary human cells, we demonstrated that the SaeR/S system reduced early monocyte production of TNF-alpha and showed that this modulation influenced the neutrophil priming and subsequent staphylocidal activity. These results demonstrated that S. aureus could reduce TNF-alpha early during infection to diminish neutrophil production of reactive oxygen species. As increased TNF-alpha is associated with morbidity and mortality during systemic infections, we propose that SaeR/S modulation of monocyte-derived TNF-alpha is important for reducing protective immune responses during localized infections. These data add to our understanding of how S. aureus disrupts early inflammatory responses to initiate infection.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 Hydrodynamic analysis of human neutrophil N-formyl chemotactic receptor-G protein interactions : mapping of interfacial domains with receptor-mimetic peptides(Montana State University - Bozeman, College of Letters & Science, 1994) Bommakanti, Rajani KanthItem Production and purification of formyl peptide receptor : explorations of protein-protein interactions(Montana State University - Bozeman, College of Agriculture, 1997) Kohler, Maria RenataItem Modulation of adherent bovine neutrophil responses by extracellular matrix proteins(Montana State University - Bozeman, College of Agriculture, 2002) Borgquist, Jessica James DavantItem 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 Franklin
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