Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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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.