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 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 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 molecular mechanisms of sensing and responding to the host by Staphylococcus aureus(Montana State University - Bozeman, College of Agriculture, 2019) Meishery-Patel, Delisha; Chairperson, Graduate Committee: Jovanka Voyich-Kane; K. B. Pallister and Jovanka Voyich were co-authors of the article, 'Role of SaeR phosphorylation in regulation of Staphylococcus aureus virulence genes' which is contained within this thesis.; Dissertation contains an article of which Delisha Meishery-Patel is not the main author.Two-component systems (TCSs) are highly conserved across bacteria and are used to rapidly sense and respond to changing environmental conditions. The human pathogen Staphylococcus aureus uses the S. aureus exoprotein expression (sae) TCS to sense host signals and activate transcription of virulence factors essential to pathogenesis. Despite its importance, the mechanism by which the sensor kinase (SaeS) recognizes a stimulus and activates its cognate response regulator (SaeR) to regulate transcription of virulence genes is incompletely defined. However, findings from our lab suggest that SaeR/S mediated transcription is unique-to and dependent-on specific host stimuli. Studies outlined in this dissertation suggest that residues in the extracellular loop may be involved in refinement of the sae regulated targets at the single amino acid level. By generating single amino acid replacement mutants in the response regulator SaeR, we identified a key aspartate residue at position 46 (D46) on SaeR to be important in SaeR mediated signaling as mutation D46A prevented the recombinant protein from binding promoter recognition sequence and subsequently influenced virulence regulation. Current studies are aimed to define the phosphorylation patterns in SaeR using SDS-PAGE analysis and mass spectrometry. Overall, these structure-function studies provide insight into the Sae- signal transduction mechanism and raise some new questions regarding the role the Sae system in the larger regulatory network S. aureus uses to control expression of its secreted virulence factors.Item Analysis of the role of iron uptake mechanisms and addition of iron-doped apatite nanoparticles in phage infections in Staphylococcus aureus and Mycobacterium smegmatis(Montana State University - Bozeman, College of Letters & Science, 2018) Rost, Linda Christina; Chairperson, Graduate Committee: Greg FrancisAntibiotic resistance has become a significant global public health issue, and phage therapy could serve as an adjuvant to traditional antibiotics. Phages are viruses that kill bacteria and produce more phages. Iron-doped apatite nanoparticles (IDANPs) have been shown to increase phage killing of bacteria. However, the mechanism of JB and Yodasoda infection of bacteria, and mechanism by which IDANPs increase phage infections, is unknown. Based on the iron composition of the IDANP, as well as extensive literature describing Staphylococcus aureus having aggressive iron uptake systems, it was hypothesized that IDANPs may affect such systems, and thereby be involved in the subsequent increase of phage-mediated bacterial death. In this work, the relationship between bacterial exposure to iron and subsequent phage infectivity was established, and IDANP effect on plaque size was determined. S. aureus cells were grown in various iron treatments, plaque assays were performed. There was a strong, positive relationship between iron treatments and plaque counts. The plaque counts were 29% higher in the 0.0004g/L iron treatment, 34% higher in 0.0008g/L, 60% higher in 0.0016 g/L and 82% higher in 0.0032g/L. When S. aureus and M. smegmatis cells were treated with IDANPs, plaque sizes were significantly larger, which may indicate increased infection in adjacent cells. Plaque sizes from IDANP-treated cells continued to increase in size as plates were incubated over 24, 48 and 96 hours. Plaque sizes also increased in size in the control cells in some time frames. S. aureus cells were also grown in 0.0016g/L iron treatment and treated with IDANPs, and there was a 65% increase in plaque counts. In higher iron treatments, it was difficult to achieve a lawn of cells to perform plaque assays. Cell growth was measured by performing serial dilutions and determining CFU/mL. There was no significant difference between cells grown in M9 minimal media or treated with IDANPs. Cells were also grown in the different iron treatments over three hours, with or without IDANPs. Less growth was observed in high iron treatments, but the differences were not significant. Cell growth was relatively slower in high iron levels in overnight treatments, and the results were significantly different. These data can be used to elucidate the relationship of iron uptake and phage killing, and therefore allow conjectures as to whether or not iron uptake mechanisms may be involved in the IDANP effect. Further research in this field can provide opportunities to develop reliable alternatives to antibiotics to treat bacterial infections.Item The role of SaeR/S in secondary Staphylococcus aureus Pneumonia(Montana State University - Bozeman, College of Agriculture, 2016) Hisey, Bennett Stephen; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Timothy R Borgogna, Kyler B. Pallister, Eli W. Sward, Fermin E. Guerra and Jovanka M Voyich were co-authors of the article, 'The role of SaeR/S in secondary Staphylococcus aureus pneumonia' submitted to the journal 'Journal of infectious diseases' which is contained within this thesis.Methicillin?resistant Staphylococcus aureus (MRSA) is a Gram?positive pathogen capable of causing diverse disease in humans. MRSA precisely controls virulence factor expression via the SaeR/S two?component gene regulatory system. While much is known about SaeR/S regulation patterns during skin infection, less is understood about the role it plays in the pulmonary environment during secondary staphylococcal pneumonia. Using an isogenic deletion mutant in pulsed field gel electrophoresis type USA300 (strain LAC) of the saeR/S two?component gene regulatory system we examined its role in mouse models of pathogenesis involving primary infection with influenza strain A/WSN/33 followed by USA300 infection. Results demonstrate SaeR/S contributes significantly to mortality during pneumonia following influenza A infection. Reverse transcription PCR and QuantiGene 2.0 assays revealed differences in both transcription of components of SaeR/S as well as virulence factors under SaeR/S control. Primary Influenza infection was seen to up regulate expression of virulence factors under control due to antecedent influenza A infection. These data underscore the importance of pathogen contribution to the pathogenesis of secondary pneumonia.Item Investigations into the activity of synthetic & natural products against methicillin-resistant Staphylococcus aureus(Montana State University - Bozeman, College of Letters & Science, 2016) Weaver, Alan James, Jr.; Chairperson, Graduate Committee: Martin Teintze; Joyce B. Shepard, Royce A. Wilkinson, Robert L. Watkins, Sarah K. Walton, Amanda R. Radke, Thomas J. Wright, Milat B. Awel, Catherine Cooper, Elizabeth Erikson, Mohamed E. Labib, Jovanka M. Voyich and Martin Teintze were co-authors of the article, 'Antibacterial activity of Tham trisphenylguanide against methicillin-resistant Staphylococcus aureus' in the journal 'PLoS ONE' which is contained within this thesis.; Abigail Van Vuren, Rakesh, Richard E. Lee, Valerie Copié and Martin Teintze were co-authors of the article, 'Exposure of methicillin-resistant Staphylococcus aureus to low levels of the antibacterial Tham-3 phi G generates a small colony drug-resistant phenotype' submitted to the journal 'PLoS ONE' which is contained within this thesis.; Amanda L Fuchs was an author, and Brian P. Tripet, Martin Teintze, Mary Cloud B. Ammons and Valerie Copié were co-authors of the article, 'Allicin identified as the principal antimicrobial compound in 1,000-year-old Bald's eyesalve' submitted to the journal 'International journal of antimicrobial agents' which is contained within this thesis.; Abigail Van Vuren, Martin Teintze, Valerie Copié and Jovanka Voyich were co-authors of the article, 'Treatment of MRSA with 18-beta-glycyrrhetinic acid reduces cell-to-cell interactions & increases production of staphyloxanthin' submitted to the journal 'Antimicrobial agents & chemotherapy' which is contained within this thesis.The studies herein investigated and characterized synthetic and natural products having efficacy against methicillin-resistant Staphylococcus aureus, which has become a significant threat to both hospital and community environments due to rapid drug resistance development. THAM-3 Phi G is a synthetic compound that showed initial promise as a novel antibacterial against S. aureus (MIC 2 mg/L) through membrane disruption. However, following sub-lethal dosing with THAM-3 Phi G, S. aureus was shown to develop resistance through a small colony variant phenotype, which was defined through 1D 1H NMR metabolomics. Natural products from age-old remedies having efficacy against S. aureus were also investigated in this study. Bald's Eyesalve has shown efficacy against S. aureus; however, the active antibacterial agent(s) remained unknown. Through molecular size and solvent fractionation, activity was isolated to the small (< 3 kDa), non-polar molecule fraction which lost activity following cysteine treatment. Following NMR spectral analysis, the organosulfur garlic-derived compound, allicin, was identified as the active antimicrobial agent. GRA is a natural product found in licorice root, which was used in ancient Chinese medicine. GRA is known to have efficacy against S. aureus and to downregulate key virulence genes. Prolonged exposure of S. aureus to GRA revealed significant increases in the pigment staphyloxanthin. Furthermore, NMR metabolomics of short-term treatments revealed a dysregulation of the TCA cycle, which collectively suggests that treatment of S. aureus with GRA results in oxidative stress. The efficacy of GRA against S. aureus biofilms was also investigated and showed GRA to be ineffective at reducing biofilm CFUs; however, GRA effected biofilm stability. Planktonic studies revealed significant reductions in cell-to-cell interactions beginning at 7.8 mg/L GRA based on optical density measurements and microscopy. Therefore, GRA may serve as part of a novel therapeutic method for treating chronic wound infections. Collectively, these studies utilized NMR to define metabolic phenotypes of bacteria in response to drug treatment and to resolve the active agent in a complex mixture of an age-old remedy. While S. aureus was able to overcome the antibacterial activity of THAM-3 Phi G, the studies of natural products from age-old remedies may provide future treatment options that require further investigation.