Theses and Dissertations at Montana State University (MSU)
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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 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 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 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 Function of non-heme-binding domains of the Staphylococcus aureus IsdB protein in heme assimilation from methemoglobin(Montana State University - Bozeman, College of Agriculture, 2014) Li, Dengfeng; Chairperson, Graduate Committee: Benfang LeiAs a hemoglobin acceptor, IsdB rapidly and efficiently acquires heme from methemoglobin (metHb) in the heme acquisition pathway of Staphylococcus aureus. The pathway of heme assimilation in S. aureus involving IsdB has been established; however, the mechanism of rapid and efficient heme assimilation of metHb heme by IsdB remains unclear. IsdB consists of five major domains: the N-terminal (ND), NEAr Transporter 1 (N1), middle (MD), heme binding NEAr Transporter 2 (N2), and C-terminal (CD) domains. The goal of this study is to elucidate the roles of these IsdB domains in the metHb-to-IsdB heme transfer reaction. Deletion of the CD region does not alter the kinetics and equilibrium of the reaction. Sequential deletions of ND and N1 of ND-N1-MD-N2 progressively reduce heme transfer rates but have no effect on the reaction equilibrium. Further deletion of MD decreases the efficiency of heme transfer from metHb to N2. The MD domain reduces heme dissociation from holo-N2 and drives the metHb/N2 reaction to the formation of holo-N2. ND-N1-MD and N2 fragments, but not ND-N1, MD, and N2, reconstitute the rapid metHb/IsdB reaction, indicating an MD/N2 interaction. Analyses of MD, N2, and MD-N2 mixture by size exclusion chromatography support an interaction between MD and N2. These results indicate that ND-N1 and MD domains critically contribute to the kinetics and equilibrium of the metHb-to-IsdB heme transfer reaction, respectively. The results also suggest that CD functions as a spacer to position IsdB in the cell wall envelope for heme relay through the cell wall. These findings support a mechanism of direct extraction of metHb heme by IsdB that involves the four structural domains of IsdB.Item Insights into the ArlR/S mediated pathogenesis of Staphylococcus aureus(Montana State University - Bozeman, College of Agriculture, 2013) Meyer, Susan Irene; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Tyler K. Nygaard, Meet Patel, and Jovanka M. Voyich were co-authors of the article, 'The ArlR/S two-component signal transduction regulatory system is essential to the pathogenesis of Staphylococcus aureus' in the journal 'Microbes and infection' which is contained within this thesis.Staphylococcus aureus (S. aureus) is a gram-positive pathogen capable of causing a wide range of disease from relatively simple soft tissue infections to severe life-threatening disease like sepsis and endocarditis. Historically, most S. aureus infections were associated with healthcare settings and a majority of cases were seen in patients with compromised immune systems. In the past decade, however, infections caused by S. aureus have become more common in healthy individuals. These community-associated strains are an even bigger problem because a large percentage are resistant to antibiotics and are have an incredible ability to incur antibiotic resistance. The ability of this bacterium to subsist and thrive in a wide range of environmental conditions is partly due to the pathogen's use of two-component signal transduction gene-regulatory systems that have the ability to sense external conditions and regulate gene transcription appropriately. This study investigates the role of one of these two-component regulatory systems, ArlR/S. An isogenic deletion mutant of the arlR/S operon was created and was tested in several in vitro assays as well as in vivo murine models of infection. Using in murine models of soft tissue infection and invasive infection, it was determined that arlR/S is important to the virulence of S. aureus. A murine model of dissemination showed that S. aureus dissemination is altered with the deletion of the ArlR/S two-component regulatory system. To determine whether the decreased pathogenicity was caused by a change in the interaction between S. aureus and immune cells of the body, in vitro assays with human whole blood and human PMNs were performed with both S. aureus and bacterial supernatants. Interestingly, no differences were seen between the wild type S. aureus and the mutant in these assays. An oligonucleotide microarray was performed and showed strong regulation of ebh (ECM-binding protein homologue), which codes for the giant staphylococcal surface protein (GSSP). Together, this study demonstrates the importance of arlR/S to the regulation of ebh and to the virulence of S. aureus in a PMN-independent manner.Item A review of plant-derived compounds and their potential for treating community-associated methicillin resistant Staphylococcus aureus(Montana State University - Bozeman, College of Letters & Science, 2013) Mead, Julia Suzanne; Chairperson, Graduate Committee: Jovanka Voyich-KaneMethicillin-resistant Staphylococcus aureus (MRSA) has been a global public health problem, especially in hospital settings, for more than fifty years. Within the last few decades, MRSA has undergone a shift in epidemiology, appearing more frequently in the community, and amongst people without traditional risk factors. Community-acquired (CA) MRSA strains contain a wide range of virulence factors and confer varying drug resistances. Infection with CA-MRSA can often lead to poor clinical outcomes, including death. Current treatments for severe infections are limited, and very few truly novel antibiotics are enrolled in late-phase clinical trials testing by the Food and Drug Administration. Vancomycin is currently the first choice of antibiotic for severe infections, however S. aureus strains with intermediate or full resistance to vancomycin have been reported since the early 2000's, thus the need for new antibiotics is urgent. This paper presents a literature review outlining the current body of knowledge regarding the use of plant-derived compounds and their activity against different strains of MRSA. Furthermore, the potential of these compounds for clinical use in treating MRSA infections will be assessed.Item Insights into the SaeR/S-mediated pathogenesis of Staphylococcus aureus(Montana State University - Bozeman, College of Agriculture, 2011) Nygaard, Tyler Kenji; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Frank R. DeLeob and Jovanka M. Voyich were co-authors of the article, 'Community-associated methicillin-resistant Staphylococcus aureus skin infections: advances toward identifying the key virulence factors' in the journal 'Current opinions in infectious disease' which is contained within this thesis.; Kyler B. Pallister, Peter Ruzevich, Shannon Griffith, Cuong Vuong and Jovanka M. Voyich were co-authors of the article, 'SaeR binds a consensus sequence within virulence gene promoters to advance USA300 pathogenesis' in the journal 'Journal of infectious disease' which is contained within this thesis.; Kyler B. Pallister, Shannon Griffith, Mark DeWald, Victor Torres, Alexander Horswill and Jovanka M. Voyich were co-authors of the article, 'SaeR/S-mediated transcription of hla by USA300 promotes human T cell plasma membrane damage and rapid human T cell proliferation' in the journal 'Journal of infectious disease' which is contained within this thesis.Staphylococcus aureus is an ubiquitous and versatile Gram-positive bacterium capable of producing a wide range of diseases in humans. Within the last ten years there has been a surge of infections caused by hyper-virulent strains of community-associated methicillin-resistant S. aureus (CA-MRSA) distinct from previously characterized healthcare-associated MRSA (HA-MRSA). In particular, pulsed-field gel electrophoresis (PFGE) type USA300 has emerged as the prominent circulating strain of CA-MRSA in the United States. How S. aureus recognizes environmental signals during infection and responds to promote bacterial survival is clearly an important aspect of pathogenesis but remains incompletely defined. It is believed S. aureus recognizes extracellular signals and subsequently alters gene transcription in response via the collective influence of 16 bacterial two-component gene-regulatory systems. This investigation examines the role of one of these two-component systems, saeR/S, in USA300 pathogenesis. An isogenic deletion mutant of saeR/S generated in USA300 (USA300 Delta saeR/S) demonstrated this two-component system is critical to USA300 infection by upregulating transcription of numerous virulence genes. 5' RACE analysis identified transcript start sites for genes strongly regulated by saeR/S. Sequence alignment upstream from transcript start sites revealed a conserved partially palindromic 17 bp sequence specific to promoter regions under strong saeR/S regulation and EMSAs confirmed direct binding of recombinant SaeR His to this conserved sequence. In particular, strong saeR/S dependent transcription of the pore-forming hla toxin and robust SaeR His binding to the hla promoter prompted further studies examining the importance of this virulence gene during USA300 pathogenesis. An isogenic deletion mutant of hla in USA300 (USA300 Delta hla) was generated and used to demonstrate saeR/S-dependent transcription of hla is critical to USA300 pathogenesis during murine models of soft-tissue infection. Though hla did not influence saeR/S-mediated plasma membrane damage on polymorphonuclear leukocytes, this toxin induced pore formation on human T cells and promoted rapid human T cell proliferation. Collectively this study demonstrates saeR/Sdependent transcription of virulence factors is critical to USA300 pathogenesis. In particular, the saeR/S-mediated regulation of the potent pore-forming hla toxin is a primary effector of USA300 pathogenesis that causes human T cell plasma membrane damage and induces rapid human T cell proliferation.Item 18-beta-glycyrrhetinic acid and methicillin resistant Staphylococcus aureus : from lytic activity to reduced pathogenesis(Montana State University - Bozeman, College of Agriculture, 2012) Long, Danyelle Renee; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Julia Meade, Jay Hendricks, Michele E. Hardy and Jovanka M. Voyich were co-authors of the article, '18-beta-glycyrrhetinic acid inhibits MRSA survival and attenuates virulence gene expression' in the journal 'Antimicrobial agents and chemotherapy' which is contained within this thesis.Methicillin-resistant Staphylococcus aureus (MRSA) is Gram-positive pathogen known to cause severe disease in humans. MRSA's resistance to the beta-lactam class of antibiotics makes it an increasing healthcare concern. Over the last two decades there has been a rise in the incidence of community-associated MRSA, specifically pulsed-field gel electrophoresis type USA300 (known to cause severe skin and soft tissue infections). The development of drug therapies against MRSA infections that do not induce resistance and have efficacy against MRSA is a pressing matter. In this study, we investigate the potential of two components in licorice extracts, Glycyrrhizic acid (GA) and 18-beta-Glycyrrhetinic acid (GRA), as effective antimicrobials against MRSA. Using in vitro survival assays, we determined that GRA is directly bactericidal to MRSA. Using a murine model of skin and soft tissue infection, we discovered that topical treatment with GRA reduced severity of MRSA skin and soft tissue infections more rapidly than treatment with GA or a control. The increase in infection clearance was not due to a reduction in bacterial burden, but results indicate that GRA may decrease severity of the infection via an effect on the immune system. Treatment of an MRSA skin infection with GRA reduced expression of KC and G-CSF. To further investigate how GRA treatment led to a more rapid clearance of infection, we analyzed the expression of five S. aureus virulence genes (saeR, hla, RNAIII transcript, mecA, and sbi) after treatment with GRA or GA. GRA rapidly down-regulated four of the five virulence genes in vitro and all five virulence genes in vivo in the skin infection model. The data presented here shows that GRA is bactericidal, assists in decreasing the severity of MRSA infection via down-regulation of virulence genes, and can induce an altered immune response in the host.