Theses and Dissertations at Montana State University (MSU)
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Item Honey bee antiviral defense mechanisms at the individual and cellular level(Montana State University - Bozeman, College of Agriculture, 2021) Parekh, Fenali Mukesh; Chairperson, Graduate Committee: Michelle Flenniken; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Chemical stimulants and stressors impact the outcome of virus infection and immune gene expression in honey bees (Apis mellifera)' in the journal 'Frontiers in immunology' which is contained within this dissertation.; Alexander J. McMenamin was an author and Verana Lawrence and Michelle L. Flennikenwas were co-authors of the article, 'Investigating virus-host interactions in cultured primary honey bee cells' in the journal 'Insects' which is contained within this dissertation.; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Honey bee antiviral response to flock house virus infection' which is contained within this dissertation.; This dissertation contains an article of which Fenali Mukesh Parekh is not the main author.Honey bees are important pollinators of fruit, nut, and vegetable crops that constitute a large proportion of the human diet. Unfortunately, annual honey bee colony losses are high, averaging 38% from 2008-2018 in the United States. Honey bee colony losses are attributed to multiple factors, including pathogens and chemical exposure. Virus incidence and abundance have been associated with colony losses. The majority of honey bee viruses are positive-sense single stranded RNA viruses. Honey bees antiviral defense include RNA interference (RNAi), a double-stranded RNA (dsRNA) triggered sequence-specific post-transcriptional silencing mechanism and a non-sequence specific dsRNA-triggered pathway. In addition, signal transduction cascades include the Toll, Imd, and Jak/STAT pathways that promote the expression of honey bee immune response genes that are also induced in response to virus infections. To investigate the impact of chemical exposure on honey bee immune responses and virus infections, we infected bees with a panel of viruses including two model viruses (i.e., Flock House virus (FHV) and Sindbis-GFP) and a naturally infecting honey bee virus, deformed wing virus (DWV) and fed them sucrose syrup containing either thyme oil, a beekeeper applied fungicide Fumagilin-B ®, or the insecticide clothianidin. We determined that bees fed thyme oil augmented sucrose syrup exhibited greater expression of key immune genes, i.e., ago2, dcr-like, abaecin, hymenoptaecin, and vitellogenin and reduced virus abundance compared to virus-infected bees fed sucrose syrup. Whereas, virus-infected honey bees fed diets containing fumagillin or clothianidin exhibited reduced expression of key immune genes and higher virus abundance suggesting that chemical stressors act as immunosuppressors in honey bees. To understand the interplay of viruses and host cell gene expression more precisely, we cultured primary honey bee cells derived from larvae (i.e., hemocytes, immune cells) or pupae (i.e., mixed cell population including epithelial cells, adipocytes, muscle cells, hemocytes) and demonstrated that these cells supported replication of sacbrood virus, DWV, and FHV. Expression of select immune genes, including bap1, ago2, and dcr-like, in virus-infected honey bee cells was similar to expression in individual bees and varied for each virus. Together, these data further our understanding of the honey bee antiviral defense network and provide new tools for studying honey bee host-virus interactions.Item Understanding resistance and transcriptional responses to potato virus Y infection in potato plants(Montana State University - Bozeman, College of Agriculture, 2021) Ross, Brian Thomas; Chairperson, Graduate Committee: Michelle Flenniken; Nina Zidack and Michelle L. Flenniken were co-authors of the article, 'Extreme resistance to viruses in potato and soybean' in the journal 'Frontiers in plant science' which is contained within this dissertation.; Nina Zidack and Michelle L. Flenniken were co-authors of the article, 'Transcriptional responses to potato virus Y infection in resistant and susceptible potato cultivars' submitted to the journal 'Cultivars' which is contained within this dissertation.The potato is one of the world's most important crops. Cultivation of potatoes occurs on every continent except Antarctica and in a wide variety of climates. Potatoes are susceptible to a multitude of pathogens that can decrease yield and market quality. Viruses are particularly problematic for potato growers, as most potato production involves the replanting of tubers grown the previous year. Because virus-infected potato plants can harbor virus in their tubers, these tubers can in turn be the source of infection in the next generation of plants. Strains of Potato virus Y are the most economically burdensome viruses for potato growers worldwide. In field settings, Potato virus Y is primarily transmitted to plant by aphids feeding on leaves, but PVY can also be transmitted mechanically through infected plant sap. The use of insecticides and the application of mineral oil to leaves can help limit aphid populations and prevent infection to an extent but are generally both less effective and more environmentally impactful than genetic antiviral resistance mechanisms. The incorporation of genes that provide durable resistance to Potato virus Y into commercial potatoes is a major focus of potato breeders. One form of resistance, called extreme resistance, is characterized by a lack of symptoms and little to no virus replication occurring at the site of infection, but the molecular mechanisms of this response are not well understood. A comprehensive analysis of the extreme resistance literature indicates that movement of the resistance protein from the cytoplasm to the nucleus of the cell directly after virus infection may be a key aspect of this immune response. The downstream, transcriptional aspects of the extreme resistance response are also not well understood. We analyzed the gene expression from a Potato virus Y-resistant potato variety, Payette Russet, and a commonly grown susceptible variety, Russet Burbank, at a series of time points after virus infection using RNA sequencing. Results of these analyses indicate that an immune response likely occurs in Payette Russet quickly after virus inoculation. These analyses also indicate that the virus-susceptible variety, Russet Burbank, exhibits changes in gene expression that are similar to other susceptible potato varieties during asymptomatic or tolerant infection. Furthering our understanding of the molecular mechanisms controlling resistance and severity of virus infections will help inform future breeding and genetic engineering efforts, which require detailed knowledge of the mechanisms of virus resistance.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 Insect microbe interactions: honey bee antiviral defense mechanisms and characterization of Spiroplasma colonizing wheat stem sawfly(Montana State University - Bozeman, College of Agriculture, 2017) Brutscher, Laura Marie; Chairperson, Graduate Committee: Michelle Flenniken; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Virus and DSRNA-triggered transcriptional responses reveal key components of honey bee antiviral defense' in the journal 'Scientific reports' which is contained within this thesis.; Curtis Fowler, David K. Weaver and Carl J. Yeoman were co-authors of the article, 'Identification and characterization of a Spiroplasma sp. (Ixodetis clade) associated with the wheat stem sawfly (Cephus cinctus)' submitted to the journal 'Microbial ecology' which is contained within this thesis.Insects play important roles in ecosystems throughout the world. There are many beneficial insects, including those that pollinate plants in diverse landscapes, while other insects are considered agricultural pests. Regardless of ecological role, insects are hosts for microbial symbionts and pathogens. Some microorganisms (e.g., viruses) are harmful to insect health, but many microbial symbionts aid in host biological processes. The projects herein describe the interplay between insects and microbes; specifically (1) honey bee host - virus interactions and (2) identification and characterization of a wheat stem sawfly-associated Spiroplasma. Honey bees (Apis mellifera) are pollinators of numerous agricultural crops and other plant species. Since 2006, there have been high annual losses of honey bee colonies in the U.S. (~33%) and throughout the world. Colony deaths are influenced by multiple factors including RNA virus infections. Honey bee antiviral defense involves several immune pathways, including dsRNA mediated responses, (i.e., RNA interference and non-sequence-specific dsRNA-triggered responses), but their relative importance in antiviral defense is not well understood. To investigate honey bee antiviral defense, bees were infected with model virus in the absence or presence of dsRNA, which reduced virus abundance. Transcriptome-level analysis determined hundreds of genes were differentially expressed in response to co-treatment of dsRNA and virus, including immune-related genes. RNAi-mediated gene knockdown of two putative antiviral genes increased virus abundance and supported their antiviral role. Additional investigation of these and other genes will improve our understanding of dsRNA-mediated antiviral defense in honey bees. In contrast, wheat stem sawflies (Cephus cintus) are major wheat pests in the Northwest United States. Strategies that target endosymbionts of sawflies could reduce wheat crop losses. Hereunto, the microbes that colonize wheat stem sawfly have not been explored. Targeted DNA sequencing determined sawflies were colonized by a Spiroplasma species that has greatest 16S rRNA sequence identity with Ixodetis clade species. Metagenomic sequencing identified several Spiroplasma encoded genes involved in metabolism, which may be important to the sawfly host. Further characterization of honey bee-virus interactions and the role of Spiroplasma in sawfly health may contribute to limiting threats to global crop production and will further scientific understanding of non-model insect-microbe interactions.Item The roles of interleukin-1 and leukotriene-B4 in the innate immune response to pulmonary Aspergillus fumigatus infection(Montana State University - Bozeman, College of Letters & Science, 2017) Caffrey-Carr, Alayna Katherine; Chairperson, Graduate Committee: Mark T. Quinn; Margaret M. Lehmann, Julianne M. Zickovich, Vanessa Espinosa, Kelly M. Shepardson, Christopher P. Watschke, Kimberly M. Hilmer, Arsa Thammahong, Bridget M. Barker, Amariliz Rivera, Robert A. Cramer and Joshua J. Obar were co-authors of the article, 'IL-1A signaling is critical for leukocyte recruitment after pulmonary Aspergillus fumigatus challenge' in the journal 'PLoS pathogens' which is contained within this thesis.; Joshua J. Obar was a co-author of the article, 'Alarmin(G) the innate immune system to invasive fungal infections' in the journal 'Current opinion in microbiology' which is contained within this thesis.; Caitlin H. Kowalski, Sarah R. Beattie, Nate A. Blaseg, Chanell R. Upshaw, Arsa Thammahong, Hannah E. Lust, Yi-Wei Tang, Tobias M. Hohl, Robert A. Cramer, Joshua J. Obar were co-authors of the article, 'IL-1A signaling is critical for resistance against highly virulent Aspergillus fumigatus strains' submitted to the journal 'Infection and Immunity' which is contained within this thesis.; Kimberly M. Hilmer and Joshua J. Obar were co-authors of the article, 'Host-derived leukotriene B4 is critical for resistance against invasive pulmonary Aspergillosis' submitted to the journal 'Microbes and Infection Short Communication' which is contained within this thesis.Aspergillus fumigatus is a ubiquitous environmental mold, and even though most individuals are regularly exposed to fungal spores, clinical invasive disease is a rare manifestation. However, in the growing population of individuals with weakened immune systems, for example due to prolonged corticosteroid treatment or chemotherapeutic interventions, A. fumigatus exposure can cause severe, invasive aspergillosis (IA). Overall, invasive fungal infections are estimated to kill at least 1.5 million people annually (Brown et al. 2012), with IA being the most common and deadly invasive respiratory fungal infection. Thus, it is critical to better understand the host-pathogen interactions after A. fumigatus exposure in order to develop novel treatment options which harness the power of the host's immune response. Defining key immunological events that are needed for the prevention of Aspergillus growth within the pulmonary environment of immune competent individuals is an essential step toward a better understanding of how the immune response is altered within the immune compromised populations that are at risk of developing IA. Utilizing an immune competent murine model of IA, we have shown that signaling through both the Interleukin-1 receptor, type I (IL-1RI) and the Leukotriene B4 receptor (BLT1) are both critical pathways for host resistance against IA through timely neutrophil recruitment which ultimately control fungal germination. More recently, we have found that different environmental and clinical strains of A. fumigatus lead to different inflammatory profiles as well as different disease pathology. Strains that are able to germinate within the lung environment are more virulent, and lead to enhanced lung damage, vascular leakage and inflammation. Furthermore, the more virulent strains induce neutrophil recruitment and subsequent fungal clearance that is dependent on the alarmin IL-1alpha, while clearance of the less virulent strains are independent of IL-1alpha signaling. With this research we will better understand the fungal component(s) that are important in virulence determination, which immune pathways are contributing to the different disease pathologies observed, as well as understand the mechanism through which a healthy immune system can resist A. fumigatus exposure on a daily basis.Item Induction of cell-mediated immune responses with effector functions by antigens of Tritrichomonas foetus(Montana State University - Bozeman, College of Agriculture, 2001) Voyich, Jovanka Marija (Vujadinovich)Item Induction of innate immune responses by plant-derived procyanidins and polysaccharides(Montana State University - Bozeman, College of Agriculture, 2012) Holderness, Jeff Scott; Chairperson, Graduate Committee: Mark Jutila; Katie F. Daughenbaugh was a main author, and Jill C. Graff, Jodi F. Hedges, Brett Freedman, Joel W. Graff and Mark A. Jutila were co-authors of the article, 'Contribution of transcript stability to a conserved procyanidin-induced cytokine response in gamma delta T cells' in the journal 'Genes and immunity' which is contained within this thesis.; Mark A. Jutila was a co-author of the article, 'Oligomeric procyanidin-mediated transcript stability is dependent upon SYK and ERK kinases' which is contained within this thesis.; Igor A. Schepetkin, Brett Freedman, Liliya N. Kirpotina, Mark T. Quinn, Jodi F. Hedges and Mark A. Jutila were co-authors of the article, 'Polysaccharides isolated from acai fruit induce innate immune responses' in the journal 'PLoS ONE' which is contained within this thesis.; Brett Freedman, Jerod Skyberg, Sharon Kemoli, Jodi F. Hedges and Mark A. Jutila were co-authors of the article, 'Acai polysaccharides activate the innate immune system via combined TLR4 and DECTIN-1 activation' which is contained within this thesis.Plants contain most of the basic metabolic systems utilized by mammals, but also contain unique structures to interact with self and with non-self biomolecules. It is of little surprise, that many of these plant biomolecules impact mammalian systems. Numerous plant products are used for treating human disease and are critical for the most fundamental aspects of medicine including pain control and cancer therapy. In addition to these drugs, plant products have been used for millennia to improve disease resistance. Our understanding of how these plants activate the innate immune system to fight off infection is tenebrous, with very little understood about receptor-mediated responses. The following studies elaborate upon our current understanding of two common, plant-derived compounds with innate stimulatory activity: procyanidins and polysaccharides. Procyanidins are a class of polyphenols and flavonoids. The research described herein shows that procyanidins directly activated gamma delta T cells to enter a primed state and stabilized select gene transcripts via ERK- and syk-mediated processes. The second class of plant products discussed are polysaccharides from Acai. The innate immune response induced by Acai polysaccharides was mediated by TLR4 and the phagocytic response, possibly mediated by Dectin-1. These studies have improved our understanding of host responses to plant products, which have implications for consumption of both foods and nutritional supplements.Item Proteomic and systems biology analysis of the response of monocytes to infection by Coxiella burnetii and exposure to innate immune adjuvants(Montana State University - Bozeman, College of Letters & Science, 2010) Shipman, Matthew Richard; Chairperson, Graduate Committee: Edward Dratz.Coxiella burnetii is an obligate intracellular pathogen that infects human monocytes, specifically inhabiting the phagolysosome. C. burnetii is a potential bioterror agent and is classified by the National Institute for Allergies and Infectious Diseases (NIAID) as a category B pathogen. This bacterium is remarkably infectious, requiring as little as one bacterium to cause infection. We used phase II C. burnetii, an avirulent laboratory strain that acts as a model for wild type phase I strains. Our research was directed towards a deeper understanding of the monocyte proteome in response to a) infection by phase II C. burnetii, and b) exposure to immune adjuvants known to increase monocyte resistance to infection by C. burnetii. Monomac I cells were infected with phase II C. burnetii and aliquots were taken at 24, 48, and 96 hours postinfection. Experiments with immune adjuvants that increase monocyte killing of C. burnetii, involved Monomac I cells treated with Securinine, E. coli lipopolysaccharide (LPS), and monophosphoryl lipid A (MPL). Securinine is a GABA A receptor antagonist that is being developed at Montana State University for biodefense purposes, and triggers an innate immune response that differs from classic Toll-like receptor (TLR) stimulation of innate immunity represented by LPS and MPL. We employed multiplex 2D gel electrophoresis (m2DE) using ZDyes, a new generation of covalent fluorescent protein dyes being developed at Montana State University, coupled with MS/MS analysis and bioinformatics to determine the proteome changes in Monomac I cells in response to the conditions described above, and to develop a preliminary mechanistic model using a systems biology approach to account for the observed changes and propose multiple testable hypotheses to focus downstream research efforts. We also tested the effects on Monomac I cells infected with phase II C. burnetii +/- Securinine. We observed a high proportion of cell death in the + Securinine samples, using a dosage of Securinine higher than the optimal effective dosage. The information derived from this experiment will be useful in monitoring the tendency towards cell death in Securinine treated samples both from C. burnetii infected monocytes and other cell types (e.g. neurons) that contain GABA A receptors.Item Comprehensive transcriptional profiling of gamma delta T cells(Montana State University - Bozeman, College of Agriculture, 2005) Graff, Jill Christin; Chairperson, Graduate Committee: Mark Jutila.gamma delta T cells have been conserved since the adaptive immune system arose, yet their importance is still unclear. In an attempt to compensate for the lack of a broad knowledge-base of gamma delta T cells across species, global analyses of gamma delta T cell transcriptomes have been performed using serial analysis of gene expression (SAGE). Twelve new SAGE libraries were generated from the following bovine lymphocyte populations: magnetic bead-sorted blood gamma delta T cells, spleen gamma delta T cells and enriched alpha beta T cells from a single calf, both rested and ConA/IL-2 stimulated, and flow cytometry-sorted blood gamma delta and alpha beta T cells each either rested, ConA/IL-2, or PMA/ionomycin stimulated. These databases were analyzed using new web-based bioinformatic tools, which allow the user to rapidly compare gene expression patterns within these and other SAGE and standard EST libraries generated from different cell types and different species. These analyses revealed striking differences between blood and spleen gamma delta T cells and how these cells respond to various mitogenic stimulation. These analyses also confirm previous studies that suggest that global gene expression in gamma delta and alpha beta T cells is quite similar; however, a five-fold increase in gamma delta T cell-specific transcripts could be induced by ConA/IL-2 stimulation of spleen cells. Even greater differences were seen between the two lymphocyte populations isolated from blood, regardless of activation state. These new public databases provide additional resources for the annotation/analysis of global gene expression in gamma delta T cells, which will facilitate studies of the biology of this enigmatic lymphoid cell.