Theses and Dissertations at Montana State University (MSU)
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Item The paradoxical role of ATP in viral infections and cellular defense(Montana State University - Bozeman, College of Agriculture, 2024) Zahl, Trevor Ronald; Chairperson, Graduate Committee: Blake Wiedenheft; This is a manuscript style paper that includes co-authored chapters.Conflict between prokaryotes and viruses has driven the evolution of diverse defense systems. The study of these systems has not only expanded our knowledge of the mechanisms underlying genetic conflict, but has also revolutionized biotechnologies. I joined the Wiedenheft lab in early 2021 during the height of the SARS-CoV-2 pandemic. As such, the lab was focused on developing technologies to aid in the identification and study of SARS-CoV-2. I joined a team that was actively repurposing a type III CRISPR system as a molecular diagnostic. Type III CRISPRs are RNA targeting systems that unleash antiviral signaling cascades upon recognition of a target. Previous work in the lab had shown that this activity could be repurposed as a molecular diagnostic, but the first generation technology was not sensitive enough for clinical use. To address this need, we developed a technology termed Capture and Concentrate, which utilized 'CRISPR magnets' to concentrate target RNAs from complex mixtures. Using this technology, we increased diagnostic sensitivity by 1,000 fold. In addition to my work at the bench, I developed an in silico model of the diagnostic to inform future development efforts. While type III CRISPRs are unique due to their ability to generate antiviral signaling cascades, they also contain intrinsic endonuclease activity. In 2023 we demonstrated that this activity could be repurposed as an RNA editing technology. Using the type III system from Streptococcus thermophilus, we introduced precise genomic modifications in a recombinant sindbis virus. This technology removed the need for reverse genetics systems, which hampered the study of the rapidly evolving SARS-CoV-2 virus. In the final year of my graduate studies, I worked with a team focused on determining how, PARIS, a bacterial innate immune system, induced host death upon viral infection. PARIS is a two-component system comprised of the ATPase, AriA, and a nuclease, AriB. Previous studies had shown that PARIS induced abortive infection in response to viral proteins, but the mechanism was not known. We determined that upon antigen recognition, AriB is activated and cleaves host tRNAs to induce cell death and prevent community spread.Item Identification of novel ssDNA and RNA coliphage in wastewater(Montana State University - Bozeman, College of Agriculture, 2024) Little, Agusta Rio; Chairperson, Graduate Committee: Blake WiedenheftBacteriophages (phages) are the most abundant biological entities on Earth. However, our understanding of their diversity is limited, with a vast gap in knowledge regarding single- stranded DNA (ssDNA) and RNA phages. This study addresses this gap by isolating and characterizing ssDNA and RNA coliphages from wastewater, a suspected rich source of these understudied phages. Traditional phage isolation methods favor double-stranded DNA (dsDNA) phages, resulting in the underrepresentation of ssDNA and RNA phages. To overcome this bias, we employed enrichment strategies using small molecules that inhibit dsDNA phage replication. Additionally, we utilized an RNase-A assay to identify potential RNA phage candidates. These enrichment techniques led to the isolation of a circular ssDNA phage (POI 1) and a ssRNA phage (POI 8). A combination of biochemical assays, sequencing, and microscopy techniques were utilized to characterize these phages. Overall, this work demonstrates the effectiveness of enrichment strategies for isolating ssDNA and RNA phages and underscores the importance of developing optimized techniques to unlock the true diversity of these understudied phage populations.Item Study of diverse host immune responses to viral and bacterial pathogens(Montana State University - Bozeman, College of Agriculture, 2023) Plewa, Jack Bruno; Chairperson, Graduate Committee: Mark Jutila; This is a manuscript style paper that includes co-authored chapters.Brucella abortus is the bacterium that causes brucellosis, an infection transmitted from cattle to people, often through consumption of raw milk and contact with aborted materials. With antibiotic resistance on the rise, phage therapy for bacterial infection may become a useful approach. The direct effects of phage on mammalian cells is important to understand, yet understudied. In vivo delivery of low phage MOI to the mouse lung was more effective at diminishing Brucella burden than higher doses of phage. In an in vitro model of intracellular Brucella infection, low phage MOI was capable of minimizing human THP-1 monocyte infection, but, unexpectedly, use of higher phage MOI diminished this effect. We hypothesized that recognition of these phage preparations may induce an antiviral immune suppressive response that may counteract their anti-bacterial effects. Indeed, when the type I IFN signaling pathway was disrupted in mice, phage treatment was more effective. However, when attempting to induce type I IFN in vitro using both human monocyte and mouse macrophage cell lines, we were unable to stimulate expression of type I IFN with Brucella phage, including in response to a combination of phage and bacteria. We then examined the effect of phage treatment on macrophage cell surface markers that are indicative of activation/differentiation. Interestingly, while Brucella LPS induced expression of CD71 and CD206, the addition of phage suppressed upregulation of these markers. Our discovery of immune suppressive effects of Brucella bacteriophage is an important consideration for using phage as a treatment.