Scholarship & Research
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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.