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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.Item Human gut phages in health and disease(Montana State University - Bozeman, College of Letters & Science, 2018) Manrique Ronquillo, Maria del Pilar; Chairperson, Graduate Committee: Mark J. Young; Michael S. Dills and Mark J. Young were co-authors of the article, 'The human gut phage community and its implications for health and disease' in the journal 'Viruses' which is contained within this dissertation.; Benjamin Bolduc, Seth T. Walk, John van der Oost, Willem M. de Vos and Mark J. Young were co-authors of the article, 'Healthy human gut phageome' in the journal 'Proceedings of the National Academy of Sciences of the United States of America' which is contained within this dissertation.; Mark J. Young was a co-author of the article, 'Interactions of the healthy gut phage community (HGP) with the core gut bacterial community' submitted to the journal 'PLOS computational biology' which is contained within this dissertation.; Yifan Zhu, John van der Oost, Willem M. de Vos and Mark J. Young were co-authors of the article, 'Gut bacteriophages and fecal microbial transplantation outcome in subjects with metabolic syndrome' which is contained within this dissertation.; Seth T. Walk and Mark J. Young were co-authors of the article, 'Bacteriophage-enriched filtrates: a potential tool to modify the structure of the gut-associated bacterial community' which is contained within this dissertation.The human body is colonized by a diverse microbial community known as the human microbiota. Most of these microbes, reside in the human intestinal tract. The gut microbiota has coevolved with humans and has become essential for multiple physiological functions that range from digestion, to development of the immune system, protection for pathogens, and even behavior. The gut microbial community is primarily dominated by Bacteria and their viruses- bacteriophages (or phages for short). Even though our knowledge of the contribution of the former to human health is extensive, the role of bacteriophages in human health and disease has been explored considerably less. Study of bacteriophages in other microbial environments has highlighted their importance in influencing the structure and function of their host community. Therefore, understanding the role of bacteriophages in the human gut ecosystem, and overall, in human health, has become a focus of current research. The main overarching hypothesis of this thesis is that human gut bacteriophages contribute to human health. To test this hypothesis, viral metagenomic surveys of healthy and disease individuals, together with experiments in a gnotobiotic mouse model system were performed. A group of bacteriophages shared among healthy individuals and significantly depleted in individuals with IBD was identified. Moreover, a host reservoir for these phages was identified in the core gut bacterial community of healthy subjects. Study of phage dynamics during an FMT treatment in patients with metabolic syndrome further highlighted the association of bacteriophages with human health. Patients that showed significant clinical improvement harbored a richer community, and a community more similar to healthy donors than patients that did not respond to the treatment. Moreover, a set of potential phage biomarkers associated with health and treatment outcome were identified. Lastly, experiments in gnotobiotic mice demonstrated the ability of bacteriophage-enriched filtrates to modify the microbial community structure. This result highlights the potential use of bacteriophages to manipulate the human gut microbiota, and potentially restore human health.Item Mechanisms of CRISPR-mediated immunity in Escherichia coli(Montana State University - Bozeman, College of Letters & Science, 2019) van Erp, Paul Bertram Geert; Chairperson, Graduate Committee: Blake Wiedenheft; Gary Bloomer, Royce Wilkinson and Blake Wiedenheft were co-authors of the article, 'The history and market impact of CRISPR RNA-guided nucleases' in the journal 'Current opinion in virology' which is contained within this thesis.; Ryan N. Jackson and Joshua Carter were authors and Sarah M. Golden, Scott Bailey and Blake Wiedenheft were co-authors of the article, 'Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli' in the journal 'Nucleic acids research' which is contained within this thesis.; Angela Patterson was an author and Ravi Kant, Luke Berry, Sarah M. Golden, Brittney L. Forsman, Joshua Carter, Ryan N. Jackson, Brian Bothner, and Blake Wiedenheft were co-authors of the article, 'Conformational dynamics of DNA binding and CAS3 recruitment by the CRISPR RNA-guided cascade complex' in the journal 'ACS chemical biology' which is contained within this thesis.; Tanner Wiegand, Royce A. Wilkinson, Laina Hall, Dominick Faith and Blake Wiedenheft were co-authors of the article, 'Protein overexpression reduces specific phage infectivity in prokaryotic argonaute screen' which is contained within this thesis.; Dissertation contains three articles of which Paul Bertram Geert van Erp is not the main author.Prokaryotes are under constant threat from foreign genetic elements such as viruses and plasmids. To defend themselves against these genetic invaders prokaryotes have evolved extensive defense mechanisms. In this thesis I explore two such defense systems: prokaryotic Argonautes and CRISPR-systems. CRISPR-systems acquire short sequences derived from foreign genetic elements and store them in the CRISPR locus. In subsequent rounds of infection these stored sequences are used as guides by Cas proteins to target the invaders. Escherichia coli K-12 contains a type I-E CRISPR system, consisting of two CRISPR loci and eight cas genes. five of these cas genes, together with and 61-nucleotide CRISPR-RNA guide form the RNA-guided surveillance complex Cascade. This complex finds and binds foreign DNA targets that are complementary to its RNA guide. After target binding the helicase/nuclease Cas3 is recruited to the Cascade-DNA complex for destruction of the target. The goal of this research is to understand the molecular mechanisms that lead to target recognition and destruction in the type I-E CRISPR systems. Atomic resolution structures of the proteins involved in these CRISPR systems provide the blueprints of these proteins machines. Structure guided mutational analysis coupled with in vivo and in vitro biochemical experiments are used to investigate the underlying molecular mechanisms of this CRISPR system. Together, these results explain the rules of target recognition and Cas3 recruitment. Prokaryotic Argonautes have been hypothesized to defend against mobile genetic elements such as plasmids and viruses through guided nuclease activity. To test this hypothesis, we overexpressed 8 phylogenetically diverse prokaryotic Argonautes proteins in Escherichia coli and challenged them with seven bacteriophages. This resulted in robust protection against phage Lambda and phage P1 by four of the tested Argonautes, while little impact on phage infectivity was observed for the other phages tested. However, control experiments with a nuclease inactive Argonaute mutant and expression of an unrelated control protein showed similar protection against phage Lambda and phage P1. Collectively, our data suggest that protein overexpression in general, rather than Argonaute expression in particular, results in protection against 2 specific phages.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 Bacteriophage in host associated microbial communities examined with continuous culture systems(Montana State University - Bozeman, College of Letters & Science, 2018) Dills, Michael Stefan; Co-Chairs, Graduate Committee: Mark J. Young and Seth WalkMechanistic understanding of the role of extracellular and parasitic elements in host ecosystems is currently lacking. Extensive surveys have catalogued a large diversity of bacteriophage which associate differentially with definable host states. This work is an attempt to aid in the development of a coherent model for complex symbiosis within mammalian host ecosystems by investigating the role of bacteriophage in microbial community structure. It details an investigation of continuous culture systems as a platform to study bacteriophage within polymicrobial communities of the human GI tract. It then describes an experiment testing an extracellular community's ability to modulate bacterial community structure.Item Biophysical characterization of P22 bacteriophage and adenoassociated viruses(Montana State University - Bozeman, College of Letters & Science, 2016) Kant, Ravi; Chairperson, Graduate Committee: Brian Bothner; Aida Llauro, Vamseedhar Rayaprolu, Shefah Qazi, Pedro J. de Pablo, Trevor Douglas and Brian Bothner were co-authors of the article, 'Stability, biomechanics and structural changes in P22 bacteriophage during maturation' which is contained within this thesis.; Vamseedhar Rayaprolu and Brian Bothner were co-authors of the article, 'Understanding of P22 bacteriophage maturation by QCM-D' which is contained within this thesis.; Navid Movahed, Dewey Brooke, Antonette Bennett, Mavis Agbandje-McKenna and Brian Bothner were co-authors of the article, 'Prolonged incubation with liposome leads to PLA2 activation in adeno-associated viruses' which is contained within this thesis.; Vamseedhar Rayaprolu and Brian Bothner were co-authors of the article, 'Comparison of the visco-elastic properties of viruses, virus based nanomaterials and active protein cages' which is contained within this thesis.The dsDNA tailed bacteriophages comprise the largest evolving life form in the biosphere. They are not only the most abundant organism on Earth, but also plausibly the most ancient. The ancient origin of phage suggests that they have had the ample opportunity to undergo the evolutionary changes necessary to perform intricate coordinated biological functions. Therefore, characterizing a tailed bacteriophage will help not only to understand biology, but also help us to establish a relationship between structure and function. Viruses display a dynamic equilibrium between structural conformations, stability, flexibility and rigidity which is essential for the perpetuation of life cycle. Understanding this complex biophysical relationship is a daunting task and requires a combination of multidimensional approaches. P22 is a tailed bacteriophage and displays a series of structural transitions during maturation. To understand the important biophysical changes in the P22 at different stages of maturation, we have introduced a suite of orthogonal techniques to address the distinct properties of intermediates. These include Differential Scanning Fluorimetry which probes the thermal stability of P22 capsids, Hydrogen-Deuterium Mass Spectrometry, which probes the conformational flexibility and Atomic Force Microscopy and Quartz Crystal Microbalance with dissipation, which probe the biomechanical transformation in the capsids. P22 investigation using these techniques reveals the large scale structural arrangements along with the expansion. Global rearrangement results in an increase in stability, rigidity and reduced dynamics. The sum results of these studies indicate that expansion is accompanied by large scale inter-subunit rearrangements which lead to the enhanced hydrophobic core at different quasi-equivalent axes. We have also studied Adeno-associated viruses, which is used as a gene delivery vehicle for the treatment of genetic disorders. AAVs lipase contains a lipase domain and its activation is important for the successful infection. Activation mechanism of lipase domain is not thoroughly understood. To understand the mechanism, we have developed a Liquid Chromatography-Mass Spectrometry assay sensitive enough to measure lipase products. This assay confirms that prolonged incubation of AAVs with liposome is able to activate the lipase domain without the involvement of receptors and co-receptors.Item The development of hybrid biomaterials using the virus-like particle (VLP) from bacteriophage P22(Montana State University - Bozeman, College of Letters & Science, 2016) Edwards, Ethan James; Chairperson, Graduate Committee: Trevor Douglas; Rajarshi Roychoudhury, Benjamin Schwarz, Paul Jordan, John Lisher and Trevor Douglas were co-authors of the article, 'Co-localization of catalysts within a protein cage leads to efficient photochemical NADH and/or hydrogen production' which is contained within this thesis.; Dissertation contains several articles of which Ethan James Edwards is not the main author.A broad range of bio-composite materials have been developed through inspiration from biology. In particular, natural systems that confine, co-localize and protect their contents has inspired the design and synthesis of the P22 virus-like particle (VLP) to effect a suite of biomaterials. These materials were realized by taking advantage of the native protein architecture of P22 as an initiation point and platform for material synthesis. Introducing a reactive cysteine on the P22 coat protein provided an initiation point for polymer synthesis. Atom transfer radical polymerization (ATRP) was initiated creating a polymer framework on the interior of the P22 VLP. Using this polymerization technique (ATRP) a photocatalytic crosslinker was successfully incorporated for reduction of methyl viologen. Next, a manganese porphyrin imaging agent was loaded creating a T 1-enhanced MRI contrast agent, as an alternative to the highly toxic Gadolinium currently used. Inspired by photosynthetic machinery, the P22-xAEMA system was labeled with a co-catalyst system, creating a co-localized photocatalytic nanoparticle capable of photochemically producing NADH/hydrogen. The production was controlled by labeling density of catalysts resulting in a tunable biomaterial. The design of a complex bio-hybrid material was developed by combining both synthetic and genetic approaches. Coupling the enzyme Alcohol Dehydrogenase D from Pyrococcus furiosis with a small molecule catalyst led to a coupled catalytic system between a synthetic catalyst and biologically derived enzyme. The P22 VLP system was studied by atomic force microscopy (AFM) and cryoelectron microscopy (cryo-EM) unraveling its biophysical properties and providing insights for further material design. 2D-crystal arrays were formed from a variety of P22-protein biomaterials, for the development of functional P22 arrays. Lastly, the P22 VLP was monitored by charge detection mass spectrometry, giving insight into the stability of the scaffolding protein. These studies show the versatility of this system for both material synthesis and fundamental biochemical understandings. Overall, the work here continues to progress and push the boundaries of protein cage nanoparticles as platforms for material synthesis. The development of hybrid biomaterials from VLPs serve to improve our basic understandings of the natural systems they are derived from and provide additional design principles for improved complex biohybrid materials.Item Expression and purification of two CRISPR-CAS proteins, Csm3 and Csm5 from Mycobacterium tuberculosis(Montana State University - Bozeman, College of Letters & Science, 2015) Hashimi, Marziah; Chairperson, Graduate Committee: C. Martin LawrenceOne third of the World's population is infected with tuberculosis (TB). TB disease is caused by bacterium called Mycobacterim tuberculosis, which is a facultative intracellular parasite that is transferred through the air from one person to another in close contact. A six month course of four antimicrobial drugs is the only current treatment for drug-sensitive TB. However, multi-drug resistance TB is difficult to treat. Phage therapy might be one answer as a treatment for multi-drug resistance TB. In order for phage therapy to have a chance against TB, the immune system of bacteria, known as CRISPR/Cas needs to be inhibited. Our lab has taken a structural and biochemical approach to try to understand the CRISPR/Cas system in M. tuberculosis. We have cloned, expressed, and purified individual Csm proteins from the H37Rv M. tuberculosis strain. Two Csm protein, Csm3 and Csm5 were successfully purified to homogeneity in yields suitable for structure and biochemical studies. While to date, each has failed to produce crystals, the ability to the express and purify each of these proteins will allow further biochemical characterization of Csm3 and Csm5.Item Bacteriocins of Vibrio cholerae in relation to defective bacteriophage(Montana State University - Bozeman, College of Agriculture, 1970) Schalie, John Vander