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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 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 Bacteriocins of Vibrio cholerae in relation to defective bacteriophage(Montana State University - Bozeman, College of Agriculture, 1970) Schalie, John VanderItem Studies of peptide mimicry of the group B Streptococcus type III capsular polysaccharide antigen(Montana State University - Bozeman, College of Letters & Science, 2007) Pomwised, Rattanaruji; Chairperson, Graduate Committee: Mark Young; Seth Pincus (co-chair)Capsular polysaccharide (CPS) of Streptococcus group B (GBS) is a poor immunogen and functions as T cell independent antigen, eliciting low IgG antibody with deficient immunologic memory. We previously identified a peptide, S9, which mimics CPS of type III GBS. Here we have taken steps to develop the mimetic peptide as a vaccine against GBS group III. We enhanced the immunogenicity of the peptide by presenting it on the coat protein of Cowpea Chlorotic Mottle Virus (CCMV). And we searched for better mimetic peptides by constructing a secondary phage display library. To accomplish the first goal, DNA encoding S9 was cloned into five constructions CCMV coat protein loops using recombinant DNA techniques. The results indicated that inserting the S9 peptide sequence into CCMV coat protein loops disrupted virus and virus-like particle assembly. Therefore the S9 peptide was conjugated to CCMV coat protein using chemical linkers. The CCMV-S9 conjugation products remained intact as monomer virions.