Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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Item Structural characterization of the Csa3/cA4 complex - a nexus for class 1 CRISPR-Cas immune response coordination & establishing a cure for highly efficient galectin expression(Montana State University - Bozeman, College of Letters & Science, 2024) Charbonneau, Alexander Anthony; Chairperson, Graduate Committee: C. Martin Lawrence; This is a manuscript style paper that includes co-authored chapters.Though Class I CRISPR-Cas systems, primarily Type I and Type III, are the most abundant CRISPR systems in archaea and bacteria, mechanisms driving their immune response regulation are not well understood. Csa3 family transcription factors, composed of N-terminal CARF and C-terminal winged helix-turn-helix domains, are frequently encoded within Type I CRISPR-Cas systems. Csa3 transcription factors are hypothesized to bind cyclic oligoadenylate (cOA) second messengers produced by Type III interference complexes, likely modulating their DNA-binding activity. Therefore, we investigated the interaction between Csa3a and cyclic tetra-adenylate (cA4). Isothermal titration microcalorimetry showed S. solfataricus Csa3a binds cA4 at biologically relevant concentrations in an entropically driven interaction. Ring nuclease assays revealed Csa3a lacks self-regulatory phosphodiesterase activity exhibited by other CARF domain proteins. We crystallized and solved the structure of the Csa3/cA4 complex, which revealed conserved motifs are responsible for cA4 binding and illuminated significant conformational changes induced by the interaction. We also identified an 18-bp palindromic motif, which we designated CAPPa, that is conserved in the 27 sequenced members of the order Sulfolobales, and shows synteny with Csa3a and acquisition genes in these genomes. We found Csa3a binds CAPPa in a nonspecific, cooperative, and cA4-independent manner. These characteristics suggest a more complex method of transcriptional regulation than previously hypothesized. However, the interaction between Csa3a and cA4 confirmed here signifies a nexus between Type I and Type III systems; we thus propose a model in which this interaction coordinates the two arms of an integrated immune system to mount a synergistic, highly orchestrated, adaptive immune response. We applied the workflow designed to produce significant protein quantities for crystallographic studies of Csa3a to the study of Homo sapiens galectin proteins, a family of beta-galactoside-binding proteins. Here, we identified a putative autoinhibitory mechanism affecting traditional IPTG-induction methods by characterizing IPTG-binding capabilities of galectins and quantifying basal protein expression over various IPTG concentrations. To bypass this predicted feedback loop, we employed a highly efficient and approachable autoinduction method, resulting in a 7-fold increase in protein expression. Much of this work was done in the context of a course-based undergraduate research experience with great success.Item Quantifying robustness of the gap gene network(Montana State University - Bozeman, College of Letters & Science, 2024) Andreas, Elizabeth Anne; Chairperson, Graduate Committee: Tomas Gedeon; Bree Cummins (co-chair)Early development of Drosophila melanogaster (fruit fly) facilitated by the gap gene network has been shown to be incredibly robust, and the same patterns emerge even when the process is seriously disrupted. We investigate this robustness using a previously developed computational framework called DSGRN (Dynamic Signatures Generated by Regulatory Networks). Our mathematical innovations include the conceptual extension of this established modeling technique to enable modeling of spatially monotone environmental effects, as well as the development of a collection of graph theoretic robustness scores for network models. This allows us to rank order the robustness of network models of cellular systems where each cell contains the same genetic network topology but operates under a parameter regime that changes continuously from cell to cell. We demonstrate the power of this method by comparing the robustness of two previously introduced network models of gap gene expression along the anterior-posterior axis of the fruit fly embryo, both to each other and to a random sample of networks with same number of nodes and edges. We observe that there is a substantial difference in robustness scores between the two models. Our biological insight is that random network topologies are in general capable of reproducing complex patterns of expression, but that using measures of robustness to rank order networks permits a large reduction in hypothesis space for highly conserved systems such as developmental networks.Item Investigating the regulation of virulence by Sae in Staphylococcus aureus(Montana State University - Bozeman, College of Agriculture, 2020) Collins, Madison Paige Martin; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Ranjan K. Behera, Kyler B. Pallister, Tyler J. Evans, Owen Burroughs, Caralyn Flack, Fermin E. Guerra, Willis Pullman, Brock Cone, Jennifer G. Dankoff, Tyler K. Nygaard, Shaun R. Brinsmade and Jovanka M. Voyich were co-authors of the article, 'The accessory gene saeP of the saeR/S two-component gene regulatory system impacts Staphylococcus aureus virulence during neutrophil interaction' in the journal 'Frontiers in microbiology' which is contained within this dissertation.; Kyler Pallister and Jovanka M. Voyich were co-authors of the article, 'Differential analysis of host/pathogen RNA expression via next generation sequencing reveals Staphylococcus aureus utilizes saeR/S-mediated factors to inhibit human neutrophil functions following phagocytosis' which is contained within this dissertation.Staphylococcus aureus (S. aureus) is a common commensal bacterium known to colonize, at minimum, 30% of the human population. It is also capable of causing a range of diseases that span from minor skin- and soft-tissue infections to life-threatening diseases. The diversity of S. aureus infections is due to the ability of the bacteria to sense and respond to environmental change. Virulence regulation in S. aureus can be attributed to the use of two-component gene regulatory systems (TCS). TCS can sense a variety of encounters including: antibiotics, heat stress, or immune cell encounter. Neutrophils are a key leukocyte involved in bacterial clearance in the human host. It follows that S. aureus has evolved mechanisms to sense and respond to neutrophils. The Sae TCS, is immediately up-regulated after neutrophil phagocytosis and has been demonstrated to be critical in the success of S. aureus both in vitro and in vivo. SaeS, the histidine kinase, and the respective response regulator, SaeR, are established components of the Sae TCS and their importance during neutrophil evasion and pathogenesis is well established. However, little is known about two accessory genes, saeP and saeQ. Results described herein using human neutrophil and murine models of infection provide evidence that SaeP modulates the Sae-mediated response of S. aureus against human neutrophils and suggest that saeQ and saeP together impact pathogenesis in vivo. To identify additional host and pathogen factors important during neutrophil interaction, we used differential analysis of host/pathogen RNA expression via Next Generation Sequencing to define the influence of SaeR/S on the host-pathogen transcriptome following neutrophil phagocytosis. Results determined that in the early stages of S. aureus infection, SaeR/S-dependent factors significantly modulate neutrophil processes involved in several pathways including autophagy, TNF-alpha signaling, and NF-kappaB signaling. These results suggest S. aureus uses SaeR/S-regulated virulence factors to hijack human neutrophil function at the transcriptional level to inhibit proper killing by neutrophils and allow for S. aureus persistence within the host.