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Browsing by Author "Slayden, Richard, advisor"

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    Bioinformatic identification and characterization of cytokinetic regulators in Mtb
    (Colorado State University. Libraries, 2013) Crew, Rebecca M., author; Slayden, Richard, advisor; Jackson, Mary, committee member; Hanneman, William, committee member
    A fundamental lack of understanding of Mtb regulation during latent tuberculosis infections (LTBI), which comprises the vast majority of tuberculosis cases, has hindered global eradication efforts. To elucidate mechanisms associated with transition to the non-replicating persistent (NRP) state associated with LTBI, we set out to identify regulators involved in cell division control in Mtb. Bioinformatic analysis identified rv1708 as encoding a MinD-like protein putatively involved in septum placement, and rv2216 as encoding a potential SOS-associated cell division inhibitor, SulA. Bioinformatic-based assessments of orthology revealed a differential lineage than anticipated for the proteins encoded by both open reading frames (ORFs). We describe these two novel regulators in Mtb here for the first time. It was found that Rv1708 lacks regions vital for MinD function and shows greater similarity with the Soj protein from Bacillus sp. involved in the regulation of sporulation and timing of division. Due to these similarities we have re-named Rv1708 as SojMtb. Significantly, SojMtb shows potential as a cytokinetic and dormancy regulator both by homology, morphology, and growth kinetic analysis. Overexpression of sojMtb attenuates growth and elicits filamentation characteristic of a disruption in early division, similar to Soj activity in other organisms. Given the role of Soj in the control of dormancy phenotypes in Bacillus sp. we believe SojMtb serves as an important regulator during dormancy transitions in mycobacteria, as associated with the development of LTBI. Although Rv2216 was initially identified by homology to SulA proteins, analysis of orthology indicates greater similarity with a separate group of widely conserved yet poorly defined cell division regulatory proteins. Thusly, we have re-name rv2216 as cdr for cell division regulator. Cdr proteins share limited similarity to SulA: enough to be mis-identified in organisms lacking a true SulA but insufficient to infer similar functionalities. Cdr proteins are present in hundreds of organisms through different walks of life, yet this work presents the first characterization of their effects on cellular activity. Induction of the SOS response by Mitomycin C treatment did not induce cdr expression, supporting our classification Cdr proteins separate from SulA. Overexpression of cdr resulted in a bimodal increase in cell length without an apparent effect on growth kinetics, suggesting Cdr stimulation of cellular elongation relative to division. Profiling of cell cycle discriminant genes in response to cdr overexpression corroborates this hypothesis, showing an induction of late division events associated with the production of new plasma membrane and cell wall components. Sub-cellular localization studies using an inducible Cdr-GFP fusion protein revealed cell cycle-dependent localization to the inner membrane at sites involved in cell wall and plasma membrane growth and remodeling. Furthermore, global transcriptional analysis revealed a unique profile of adaptive programs associated with hypoxia-associated NRP, de novo lipid synthesis and phospholipid/triacylglycerol turnover. These processes are required for normal growth and promote homeostasis during times of stress by preventing and repairing oxidative damage to membrane constituents in diverse organisms. Importantly, Cdr represents a novel regulatory class of proteins with broad representation in all classifications of life, potentially involved in division and stress responses associated with dormancy, and is described here for the first time in Mtb. The foundation provided here, both for SojMtb and Cdr, provides insight into the regulatory mechanisms employed during NRP transitions associated with LTBI, and will aid in the development and implementation of more targeted studied in the future.
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    Drug discovery for Francisella tularensis and Mycobacterium tuberculosis
    (Colorado State University. Libraries, 2009) Brostrom, Kathleen England, author; Slayden, Richard, advisor
    Today, we are faced with the challenges of fighting infectious organisms that have either been neglected or have developed ways to resist current treatments. Longstanding public health problems, such as tuberculosis, have evolved into mulitdrug-resistant bacilli, tolerant to current drug regimens. In addition, neglected diseases such as F. tularensis have concerns regarding their use in bioterrorism. Current research initiatives search for novel chemotherapeutics to alternative targets in order to resolve the problem of resistance and the continued battle against infectious disease. The research presented utilizes both genomic and structure-based approaches to identify new drug targets for Mycobacterium tuberculosis and optimize a series of diphenyl ether compounds with specific inhibitory activity against ftuFabI, the enoyl ACP-reductase enzyme of Francisella tularensis.
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    Investigation of RelBE1 toxin-antitoxin function in the carbon-dependent metabolic adaptation of Mycobacterium tuberculosis
    (Colorado State University. Libraries, 2022) Starkey, Julie M., author; Slayden, Richard, advisor; Dobos, Karen, committee member; Abdo, Zaid, committee member; Tjalkens, Ron, committee member
    Tuberculosis (TB) is a devastating disease with suboptimal treatment regimens and a single vaccine with variable efficacy. Reducing the global burden of TB requires a refined arsenal of methods to prevent and treat the disease, which necessitates a better understanding of M. tuberculosis (Mtb) pathogenesis during infection. Mtb undergoes continuous metabolic reprogramming throughout acute and chronic stages of infection in order to survive and persist harsh host conditions, and the regulatory network responsible for mediating metabolic adaptation has not been fully defined. Mtb harbors at least 88 Toxin-antitoxin (TA) loci that have been proposed to function as regulatory modules in response to stress. TA systems are uniquely abundant in Mtb, making them viable targets for the treatment of both active and latent infection. Several RelBE TA systems are present in Mtb, and the RelE toxins function as ribonucleases to inhibit translation when not bound to RelB antitoxins. The genes encoding relBE1 are adjacent to a gene that encodes an enzyme involved in central carbon metabolism, which could suggest a regulatory role for RelBE1 in carbon metabolism. We aimed to explore the relationship between the RelBE1 TA system and carbon-mediated metabolic adaptation. This work incorporated in vitro transcriptional and genetic studies under defined carbon sources to investigate the activity of RelBE1 and the requirement of RelE1 in Mtb metabolism, growth, and viability in the presence of different carbon sources. We observed transcriptional and physiological trends consistent with the hypothesis that RelBE1 contributes to iii adaptation of Mtb metabolism in the presence of cholesterol and oleate. Additionally, we found evidence that supports the necessity of RelE1 in Mtb metabolism under conditions depleted of nutrients. To investigate if multiple RelBE systems work redundantly or cooperatively in Mtb metabolic adaptation, we applied CRISPRi to simultaneously silence three RelBE TA loci. CRISPRi construction of knockdown mutants resulted in variable success but did not fully resolve the question regarding the cooperative or redundant functions of RelBE systems in Mtb metabolism. Nonetheless, the study provided the building blocks for efficient genetic manipulation of multiple TA systems in Mtb that are essential for exploring the coordination of TA systems in their contribution to Mtb pathogenesis. This thesis work contributes to the debate regarding TA system function in Mtb stress response and adaptation during infection. Given the limitations of the presented studies, further work is warranted to elucidate the relationship between TA systems and Mtb pathogenesis. Expanding our understanding of TA systems in TB disease would provide novel avenues in research to improve treatments against TB.