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Title: Meta-analyses of gene expression in age-dependent diseases
Creator: Rogers, Lavida Rashida Kenera
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Physiological changes with age such as immune system decline and brain aging cause an increased risk for diseases. Age-related diseases are of major concern especially in the elderly population due to there being an increase in the average lifespan. This dissertation explores neurodegenerative and respiratory diseases and how gene expression varies due to disease status, age tissue and sex.Alzheimer's disease (AD) has been categorized by the Centers for Disease Control and Prevention (CDC) as...
Show morePhysiological changes with age such as immune system decline and brain aging cause an increased risk for diseases. Age-related diseases are of major concern especially in the elderly population due to there being an increase in the average lifespan. This dissertation explores neurodegenerative and respiratory diseases and how gene expression varies due to disease status, age tissue and sex.Alzheimer's disease (AD) has been categorized by the Centers for Disease Control and Prevention (CDC) as the 6th leading cause of death in the United States. AD is a significant health-care burden because of its increased occurrence (specifically in the elderly population), and the lack of effective treatments and preventive methods. AD targets neuronal function and can cause neuronal loss due to the buildup of amyloid-beta plaques and intracellular neurofibrillary tangles.The respiratory disease, chronic obstructive pulmonary disease (COPD), was classified by the Centers for Disease Control and Prevention in 2014 as the 3rd leading cause of death in the United States. The main cause of COPD is exposure to tobacco smoke and air pollutants. In addition to exploring genetic variation due to disease state, sex and age we also explored the role of smoking status on expression profiles.Additionally, the respiratory infections, influenza and pneumonia affect thousands of people worldwide. Young children, elderly and immunocompromised individuals are at higher risk for being infected by the influenza virus and Streptococcus pneumoniae. Host responses to these pathogens and vaccinations vary by the state of one's immune system.This dissertation includes multiple meta-analyses to assess genetic variation in Alzheimer's disease, COPD and Influenza, and an assessment of pneumococcal disease and aging. To identify significant differentially expressed genes we ran an analysis of variance with a linear model with disease state, age, sex, tissue, smoking status and study as effects that also included binaryinteractions.Our meta-analysis approach effectively combined multiple publicly available microarray datasetsto identify gene expression differences across diseases including full age, sex, smoking status and tissue type considerations. Our findings provide potential gene and pathway associations that can be targeted to improve treatment and prevention of diseases.
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Title: Reducing iron on a knife's edge - genomic and mechanistic studies of (hyper)thermophilic dissimilatory iron-reducing bacteria and archaea
Creator: Manzella, Michael P.
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Some members of the Bacteria and the Archaea have the rare ability to transfer electrons beyond the surface of their cells. This extracellular electron transfer permits the reduction of otherwise inaccessible electron acceptors such as insoluble Fe(III) oxides. The mechanisms that enable this ability have direct implications for geochemical cycles today and for life on early Earth. The physical settings present on early Earth, hot and influenced primarily by hydrothermal activity, can be...
Show moreSome members of the Bacteria and the Archaea have the rare ability to transfer electrons beyond the surface of their cells. This extracellular electron transfer permits the reduction of otherwise inaccessible electron acceptors such as insoluble Fe(III) oxides. The mechanisms that enable this ability have direct implications for geochemical cycles today and for life on early Earth. The physical settings present on early Earth, hot and influenced primarily by hydrothermal activity, can be found in rare sites on modern Earth. These sites most often surround deep-sea hydrothermal vents. Organisms present at these sites thrive, in most cases, absent of the sun’s influence. Instead of primary production from photosynthetic microorganisms, these communities rest on the shoulders of chemoautolithotrophic bacteria and archaea. Two of these organisms, the thermophilic bacterium Geothermobacter ehrlichii and the hyperthermophilic archaeon Geoglobus ahangari were selected to undergo genome and physiological characterization to determine how they interact with the abundant insoluble Fe(III) oxides found at hydrothermal vents. Both genomes were sequenced and, while only the genome of G. ahangari was complete, this permitted identification of critical components for iron respiration. In addition, mechanistic studies were performed on G. ahangari to elucidate a direct-contact mechanism of iron reduction. Finally, the extracellular filaments from these microorganisms were characterized and the more abundant filaments, in both organisms, were found to be conductive. These are the first examples of nanowires discovered outside of the mesophilic bacteria. In addition, the phylogenetic and geographic diversity between these isolates suggests that microbial nanowires are more widespread than previously thought.
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Title: The role of the second messenger cyclic di-GMP in light-dependent responses in cyanobacteria
Creator: Agostoni, Marco
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Second messengers are intracellular substances regulated by specific external stimuli globally known as first messengers. Cells rely on second messengers to generate rapid responses to environmental changes and the importance of their roles is becoming increasingly realized in cellular signaling research. Cyanobacteria are photooxygenic bacteria that inhabit most of Earth's environments. The ability of cyanobacteria to survive in ecologically diverse habitats is due to their capacity to adapt...
Show moreSecond messengers are intracellular substances regulated by specific external stimuli globally known as first messengers. Cells rely on second messengers to generate rapid responses to environmental changes and the importance of their roles is becoming increasingly realized in cellular signaling research. Cyanobacteria are photooxygenic bacteria that inhabit most of Earth's environments. The ability of cyanobacteria to survive in ecologically diverse habitats is due to their capacity to adapt and respond to environmental changes. Chapter 1 will review known second messenger-controlled physiological processes in cyanobacteria. Second messengers used in these systems include the element calcium (Ca2+), the nucleotide-based guanosine tetraphosphate or pentaphosphate (ppGpp or pppGpp, represented as (p)ppGpp), the cyclic adenosine 3′,5′-monophosphate (cAMP), the c-di-GMP, the cyclic guanosine 3′,5′-monophosphate (cGMP), and the cyclic dimeric AMP (c-di-AMP), and the gaseous nitric oxide (NO). The discussion will focus on processes central to cyanobacteria such as nitrogen fixation, light perception, photosynthesis-related processes, and gliding motility. Chapter 2 will confirm that many putative c-di-GMP synthesis or degradation domains are found in genes that also harbor light-responsive signal input domains, suggesting that light is an important signal for altering c-di-GMP homeostasis. I will also compare intracellular levels of c-di-GMP in Synechocystis sp. PCC 6803 and Fremyella diplosiphon under different light qualities, confirming that light is an important factor for regulating this second messenger in vivo. Chapter 3 will demonstrate that biofilm formation, cellular aggregation or flocculation, and cellularbuoyancy are under the control of c-di-GMP in Synechocystis sp. PCC 6803 and F. diplosiphon. In this chapter, I present data on Synechocystis and F. diplosiphon transformed with a plasmid for constitutive expression of genes encoding diguanylate cylase (DGC) and phosphodiesterase (PDE) proteins from Vibrio cholerae and Escherichia coli, respectively. Engineering of these strains allowed modulation of intracellular c-di-GMP levels. Chapter 4 will confirm the role of c-di-GMP in F. diplosiphon in regulating pigments. I show that c-di-GMP is involved in light signalling networks and correctly tunes the pigments of cells under changes to ambient light. In chapter 5, I conclude by addressing future research trajectories needed to better understand the signaling networks and cross talk in the signaling pathways of these molecules in cyanobacteria. Second messengers have significant potential to be adapted as technological tools and possible novel and practical applications are highlighted based on current understanding of these molecules and the signaling networks that they control.
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Title: Defining the role of ballast water in the transport of viruses in aquatic environments through metagenomic approaches
Creator: Kim, Yiseul
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Global shipping activities transport 12 billion tons of water across regions each year. This so called ballast water contains a variety of biological materials and has been considered to transfer non−native species between biomes, resulting in potential ecological, economic, and public health problems in major ports worldwide. Despite the large amount of ballast water transported around the globe and its negative impact on native ecosystems, relatively little attention has been paid to viral...
Show moreGlobal shipping activities transport 12 billion tons of water across regions each year. This so called ballast water contains a variety of biological materials and has been considered to transfer non−native species between biomes, resulting in potential ecological, economic, and public health problems in major ports worldwide. Despite the large amount of ballast water transported around the globe and its negative impact on native ecosystems, relatively little attention has been paid to viral invasions via ballast water due to technical challenges in detecting the wide range of viruses. The limitations of virus discovery using traditional approaches can now be overcome with the emergence of metagenomics, which enables unprecedented views of viral diversity and functions. This dissertation brought together environmental virology, metagenomics, and bioinformatics for the first time in order to examine taxonomic composition and diversity of viruses in ballast and harbor waters collected from a freshwater system, and to investigate global transport of viruses through ballast water and effect of engineered, management, and environmental parameters associated with ballast water on ocean viruses. Viral communities in ballast water in the Great Lakes were examined due to the long history of non−native species invasions in this region of the world. Five ballast and three harbor waters were collected from the Port of Duluth−Superior on May 2013. Bioinformatics analyses of over 550 million Illumina reads showed that the viral sequences had mostly no homologs in the public database, indicating that our knowledge about viral diversity is still very limited. Among the sequences homologous to known viruses (22.3 ± 6.2%), ballast and harbor waters contained a diversity of viruses, which were largely dominated by double–stranded (ds) DNA phages, including Myoviridae, Podoviridae, and Siphoviridae. Along with these phage families, viruses that could infect a broad range of hosts, including archaea, fungi, invertebrate, plant, protist, and vertebrate, some of which are highly pathogenic to fish and shrimp, were present at different levels in the viral metagenomes (viromes). Comparative virome analyses showed that viromes were distinct among the Great Lakes and formed a specific group of temperate freshwater viromes, separate from viromes associated with marine environments and engineered freshwater systems.The scope of this research was expanded to examine viral communities in marine environments. Sixteen ballast and eight harbor waters were collected from the Port of Los Angeles/Long Beach and the Port of Singapore from March through May 2014. Bioinformatics analyses of 3.8 billion Illumina reads revealed that taxonomic profile of the sequences homologous to known viruses (30.6 ± 0.03%) was similar to that observed in the Great Lakes viromes, which were largely dominated by dsDNA phages. Moreover, this research was able to detect sequences most similar to viruses infecting human, fish, and shrimp, which are related to significant public health problems or direct economic impact. Variations in virome composition of ballast and harbor waters were found between geographic locations, suggesting that the movement of ballast water across the global shipping network transports the ocean viromes. Importantly, this research showed that virus richness (type of viruses) in ballast water was not governed by engineered or management variables but by conditions of local environment where viruses arise from showing associations with latittude. Outcomes of the present research represent the most detailed characterization to date of viruses in ballast water, defining the role of ballast water in the transport of freshwater and ocean viromes and an increased risk of exposure of aquatic fauna and flora to viruses. The present findings emphasize the need for implementing ballast water discharge limits for viruses and treatment. More research is needed on host population structure to better understand the impact of the transport of viruses between biomes.
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Title: Phenotypic and genotypic determinants of colonization and pathogenesis in group B Streptococcus
Creator: Parker, Robert Edward
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Group B Streptococcus (GBS) is a leading cause of sepsis and meningitis in neonates, an important factor in premature and still births worldwide, and a threat to immunocompromised and elderly adults. The prevention strategy for neonatal invasive disease targets maternal colonization, the primary risk factor, through late-gestational screening and intrapartum antibiotics. This approach has resulted in a significant decline in disease rates; however, this decrease has stagnated in the 2000s,...
Show moreGroup B Streptococcus (GBS) is a leading cause of sepsis and meningitis in neonates, an important factor in premature and still births worldwide, and a threat to immunocompromised and elderly adults. The prevention strategy for neonatal invasive disease targets maternal colonization, the primary risk factor, through late-gestational screening and intrapartum antibiotics. This approach has resulted in a significant decline in disease rates; however, this decrease has stagnated in the 2000s, and maternal re-colonization following treatment is common. For my doctoral work, I examined phenotypic and genotypic factors which facilitate colonization, antibiotic tolerance, and persistence of GBS including biofilm production, quorum sensing, and phenotypic heterogeneity. The assessment of biofilm formation across a diverse set of isolates, including colonizing and invasive clinical human strains, found that weak biofilm production correlated with genotype, pilus profile, and invasive disease. Furthermore, asymptomatic colonization was associated with strong biofilm production suggesting a colonization advantage for strong biofilm producers. The role of a putative quorum-sensing auto-inducing peptide, RgfD, was investigated through the creation of a deletion mutant through homologous recombination. In this work, rgfD was found to drive adherence to decidualized human endometrial cells through the upregulation of the regulator of fibrinogen-binding two-component system suggesting quorum-sensing in GBS is important for colonization. Lastly, the importance of phenotypic diversification in a single strain of GBS was assessed through the identification and characterization of a locked mutant small colony variant (SCV) derived from a clinical isolate. This is the first demonstration of SCV formation in GBS. The mutant SCV displayed increased penicillin tolerance and biofilm production, but reduced phagocytic uptake by THP-1 macrophages. Furthermore, the SCV phenotype was inducible when treated with antibiotics or exposed to acidic pH, which, alongside whole transcriptome analysis, suggests variant-formation to be driven by stress response in GBS. The work contained herein furthers the understanding of GBS colonization and identifies key phenotypic and genotypic characteristics driving colonization and persistence that must be considered in the development of future therapeutics.
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Title: Roles of hfq-dependent srnas in e. amylovora regulation of virulence
Creator: Schachterle, Jeffrey Kent
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Erwinia amylovora is the causative agent of fire blight disease of apple and pear trees, causing annual losses of over 100 million USD in the USA. E. amylovora cells are disseminated to new hosts by insects, wind, and rain, and then invade susceptible tissues and migrate systemically throughout the host, requiring coordinate regulation of several virulence factors, including production of the exopolysaccharides amylovoran and levan, biofilm formation, flagellar motility, and type III...
Show moreErwinia amylovora is the causative agent of fire blight disease of apple and pear trees, causing annual losses of over 100 million USD in the USA. E. amylovora cells are disseminated to new hosts by insects, wind, and rain, and then invade susceptible tissues and migrate systemically throughout the host, requiring coordinate regulation of several virulence factors, including production of the exopolysaccharides amylovoran and levan, biofilm formation, flagellar motility, and type III secretion. Complex regulatory mechanisms have evolved in E. amylovora that occur at the transcriptional, post-transcriptional, and post-translational levels to control these virulence factors. In my work, I analyze the role of small RNAs (sRNAs) as post-transcriptional regulators of virulence-associated traits in E. amylovora.The Hfq chaperone protein stabilizes sRNAs in the cell, allowing them to interact with and regulate mRNA targets. An hfq mutant differs from wild-type cells in several virulence-associated phenotypes including production of the exopolysaccharides amylovoran and levan, biofilm formation, flagellar motility, and type III secretion. E. amylovora encodes at least 40 Hfq-dependent sRNAs; in my work, I have systematically made deletion mutants of each sRNA singly, as well as constructed inducible expression vectors for each sRNA. Screening of this sRNA library has shown that several sRNAs contribute to regulation of each virulence phenotype, indicating complex regulation of the traits assessed. Of particular interest, the ArcZ sRNA regulates several of the virulence-associated traits we have assessed, and an arcZ deletion mutant loses virulence in both immature pear and apple shoot infection models.Flagellar motility, which enables E. amylovora cells to swim through flower nectar to invade natural openings in host flowers, is regulated by ArcZ. We have shown that ArcZ regulates motility by regulating the flagellar transcription factor FlhD at both the transcriptional and post-transcriptional levels. Because the ArcZ regulation of FlhD at the transcriptional and post-transcriptional levels has a contradiction in sign, we searched for additional layers of regulation between ArcZ and FlhD. We did so by conducting a transposon screen in the arcZ mutant background for suppressor mutants that restored flagellar motility. This screen yielded as the most common suppressor mutation the leucine responsive regulator protein (Lrp), a global transcription factor known for regulation of amino acid metabolism. We have found that Lrp not only acts as a regulator of flagellar motility between ArcZ and FlhD, but that it also reverses the regulatory effects of arcZ deletion on amylovoran and levan production, as well as biofilm formation. Our work shows that Lrp is a novel virulence regulator that plays an important role in regulating several virulence-associated traits in conjunction with the sRNA ArcZ.Transcriptomic comparison between the arcZ mutant and wild-type cells confirmed that ArcZ regulates several genes known to also be regulated by Lrp, and also indicated that ArcZ regulates several genes involved in mitigating the threat of reactive oxygen species, including genes encoding a catalase, a thiol-peroxidase, and a peroxiredoxin. We found that catalase makes the greatest contribution to diminishing the threat of exogenous hydrogen peroxide. Additional analysis suggests that ArcZ participates in regulation with an oxidative sensing transcription factor network that includes the transcription factors ArcA, Fnr, and Fur.This work shows that several sRNAs make small contributions to virulence trait regulation, and that a few sRNAs, like ArcZ, make major contributions to E. amylovora virulence. ArcZ regulates several virulence-associated traits through the global transcription factor Lrp, which we have found to be a novel virulence regulator. ArcZ also regulates genes involved in mitigating the threat of reactive oxygen species, which can protect E. amylovora cells from host defenses during infection. Thus, ArcZ plays an integral role in modulating phenotypic expression during fire blight disease progression that enables E. amylovora to successfully colonize and infect host plants. Mechanistic understanding of E. amylovora gene regulation moves us closer to understanding weaknesses that can be exploited for development of novel disease control strategies.
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Title: Pancreatic secretions and their effect on circulatory and immune cells
Creator: Entwistle, Kristen
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia and insulin dysfunction, which often results in secondary complications associated with blood flow. Four major types of diabetes are defined by the American Diabetes Association: type 1 diabetes (T1D, insulin insufficiency), type 2 diabetes (T2D, insulin resistance), gestational diabetes (occurring during pregnancy), and other types, which includes cystic fibrosis related diabetes (CFRD). Although each of these...
Show moreDiabetes mellitus is a metabolic disease characterized by chronic hyperglycemia and insulin dysfunction, which often results in secondary complications associated with blood flow. Four major types of diabetes are defined by the American Diabetes Association: type 1 diabetes (T1D, insulin insufficiency), type 2 diabetes (T2D, insulin resistance), gestational diabetes (occurring during pregnancy), and other types, which includes cystic fibrosis related diabetes (CFRD). Although each of these types of diabetes is associated with insulin dysfunction, it is important to note that the treatment of oral medications or exogenous insulin, although sufficient to effectively manage blood glucose levels, is not sufficient to completely cure diabetes. Secondary complications resulting from diabetes include retinopathy, nephropathy and neuropathy, along with dysfunctions in immune response. In recent years, a surge in the research of C-peptide, the 31 amino acid peptide co-secreted with insulin, has revealed that C-peptide may actually help to ameliorate some of these secondary complications, including neuropathy and immune response. Although initial studies revealed promising results, a phase 2b clinical trial was shut down in 2014 due to indistinguishable results in placebo and treatment groups in T1D patients. To enhance our knowledge of these downstream problems, we here report the effect of C-peptide/zinc/albumin on RBCs and peripheral neutrophils (an immune cell) that mimic those from CF/CFRD patients using a chemical inhibitor (CFTRinh-172). C-peptide and zinc binding to RBCs confirms previously published data, and binding to CFTR-inhibited RBCs is increased from the control, however, the molar binding ratio of C-peptide to zinc remains 1:1. ATP release is increased in control and CFTR-inhibited cells only in the presence of the ensemble of C-peptide/zinc/albumin, but is abolished when any one of these components is missing. Similarly, in neutrophils, we observe a 1:1 molar binding ratio of C-peptide to zinc when albumin is present in both control and CFTR-inhibited cells. We also observed a significant increase in intracellular calcium, intracellular NO, extracellular NO, glucose uptake, and rate of chemotaxis in neutrophils that had been treated with the ensemble, but not when any one component was missing. In both RBCs and neutrophils, we observed an increase in membrane expression of glucose transporter 1 (GLUT1) only when C-peptide, zinc and albumin were present. Insulin did not have this effect. The results reported here are the first to indicate that C-peptide may be acting on cells that contain primarily GLUT1, and that the mechanism of action may be similar to the way that insulin acts in muscle and fat cells, by translocating GLUT4 to the plasma membrane. These results are also the first to suggest that C-peptide may directly be affecting peripheral neutrophils, which could have significant implications in the treatment of immune complications in all forms of diabetes. Additionally, these results are the first to examine the effect this peptide and its metal may have on patients who have CF, and may point to its use a potential therapy in both T2D and CFRD.
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Title: Genetic and chemical biology studies of Mycobacterium tuberculosis pH-driven adaptation
Creator: Dechow, Shelby J.
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Mycobacterium tuberculosis (Mtb) endures robust immune responses by sensing and adapting to its host environment. One of the first cues the bacterium encounters during infection is acidic pH, a characteristic of its host niche – the macrophage. Targeting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. A high throughput screen of a >220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb...
Show moreMycobacterium tuberculosis (Mtb) endures robust immune responses by sensing and adapting to its host environment. One of the first cues the bacterium encounters during infection is acidic pH, a characteristic of its host niche – the macrophage. Targeting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. A high throughput screen of a >220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb growth at acidic pH. From this screen, AC2P20 was identified as a chemical probes that kills Mtb at pH 5.7 but is inactive at pH 7.0. Through a combination of transcriptional profiling, mass spectrometry, and free thiol abundance and redox assays, I show that AC2P20 likely functions by depleting intracellular thiol pools and dysregulating redox homeostasis. Findings from this study have helped define new pathways involved in Mtb’s response to acidic pH using a chemical genetic approach.Upon sensing acid stress, Mtb can adapt accordingly by entering a nonreplicating persistent state, resulting in increased tolerance to host immune pressures and antibiotics. During growth in vitro, when given glycerol as a sole carbon source, Mtb responds to acidic pH by arresting its growth and entering a metabolically active state of nonreplicating persistence, a physiology known as acid growth arrest. To answer how Mtb regulates and responds to acidic pH, I performed genetic selections to identify Mtb mutants defective in acid growth arrest. These selections identified enhanced acidic growth (eag) mutants which all mapped to the proline-proline-glutamate ppe51 gene and resulted in distinct amino acid substitutions: S211R, E215K, and A228D. I demonstrated that expression of the PPE51 variants in Mtb promotes significantly enhanced growth at acidic pH showing that the mutant alleles are sufficient to cause the dominant gain-of-function, eag phenotype. Furthermore, I performed single carbon source experiments and radiolabeling experiments showing that PPE51 variants preferentially uptake glycerol at an enhanced rate, suggesting a role in glycerol acquisition. Notably, the eag phenotype is deleterious for growth in macrophages, where the mutants have selectively faster replication but reduced virulence in activated macrophages as compared to resting macrophages. This supports that acid growth arrest is a genetically controlled, adaptive process that could act as a potential targetable physiology in future TB therapeutics. My work with the carbonic anhydrase inhibitor, ethoxzolamide, sought to combine genetic and chemical biology to better understand pH-adaptation in Mtb. Ethoxzolamide is a potent inhibitor of Mtb carbonic anhydrase activity and the PhoPR regulon, suggesting a previously unknown link between carbon dioxide and pH-sensing. We hypothesized that the production of protons from carbonic anhydrase activity could be modulating PhoPR signaling. Mtb has three carbonic anhydrases (CanA, CanB, and CanC), and by using CRISPRi and gene knockout, I show that CanB is required for pathogenesis in macrophages, but I did not observe a function in controlling PhoPR signaling. However, transcriptional profiling at different pH and CO2 concentrations show that PhoPR is induced by high CO2 and also revealed a core subset of CO2 responsive genes independent of PhoPR or acidic pH regulation. Overall, these studies defined new functions for thiol- and redox-homeostasis, glycerol uptake, and CO2-concentration in regulating Mtb adaptation to acidic environments and provide new targets for the development of acidic pH-dependent therapeutics.
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Title: ECOPHYSIOLOGY OF (PERI)ORAL BACTERIA AND IMPACT OF OTIC COLONIZATION
Creator: Jacob, Kristin Marie
Date: 2022
Collection: Electronic Theses & Dissertations
Description: The middle ear is typically assumed to be sterile in health due to its secluded location, closed off from external forces by the tympanic membrane (ear drum) and from the naso/oropharynx by a collapsed Eustachian tube. However, the periodic opening of the Eustachian tube to the naso/oropharyngeal space, which releases pressure across the eardrum and drains otic fluids, could introduce bacteria. Previous studies have tested for the presence of bacteria in the uninfected otic cavity using...
Show moreThe middle ear is typically assumed to be sterile in health due to its secluded location, closed off from external forces by the tympanic membrane (ear drum) and from the naso/oropharynx by a collapsed Eustachian tube. However, the periodic opening of the Eustachian tube to the naso/oropharyngeal space, which releases pressure across the eardrum and drains otic fluids, could introduce bacteria. Previous studies have tested for the presence of bacteria in the uninfected otic cavity using samples collected via invasive surgeries (through or around the eardrum). Findings from these studies are controversial due to contradictory results between studies, lack of critical experimental controls, and sampling of participants with underlying ailments (i.e., cochlear implant surgery) that could impact the microbiology of the otic mucosa. The studies reported herein bypass these limitations by using samples of otic secretions collectively non-invasively (through the mouth) in a cohort of healthy young adults. This dissertation describes cultivation-dependent methods to investigate the microbiology of the middle ear in health. The study used an IRB-approved protocol (#17-502) to collect otic secretions in order to 1) sequence their microbiome (contribution by Dr. Joo-Young Lee) and 2) recover in pure culture otic bacteria for further characterization (my contribution). As controls, we also collected buccal (top palate and inside of cheeks) and oropharyngeal swabs from each participant. Of the collected secretions, samples from 19 individuals were used for culture independent studies, while samples from the remaining 3 participants were subjected to culture dependent studies. 16S rRNA-V4 sequencing detected a diverse and distinct microbiome in otic secretions comprised primarily of strictly anaerobic bacteria belonging to the phyla Bacteroidetes, Firmicutes and Fusobacteria, and to a lesser extent facultative anaerobes (Streptococcus). I recovered from the otic, oropharyngeal, and buccal secretions 39 isolates of predominantly facultative anaerobes belonging to Firmicutes (Streptococcus and Staphylococcus), Actinobacteria (Micrococcus and Corynebacterium), and Proteobacteria (Neisseria) phyla, and used partial 16S rRNA amplicon sequences to demonstrate the distinct phylogenetic placement of otic streptococci compared to the oral ancestors (Chapter 2). This finding is consistent with the ecological diversification of oral streptococci once in the middle ear microenvironment. The recovery of streptococci and transient migrants (Staphylococcus, Neisseria, Micrococcus and Corynebacterium) from otic secretions prompted us to study the adaptive responses that give the streptococcal migrants a competitive advantage during the colonization of the middle ear (Chapter 3). For these studies, I sequenced and partially assembled the genomes of the otic isolates and used the full length 16S rRNA sequences for taxonomic demarcation at the species levels. Phylogenetic analyses demonstrated the oral ancestry of the otic streptococci, which retained from the otic adaptive traits critical for growth and reproduction in the middle ear mucosa (biofilm formation, mucolytic and proteolytic activity, robust growth under redox fluctuations, and fermentative production of lactate, a key metabolic intermediate in the otic trophic webs). These adaptive traits give oral streptococci a colonization advantage over competing (peri)oral migrants such as Staphylococcus. Furthermore, the otic streptococci inhibited the growth of otopathogens, including Staphylococcus aureus. These antagonistic interactions give streptococci a competitive advantage during the colonization of the middle ear and suggest a role for these commensals in promoting mucosal health. The ability of staphylococcal migrants to breach the middle ear mucosal barrier and cause infections prompted us to study the environmental factors that facilitate the spreading of staphylococci from the nasal to the middle ear mucosa. Allergies, respiratory maladies (cold, flu), or (peri)oral bacterial infections (sinus, adenoids, tonsils, etc.) lead to inflammation of the Eustachian tube and changes in the rheological properties of the otic mucus that increase the risk of infections. Thus, we examined the spreading of staphylococci on mucus-like viscous surfaces (semisolid agar plates). In Chapter 4, I show that mucins, the mucosal glycoproteins that control the viscosity and wettability of the mucus layer, induce the rapid spreading and dendritic expansion of clinical isolates closely related to S. aureus and Staphylococcus epidermidis but not of Staphylococcus hominis. Mucin glycosylation controlled the hydration of the mucoid surface and the ability of the cells to spread rapidly, in a process that was dependent on the secretion of surfactant-active, phenol-soluble modulins via the agr-quorum sensing two-component system. These results provide a plausible explanation for the rapid spreading of staphylococcal otopathogens from the nasopharynx to the middle ear through a swollen, and mucin-rich Eustachian tube. The work described in this dissertation provides much needed understanding of the adaptive responses that allow (peri)oral bacteria to colonize the middle ear. The studies add to the accumulating evidence that the middle ear mucosa is not sterile but rather harbors a commensal microbiota in health. These commensal community shares many metabolic similarities with ancestors in oral biofilms and retain adaptive traits critical for growth in the otic mucosa and inhibition of otopathogens. Additionally, this work identifies environmental factors that could contribute to staphylococcal virulence, broadening the understanding of newly identified motility phenotypes in the genus that could provide novel pharmaceutical targets.
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Title: MEMBRANE-LOCALIZED TRANSCRIPTION REGULATORS : UNDERSTANDING POST-TRANSLATIONAL REGULATION AND SINGLE-MOLECULE DYNAMICS OF TCPP IN VIBRIO CHOLERAE
Creator: Demey, Lucas Maurice
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is a Gram-negative gastrointestinal pathogen that has evolved an elegant regulatory system to precisely time production of essential virulence factors. A key step in this regulatory system is the transcription of a soluble AraC-like transcription factor, ToxT. ToxR and TcpP, two membrane-localized transcription regulators (MLTRs), positively regulate toxT. Much work has contributed to our understanding of TcpP and ToxR regulation, yet major gaps remain in our knowledge of...
Show moreVibrio cholerae is a Gram-negative gastrointestinal pathogen that has evolved an elegant regulatory system to precisely time production of essential virulence factors. A key step in this regulatory system is the transcription of a soluble AraC-like transcription factor, ToxT. ToxR and TcpP, two membrane-localized transcription regulators (MLTRs), positively regulate toxT. Much work has contributed to our understanding of TcpP and ToxR regulation, yet major gaps remain in our knowledge of these MLTRs. MLTRs are unique one-component signal transduction systems because they respond to extracellular stimuli by influencing gene transcription from their location in the cytoplasmic membrane. In Chapter 2, I explore the prevalence and diversity of MLTRs within prokaryotes to enhance our understanding of TcpP and ToxR. I show that MLTRs are far more common among prokaryotes than previously anticipated and that MLTRs are an understudied class of transcription regulators. In Chapter 3, I describe the use of super-resolution single-molecule tracking to investigate how TcpP, a model MLTR, identifies the toxT promoter. I provide evidence that TcpP binds to the toxT promoter independent of ToxR, and TcpP transitions to a specific diffusion state. The data support the first biophysical model for how TcpP-like MLTRs locate their target promoters. TcpP is subject to a form of post-translational regulation known as regulated intramembrane proteolysis (RIP). RIP of TcpP results in its complete inactivation, resulting in loss of virulence factor production. TcpH inhibits RIP of TcpP under certain pH and temperature conditions. In Chapter 4, I describe the mechanism TcpH employs to inhibit TcpP RIP while V. cholerae is present in the mouse gastrointestinal tract. I demonstrate that the dietary fatty acid α-linolenic acid enhances inhibition. I also show that α-linolenic acid promotes TcpH-mediated inhibition of TcpP RIP by increasing association of both proteins with detergent-resistant membrane (DRM) domains. My work provides the first evidence that DRMs influence virulence factor transcription in V. cholerae and that a dietary fatty acid promotes V. cholerae pathogenesis.
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Title: Systematic analysis of the signal responsive gene regulatory network governing Myxococcus xanthus development
Creator: Saha, Shreya
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Studies of signal-induced gene expression in bacteria have contributed to understanding of how bacteria cope with environmental stress. As an extensively studied model, Myxococcus xanthus provides fascinating insights into how changes at the level of gene expression enable which bacteria to survive environmental insults such as nutrient limitation. Upon starvation M. xanthus cells glide into aggregates and form mounds that mature into fruiting bodies as some cells form spores. Previously, our...
Show moreStudies of signal-induced gene expression in bacteria have contributed to understanding of how bacteria cope with environmental stress. As an extensively studied model, Myxococcus xanthus provides fascinating insights into how changes at the level of gene expression enable which bacteria to survive environmental insults such as nutrient limitation. Upon starvation M. xanthus cells glide into aggregates and form mounds that mature into fruiting bodies as some cells form spores. Previously, our group defined 24-30 h poststarvation as the critical period for commitment to spore formation, when cells commit to form spores despite perturbation of the starvation signal by nutrient addition. The process of multicellular development that culminates in sporulation is governed by a network of signal-responsive transcription factors that integrate signals for starvation and cellular alignment. In this dissertation I present the first systematic approach to elucidate the network dynamics during the commitment period.In the network, MrpC is a starvation-responsive transcription factor, whereas FruA is a transcription factor that responds to cellular alignment conveyed by C-signaling. Transcription of fruA is dependent on MrpC binding, and FruA activity is proposed to be posttranslationally regulated by C-signaling, although the mechanism is unknown. FruA and MrpC cooperatively regulate transcription of the dev operon. My systematic analysis of the network dynamics supported a model in which posttranslational activation of FruA by C-signaling is critical for dev transcription and for commitment to spore formation. Similar to dev, MrpC and C-signal-activated FruA combinatorially controlled transcription of the late-acting fadIJ operon involved in spore metabolism. Regulation of late-acting operons implicated in spore coat biogenesis (exoA-I, nfsA-H, MXAN_3259-MXAN_3263) was discovered to be under complex control by MrpC and FruA. My evidence suggests that transcription of these operons depends at least in part on a C-signal-dependent switch from negative regulation by unactivated FruA to positive regulation by activated FruA during the period leading up to and including commitment to sporulation. MrpC negatively regulated exo and MXAN_3259 during mound formation, but positively regulated nfs. During commitment to sporulation, MrpC continued to positively regulate nfs, switched to positive regulation of MXAN_3259, and continued to negatively regulate exo. A third transcription factor, Nla6, appeared to be a positive regulator of all the late genes. We propose that in combination with regulation by Nla6, differential regulation by FruA in response to C-signaling and by MrpC controls late gene expression to ensure that spore resistance and surface characteristics meet environmental demands.
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Title: Variation in host-pathogen interactions among genetically diverse strains of Group B Streptococcus
Creator: Korir, Michelle Lynn
Date: 2016
Collection: Electronic Theses & Dissertations
Description: "Streptococcus agalactiae, or Group B Streptococcus (GBS), is a highly diverse species that can be found asymptomatically colonizing the gastrointestinal and genital tracts of healthy adults, but is also capable of causing severe invasive disease. GBS is a leading cause of sepsis and meningitis in neonates and the only preventative measure is antibiotic therapy given to pregnant mothers during labor to prevent transmission. Although this method was effective at reducing case rates upon...
Show more"Streptococcus agalactiae, or Group B Streptococcus (GBS), is a highly diverse species that can be found asymptomatically colonizing the gastrointestinal and genital tracts of healthy adults, but is also capable of causing severe invasive disease. GBS is a leading cause of sepsis and meningitis in neonates and the only preventative measure is antibiotic therapy given to pregnant mothers during labor to prevent transmission. Although this method was effective at reducing case rates upon implementation, case rates have remained unchanged since the initial decline and some mothers remain persistently colonized by GBS. Due to the high level of diversity among strains it is important to understand how strains differ at the various stages of disease progression in order to have a more complete understanding of GBS pathogenesis. Here, I examined how genotypically diverse strains differ in their interactions with human cells. The examination of strains of the same serotype in ability to associate with decidual cells and lung epithelial cells revealed that strains within the same serotype, and even the same sequence type (ST) differed in attachment and invasion, but this variation was dependent on host cell type. More specifically, strains of the hypervirulent lineage, ST-17 associated with decidual cells significantly more than the other STs, but the opposite was true for lung epithelial cell attachment. Mechanisms of persistent colonization was explored by comparing antibiotic tolerance and macrophage survival between ST-17 and ST-12 strains, which persisted and was eradicated after antibiotic prophylaxis, respectively. This study revealed that although the ST-17 strain was not tolerant to antibiotics, subinhibitory antibiotics enhanced phagocytic uptake of this strain where it was able to survive for an extended period of time. Additionally, intracellular survival of the ST-17 strain was dependent on acidification of the phagosome, whereas altered pH had no effect on survival of the ST12 strain, suggesting GBS can use different mechanisms of survival. Moreover, serotype III GBS strains were better able to survive phagosomal stress compared to other serotypes. Lastly, transcriptome analysis of the ST-17 strain during intracellular survival revealed temporal gene expression responses to long term survival and identified a large number of factors important for intracellular survival. Through mutagenesis studies, the roles of NADH peroxidase (Npx) and cadmium resistance protein (CadD) in GBS intracellular survival was examined. These studies demonstrated that Npx promotes resistance to reactive oxygen stress through detoxification of hydrogen peroxide and CadD serves as a heavy metal efflux pump to confer resistance to intoxication by certain divalent metal cations. The work described here reveals new insights in GBS pathogenesis and helps identify key virulence factors that can serve as targets for alternative therapeutics and vaccine development."--Pages ii-iii.
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Title: Characterizing single-cell behavior of flagellated pathogenic bacteria in mucus and visco-elastic environments
Creator: Nguyen, Nhu Thi Quynh
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Flagellar motility is required for some enteric pathogens to colonize the intestine. Understanding how these pathogens overcome the mucus layer protecting the epithelial tissue is necessary for disease prevention. In this thesis project I examined bacterial motility in mucus to understand factors that facilitate mucus penetration and contrasted this with the motility in different visco-elastic materials.In this thesis, I used single cell tracking to characterize the flagellar motility of...
Show moreFlagellar motility is required for some enteric pathogens to colonize the intestine. Understanding how these pathogens overcome the mucus layer protecting the epithelial tissue is necessary for disease prevention. In this thesis project I examined bacterial motility in mucus to understand factors that facilitate mucus penetration and contrasted this with the motility in different visco-elastic materials.In this thesis, I used single cell tracking to characterize the flagellar motility of Vibrio cholerae and Salmonella enterica in different visco-elastic environments. First, I tested if V. cholerae and S. enterica were still motile in unprocessed pig intestinal mucus. Second, I studied factors that supported their motility in mucus. The first factor I investigated was the effect of pH on V. cholerae motility. I also studied the role of curvature in V. cholerae motility within mucus and some other visco-elastic environments, including liquid and agar. Last, I began investigation of the role of flagellar number in S. enterica motility. In these studies, I focused on analyzing the effective diffusion of bacteria and factors promoting the diffusion in each environment. My findings demonstrate that V. cholerae and S. enterica are able to swim in mucus, and that the torque, and the curvature of V. cholerae, and the flagellar number of S. enterica, play a significant role in bacteria motility in mucus and liquid. Moreover, my findings help elucidate the significance of motility in pathogenesis.
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Title: CHEMICAL BIOLOGY AND GENETIC STUDIES TARGETING THE MYCOBACTERIAL CELL ENVELOPE
Creator: Williams, John Tison
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Mycobacterium tuberculosis is one of the leading causes of death due to a single infectious pathogen. The evolution and spread of drug resistant strains requires new antibiotics to control the TB pandemic. Over the last decade, the lipid flippase MmpL3 has been identified as a potential drug candidate based on its essential nature for cell viability and repeated identification as the lead target of small molecule inhibitors of Mtb growth. Using a combined untargeted and targeted whole cell...
Show moreMycobacterium tuberculosis is one of the leading causes of death due to a single infectious pathogen. The evolution and spread of drug resistant strains requires new antibiotics to control the TB pandemic. Over the last decade, the lipid flippase MmpL3 has been identified as a potential drug candidate based on its essential nature for cell viability and repeated identification as the lead target of small molecule inhibitors of Mtb growth. Using a combined untargeted and targeted whole cell phenotypic screen I identified novel inhibitors of this valued target. A combination of lipid profiling and an innovative competitive binding assay supported MmpL3 as the target of these inhibitors. Cross resistance profiling of MmpL3 inhibitors against twenty-four unique mmpL3 Mtb mutants demonstrated that the level of resistance is associated with the proximity of resistant mutants to essential residues for protein function. Further, these resistance profiles suggested that MmpL3 inhibitors fall into separate clades depending on their chemical scaffolds. The results of this screen led to the development of novel potent analogs for one of the identified MmpL3 inhibitors, HC2099. These analogs were active against clinically relevant drug resistant Mtb strains that cause treatment failure in patients. Active analogs were able to kill Mtb inside of infected macrophages, an infectious niche of Mtb, without inducing cytotoxicity against these important immune cells. One of these analogs, MSU-43085, was orally bioavailable and successfully inhibited Mtb growth in infected mice, supporting further development and highlighting the therapeutic potential of this series. High throughput screens are often used to identify new inhibitors of Mtb growth. However, prioritized hits form these screens often identify similar targets such as MmpL3, lipid synthesis enzymes, redox cyclers, as well as inhibitors of the electron transport chain. Follow up studies of these inhibitors are often time consuming, costly and result in the rediscovery of previously identified targets. While this is not necessarily detrimental to Mtb drug discovery, as these reoccurring targets have therapeutic potential. The continued prioritization of inhibitors for these common metabolic pathways potentially limits the identification of inhibitors for novel targets. Therefore, additional steps that identify inhibitors of these common pathways could reshape how high throughput screen hits are prioritized. By applying the targeted mutant screen used to identify MmpL3 inhibitors to a non-prioritized library of hits from a high throughput screen, we identified more than fifty new potential MmpL3 inhibitors. Using an iterative strategy of applying additional mutants of commonly identified targets, this strategy promises to lead to parallel follow-up studies of inhibitors with known and unknown mechanisms of action. The ability of Mtb to enter into quiescent states in response to host stresses is one of the leading causes for the extended time to cure and evolution of drug resistance. These states can be induced by several environmental stresses including acidic pH, hypoxia, and others. In an effort to study this adaptation in the rapidly growing mycobacterial species M. smegmatis, we identified a lethal sodium citrate phenotype. Transcriptional profiling and genetic screening of mutants tolerant to sodium citrate indicated that this phenotype was due to the combined action of both chelation and osmotic stresses. Cell viability could be reduced from sodium citrate killing by cation and osmoprotectant supplementation. From these experiments we propose a model that can be applied to study carbon source uptake and probe the role of genes identified from the forward genetic screen with unknown function
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Title: DISSECTING THE MULTIMODAL SIGNALING NETWORK MEDIATED BY CYCLIC-DI-GMP IN ERWINIA AMYLOVORA
Creator: Kharadi, Roshni Russi
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Erwinia amylovora is the bacterial phytopathogenic causal agent of fire blight, an economically impactful disease that affects apple and pear production worldwide. The successful orchestration of infection by E. amylovora within the host entails a coordinated implementation of several different virulence strategies. A key step in the disease cycle of E. amylovora is the transition from a primarily Type III secretion system (T3SS) effectors-dependent phase in the leaf apoplast to a biofilm...
Show moreErwinia amylovora is the bacterial phytopathogenic causal agent of fire blight, an economically impactful disease that affects apple and pear production worldwide. The successful orchestration of infection by E. amylovora within the host entails a coordinated implementation of several different virulence strategies. A key step in the disease cycle of E. amylovora is the transition from a primarily Type III secretion system (T3SS) effectors-dependent phase in the leaf apoplast to a biofilm-dependent phase within xylem vessels. Cyclic-di-GMP (c-di-GMP), a ubiquitous bacterial second messenger mediates this phase transition into a sessile, attached lifestyle within biofilms in E. amylovora. This body of work encompasses several aspects of the complex and multifactorial signaling network dependent on c-di-GMP in E. amylovora. Diguanylate cycles enzymes (encoded by edc genes) dimerize GTP subunits to synthesize c-di-GMP and phosphodiesterase enzymes (encoded by pde genes) hydrolyze c-di-GMP. We found that the deletion of the three active pde genes in E. amylovora, singly and in combinations of two and all three genes, led to a measurable increase in intracellular c-di-GMP levels. In addition, the elevated c-di-GMP levels correlated with increased production of amylovoran, which is the most abundant exopolysaccharide (EPS), and, a pathogenicity factor in E. amylovora. The expression of T3SS, quantified by the transcriptional level of hrpL as well as by virulence measurements in the apple and pear models, was found to be negatively regulated by c- di-GMP. While biofilm formation generally increased with elevated levels of c-di-GMP, the total pde deletion mutant, ΔpdeABC, showed a relative depreciation in the ability to form biofilms, owing to the physical autoaggregative characteristic of this strain when grown in liquid media. Autoaggregation also impaired cell separation post division, leading to the presence of filamentous cells within cellular aggregates. In addition to the EPSs amylovoran and cellulose, EagA, a peptidoglycan hydrolase was found to be a major contributor to the facilitation of autoaggregation in E. amylovora. The eagA/znuABC zinc uptake gene cluster, was found to be transcriptionally regulated by c-di-GMP and the zinc-dependent repressor Zur. Further, we evaluated the impact of a systemic deletion of all genetic components involved in c-di-GMP metabolism, including active and degenerate dgc and pde encoding genes. The resulting mutant, Ea1189Δ12 was found to be impaired in surface sensing and attachment, which are key steps in the initiation of biofilm formation both in-vitro and in-planta. The transcriptomic profile of WT Ea1189 and Ea1189Δ12 at various stages of biofilm development revealed marked differences in critical metabolic and signal transduction pathways. The correlational clustering of phenotypic data gathered from single gene complemented strains generated from Ea1189Δ12, enabled the functional categorization of each of the systemic components.
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Title: EXAMINING INHIBITION AND SUBSTRATE INTERACTION OF SPOIVFB, AN INTRAMEMBRANE METALLOPROTEASE OF BACILLUS SUBTILIS
Creator: Olenic, Sandra D
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Intramembrane proteases (IPs) regulate diverse signaling pathways in all three domains of life by cleaving membrane-associated substrates. Currently, there are four known IP families, including intramembrane metalloproteases (IMMPs), which contain characteristic HEXXH and NPDG motifs that coordinate a zinc ion at the active site. The study of IMMPs, including RseP (Escherichia coli) and SpoIVFB (Bacillus subtilis) have provided insights into potential mechanisms for substrate interaction and...
Show moreIntramembrane proteases (IPs) regulate diverse signaling pathways in all three domains of life by cleaving membrane-associated substrates. Currently, there are four known IP families, including intramembrane metalloproteases (IMMPs), which contain characteristic HEXXH and NPDG motifs that coordinate a zinc ion at the active site. The study of IMMPs, including RseP (Escherichia coli) and SpoIVFB (Bacillus subtilis) have provided insights into potential mechanisms for substrate interaction and cleavage, which could guide efforts to develop inhibitors of IMMPs that regulate virulence in bacterial pathogens. Work described in this dissertation has advanced the knowledge of inhibition and substrate interaction of SpoIVFB, which cleaves Pro-σK during endosporulation. Improved methods are presented for heterologous production in E. coli of SpoIVFB, Pro-σK, and the inhibitory proteins BofA and SpoIVFA. Three conserved BofA residues (N48, N61, T64) in or near transmembrane segment (TMS) 2 are required for SpoIVFB inhibition. Cross-linking indicates that BofA TMS2 occupies the SpoIVFB active site region. Interestingly, BofA and SpoIVFA do not prevent interactions between portions of Pro-σK and SpoIVFB, so all four proteins can exist in a complex. A structural model of SpoIVFB with BofA and parts of SpoIVFA and Pro-σK was built using partial homology and constraints from cross-linking and co-evolutionary analyses. The model suggests that SpoIVFA stabilizes BofA in the SpoIVFB active site region and leads us to propose that BofA TMS2 sterically hinders access of the substrate. Our work has also advanced knowledge of interactions between Pro-σK and the broadly conserved SpoIVFB NPDG motif, which is located in a predicted short loop that interrupts TMS4 and faces the active site. Three highly conserved residues (N129, P132, P135) of SpoIVFB were found to be important for substrate interactions and cleavage, and we propose that P135 is necessary to position D137 to act as a zinc ligand. More work is needed to fully understand how IMMPs interact with their substrates and whether the insights from BofA inhibition of SpoIVFB can be applied to other IMMPs. Outstanding questions and future directions related to these two projects are described.
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Title: The Control of Phenotypic Diversity in Vibrio cholerae during the Transition between Motility and Attachment
Creator: Lee, John Seungwu
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is a causative agent of human intestinal disease, cholera. It requires both flagellar-based motility and biofilm formation to colonize the small intestine. The secondary messenger molecule, c-di-GMP, plays a central role in controlling transition between motility and biofilm formation. However, the switch between these two lifestyles has been studied on an average population scale, overlooking the heterogeneous phenotypic response that can occur at a single-cell level. V....
Show moreVibrio cholerae is a causative agent of human intestinal disease, cholera. It requires both flagellar-based motility and biofilm formation to colonize the small intestine. The secondary messenger molecule, c-di-GMP, plays a central role in controlling transition between motility and biofilm formation. However, the switch between these two lifestyles has been studied on an average population scale, overlooking the heterogeneous phenotypic response that can occur at a single-cell level. V. cholerae infections are characterized by the co-occurrence of cells with motile and sessile behaviors, but the determinant of this phenotypic diversity remains poorly understood. We used single-cell tracking to examine the motile behaviors of two V. cholerae strains (El Tor C6706 and Classical O395) in response to direct manipulations of c-di-GMP concentration. Both motile and non-motile cells are present in a well-mixed batch culture with distributions of these phenotypes that change depending on the growth phase. We determined that the proportion of motile cells differs between El Tor and Classical strains because they maintain different levels of c-di-GMP. However, even in conditions that promote biofilm formation, V. cholerae still generates a sub-population of motile cells. C-di-GMP is known to inversely regulate assembly of mannose-sensitive haemagglutinin (MSHA) pili and flagella. We found that the most cells in clonal populations are flagellated, but MSHA piliation affords the variable opportunity to attach at single-cell level, driving behavioral switching between motile and sessile behaviors. Our results support the hypothesis that c-di-GMP regulates phenotypic diversity in V. cholerae, and it does so by simultaneous elaboration of MSHA pili and flagellum.
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Title: Deconstructing the correlated nature of ancient and emergent traits : an evolutionary investigation of metabolism, morphology, and mortality
Creator: Grant, Nkrumah Alions
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Phenotypic correlations are products of genetic and environmental interactions, yet the nature of these correlations is obscured by the multitude of genes organisms possess. My dissertation work focused on using 12 populations of Escherichia coli from Richard Lenski's long-term evolution experiment (LTEE) to understand how genetic correlations facilitate or impede an organism's evolution. In chapter 1, I describe how ancient correlations between aerobic and anaerobic metabolism have...
Show morePhenotypic correlations are products of genetic and environmental interactions, yet the nature of these correlations is obscured by the multitude of genes organisms possess. My dissertation work focused on using 12 populations of Escherichia coli from Richard Lenski's long-term evolution experiment (LTEE) to understand how genetic correlations facilitate or impede an organism's evolution. In chapter 1, I describe how ancient correlations between aerobic and anaerobic metabolism have maintained - and even improved - the capacity of E. coli to grow in an anoxic environment despite 50,000 generations of relaxed selection for anaerobic growth. I present genomic evidence illustrating substantially more mutations have accumulated in anaerobic-specific genes and show parallel evolution at two genetic loci whose protein products regulate the aerobic-to-anaerobic metabolic switch. My findings reject the "if you don't use it, you lose it" notion underpinning relaxed selection and show modules with deep evolutionary roots can overlap more, hence making them harder to break. In chapter 2, I revisit previous work in the LTEE showing that the fitness increases measured for the 12 populations positively correlated with an increase in cell size. This finding was contrary to theory predicting smaller cells should have evolved. Sixty thousand generations have surpassed since that initial study, and new fitness data collected for the 12 populations show fitness has continued to increase over this period. Here, I asked whether cell size also continued to increase. To this end, I measured the size of cells for each of the 12 populations spanning 50,000 generations of evolution using a particle counter, microscopy, and machine learning. I show cell size has continued to increase and that it remains positively correlated with fitness. I also present several other observations including heterogeneity in cell shape and size, parallel mutations in cell-shape determining genes, and elevated cell death in the single LTEE population that evolved a novel metabolism - namely the ability to grow aerobically on citrate. This last observation formed the basis of my chapter 3 research where my collaborators and I fully examine the cell death finding and the associated genotypic and phenotypic consequences of the citrate metabolic innovation.
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Title: The effects of genetic background on the evolution of antibiotic resistance and its fitness costs
Creator: Card, Kyle Joseph
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Antibiotic resistance is a growing public-health concern. Efforts to control the emergence and spread of resistance would benefit from an improved ability to forecast when and how it will evolve. To predict the evolution of resistance with accuracy, we must understand and integrate information about many factors, including a bacterium's evolutionary history. This dissertation centers on the effects of genetic background on the evolution of phenotypic resistance, its genetic basis, and its...
Show moreAntibiotic resistance is a growing public-health concern. Efforts to control the emergence and spread of resistance would benefit from an improved ability to forecast when and how it will evolve. To predict the evolution of resistance with accuracy, we must understand and integrate information about many factors, including a bacterium's evolutionary history. This dissertation centers on the effects of genetic background on the evolution of phenotypic resistance, its genetic basis, and its fitness costs. To address these issues, I used Escherichia coli strains from the long-term evolution experiment (LTEE) that independently evolved for multiple decades in an environment without antibiotics.First, I examined how readily these LTEE strains could overcome prior losses of intrinsic resistance through subsequent evolution when challenged with antibiotics. Second, I investigated whether lineages founded from different genotypes take parallel or divergent mutational paths to achieve increased resistance. Third, I tested whether fitness costs of resistance mutations are constant across different genetic backgrounds. In these studies, I focused attention on the interplay between repeatability and contingency in the evolutionary process. My findings demonstrate that genetic background can influence both the phenotypic and genotypic evolution of resistance and its associated fitness costs. I conclude this dissertation with a broader discussion about these and other factors that can influence the evolution of antibiotic resistance, and their clinical and public-health implications.
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Title: Genetic diversity of clinical and bovine non-o157 shiga toxin-producing escherichia coli (stec
Creator: Blankenship, Heather Marie
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infection resulting in 265,000 illnesses and more than 3,600 hospitalizations annually. Since its identification in 1982 associated with an outbreak of haemorrhagic colitis, serotype O157:H7 has been the primary focus of research and surveillance. However, the increasing incidence of other serogroups, or non-O157 STEC, that are associated with clinical illness has since surpassed the incidence of O157 and has raised...
Show moreShiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infection resulting in 265,000 illnesses and more than 3,600 hospitalizations annually. Since its identification in 1982 associated with an outbreak of haemorrhagic colitis, serotype O157:H7 has been the primary focus of research and surveillance. However, the increasing incidence of other serogroups, or non-O157 STEC, that are associated with clinical illness has since surpassed the incidence of O157 and has raised questions about the genetic diversity of this pathogen population. Six serogroups, O26, O45, O103, O111, O121, and O145, have been denoted as "big six" non-O157 STEC serogroups since they are frequently associated with clinical outcomes.In this dissertation, 895 non-O157 STEC isolates recovered from patients in Michigan between 2001-2018 were analyzed using whole genome sequencing (WGS) to identify virulence gene profiles and apply new typing methods to better discriminate closely related strains. The recovery of a wide range of serogroups from cases presenting with symptoms ranging from mild diarrhea to hemorrhagic colitis, indicates that genetic diversity and variation may have an impact on disease outcomes. The number and richness of serogroups identified over the past 18 years has been steadily increasing and serogroup alone lacks the discriminatory capabilities to classify related isolates. Indeed, strains representing the same sequence types (ST) were often found to be unrelated by serogroup. Notably, some serogroups, STs, virulence gene profiles and alleles were associated with clinical outcomes and patient demographics. Contrast to national surveillance, cases between 11 and 29 years of age had the highest frequency of STEC infections in Michigan.Additionally, a subset of 44 non-O157 STEC recovered from Michigan patients between 2000 and 2006 were examined more comprehensively while making comparisons to 114 clinical STEC isolates from Connecticut to examine the impact of geographic location on risk factors for non-O157 STEC infections. Lastly, a subset of STEC isolates associated with outbreaks in Michigan were examined to identify the impact of WGS on identification of strain relatedness for surveillance compared to pulsed-field gel electrophoresis.While most of the work outlined in this dissertation focused on characterizing clinical non-O157 STEC isolates, a comparative analysis of cattle isolates was also performed since cattle are an important reservoir of STEC. Indeed, numerous outbreaks and illnesses have been traced back to contaminated cattle-based food products or fecal contamination of water and crops. The ability of STEC to persist in the cattle reservoir and farm environment may give rise to more pathogenic strains due to the accumulation of horizontally acquired genes. 66 STEC isolates recovered from a beef herd over four samplings were examined to identify the genetic diversity within the cattle population and longitudinal persistence. The ability of a strain to form a strong biofilm was associated with the ability to persist and be recovered at multiple sampling phases from the same animal. Further, to better understand the genetic diversity of STEC recovered from the cattle reservoir, an additional 12 STEC isolates from three bovine herds (n = 78) and 241 clinical O157 STEC isolates (n = 1,135) were included to identify shared profiles. The similarity in serogroups and virulence gene profiles warrant a continued surveillance of the cattle environment to better understand crossover events and the ability of strains to evolve into new virulent STEC lineages. The work described in this dissertation helped to elucidate the genetic characteristics important for clinical outcomes and identified targets for future surveillance to better understand lineages that may be important for disease.
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