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Investigating The Impact Of Nonenzymatic Lysine Acetylation On The Function Of The Bacterial Ribosome, Sarah Caldwell Feid

Dissertations

An under-investigated target of lysine acetylation is the bacterial ribosome. Although lysine acetylations on the ribosome are common and conversed in diverse bacterial species, little work has been done to understand how lysine acetylation affects the bacterial ribosome. The goal of this work is to determine if lysine acetylation has functional impact on the bacterial ribosome.I have identified in vitro and in vivo effects of nonenzymatic, AcP-dependent lysine acetylation on translation and the ribosome. In vitro acetylation of transcription-translation reactions causes a translation-specific defect that is unaffected by the addition of the CobB deacetylase. This suggests certain AcP-sensitive residues that …

Characterization Of Mroq-Dependent Regulation Of Staphylococcus Aureus Quorum Sensing, Madison Ruth Stock

Dissertations

Gram-positive bacteria produce small autoinducing peptides (AIPs), which act to regulate expression of genes that promote adaptive traits including virulence. The Gram-positive pathogen Staphylococcus aureus generates a cyclic AIP that controls expression of virulence factors via the accessory gene regulatory (Agr) system. S. aureus strains belong to one of four Agr groups (I, II, III, and IV), and each group harbors allelic variants of AgrD, the precursor of AIP. In a prior screen for S. aureus virulence factors, the Alonzo lab identified MroQ, a putative peptidase. A ΔmroQ mutant closely resembled a Δagr mutant and had significant defects in AIP …

Identification Of Conditions That Promote Biofilm Formation By Vibrio Fischeri And Elucidation Of The Underlying Signal Transduction Pathway, Courtney Noelle Dial

Dissertations

Biofilms are communities of microorganisms that have intrinsically antimicrobial properties and are difficult to treat in the clinical setting. The marine bacterium Vibrio fischeri efficiently colonizes its symbiotic squid host, Euprymna scolopes, by producing a transient biofilm dependent on the symbiosis polysaccharide (SYP), making it the perfect model system to study biofilm dynamics. In vitro, however, wild-type (WT) strain ES114 fails to form SYP – dependent biofilms. Instead, genetically engineered strains, such as those lacking the negative regulator BinK, have been developed to study SYP biofilms. Historically, V. fischeri has been grown using LBS, a complex medium containing tryptone and …

The Expanding Marine Built Environment And Biofilm Formation Processes, Rachel Mugge

Dissertations

The marine built environment (i.e., places or things made or modified by humans) in the northern Gulf of Mexico is rapidly expanding and includes over 2,000 known historic shipwrecks, nearly 4,100 artificial reefs, and other built structures for natural resource extraction, marine aquaculture, renewable energy generation, munitions dumping areas, and commercial and recreational activities. While these structures have the potential to develop into artificial reefs, acting as biodiversity hotspots that provide food and shelter and facilitate organism transport, it is unclear how they affect biodiversity and ecosystem function in the marine environment. The success of artificial reefs to become ecosystems …

An Investigation On The Impact Of Intestinal Oxygen Availability On Survival And Regulation Of Virulence In Listeria Monocytogenes, Damayanti Chakravarty

Dissertations

The deadly foodborne pathogen Listeria monocytogenes is a gram positive facultative anaerobic bacterium. It is the third leading cause of death from food-borne illnesses. Once ingested, it encounters various stressors in the gastrointestinal (GI) tract, including acidic pH, bile, and alterations in oxygen availability. Various studies have been done regarding the pathogen’s survival mechanism against acid and bile. Since the lower parts of the GI tract are anaerobic, it is imperative to investigate how physiologically relevant anaerobic conditions impact L. monocytogenes’s survival. Transcriptomic analysis of L. monocytogenes under conditions mimicking the GI tract was performed. A large number of genes …

Interleukin-17a Facilitates Chikungunya Virus Infection By Inhibiting Ifn-Α2 Expression, Biswas Neupane

Dissertations

Interferons (IFNs) are the key components of innate immunity and are crucial for host defense against viral infections. Here, we report a novel role of interleukin-17A (IL-17A) in inhibiting IFN-α2 expression, thus promoting chikungunya virus (CHIKV) infection. CHIKV infected IL-17A deficient (Il17a^-/-) mice expressed a higher level of IFN-α2 and developed diminished viremia and milder footpad swelling in comparison to wild-type (WT) control mice, this was also recapitulated in IL-17A receptor-deficient (Il17ra^-/-) mice. Interestingly, IL-17A selectively blocked IFN-α2 production during CHIKV, but not West Nile virus (WNV) or Zika virus (ZIKV), infections. Recombinant IL-17A …

The Role Of The Msaabcr Operon In Cell Wall Integrity And Programmed Cell Death During Biofilm Development, Bibek G C

Dissertations

Staphylococcus aureus is an important human pathogen in both community and health care settings. Biggest challenges with S. aureus as a pathogen is its ability to acquire antibiotic resistance and produce robust biofilms. In this work, we investigated the nature of the cell wall defect in the msaABCR operon mutant in the Mu50 (VISA) and USA300 LAC methicillin-resistant Staphylococcus aureus (MRSA) strains. Results showed that msaABCR-mutant cells had decreased cell wall thickness and cell wall crosslinking in both strains. These defects are most likely due to increased murein hydrolase activity and/or nonspecific processing of murein hydrolases mediated by increased …

Elucidating The Role Of The Msaabcr Operon In Persister Cells Formation And Stress Response In Staphylococcus Aureus, Shanti Pandey

Dissertations

Staphylococcus aureus is a major human pathogen that causes lethal systemic conditions such as infective endocarditis, osteomyelitis, sepsis and pneumonia. Chronic in nature, these diseases are often refractory to the antibiotic treatment. Such recalcitrance is mainly caused due to the presence of persister cells, which are a bacterial subpopulation that exhibits extreme, yet transient antibiotic tolerance accompanied by a transient halt in the growth. However, upon cessation of antibiotic treatment, resumption in growth of persister cells causes recurrence of infections and treatment failure, displaying tremendous clinical significance. In this study, we show the involvement of the msaABCR operon in persister …

Investigating The Roles Of Lipids In Staphylococcus Aureus Infection, Xi Chen

Dissertations

The Gram-positive bacterium Staphylococcus aureus is a major threat to public health and causes a multitude of infections, ranging from mild skin and soft infection, to more severe diseases including sepsis, osteomyelitis and infective endocarditis. As a successful pathogen, S. aureus employs various mechanisms to invade host tissues, evade immune responses, and survive in the host environment. One critical adaptive trait of S. aureus that promotes virulence and survival is the ability to maintain membrane homeostasis via flexible modifications to its lipid composition in response to the surrounding lipid environment. Phospholipids are the major component of the bacterial membrane and …

F Plasmids In Escherichia Coli Decrease Permissivity To Coliphage, Cesar Eber Montelongo Hernandez

Dissertations

The urinary tract contains a community of bacteria called the urinary microbiota (urobiota) thatmay be relevant to health; the genomic component of the urobiota is the urinary microbiome (urobiome). Urinary bacteria have been associated with both asymptomatic states and disease conditions, such as urinary tract infection (UTI), overactive bladder (OAB), and urge urinary incontinence (UUI). Some bacteria, such as E. coli, are considered urinary pathogens (uropathogens) but also can be commensals. Bacteriophage (phage) are ubiquitous in nature and likely shape bacterial populations in every niche; thus, phage may be one factor that modulates the urobiota. Phages have a specific host …

Identification And Characterization Of Calcium As A Biofilm Promoting Signal By Vibrio Fischeri, Alice Hannah Tischler

Dissertations

The marine bacterium, V. fischeri actively engages in an exclusive partnership in the light organ of its squid host, Euprymna scolopes. A critical step in this colonization process is the formation of a bacterial aggregate, or biofilm, which is a community of bacteria embedded within a protective extracellular matrix. While V. fischeri readily forms biofilms in nature, genetic overexpression must be utilized to achieve biofilm formation in the laboratory. Recent work investigating media composition led myself and others to evaluate how seawater salts impacted growth and biofilm formation in a number of mutant backgrounds, leading to the identification of calcium …

1,4-Dioxane Biodegradation In Propanotrophs: Molecular Foundations And Implications For Environmental Remediation, Li Fei

Dissertations

1,4-Dioxane (dioxane) has emerged with an escalating concern given its human carcinogenicity and widespread occurrence in groundwater. Bioremediation is promising as an effective and cost-efficient treatment alternative for in situ or ex situ cleanup of dioxane and co-existing pollutants in the field. Soluble di-iron monooxygenases (SDIMOs) are reputed for their essential roles in initiating the cleavage of dioxane and other pollutants. In this doctoral dissertation, molecular foundations for SDIMOs-mediated dioxane biodegradation are untangled to promote the development and implication of site-specific bioremediation and natural attenuation strategies. This dissertation focused on propanotrophic bacteria given their pivotal roles in dioxane metabolism and …

Antibiotic Resistant Bacteria, Antibiotic Resistance Genes And Potential Drivers In The Aquatic Environments, Shuo Shen

Dissertations

As antibiotic resistance genes in aquatic environment have been increasing across the world, affecting water quality and public health, many studies documented concentrations of antibiotic resistance genes and some studies discussed their potential drivers. However, systematic and quantitative reviews that link antibiotic resistance genes (ARGs) to anthropogenic and environmental factors are limited. Nevertheless, this information will be important for developing regulation policy on controlling antibiotic use and therefore reducing potential risks to antibiotic resistance. I conducted meta-analysis of ARGs concentration at a global scale using Bayesian inference to explore climatic and socio-economic factors as drivers. I found local-scale climatic variables …

Investigating The Mechanism Of The Coronavirus Endoribonuclease In Antagonizing Innate Immune Signaling, Matthew Hackbart

Dissertations

Coronaviruses (CoVs) are positive-sense RNa viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease (EndoU) that cleaves RNa in biochemical assays, but the target and function of EndoU activity during viral replication was not known. My work focused on characterizing the functions of EndoU during infection. I report that EndoU is an innate immune antagonist. to function as an immune antagonist, EndoU cleaves the 5'-Poly-Uridines from Negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNa synthesis. Using a virus containing an EndoU catalytic-inactive mutation, I detected …

Elucidating The Impact And Mechanism Of Hepatitis C Virus Cell-To-Cell Spread, Karina Durso-Cain

Dissertations

Virus cell-to-cell spread has been implicated in the establishment of persistent infection and has been shown to be involved in the transmission of antiviral resistant mutants. However, relatively little is known about how virus cell-to-cell spread impacts infection or the specific mechanisms by which cell-to-cell spread occurs. as such, this dissertation focused on investigating HCV cell-to-cell spread not only to learn more about this medically important virus, but also to determine the broader impact cell-to-cell spread has on viral infection dynamics, identify the cellular factors involved, and perhaps ultimately provide insight into antiviral strategies that might enhance the barrier to …

Distinct Roles For Carbohydrate And Protein Receptors In Coronavirus Infection, Enya Qing

Dissertations

Coronaviruses (CoVs) are common human and animal pathogens. in humans, four endemic CoV species together account for one third of mild respiratory infections worldwide. More severe and frequently fatal respiratory pathologies are caused by recent CoV outbreaks that resulted from occasional zoonotic spillover from animal CoV reservoirs, namely, SARS-CoV in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019. Because CoVs threaten global health, any chance of relieving CoV's threat on human populations would rely heavily on our understanding of the mechanistic requirements for CoV tropism, whose major determinant is at the level of viral entry. CoVs have evolved to use …

Elucidating The Targets And Function Of The Mlr Compass-Like Complex During Development, David Joseph Ford

Dissertations

MLR COMPASS-like complexes are highly-conserved epigenetic regulators required for enhancer establishment and subsequent reprogramming during differentiation and development. Mutation of MLR complex subunits in humans is associated with cancer and developmental disorder, yet much remains to be determined concerning both the healthy and disease-altered functions of these complexes. Using the developmental model Drosophila melanogaster, I further elucidate the functions of the MLR complex during in vivo organ development as well as stress response. I characterize the miRNa bantam as a regulatory target of the complex, required for proper tissue patterning during wing and compound eye formation. in the same systems …

The Histoplasma Capsulatum Ddr48 Gene Is Required For Survival Within Macrophages And Resistance To Oxidative Stress And Antifungal Drugs, Logan Blancett

Dissertations

Histoplasma capsulatum(Hc)is a systemic, dimorphic fungal pathogen that affects upwards of 500,000 individuals in the United States annually. Hc grows as a multicellular mold at environmental temperatures; whereas, upon inhalation into a human or other mammalian host, it transforms into a unicellular, pathogenic yeast. The research presented in this dissertation is focused on characterizing the DNA damage-responsive gene HcDDR48. HcDDR48was originally isolated via a subtractive DNA library enriched for transcripts enriched in the mold-phase of Hcgrowth. Upon further analysis we found that HcDDR48is not just expressed in the mold morphotype, but both growth programs …

The Antimicrobial Activity And Cellular Targets Of Plant Derived Aldehydes And Degradable Pro-Antimicrobial Networks In Pseudomonas Aeruginosa, Yetunde Adewunmi

Dissertations

Essential oils (EOs) are plant-derived products that have been long exploited for their antimicrobial activities in medicine, agriculture, and food preservation. EOs represent a promising alternative to conventional antibiotics due to the broad-range antimicrobial activity, low toxicity to human commensal bacteria, and the capacity to kill microorganisms without promoting resistance. Despite the progress in the understanding of the biological activity of EOs, many aspects of their mode of action remain inconclusive. The overarching aim of this work was to address these gaps by studying molecular interactions between antimicrobial plant aldehydes and the opportunistic human pathogen Pseudomonas aeruginosa. We initiated …

The Role Of Multidrug Resistance Regulators Mara, Soxs, Rob And Rama In Regulating Virulence Traits In Salmonella Enterica, Srinivas Thota

Dissertations

Enteric pathogens sense numerous signals specific to the anatomical location in the intestine and integrate them with the complex regulatory networks to temporally and spatially regulate their virulence genes. MarA, SoxS, Rob and RamA are homologous transcription factors that belong to AraC family of proteins in Salmonella enterica that primarily were thought to be involved in rendering antibiotic resistance to bacteria by up regulating efflux pumps and down regulating outer membrane porins. The fact that these transcription factors respond to the same intestinal compounds that regulate virulence genes in Salmonella motivated us to look for other roles of these transcription …

Regulation Of Acetyl Phosphate-Dependent Acetylation And Identification Of Novel Lysine Acetyltransferases In Escherichia Coli, David George Christensen

Dissertations

Over billions of years, organisms have organized chemical reactions into metabolic pathways to sustain life. However, metabolic substrates can undergo many uncatalyzed, extra-metabolic reactions. Acetyl phosphate (AcP), an intermediate of the acetate fermentation pathway in E. coli, is one such metabolite that has been shown to non-enzymatically acetylate hundreds of proteins. This diverse set of targets suggests that acetylation could be a way for the cell to sense its nutritional status and regulate protein activity accordingly. However, how E. coli regulates acetylation, if at all, is unknown.Previous work showed that acetylation becomes pronounced in stationary phase cells. I determined that …

Membrane Microdomains As Platforms For Extra-Cellular Fusions, Michael Hantak

Dissertations

Life requires biological membranes. Membrane-enclosed compartments separate and unite through dynamic fission and fusion reactions. These are catalyzed processes that are central in organismal biogenesis. This dissertation focuses on extracellular membrane fusions, which are central to several processes. (1) Enveloped viruses enter cells through membrane fusions. (2) Extracellular vesicles (EVs) also deliver molecules into cells through membrane fusions. (3) Entire cells also fuse together, generating fertilized zygotes, skeletal muscles, and giant cell macrophages.Mechanisms of extracellular membrane fusion are poorly understood. This dissertation aimed to further define these mechanisms. We focused on regulatory cofactors, including tetraspanins, transmembrane proteins that cluster into …

Staphylococcus Aureus Evasion Of The Innate Immune System, James Paul Grayczyk

Dissertations

Upon entry into the host, pathogens must overcome innate immunity in order to cause disease. The innate immune system represents a fast-acting initial line of defense to prevent infection. In order to withstand innate defenses, bacterial pathogens like the Gram-positive bacterium Staphylococcus aureus, produce a wide array of virulence factors that can inhibit innate immune cell recruitment and antimicrobial activity, or directly target and kill phagocytic leukocytes thereby facilitating pathogenesis. Infection with S. aureus can cause disease in virtually any tissue site and is a significant burden to human health. In this dissertation, we sought to understand how S. aureus …

Aerococcus Urinae: Establishing The Pathogenesis Of An Emerging Uropathogen, Evann E. Hilt

Dissertations

Urinary tract infection (UTI) is the world's most common bacterial infection. Much is known about the infectious process (pathogenesis) of a few of the bacteria that cause these infections, especially E. coli. Unfortunately, the pathogenesis of E. coli and other uropathogenic bacteria was explored almost exclusively in the belief that the bladder is supposed to be sterile. Our recent evidence, however, debunks this dogma. We used modern methods to reveal diverse bacterial communities in the bladders of adult women. These communities differ in women with and without lower urinary tract symptoms (LUTS), including UTI and urinary incontinence (UI). Many bacteria …

Assessing The Temporal Dynamics Of The Lower Urinary Tract Microbiota And The Effects Of Lifestyle, Travis Kyle Price

Dissertations

Urinary tract infections (UTIs) are among the most common bacterial infections in humans, accounting for $3.5 billion in health care expenditures yearly in the United States alone. Yet, treatments for UTI have seen little innovation over the past decade. As demonstrated in other body sites, such as the vagina and gastrointestinal (GI) tract, acute and infectious diseases often have indirect microbial contributions which serve as intriguing new targets for therapies. The recent discovery of the existence of a resident community of bacteria (i.e., microbiota) in the bladders of both women and men, represents a novel avenue for targeting UTIs. However, …

Generating Biologic And Genetic Research Tools To Investigate Serotype I Feline Coronaviruses, Robert Christian Mettelman

Dissertations

Serotype I feline infectious peritonitis virus (FIPV) is an alphacoronavirus of high veterinary importance due to the 99% mortality rate observed in cats. Since the 1980s, robust experimentation on these viruses has been limited by availability of culturable pathogenic strains, few permissive cell lines, and a lack of standardized methods to study their basic virology. These complications have resulted in variable and conflicting literature reports, have slowed study of clinical strains and hindered effective vaccine design. The goal of this dissertation was to develop a laboratory "toolbox" containing standardized methods, permissive cell lines and genetic techniques to alleviate some of …

Stress Response Mechanisms Of Listeria Monocytogenes, Oindrila Paul

Dissertations

Listeria monocytogenes is a Gram-positive, facultative intracellular food-borne pathogen that causes the disease listeriosis. In order to establish an infection, L. monocytogenes must survive multiple stressors encountered within the gastrointestinal tract, including alterations in pH, bile, salt, and oxygen availability. This dissertation focused on understanding the stress response of L. monocytogenes to bile. Bile acts as a bactericidal agent by disrupting the membrane integrity and causing instability to macromolecules like DNA. Thus, a bacterium must be able to maintain its membrane architecture, composition and integrity.

Often times, bacteria will modulate their fatty acid composition in the membrane to cope with …

The Characterization Of The Transcription Factor Msab And Its Role In Staphylococcal Virulence, Justin Batte

Dissertations

Staphylococcus aureus is a common human pathogen that is responsible for a wide range of infections, ranging from relative minor skin infections to life-threatening disease such as bacteremia, septicemia, and endocarditis. S. aureus possesses many different virulent factors that aid in its ability to cause this wide array of infections. One major virulence factor includes the production of capsular polysaccharide (CP). The production of CP plays a major role in the virulence response during infection specifically by providing S. aureus an antiphagocytic mechanism that allows the pathogen to evade phagocytosis during an infection. S. aureus has developed complex genetic regulatory …

Molecular Determinants Of Trim5Α Restriction And Recruitment Of Autophagic Effectors, Sabrina Imam

Dissertations

TRIM5α is an anti-viral restriction factor that inhibits the lifecycle of retroviruses. TRIM5α binds to and forms a hexameric lattice around the retroviral capsid, thereby initiating its antiviral activities, which include: (1) inhibition of viral infection; (2) inhibition of viral reverse transcription; (3) disassembly of the capsid; and (4), activation of innate signaling pathways. The formation of this assembly also activates the E3 ubiquitin ligase function of TRIM5α. Ubiquitin modification is associated with directing substrates to particular cellular pathways. We and others have shown that TRIM5α cytoplasmic bodies colocalize with proteins involved in the autophagy pathway, and we hypothesized that …

Uncovering The Mechanisms Underlying The Immunogenicity Of Adenovirus Vaccine Vectors, Natalie Nidetz

Dissertations

Vaccination is historically the most effective tool for preventing infectious disease but current vaccine strategies fail to generate robust immunity to major infectious diseases such as HIV and malaria. Therefore, newer vaccine approaches are needed. Vaccines generated from viral, adenovirus based, vectors (AdVs) have proven highly immunogenic in multiple disease models. However, the clinical use of many AdVs is limited by the presence of pre-existing antibodies in human populations, which prevent expression of antigenic genes during immunization with AdVs based on common adenovirus (Ad) serotypes, such as HAdV-5C. Immunization with rare serotype based AdVs, such as HAdV-28D, are not affected …