Microbiology Commons^™

Investigating Host Factors In Coronavirus Assembly And Egress, Surovi Binte Sharmin Mohona

Dissertations

Coronavirus (CoV) structural proteins congregate at the ER-Golgi intermediate compartments (ERGIC) prior to virus budding into the organelle lumen. Subsequent trafficking and fusion of virus-containing, ERGIC-derived vesicles then release nascent viruses from infected cells. Our findings in this dissertation illuminate mechanisms by which CoV structural proteins coalesce at the virus assembly and budding sites. The cytoplasmic tails of all CoV spike (S) glycoproteins contain variants of a consensus KxKxx coatomer-binding motif. Host coatomer complexes facilitate retrograde trafficking of client cargo proteins via recognition of such motifs. Utilizing reverse genetics methodologies, we introduced variations in the S coatomer motif in a …

An Examination Of Lysine Acetylation In The Context Of Bacterial Metabolism, Translation, And Regulation Of Gene Expression, Thomas Patrick Bank

Dissertations

Post-translational modifications are a critical tool bacteria employ to survive the challenges of microbial life. Acetylation continues to gain more recognition as a modification of unique importance because the sources of acetylation – acetyl-phosphate and acetyl-coenzyme-A – are critical metabolites associated with central carbon metabolism, acetate overflow metabolism and acetate assimilation. This suggests that acetylation, indelibly coupled to metabolic flux, integrates metabolic sensing with any cellular process or pathway where an acetylation alters the function of a protein. Intuitively, this makes sense. Of course, a bacterial cell would coordinate any cellular process closely with the amount of energy available to …

Fungal Biodiversity, Microbial Communities, And Ecosystem Function In Non-Perennial Streams, Charles T. Bond

Dissertations

A global majority of steams are non-perennial, drying at some point in time and space. Despite the vital roles that fungi and other microbes play in stream ecosystems, most research on stream microbiomes comes from perennial (continuously flowing) streams. The present series of studies addresses this research gap by examining microbial community composition and function in non-perennial streams, with an emphasis on fungi. Chapter I reviews existing scientific literature on the ecological processes that shape microbiome structure and function in non-perennial streams and outlines the varieties of fungi that may live there. Chapter II uses rDNA metabarcoding to survey fungal …

Increasing Pediatric Hpv Vaccination Rates, Brooke A. Nicholas

Dissertations

Increasing Pediatric HPV Vaccination Rates

Abstract

Problem: The human papillomavirus (HPV) is a sexually transmitted infection that affects more than 80% of the population within the United States (Eisenhauer et al., 2020). HPV can develop into cervical, anogenital, and oropharyngeal cancers (Eisenhauer et al., 2020). About 4,000 individuals die from cervical cancer alone each year (O’Leary, 2022). The HPV vaccine, though recommended by the ACIP and AAP, is below national vaccination rates in comparison to other vaccines (O’Leary, 2022).

Methods: This quality improvement project is a non-randomized controlled trial that observed HPV vaccine dose completion rates for patients aged 9-14 …

Genetic And Biochemical Analyses Reveal Litr's Role In Vibrio Fischeri Biofilm Formation, Brittany Loi Fung

Dissertations

Quorum sensing controls numerous processes ranging from production of virulence factors to biofilm formation. Biofilms, communities of bacteria that are attached to one another and/or a surface, are common in nature and, when they form, can produce a quorum of bacteria. One model system to study biofilms is the bacterium Vibrio fischeri, which forms a biofilm that promotes colonization of its symbiotic host. Many factors promote V. fischeri biofilm formation in vitro, including the symbiosis polysaccharide (SYP), cellulose, and the LapV adhesin but, so far, only a handful of studies have probed connections between biofilm formation and quorum sensing. In …

Investigating The Consequences Of Conjugated And Secreted Isg15 On Coronavirus Pathogenesis, Mudassir Syed Ali

Dissertations

Coronaviruses have evolved a breadth of mechanisms to antagonize host innate immune responses. One widely studied mechanism is the ability to deconjugate ISG15 from target proteins in a process called deISGylation, which is catalyzed by the papain-like protease (PLP) domain of viral non-structural protein (nsp) 3. Since PLP activity is required for viral replication, it has been challenging to generate an infectious virus with a PLP that maintains protease activity but has lost deISGylase activity. In this study, we generated a chimeric MHV-A59 with its PLP2 domain replaced with that of the human coronavirus HKU1, which has been documented to …

Interrogating The Dispersal Process And Relevant Genes In Vibrio Fischeri, Jeremy Esin

Dissertations

Biofilms are aggregates of bacteria encased in a matrix that shields bacteria from environmental hazards. These structures are formed in response to cues from the environment and drive colonization of new niches. Biofilms can be both beneficial and detrimental. Largely, research focuses on biofilms that have negative outcomes in clinical, environmental, and economical settings. However, the value of beneficial biofilms is recognized and studied. The marine bacterium Vibrio fischeri is part of a model system for studying symbiotic biofilms in a eukaryotic host. V. fischeri produces a transient biofilm at the surface of a specialized light organ and then disperses …

Investigating The Proviral And Immunogenic Effects Of Sars-Cov-2 Accessory Protein 7a, Grant Michael Hawkins

Dissertations

SARS-CoV-2 and related sarbecoviruses encode a set of accessory proteins (3a, 3b, 6, 7a, 7b, 8, 9b, and 10) that control host responses to infection and promote virus growth. Of these accessory proteins, 7a is set apart by its intracellular localization near coronavirus (CoV) budding sites and its incorporation into secreted virions. Initial studies utilizing a virus-like particle (VLP) system identified significant proviral effects mediated by 7a, but the system lacked the context of an actual CoV infection. To investigate 7a functions during CoV infections, we constructed recombinant Mouse Hepatitis Viruses (rMHV strain A59) that express 7a genes. Comparative infections …

Discerning The Role Of Microtubule Acetylation During Hiv-1 Infection, Drew Michael Lichon

Dissertations

The host-pathogen interactions underlying Human Immunodeficiency Virus type 1 (HIV-1) infection are complex and involve the exploitation of host cellular machinery. This thesis investigates the roles of dynein adaptors, microtubule acetylation, and TRIM69-mediated restriction in HIV-1 infection. Through a series of detailed experiments, we explore how HIV-1 utilizes multiple dynein adaptors, such as BICDR1 and Hook3, for intracellular transport, and reveal that these adaptors are critical for efficient viral trafficking. Conversely, the knockdown of Ninein surprisingly enhances HIV-1 infectivity, suggesting a nuanced regulatory role for this adaptor in viral transport. Further, this thesis challenges the prevailing paradigm that microtubule acetylation …

Investigating The Role Of The Nsp15 Amino-Terminal Domain In Nsp15 Function, Viral Replication, And Pathogenesis, Monika Evdokimova

Dissertations

Identifying conserved mechanisms used by viruses to delay host innate responses can reveal potential targets for antiviral therapeutics. Here, we investigated coronavirus nonstructural protein 15 (nsp15), which encodes a highly conserved endoribonuclease (EndoU). EndoU functions as an immune antagonist by limiting the accumulation of viral replication intermediates that would otherwise be sensed by the host. Despite being a promising antiviral target, it has been difficult to develop small molecule inhibitors that target the EndoU active site. We generated nsp15 mutants of SARS-CoV-2 and mouse coronavirus MHV-A59 and identified conserved residues within the amino-terminal domain that are required for EndoU activity. …

Insights Into Female Urinary Microbiota Through Long-Read 16s Rrna Sequencing, Jerrin John

Dissertations

The human urinary microbiome—once thought to be sterile—is now recognized as a dynamic, low-biomass microbial community with potential implications for urinary tract health. Conventional short-read 16S rRNA gene sequencing targeting individual variable regions often lacks the taxonomic resolution needed to distinguish closely related species, while culture-based methods may miss fastidious or low-abundance taxa. We applied full-length 16S rRNA gene sequencing across all nine variable regions to daily urine samples collected from asymptomatic premenopausal female participants. Sequence data were processed with a modified DADA2 pipeline optimized for long reads to generate exact sequence variants. We compared species-level community profiles against both …

Interactions Between Microplastics, Biofilms, And Antimicrobials In Freshwater Streams, Ryan Thomas Krantz

Dissertations

Microplastics (MPs) are ubiquitous contaminants in freshwater ecosystems that could be hotspots for the interaction of antimicrobial compounds and surface-attached microbial biofilm communities. MPs and antimicrobials are both common in wastewater and urban freshwaters and MPs can adsorb contaminants like antimicrobials to their surface. Within aquatic habitats, MPs also support colonization by microbial biofilms. Therefore, we hypothesized that the adsorption of antimicrobials to MP surfaces would affect microbial communities colonizing MPs, altering their diversity, composition, and antimicrobial resistance. We investigated the effects of these interactions across two studies. The first study, discussed in Chapter 2, used a microcosm approach to …

Role Of Coronavirus Envelope Protein On Infectivity, Garrett E. Zaagman

Dissertations

Coronaviruses infect a wide range of hosts and have caused recent human pandemics. While much has been learned, gaps remain in understanding viral assembly and egress, particularly the role of the envelope (E) protein in specific infectivity. To study this, recombinant MHV viruses were created with E gene deletions and HiBiT-tagged spike (S) proteins to monitor virion release. In mouse cells, MHV∆E S-HiBiT secreted less spike and fewer infectious particles, indicating reduced specific infectivity. Comparisons between human HeLa-CEACAM and mouse DBT cells showed species-specific support for virus production. TMED10, a host membrane protein, was tested as a potential E-interacting partner …

Elucidating The Mechanism Of Interleukin-17a Signaling In The Pathogenesis Of West Nile Virus, Farzana Nazneen

Dissertations

West Nile Virus (WNV), a neurotropic flavivirus transmitted by mosquitoes, is a leading cause of viral encephalitis in the United States and remains a significant public health concern. Unfortunately, no vaccine or therapeutic intervention is available against WNV infection. Previous studies, including ours, demonstrated that interleukin-17A (IL-17A) signaling increases the cytotoxic potential of CD8⁺ T cells to facilitate WNV and parasite clearance; however, the molecular mechanism is not understood. IL-17 receptor C (IL-17RC) is an obligatory co-receptor with IL-17 receptor A (IL-17RA) for signaling induced by IL-17A, IL-17A/F, and IL-17F. In this study, we found that IL-17RC deficient ( …

Elucidating Enterococcal Mechanisms Underlying Recurrent Urinary Tract Infections, Baylie Hochstedler-Kramer

Dissertations

Urinary tract infection (UTI) is one of the most common bacterial infections globally and can lead to life-threatening complications. UTI disproportionately affects females, with the majority of females being treated for at least one UTI in their lifetime. Of the females who develop UTI in their lifetime, almost one third will develop recurrence within six months after initial treatment and many will struggle with UTI recurrence for long spans of their lives. Although recurrent UTI has previously been attributed to persistence of Escherichia coli, recent studies utilizing more sensitive detection methods have shown Enterococcus faecalis is strongly associated with recurrent …

Disambiguation Of Aerococcus Urinae Species Diversity, Brian Ian Choi

Dissertations

Aerococcus urinae is an uncommon inhabitant of the human urogenital tract. The Gram-positive bacterium has been implicated in a wide range of urinary tract diseases that, if left untreated or undiagnosed, can progress to severe disease such as infective endocarditis. Comparative analysis of 115 A. urinae isolates from infection episodes of urinary tract infection, bacteremia, infective endocarditis, urgency urinary incontinence, stress urinary incontinence, interstitial cystitis / painful bladder syndrome, and asymptomatic individuals reveal substantial diversity with respect to predicted virulence factors, metabolism, and observed biofilm behavior. Through whole genome sequencing analysis, these strains can be reorganized into six new taxonomic …

Characterization Of The Glyoxal Response In Pseudomonas Aeruginosa, Christopher James Corcoran

Dissertations

The toxic dialdehyde glyoxal is found in all domains of life and results as a by-product from a variety of metabolic activities such as DNA oxidation, lipid peroxidation, and autoxidation of sugars such as glucose. If allowed to accumulate, the reactive aldehyde groups of glyoxal have the ability to react with a plethora of molecules including, DNA, lipids, and protein side chains. Most notably, glyoxal can react with arginine, lysine, and cysteine residues, forming irreversible covalent modifications known as advanced glycation end products (AGEs). These glyoxal-derived AGEs can impair protein function, leading to cellular toxicity and eventual cell death. Due …

Genetic, In Vitro Phenotypic, And Clinical Characterization Of Atypical Enteropathogenic E. Coli Infection And Pathogenesis, Sarah E. Kralicek

Dissertations

Enteropathogenic E. coli (EPEC) are enteric pathogens that are non-invasive, lack Shiga toxin, and use a type 3 secretion system encoded on the locus of enterocyte effacement (LEE) pathogenicity island to translocate bacterial effector proteins into host intestinal epithelial cells leading to diarrhea. Atypical EPEC (aEPEC), in contrast to typical EPEC (tEPEC), lack bundle-forming pili, likely altering adherence and pathogenicity. Detection of aEPEC with co-infecting pathogens and in some asymptomatic individuals leads to questions regarding aEPEC virulence, especially in adults. I aimed to characterize the clinical manifestations of aEPEC infection and the genetic and in vitro phenotypic factors that contribute …

Elucidating The Underlying Mechanisms Polyamines Play On Coxsackievirus B3 Infection, Mason Richard Firpo

Dissertations

Polyamines are a class of metabolites consisting of small carbon chains that contain amine groups and are positively charged at cellular pH. They play many important roles within the cell and are necessary for cell growth, membrane function, transcription, and translation. They are also required by a myriad of viruses for a productive infection. This includes the non-polio enterovirus Coxsackievirus B3 (CVB3). CVB3 is a cardiotropic virus that can readily infect the heart and cause irreversible damage which can eventually lead to dilated cardiomyopathy and the need for a heart transplant. Unfortunately, there are no approved antivirals or vaccines that …

Shigella Flexneri Diguanylate Cyclases Regulate Pathogenesis, Ruchi Ojha

Dissertations

The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to colon invasion, this bacterium navigates through different environments within the human body, including acidic conditions in the stomach and the small intestine which contains antimicrobial molecules including bile acids. To adapt to changing environments, Shigella uses the bacterial second messenger c-di-GMP signaling system. C-di-GMP is synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella encodes 10 DGCs in its genome, but 6 of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we first expressed VCA0956, a …

Establishing A Novel Lipid-Polyamine Metabolic Connection To Virus Replication, Yazmin Edith Cruz Pulido

Dissertations

Polyamines are small positively charged alkylamines found in all living cells. These ubiquitous molecules have been found to play a variety of roles ranging from stabilizing nucleic acids (DNA and RNA) to cellular proliferation. Studies have also identified roles for polyamines in viral packaging and viral enzyme function. However, the role of polyamines on cellular components and their effect on viral replication is less known. In our efforts to identify the lipid-polyamine metabolic connection to virus replication we utilized a human hepatocyte cell line in which polyamines were depleted via pharmacological or genetic approaches and studied the effects on lipid …

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 …

Sensing The Rhizosphere: Microbial Transcriptome Responses To Root Exudates And Redox-Active Metabolites, Ankita Bhattacharyya

Dissertations

The term “rhizosphere” describes the dynamic interface between plant roots and soil influenced by root exudates. It is a hotspot of microbial activity, plant-microbe, and microbe-microbe interactions. Distinct variations in bacterial diversity are thought to be driven by plant root exudates. However, the molecular details of these processes still remain unclear.

I addressed this gap by focusing on two strains, Pseudomonas synxantha 2-79 and Burkholderia lata 383, representing diverse groups of Gammaproteobacteria comprised of species with environmental, agricultural, and medical significance. In the first part of this project, we used a combination of metabolomics and RNA-seq to characterize transcriptome responses …

Characterization Of Immunomodulatory Properties Of Manuka Honey: Potential Mitigation Against Colon Cancer, Razan Jamil Masad

Dissertations

The focus on using alternative natural products for cancer prevention and treatment has been increasing over the past decade. Among these natural products, Manuka honey (MH) has been extensively investigated for its antimicrobial, wound healing, and anticancer effects. The current study aimed to characterize the immunomodulatory capacity of MH using both in vitro and in vivo approaches and investigate its potential to modulate anti-tumor immune responses in a preclinical model of colorectal cancer. We demonstrate that MH can directly act on macrophages to induce gene expression of several inflammatory cytokines and chemokines and the production of TNF-a, a major proinflammatory …

Development Of Novel Nanocarbon Based Materials And Membranes With Enhanced Biocidal Capabilities For Clean Water Generation, Indrani Gupta

Dissertations

Water scarcity is one of the major challenges facing humanity today, driven by population growth, economic development, and dietary shifts towards more water-intensive animal products. To address this issue, efficient water resource management and recycling of wastewater are essential. However, around 80% of wastewater globally is discharged into water bodies without treatment, posing a risk to ecosystems and human health. Pathogens, or harmful microorganisms, are a primary cause of water impairment and the transmission of waterborne diseases, leading to increased mortality rates. Traditional methods of treating pathogens involve the use of radiation or chemical disinfectants. Nonetheless, these methods have drawbacks, …

Modulatory Roles Of Human Adenovirus Type 3 In Extracellular Vesicle Development, Trafficking, And Inhibition, Ayodeji Oludare Ipinmoroti

Dissertations

Respiratory human adenovirus (HAdV) has been closely linked with acute respiratory infections such as pharyngitis and coryza (common cold). While some HAdV serotypes antagonize the immune system leading to meningitis, gastroenteritis, and acute hemorrhagic cystitis among other conditions, HAdV type 3 (HAdV3) predominantly infects the upper respiratory tract. Studies have shown that the release of small, membrane-derived extracellular vesicles (EVs), termed exosomes, may offer a mechanism by which certain viruses, enter cells via receptor-independent entry. EVs play a major role in both cellular homeostasis and intercellular communication under both healthy and pathological conditions. Firstly, in this study, we demonstrated that …

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 …

Elucidating The Role Of A Staphylococcal Glucosaminidase In Host Innate Immune Responses, Zachary Resko

Dissertations

The peptidoglycan of Staphylococcus aureus is a critical cell envelope constituent and virulence factor that subverts host immune defenses and provides protection against environmental stressors. Peptidoglycan chains of the S. aureus cell wall are processed to characteristically short lengths by the glucosaminidase, SagB. It is well established that peptidoglycan is an important pathogen-associated molecular pattern (PAMP) that is recognized by the host innate immune system and promotes production of pro-inflammatory cytokines, including IL-1β. However, how bacterial processing of peptidoglycan drives IL-1β production is comparatively unexplored. Here, we tested the involvement of staphylococcal glucosaminidases in shaping innate immune responses and identified …

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 …

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 …