Microbiology Commons^™

Crispr Technology As An Antiviral In Dsdna And Ssrna Viruses, Cathryn Mayes

Theses & Dissertations

The COVID-19 pandemic highlights the necessity of emergency response and pandemic preparedness, especially for emerging viral threats. Currently, virus-specific vaccines and antivirals are the primary tools to combat viral diseases; however, broad-spectrum antivirals that target more than one virus species could provide additional protection from emerging and re-emerging viral diseases (Andersen et al. 2020; Zhu et al. 2015; Hickman et al. 2022).

Clustered regulatory interspaced short palindromic repeat (CRISPR)-associated endonucleases have become recently utilized as potential antiviral strategies due to their high specificity, efficacy, and versatility (Najafi et al. 2022). While CRISPR-based antivirals have previously been used to target specific …

Gut Commensals Modulate Siv/Shiv Pathogenesis And Therapeutics, Samuel Johnson

Theses & Dissertations

Despite significant advancements in combination antiretroviral therapy (cART), ongoing inflammation in the brain and gut remain two of the most significant hurdles in the health of people living with human immunodeficiency virus (HIV). Additionally, a viral reservoir in each compartment inhibits cure efforts by allowing rapid viral rebound following cART interruption. Emerging understanding of the gut-brain axis (GBA) implicates each compartment in the modulation of the other in a complex bi-directional interaction mediated by vagus innervation, circulating lymphocytes, and microbiome composition and biproducts. Using multiple models of the simian (and simian-human) immunodeficiency virus (SIV/SHIV) and therapeutic intervention, I present how …

Metagenome-Wide Associations And Metabolic Modeling To Predict The Biomarkers For Colorectal Cancer, Nagavardhini Avuthu

Theses & Dissertations

Colorectal cancer (CRC) is the most common malignancy and the second most common cause of cancer-related deaths in the world. It is a multifactorial disease mediated by genetic, environmental, and lifestyle factors. Over time, the accumulation of genetic and epigenetic changes results in the activation of oncogenes and deactivation of tumor suppressor genes in the colon and rectal region which finally leads to tumor development. The gut microbiome is the main factor linking the effects of diet and environmental factors to host metabolism. Several studies showed the dysbiosis of gut microbiota in CRC patients with an increased proportion of pathogenic …

Understanding The Impact Of Chlamydia Trachomatis Infection On Host Vesicular Trafficking, Lisa M. Jorgenson

Theses & Dissertations

Chlamydia trachomatis is a highly evolved obligate intracellular bacterial pathogen. During infection, organisms reside within a host epithelial cell in a membrane-bound vacuole called the inclusion. The inclusion membrane contains both pathogen and host components, including unique chlamydial type III effector proteins called Incs and host lipids like cholesterol and sphingomyelin. The inclusion membrane serves as the main barrier between the bacteria and the host cytosol, and thus, functions as the platform supporting host-chlamydial interactions. Via the inclusion membrane, C. trachomatis intercepts components of host vesicular traffic including many host SNARE proteins, which are a conserved family of eukaryotic proteins …

Metabolic Heterogeneity And The Roles Of Cody And Ccpa In Central Metabolism And S. Aureus Biofilm Formation., Logan L. Bulock

Theses & Dissertations

Staphylococcus aureus is a metabolically versatile human pathogen, causing disease in many areas of the body. Its versatility can be attributed to the fact that it utilizes a variety of tools to adapt to many different environments, including toxins to scavenge from the host and multiple transporters to compete for its preferred carbon sources. S. aureus can also survive in harsh conditions through biofilm development, which are notoriously recalcitrant to antibiotics and immune defenses. Biofilms exhibit marked heterogeneity, with division of labor for production of matrix components and differential gene expression among various niches within the biofilm.

In this study, …

The Staphylococcus Aureus Lrgab Operon Encodes A Holin-Like Protein Involved In Pyruvate Transport, Jennifer L. Endres

Theses & Dissertations

The Staphylococcus aureus cidABC and lrgAB operons encode a well-conserved family of proteins involved in programmed cell death (PCD) during biofilm development. Based on the structural similarities that CidA and LrgA share with bacteriophage holins, we have hypothesized that these proteins function by forming pores within the cytoplasmic membrane. To test this, we utilized a “lysis cassette” system that demonstrated the abilities of the cidA and lrgA genes to support bacteriophage endolysin-induced cell lysis. In addition, the CidA and LrgA proteins were shown to localize to the surface of membrane vesicles and cause leakage of small molecules, providing direct evidence …

Strainiq: An N-Gram-Based Method To Identify And Quantify Microbial Communities In Metagenomic Samples, Sanjit Pandey

Theses & Dissertations

Microbes are ubiquitous in nature, and they play vital roles in various processes associated with metabolism in the human body, photosynthesis in plants, or decomposition of waste in the environment. Hence, it is essential to understand how the composition of microbial communities affects the ecosystem of different environments ranging from ocean floors to hot springs to the human body. Microbial communities present in different human body sites are of particular importance due to their implications in the cause and prevention of human diseases. The traditional approaches limit microbial research to exclusively studying species that can be successfully cultured in the …

Role And Regulation Of Staphylococcal Cell Death, Abdulelah Ahmed Alqarzaee S

Theses & Dissertations

The transition from growth to stationary phase is a natural response of bacteria to starvation and stress. When stress is alleviated and more favorable growth conditions return, bacteria resume proliferation without a significant loss in fitness. Although specific adaptations that enhance persistence and survival of bacteria in stationary phase have been identified, mechanisms that help maintain the competitive fitness potential of non-dividing bacterial populations have remained obscure. This dissertation demonstrates that staphylococci entering stationary phase following growth in excess glucose undergo regulated cell death to maintain the competitive fitness potential of the population. Upon a decrease in extracellular pH, the …

Role Of Endocytic Machinery Regulators In Egfr Traffic And Viral Entry, Insha Mushtaq

Theses & Dissertations

STUDY 1: Role of endocytic regulator EHD1 and its binding partner RUSC2 in EGFR traffic

Abstract

Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors. Cell surface display of EGFR is essential for cellular responses to its ligands. While post activation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/pre-activation surface display of EGFR. Here, we identify a novel role of the endocytic regulator EHD1 and a potential EHD1 partner, RUSC2, in cell surface display of EGFR. EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular …

Multistrain Hiv-1 Elimination: A Crispr-Cas9 And Theranostics-Based Approach, Jonathan Herskovitz

Theses & Dissertations

A critical barrier to achieving a functional cure for infection by human immunodeficiency virus type one (HIV-1) rests in the presence of latent proviral DNA integrated in the nuclei of host CD4+ T cells and mononuclear phagocytes. Accordingly, HIV-1-infected patients must adhere to lifelong regimens of antiretroviral therapy (ART) to prevent viral rebound, CD4+ T cell decline, and progression to acquired immunodeficiency syndrome (AIDS). Gene editing using clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 technology stands as one means to inactivate integrated proviral DNA. We devised a mosaic gRNA CRISPR-Cas9 system- TatDE- that targets viral transcriptional regulator genes tat / …

Glycan-Dependent Adherence And Skin Colonization Of Staphylococcus Epidermidis Mediated By The Surface Protein Aap., Paroma Roy

Theses & Dissertations

Skin-dwelling coagulase-negative staphylococci (CoNS), a group of bacteria that includes Staphylococcus epidermidis, has been implicated to promote skin immunity and antimicrobial defense and prohibit colonization of skin by pathogens like S. aureus. As a skin inhabitant, S. epidermidis lives in tight association with corneocytes, the cells that constitute the uppermost layer of the skin epidermis. Yet the molecular mechanism responsible for adhesion of S. epidermidis to corneocytes remains poorly understood. Our study indicated that Accumulation-associated protein (Aap), a cell-wall anchored, fibrillar adhesin mediates bacterial-host interaction, demonstrated by significantly higher corneocyte binding by Aap-positive 1457 mutants as compared to …

Insights Into The Chlamydial Niche: The Dynamic Roles Of Inclusion Membrane (Inc) Proteins In Chlamydia Trachomatis Development, Macy G. Wood

Theses & Dissertations

Chlamydia trachomatis (Ctr) is the leading cause of bacterial sexually transmitted infections. Ctr, an obligate intracellular bacterium, develops within a membrane-bound vacuole called an inclusion. The inclusion membrane is modified by chlamydial inclusion membrane (Inc) proteins, the functions of which are poorly characterized. Bacterial two-hybrid analyses found some Incs (e.g., IncF) interacted with numerous Incs while others (e.g., IncA) did not. We hypothesize that some Incs organize the inclusion through Inc-Inc interactions whereas other Incs promote chlamydial-host interactions by binding eukaryotic proteins. To test our hypothesis, we implemented the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with …

Flavonoid And Cannabidiol Neural Glyoxalase Pathway Enhancement Against Aging And Alzheimer’S Disease, Joel R. Frandsen

Theses & Dissertations

Alzheimer’s Disease is a neurodegenerative condition featuring neural cell death and a decline in cognitive capacity caused by elevated inflammation and production of reactive oxygen species. The glyoxalase pathway is an endogenous antioxidant system that neutralizes reactive methylglyoxal through sequential reactions. Dysfunction of the glyoxalase pathway contributes to oxidative stress and the accumulation of inflammatory metabolic byproducts. Plant-produced compounds with antioxidant activity can enhance endogenous antioxidant pathways and protect cells from elevated ROS production. We hypothesize that flavonoids and limited Cannabis Sativa-produced cannabidiol can enhance glyoxalase pathway function through regulation of antioxidant and pro-apoptotic signaling pathways to prevent methylglyoxal-mediated …

Acid Resistance Mechanisms In Staphylococcus Aureus, Chunyi Zhou

Theses & Dissertations

Staphylococcus aureus is a leading cause of opportunistic infections in community and health care settings. To thrive in a great variety of environments, S. aureus has developed the capability of tolerating temporary pH changes, as well as resisting constant acid stress. To evaluate the impact of strong and weak acid stress on S. aureus, growth patterns of JE2 were monitored when cultured in chemically defined media (CDM) at various pH in the presence of hydrochloric acid (HCl), acetic acid, and lactic acid. Our results showed that S. aureus responds to strong and weak acids in different manners. S. aureus …

Synthesis And Characterization Of A Long-Acting Emtricitabine Prodrug Nanoformulation, Ibrahim M. Ibrahim

Theses & Dissertations

The introduction of highly active antiretroviral therapy led to a paradigm shift in the management of HIV/AIDS changing a disease considered “a death sentence” to “a manageable chronic disease”. Nevertheless, challenges exist for successful treatment of HIV, including patient adherence to the complex daily regimens and the inability of current formulations to target viral sanctuaries. Introduction of nanoformulated antiretroviral therapy (ART) is a promising alternative to tackle these challenges. Our laboratory has been focusing on developing long-acting (LA) nanoformulated antiretrovirals and has succeeded in developing LA integrase inhibitors. However, challenges for this approach extend to a range of short-acting hydrophilic …

Development Of A Long-Acting Nanoformulation Of Dolutegravir For Prevention And Treatment Of Hiv-1 Infection, Brady Sillman

Theses & Dissertations

Dolutegravir (DTG) is a potent human immunodeficiency virus type 1 (HIV-1) integrase strand-transfer inhibitor (INSTI) with a high barrier to viral drug resistance. However, opportunities to improve its profile abound. These include extending the drug’s apparent half-life, increasing penetrance to “putative” viral reservoirs, and reducing inherent toxicities. These highlight, in part, the need for long-acting, slow effective release antiretroviral therapy (LASER ART) delivery schemes. A long-acting (LA) DTG was made by synthesizing a hydrophobic and lipophilic prodrug encased with poloxamer (P407) surfactant. This modified DTG (MDTG) reduced systemic metabolism and polarity, increased lipophilicity and membrane permeability, improved encapsulation, and formed …

Transcriptional Regulation Of Icaadbc By Icar And Tcar In Staphylococcus Epidermidis, Tramy Hoang

Theses & Dissertations

Biofilm formation is the primary virulence factor in Staphylococcus epidermidis. Polysaccharide intercellular adhesin (PIA) is an adhesive molecule and a significant component of the biofilm matrix. It is synthesized by the products of the icaADBC operon whose regulation has been shown to involve environmental factors as well as many transcriptional regulators. Of these regulators, we explored the function of the repressors IcaR and TcaR and their roles in directly influencing icaADBC transcription and PIA synthesis. Based on previous observations that icaADBC positive clinical isolates of S. epidermidis are highly variable in PIA synthesis and biofilm formation, our goal was to …

Francisella Tularensis Insertion Sequence Elements Contribute To Differential Gene Expression, Amanda M. Bartling

Theses & Dissertations

This abstract is under embargo until April 26, 2020.

Redirection Of The Immune Response To Staphylococcus Aureus Biofilm Infection, Anna G. Staudacher

Theses & Dissertations

Staphylococcus aureus (S. aureus) is a leading cause of community- and healthcare-associated infections and has a propensity to form biofilms. Biofilm infections are recalcitrant to host immune-mediated clearance as well as antibiotics, making them exceptionally difficult to eradicate. The biofilm environment has been shown to skew the host immune response towards an anti-inflammatory phenotype, characterized by alternatively activated macrophages, recruitment of myeloid-derived suppressor cells (MDSCs), and minimal neutrophil and T cell infiltrates. Our laboratory has attempted to redirect the host immune response towards one that would favor bacterial clearance by employing strategies to augment pro-inflammatory mechanisms. One such …

Amino Acid Catabolism In Staphylococcus Aureus, Cortney Halsey

Theses & Dissertations

Staphylococcus aureus must rapidly adapt to a variety of carbon and nitrogen sources during invasion of a host. Within a staphylococcal abscess, preferred carbon sources such as glucose are limiting, suggesting S. aureus survives through the catabolism of secondary carbon sources. S. aureus encodes pathways to catabolize multiple amino acids including those that generate pyruvate, 2-oxoglutarate, and oxaloacetate. To assess amino acid catabolism, S. aureus JE2 and mutants were grown in complete defined medium containing 18 amino acids but lacking glucose (CDM). A mutation in glutamate dehydrogenase (gudB), which generates 2-oxoglutarate from glutamate, abrogated growth in CDM suggesting …

Role Of Stemloop D In Terminally Deleted Coxsackievirus B3 Replication, Lee K. Jaramillo

Theses & Dissertations

Coxsackievirus B3 (CVB3) is an enterovirus with no known form of latency. However, assays designed to detect enteroviral RNA have shown that CVB3 RNA can persist for weeks beyond the acute infection both naturally and experimentally. Our previous work with coxsackievirus revealed an inhibited version of enteroviral replication where the progeny virus, termed terminally deleted (TD) virus, was missing a maximum of 49 nucleotides from the beginning of the 5’ non-translated region (NTR). The largest terminally deleted virus, TD50, effaced stem a, stemloop b, and stemloop c from the secondary structure, the cloverleaf. We hypothesized that further deletion beyond those …

Regulation Of Cid-Mediated Cell Death In Staphylococcus Aureus, Ian H. Windham

Theses & Dissertations

The death and lysis of a subpopulation of cells in Staphylococcus aureus biofilms is thought to benefit the surviving population by releasing extracellular DNA, a critical component of the biofilm extracellular matrix. Although the means by which S. aureus controls cell death and lysis is not completely understood, studies implicate the role of the cidABC, alsSD and lrgAB operons in this process. This dissertation has focused on the regulation of cidABC and alsSD expression, which is mediated, primarily, by the LysR-Type Transcriptional Regulator (LTTR) known as CidR. To better define the role of CidR in regulating cidABC and alsSD transcription …

Spatiotemporal Control Of Staphylococcus Aureus Biofilm Development, Derek E. Moormeier

Theses & Dissertations

Chronic biofilm-related infections caused by the human pathogen Staphylococcus aureus often lead to significant increases in morbidity and mortality in both hospital- and community-associated settings. Typically, S. aureus biofilm development occurs in three stages: 1) attachment, 2) tower maturation, and 3) dispersal. Here, utilizing BioFlux1000 time-lapse microscopy we have expanded upon these fundamental stages of biofilm development and also unveiled and characterized two additional stages (multiplication and exodus).

The attachment and multiplication stages were shown to be protease sensitive but independent of most cell surface-associated proteins. Following multiplication, an exodus of the biofilm population that followed the transition of the …

Characterization Of The Replication Of Coxsackievirus B3 With A Mutationally Disrupted Cre(2c) And The Use Of Creatinine-Hydrochloride As An Antibacterial Agent, Shane E. Smithee

Theses & Dissertations

Following natural or experimental infection, and in cell culture, coxsackie B virus (CVB) RNA can persist for weeks in the absence of CPE yet with detectable viral RNA. Earlier studies in our laboratory demonstrated that this persistence produced viral RNA with up to 49 nucleotide deletions at the 5’ genomic terminus, partially degrading the cloverleaf (or domain I), an RNA structure that is required for efficient viral replication. A cis-acting replication element (CRE) in the 2C protein coding region [CRE(2C)] templates the addition of two uridine residues to the virus-encoded RNA replication primer, VPg, prior to genomic replication. Because …

Role Of The C-Terminus Of The Catalytic Subunit Of Translesion Synthesis Polymerase Ζ (Zeta) In Uv-Induced Mutagensis, Hollie M. Siebler

Theses & Dissertations

Cellular DNA is under constant attack by endogenous and exogenous DNA damaging agents that threaten genome integrity. Unrepaired DNA lesions often stall replicative DNA polymerases and are bypassed by translesion synthesis (TLS) to prevent replication fork collapse. TLS mechanisms are lesion- and species-specific, with prominent roles of specialized DNA polymerases with relaxed active sites. After incorporation of nucleotide(s) across from the lesion, the distorted primer termini are typically extended by DNA polymerase ζ (Pol ζ). As a result, Pol ζ is responsible for most DNA damage-induced mutations. Mechanisms of sequential polymerase switches and regulation of Pol ζ access to DNA …