Digital Commons Network^™

Persistence Of Wastewater-Associated Antibiotic Resistant Bacteria In River Microcosms, Aoife P. Mahaney

Theses and Dissertations

The spread of antibiotic-resistant bacteria (ARB) associated with wastewater is a significant environmental concern, but little is known about the persistence and proliferation of these organisms in receiving water bodies after discharge. To address this knowledge gap, we performed a series of microcosm experiments in which river water was amended with either untreated or treated wastewater, and the abundance of viable ciprofloxacin-, Bactrim-, and erythromycin-resistant bacteria was monitored for 72 hours.

Both types of wastewater amendments increased the initial abundance of ARB compared to microcosms containing only river water. The increase was greatest with untreated wastewater, but that effect decreased …

The Type Iv Pilus Secretin Bfpb: Structural Analysis And Binding Interactions, Janay I. Little

Theses and Dissertations

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. The type IV pilus (T4P) of EPEC, known as the bundle-forming pilus (BFP), plays an important role in EPEC pathogenesis. T4Ps are a family of surface appendages that are important for adhesion, colonization, biofilm formation, virulence, twitching motility and many other functions. One essential component of the BFP system is the secretin, BfpB. Secretins are a large family of integral outer membrane proteins found in T4Ps as well as type II and type III secretion systems, and filamentous phages. Details of the secretin structure have been limited to the overall …

Probing The Porphyromonas Gingivalis Nitrosative Stress Response Network, Alexandra Galina

Theses and Dissertations

Objective: To persist in the oral cavity, the perio-pathogen Porphyromonas gingivalis must have an efficient nitrosative stress response mechanism. To better understand how P. gingivalis responds to nitrite (NO₂^-) we must determine what genes are involved in the nitrosative stress response network.

Methods: Through genomic screening, a list of genes was generated that were hypothesized to be part of the nitrosative stress response network and believed to be involved in nitrate/nitrite metabolism or redox state of the cells. Protein coding regions were replaced with the ermF sequence by allelic exchange mutagenesis. 15 mutants were created …

Treponema Denticola Synthesizes C-Di-Amp And Encodes The Cdaa-Type Diadenylate Cyclase Cdaa, Claire R. O'Brien

Theses and Dissertations

Periodontitis is a form of oral disease characterized by dysbiosis of the oral microbiome, leading to inflammation, bone resorption, and in severe cases, entire tooth loss, affecting 42% of adults in the US. One of the bacteria most associated with periodontal disease progression is Treponema denticola (Td), an oral spirochete which inhabits the mouth in small quantities during health but which can dominate the biofilms that form during periodontal disease. The ability of Td to survive in a disease environment and contribute to the progression of disease requires the use of robust signaling networks. Analysis of Td cultures …

Human Papillomavirus 16 E2 Regulates Host Cell Pathways Important For Cancer Progression And Treatment Sensitivity Which May Contribute To Cancer Outcomes, Christian Fontan

Theses and Dissertations

Human papillomaviruses (HPVs) are causative agents in around 5% of all cancers, including cervical and oropharyngeal. A feature of HPV cancers is their better clinical outcome compared with non-HPV anatomical counterparts. In turn, the presence of E2 predicts a better clinical outcome in HPV-positive cancers; the reason(s) for the better outcome of E2-positive patients is not fully understood.

Previously, we demonstrated that HPV16 E2 regulates host gene transcription that is relevant to the HPV16 lifecycle in N/Tert-1 cells. One of the genes repressed by E2 and the entire HPV16 genome in N/Tert-1 cells is TWIST1. In these studies, we demonstrate …

Modification Of Culture Medium And Identification Of Microbial Contaminants For Improved In Vitro Propagation Of Freshwater Mussels, Raquel M. Wetzell

Theses and Dissertations

In vitro propagation efforts play an essential role in conserving and restoring threatened freshwater mussel populations by circumventing the need for a fish host. Across a broad range of taxa, transformation is induced with an artificial M199 medium and rabbit serum. However, such formulation may not be sufficient in culturing critical species with more specific physiological requirements. In this study, multiple serum mixtures were tested to improve in vitro transformation of two freshwater mussel species: yellow lampmussel (Lampsilis cariosa) and tidewater mucket (Atlantaconcha ochracea). These species were selected because they parasitize similar fish host species but …

Bioinformatic Pipeline For Determining Terminal Repeats In The Human Cytomegalovirus Genome Assembled With Pacbio Long Read Sequences, Ahmed Al Qaffas

Theses and Dissertations

Human Cytomegalovirus (HCMV) is a member of the betaherpesvirinae subfamily of the Herpesvirus family. HCMV infection is common among adults worldwide, with an estimated seroprevalence of 66 to 95%, depending on the geographic region (Zuhair et al., 2019). Although most of the virus genomic content has been studied extensively, the terminal repeating region sequences remain understudied. Two main challenges hindered the study of the region: a) limitations of sequencing technologies; and b) misassembly of the repeats due to its complex nature. Here I show a novel bioinformatics pipeline that takes advantage of PacBio's long reads to resolve the challenges mentioned …

The Influence Of Salinity On The Structure And Function Of Soil Prokaryotic Communities In Coastal Freshwater Wetlands, Joseph C. Morina Ii

Theses and Dissertations

Microbial communities are critical biological components of the world’s ecosystems, and their respiratory pathways are directly involved in the biogeochemical cycling of essential nutrients. As genomic technologies advance, allowing for more detailed profiling of microbial communities, efforts have successfully linked microbial community composition to ecosystem-level functions and have shown microbial communities are susceptible and resistant to disturbance events. The goal of this dissertation is to address the temporal scales in which microbial communities respond to the disturbance of salinization, and the repercussions this has on microbially-meditated carbon and nitrogen cycling. Coastal freshwater wetlands are an excellent study system to investigate …

Antiviral Effects Of Metalloshielding: Differential Antiviral Activity Of Polynuclear Platinum And Cobalt Compounds, Mary Zoepfl

Theses and Dissertations

The majority of antiviral drug development has focused on virus-specific discovery targeting discrete steps in the individual life cycles. Although great strides have been made for a number of clinically relevant diseases such as human immunodeficiency virus, influenza virus, and hepatitis B, broad spectrum antivirals do not exist. Broad spectrum antivirals would offer (1) treatment for viruses without specifically-targeted antivirals, (2) treatment for viruses which have developed resistance to their available treatments, and (3) a rapidly deployable treatment option in viral epidemics. Many viruses including human cytomegalovirus (HCMV), HIV, and SARS-CoV-2. rely on heparan sulfate (HS), a highly sulfated glycosaminoglycan …

Targeting Transferrin Binding Protein A For Vaccine Development In Neisseria Gonorrhoeae, Ashley Greenawalt

Theses and Dissertations

Neisseria gonorrhoeae, the causative agent of the sexually-transmitted infection gonorrhea, has recently been labeled a superbug. With dwindling treatment options, there is urgent need for a gonococcal vaccine. TonB-dependent transporters (TDTs) are important gonococcal virulence factors that allow the gonococcus to pirate metals directly from host proteins. The TDTs, TbpA and TbpB, are promising vaccine targets because both proteins are expressed and highly conserved in gonococcal strains, and they are not subject to high-frequency antigenic variation. Because the Tbps bind to host protein as their natural function, our immune system does not recognize them as foreign antigens; thus, immunogenicity is …

Il-36g Cytokine Induction In Oral Epithelial Cells Triggered By Treponema Denticola And Their Role In Chronic Periodontitis, Christopher Blake

Theses and Dissertations

Periodontal disease is a polymicrobial disease affecting 10-15% of the population. Periodontal disease affects the quality of life by reducing mastication and potentially causing tooth loss. Periodontal disease is also linked to more severe, systemic diseases such as cardiovascular disease and rheumatoid arthritis. Development of the diseased state occurs when the plaque biofilm shifts from healthy to dysbiotic. The shift to a dysbiotic biome brings with it a shift in bacterial presence, specifically an increase in gram-negative bacteria, such as T. denticola. T. denticola is a gram-negative, anaerobic spirochete whose relationship with oral epithelial cells is poorly understood. Preliminary data …

The Role Of Manganese In Streptococcus Sanguinis, Tanya M. Puccio

Theses and Dissertations

Streptococcus sanguinis is primarily associated with oral health as a commensal bacterium. As an opportunistic pathogen, S. sanguinis is capable of colonizing heart valve vegetations, leading to the disease infective endocarditis. Previous studies from our lab have identified the high-affinity manganese transporter SsaACB as important for endocarditis virulence. The impact that manganese depletion has on S. sanguinis had never been evaluated and a secondary manganese transporter has not been identified. Thus, we employed the use of a fermentor to control large-scale growth over time and depleted manganese in an ΔssaACB mutant using a metal chelator, EDTA. The changes in …

Development Of A Screening Assay For Type Iii Secretion System Inhibitors And High Throughput Screening Campaign Of Inhibitors Of Prp Of Staphylococcus Aureus, Heather A. Pendergrass

Theses and Dissertations

Antibiotics inhibit the growth or survival of bacteria by targeting their essential functions.¹ Due to weaknesses in traditional antibiotics and the increasing prevalence of antibiotic resistance genes, virulence factors are being targeted for therapeutic treatment of bacterial infection.² We have developed an assay to quantify and observe type III secretion system (T3SS) activity. The type III secretion system (T3SS) is a virulence factor present in some Gram-negative pathogens including enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC, respectively),³ and others.^4–9 The T3SS between EPEC and EHEC are highly conserved and share over 90% sequence identity with …

Response And Recovery Of Syntrophic And Methanogenic Activity To Saltwater Intrusion In A Tidal Freshwater Marsh Soil, David J. Berrier Jr.

Theses and Dissertations

Tidal freshwater wetland soils contain large amounts of organic carbon, some of which is mineralized to carbon dioxide (CO₂) and methane (CH₄) by a diverse consortium of anaerobic microorganisms that includes fermenters, syntrophs, and methanogens (MG). These microbial groups are tightly linked and often rely on cooperative interspecies metabolisms (i.e., syntrophy) to survive. Environmental perturbations can disrupt these interactions and thus alter the rates and pathways of carbon cycling. One environmental change of particular concern in coastal wetlands is sea level rise, which can result in increased episodic saltwater intrusion events into these ecosystems. These events …

Illumination Of The Golgi Apparatus Of Pathogenic And Nonpathogenic Naegleria Species, Tyler M. Poe

Theses and Dissertations

In this study, Naegleria fowleri, a pathogenic amoeba and the causative agent of Primary Amebic Meningoencephalitis (PAM), was utilized to determine the presence or absence of classically conserved Golgi molecules featured in the expression of a Golgi apparatus. Previous studies concluded no Golgi expression via light microscopy and transmission electron microscopy, but a recent report on Naegleria gruberi indicated the presence of dispersed Golgi tubules. Non-pathogenic species of the Naegleria genus such as Naegleria gruberi 30540 and Naegleria lovaniensis 30569 were utilized in Western immunoblot analysis compared to reduced whole-cell lysate proteins of two strains of N. fowleri and …

The Infectivity Of Naegleria Fowleri Cysts In Vivo And In Vitro, And Mediation Of Encystment By Camp, Anna L. Evdokiou

Theses and Dissertations

The free-living amoeba and causative agent of Primary Amoebic Meningoencephalitis, Naegleria fowleri, has three life stages: the trophozoite, the flagellate, and the cyst. This study examined the ability of the cyst to attach to, excyst upon, and destroy cell cultures grown to confluent monolayers, and to cause Primary Amoebic Meningoencephalitis in a murine animal model. The co-culture of cysts with P388D.1, CHME3, Vero, human nasal epithelial, and rat primary mixed glial cells resulted in destruction of the monolayer of all cell types once the cysts attached and excysted. One day post exposure to cysts, the mixed glial cells exhibited …

Acid Sphingomyelinase Is Essential For Vacuolar Development Of A. Phagocytophilum., Chelsea Cockburn

Theses and Dissertations

Obligate intracellular bacteria are significant causes of morbidity and mortality with over two hundred and fifty million infections worldwide annually. One such bacterium, Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA), a tick-transmitted febrile illness. Previous studies have shown that A. phagocytophilum lacks genes for cholesterol biosynthesis and solely relies on Niemann Pick protein type C (NPC)1-mediated low density lipoprotein (LDL)-derived cholesterol to complete its infection cycle.Acid sphingomyelinase (ASMase) is a lysosomal enzyme that is essential for diverse cellular processes including liberation of LDL-derived cholesterol from the lysosome. By first studying A. phagocytophilum, we found that …

Evolution And Niche Specialization Of Microbial Taxa In Vaginal Infection And Pregnancy, Abigail L. Glascock

Theses and Dissertations

The vaginal microbiome plays an important role in reproductive health and pregnancy. It has coevolved with humans and has direct effects on reproductive success, rendering selective pressure more pronounced at this site. Herein, we probe coevolution of the vaginal microbiome using a systems-level approach. In Chapter 2, we examine the evolutionary trajectory of two vaginal Veillonellaceae phylotypes evolved from an ancestral gastrointestinal lineage to inhabit the vaginal niche. We present evidence of their divergence and subniche specification and describe their differential associations with vaginal infection and pregnancy. In Chapter 3, we identify ten bacterial taxa, predicted to contribute to the …

Assessment Of A Predicted Diguanylate Cyclase In Treponema Denticola, Dhara T. Patel

Theses and Dissertations

Periodontal disease is a progressive inflammatory condition that is characterized by the reabsorption of alveolar bone, the destruction of connective tissue, and edentulism. It is caused by a dysbiosis in the oral microbiome as a result of a shift from a Gram-positive aerobic bacterial population, to one that becomes more Gram-negative and anaerobic. Treponema denticola is thought to drive this diseased state based on its role as a keystone periopathogen. A major component of T.denticola’s invasiveness is its motility, which allows the spirochete to penetrate and disseminate through tissues. This motility, which has been seen to be crucial to …

Orientia Tsutsugamushi Modulates Endoplasmic Reticulum Stress To Benefit Its Intracellular Growth And Targets Nlrc5 To Inhibit Major Histocompatibility Complex I Expression, Kyle G. Rodino

Theses and Dissertations

Scrub typhus, caused by the obligate intracellular bacterium Orientia tsutsugamushi, afflicts one million people annually. Despite being a global health threat, little is known about O. tsutsugamushi pathogenesis. Here, we demonstrate that O. tsutsugamushi modulates the ER and ER-associated processes as mechanisms of nutritional virulence and immune evasion. To obtain amino acids to fuel replication, O. tsutsugamushi simultaneously induces ER stress and the unfolded protein response (UPR) while inhibiting ER-associated degradation (ERAD) during early infection time points. During exponential growth, the bacterium releases the ER bottleneck, resulting in generation of ERAD-derived amino acids that it parasitized for replication. …

Discovery And Characterization Of Bile Acid And Steroid Metabolism Pathways In Gut-Associated Microbes, Spencer Harris

Theses and Dissertations

The human gut microbiome is a complex microbial ecosystem residing in the lumen of our gastrointestinal tract. The type and amounts of microbes present in this ecosystem varies based on numerous factors, including host genetics, diet, and environmental factors. The human gut microbiome plays an important role in normal host physiological functions, including providing energy to colonocytes in the form of short-chain fatty acids. However, gut microbial metabolites have also been associated with numerous disease states. Current tools for analyzing the gut microbiome, such as high-throughput sequencing techniques, are limited in their predictive ability. Additionally, “-omic” approaches of studying the …

Come Fly With Me: Using Amixicile To Target Periodontal Pathogens And Elucidating The Innate Immune Response In Drosophila Melanogaster, Kathryn Sinclair

Theses and Dissertations

Periodontal diseases (PD) affect 46% of American adults over age 30. These diseases cause symptoms including bleeding and swelling of the gums, bone resorption, and tooth loss, that affect quality of life and have a high economic burden. Periodontal diseases are caused by an imbalance in the oral microbiome, from a healthy state that contains anti-inflammatory commensals like Streptococcus gordonii and mitis, to a diseased state that has pro-inflammatory anaerobic pathogens including Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Tannerella forsythia. The latter initiate disease progression in the oral cavity. However, it’s the host immune …

Hub Proteins, Paralogs, And Unknown Proteins In Bacterial Interaction Networks, Neha Sakhawalkar

Theses and Dissertations

Proteins are the functional units of cells. However, a major portion of the proteome does not have a known functional annotation. This dissertation explores protein -protein interactions, involving these uncharacterized or unknown function proteins. Initially, protein – protein interactions were tested and analyzed for paralogous proteins in Escherichia coli. To expand this concept further and to get an overview, protein – protein interactions were analyzed using ‘comparative interactomics’ for four pathogenic bacterial species including Escherichia coli, Yersinia pestis, Vibrio cholerae and Staphylococcus aureus. This approach was used to study unknown function protein pairs as well as to …

Orientia Tsutsugamushi Secretes Two Ankyrin Repeat-Containing Effectors Via A Type 1 Secretion System To Inhibit Host Nf-Κb Function, Sean M. Evans

Theses and Dissertations

Scrub typhus is a potentially fatal infection that threatens one billion persons in the Asia-Pacific region and is caused by the obligate intracellular bacterium, Orientia tsutsugamushi. How this organism facilitates its intracellular survival and pathogenesis is poorly understood. Intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) into the host cell to modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank libraries as well as Type 1 and Type 4 secretion systems (T1SS and T4SS), which are expressed during infection. In …

Amixicile Inhibits Anaerobic Bacteria Within An Oral Microbiome Derived From Patients With Chronic Periodontitis, Kane Ramsey

Theses and Dissertations

Periodontitis is a chronic inflammatory disease caused by pathogenic bacteria residing in a complex biofilm within a susceptible host. Amixicile is a non-toxic, readily bioavailable novel antimicrobial that targets strict anaerobes through inhibition of the activity of Pyruvate Ferredoxin Oxidoreductase (PFOR), a major enzyme mediating oxidative decarboxylation of pyruvate. Our study aimed to evaluate the efficacy of amixicile, when compared to metronidazole, in inhibiting the growth of bacteria present in a microbiome harvested from patients with chronic periodontitis.

Plaque samples were harvested from patients with severe chronic periodontitis and cultured under anaerobic conditions. The microbiomes were grown in the presence …

In Search Of A Function For An Uncharacterized Conserved Protein In Streptococcus Sanguinis Sk36, Ayana Scott-Elliston

Theses and Dissertations

With the number of fully sequenced bacterial genomes increasing in the past 7 years, it has been discovered that a large percentage of the putative protein coding genes have no known function. This lack of knowledge leaves scientists with an incomplete understanding of bacteria. In this study, conserved hypothetical protein mutants from Streptococcus sanguinis SK36 were screened on solid media with various environmental conditions. From these screens, the candidate protein, SSA_2372, displayed a sensitivity to acidic conditions. Its homolog in Bacillus subtilis 168, BSU00030, also displayed a sensitivity to pH conditions at its acid tolerance extremes unlike its other homolog …

A Systems Biology Approach For Predicting Essential Genes And Deciphering Their Dynamics Under Stress In Streptococcus Sanguinis, Fadi El-Rami

Theses and Dissertations

Infectious diseases are the top leading cause of death worldwide. Identifying essential genes, genes indispensable for survival, has been proven indispensable in defining new therapeutic targets against pathogens, major elements of the minimal set genome to be harnessed in synthetic biology, and determinants of evolutionary relationships of phylogenetically distant species. Thus, essentiality studies promise valuable revenues that can decipher much of biological complexities.

Taking advantage of the available microbial sequences and the essentiality studies conducted in various microbial models, we proposed a framework for the prediction of essential genes based on our experimentally verified knowledge of the pathways involved in …

Contribution Of A Class Ii Ribonucleotide Reductase To The Manganese Dependence Of Streptococcus Sanguinis, John L. Smith

Theses and Dissertations

Manganese-deficient Streptococcus sanguinis mutants exhibit a dramatic decrease in virulence for infective endocarditis and in aerobic growth in manganese-limited media. Loss of activity of a manganese-dependent, oxygen-dependent ribonucleotide reductase (RNR) could explain the decrease in virulence. When the genes encoding this RNR are deleted, there is no growth of the mutant in aerobic broth culture or in an animal model. Testing the contribution of the aerobic RNR to the phenotype of a manganese transporter mutant, a heterologous class II RNR from Lactobacillus leichmannii called NrdJ that requires B₁₂ rather than manganese as a cofactor was previously introduced into an …

Expression Of Matrix Metalloproteinases In Naegleria Fowleri And Their Role In Degradation Of The Extracellular Matrix, Charlton Lam

Theses and Dissertations

Naegleria fowleri is a free-living amoeba found in freshwater lakes and ponds that is the causative agent of Primary Amoebic Meningoencephalitis (PAM). Matrix metalloproteinases (MMPs) have been described in protozoa, such as Plasmodium falciparum, Trypanosoma brucei, and Balamuthia mandrillaris, and have been linked to their increased motility and invasive capability by degrading components of the extracellular matrix (ECM). In addition, MMPs are often upregulated in tumorigenic cells and have been attributed as responsible for the metastasis of certain cancers. In the present study, in vitro experiments indicated that MMPs are linked functionally to the ECM degradation process. …

The Effects Of The Hiv-1 Tat Protein And Morphine On The Structure And Function Of The Hippocampal Ca1 Subfield, William D. Marks

Theses and Dissertations

HIV is capable of causing a set of neurological diseases collectively termed the HIV Associated Neurocognitive Disorders (HAND). Worsening pathology is observed in HIV+ individuals who use opioid drugs. Memory problems are often observed in HAND, implicating HIV pathology in the hippocampus, and are also known to be exacerbated by morphine use. HIV-1 Tat was demonstrated to reduce spatial memory performance in multiple tasks, and individual subsets of CA1 interneurons were found to be selectively vulnerable to the effects of Tat, notably nNOS+/NPY- interneurons of the pyramidal layer and stratum radiatum, PV+ neurons of the pyramidal layer, and SST+ neurons …