Life Sciences Commons^™

Identification Of New Substrates From Pseudomonas Aeruginosa Exos, Adam V. Thota

Master's Theses

Pseudomonas aeruginosa is a gram-negative environmental bacterium that uses its intrinsic and acquired antibiotic resistance to infect patients with compromised epithelium, such as cystic fibrosis and burn wounds, and corneal keratitis. In order to cause disease, most strains of P. aeruginosa use the type three secretion system (T3SS) to disrupt cellular signaling and integrity. However, P. aeruginosa may also secrete exotoxins after invading host cells, which prolongs host cell death and maintains an intracellular niche. Of the T3 secreted effector toxins, ExoS, specifically the ADP-ribosyltransferase activity of ExoS, was shown to delay the death of invaded corneal cells, but it …

Role Of Tetraspanins In Sars-Cov-2 Fusion And Entry, Marcos Saul Santiago Figueroa

Master's Theses

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the pandemic the world has been facing in recent years. Even as new countermeasures are developed, there is still much we don’t know in terms of its entry into host cells. Tetraspanins are transmembrane proteins that are near ubiquitous amongst cell types. They fulfill numerous roles, including that of a viral co-receptor. Here, we considered whether tetraspanins, specifically CD9, CD63, and CD81 influence SARS-CoV-2 fusion and entry. Using ACE2-LgBit and tetraspanin overexpressing EVs and HeLa cells, we find that the presence of excess tetraspanins inhibit fusion and entry. However, we …

Interrogating Antagonistic Differences Among Pseudomonas Aeruginosa Cdia Alleles, Haotian Yang

Master's Theses

Bacteria are complex social organisms that live within mixed communities where microorganisms compete for limited resources. We studied Contact-Dependent Growth Inhibition (CDI), a particular type of competition mediated by a type 5b, or two-partner, secretion system that is widely spread among Gram-negative bacteria. CdiB and CdiA make up the two-partner secretion system that mediates CDI. CdiB translocates CdiA to the cell surface, and CdiA delivers its C-terminal toxin domain to the target cell. The C-terminal toxin (Tox) domain of CdiA is highly diverse. We studied Tox domains of various Pseudomonas aeruginosa clinical isolates. We found different strains that contain unique …

Bisacodyl Limits Chikungunya Virus In Vitro And Is Broadly Antiviral, Natalie June Lomascolo

Master's Theses

Identifying novel antivirals requires significant time and resource investment, and the continuous threat of viruses to human health necessitates commitment to antiviral identification and development. Developing antivirals requires years of research and validation, and recent outbreaks have highlighted the need for preparedness in counteracting pandemics. One way to facilitate development is to repurpose molecules already used clinically. By screening such compounds, we can accelerate antiviral development. Here, we screened compounds from the National Institutes of Health’s Developmental Therapeutic Program for activity against chikungunya virus, an alphavirus that is responsible for a significant outbreak in the Americas in 2013. Using this …

Investigating The Role Of Nagc Reveals It To Be A Global Regulator, Natasha Peterson

Master's Theses

Bacterial colonization of host surfaces can result in symbiotic or pathogenic associations.As bacteria becomes more resistant to antibiotics, pathogenic bacterial colonization poses an increasing risk to human health. Symbiotic associations that have beneficial outcomes for both the host and the microbe can serve as models to investigate how bacterial processes affect colonization of host surfaces. The symbiotic relationship between the Gram-negative, bioluminescent bacterium Vibrio fischeri and the Hawaiian bobtail squid is one such model (E. V. Stabb & Visick, 2013). The study of this exclusive relationship has developed in a way where in vitro phenotypes can predict the ability of …

Calcium-Mediated Induction Of Vibrio Fischeri Es114 Biofilms, Katia Elizabeth Semenchuk

Master's Theses

Vibrio fischeri bacteria form biofilms that facilitate symbiotic colonization of the Hawaiian bobtail squid, Euprymna scolopes. These host-associated biofilms require production of the SYP polysaccharide. However, it has been difficult to observe robust biofilm formation in vitro in a laboratory setting using wild-type strain ES114; instead, most work investigating syp-dependent biofilm formation has relied on genetically altered strains. Guided by recent findings that increasing calcium induces c-di-GMP (Tischler et al., 2021), a molecule known in other systems to promote biofilm formation, the question was posed as to whether high levels of calcium alone could promote syp-dependent biofilm formation by ES114. …

Characterizing A Novel Cocksackievirus B3 Protease Mutant And Its Response To Polyamine Depletion, Bridget Hulsebosch

Master's Theses

Enteroviruses, including Coxsackievirus B3 (CVB3), are pervasive pathogens that cause significant disease, including cardiomyopathies. Unfortunately, no treatments or vaccines are available for infected individuals. We identified the host polyamine pathway as a potential drug target, as inhibiting polyamine biosynthesis significantly reduces enterovirus replication in vitro and in vivo. Here, we show that CVB3 is sensitive to polyamine depletion through the polyamine analog diethylnorspermidine (DENSpm) which enhances polyamine catabolism through induction of polyamine acetylation. We demonstrate that CVB3 acquires resistance to DENSpm via mutation of the 2A protease, which enhances proteolytic activity in the presence of DENSpm. Resistance to DENSpm occurred …

Assessing The Role Of The Interaction Between Polyamines And Heparan Sulfate In Coxsackievirus B3 Viral Infection, Oreoluwa Omoba

Master's Theses

Non-polioenteroviruses are highly infectious viruses that typically cause mild asymptomatic cases but can cause severe disease such as aseptic meningitis and dilated cardiomyopathy. Coxsackievirus B3 (CVB3) is a member of this group and is prevalent in the United States and Eastern Asia. Viruses like CVB3 rely on the host cell for many molecules in order to replicate. One such set of molecules is the polyamines. Polyamines are small, positively charged molecules that play a role in a multitude of cellular processes including RNA/DNA stabilization, gene expression, translation, and regulating membrane fluidity. Previous studies have shown CVB3 relies on polyamines for …

Elucidating Regulatory Connections Between The Second Messenger Cyclic-Di-Gmp With Biofilm And Motility Phenotypes Of Vibrio Fischeri, Ali Razvi

Master's Theses

The symbiosis between the gram-negative marine bacterium Vibrio fischeri and its host Euprymna Scolopes occurs when the bacteria reaches and colonizes the light organ. This pathway to colonization begins in the outer marine environment and finishes within the deep crypts of the light organ, with states of biofilm formation and dispersal in between. This switch between biofilm formation to dispersal is important as the bacteria transitions from the surface on the light organ into a pore leading to the interior of the light organ. This dispersal is regulated by LAP system which is dependent on the second messenger c-di-GMP. Through …

Chga-Depleted Urothelial Amp And Immune Response During Urinary Tract Infection, Theadora Jane Ceccarelli

Master's Theses

Women are 8 times more likely than men to experience a urinary tract infection (UTI) with up to 60% of women reporting a UTI in their lifetime. This significant healthcare burden is caused by the infection of uropathogenic Escherichia coli in the urinary tract. At this time, treatment is limited to antibiotic therapy, which is challenged by antibiotic-resistance. These factors make UTIs an NIH priority and are motivation for our research.CgA is a protein of the granin family co-released with catecholamines from neuroendocrine cells throughout the body. Pro-hormone convertases post-translationally cleave CgA into peptides, like catestatin, which can have antimicrobial …

Investigating The Role Of Hypusine In Viral Translation And Infection, Jeremy Joseph Esin

Master's Theses

Polyamines are small, positively charged, molecules found in eukaryotic cells with roles in cellular function and viral infection. in previous work we have shown RNa viruses, including bunyaviruses, to be sensitive to polyamine depletion, but exact mechanisms are unknown. Bunyaviruses are emerging pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. Rift Valley Fever Virus is a bunyavirus that infects both humans and livestock with severe mortality and morbidity. in previous work done with Rift Valley Fever Virus polyamine depleted results in noninfectious interfering particles without affecting the shape or size of the particles. Here, I investigate a specific metabolic pathway …

Zika Virus Inactivates Polyamine Catabolism Via Alternative Splicing To Enhance Infection, Marion Lea Graham

Master's Theses

During viral infection, the virus and host must compete for resources inside the cell. One of these resources is polyamines. Polyamines are small, positively charged molecules that are found in all eukaryotic cells. They play a key role in several cellular functions including growth and proliferation, transcription and translation, and membrane stability. Viruses also rely polyamines for productive replication, utilizing them during DNA/RNa polymerization, nucleic acid packaging, and protein synthesis. in response to a virus infecting a host cell, the host cell will begin to regulate polyamine levels as a way to combat the infection. Polyamine levels are regulated by …

A Susceptibility Study Of Urinary Isolates To Methenamine Hippurate And Its Metabolites, Nancy Sloan

Master's Theses

Urinary Tract Infection (UTI) is one of the most common bacterial infections in adult women. Treatment is one of the most common reasons for antibiotic prescription. the rise of antibiotic-resistant infections coupled with the shortage of new antibiotics creates an urgent need for alternative non-antibiotic treatments. One alternative is methenamine hippurate (MH), which is a non-antibiotic prophylaxis. It is reported to be active against some common uropathogens including Escherichia coli and Enterococcus species, but its effects on the newly discovered bladder microbiota (urobiome) are undocumented. Given this knowledge gap, this study aimed to evaluate the drug's spectrum of susceptibility on …

Determining Interactions Between Bacteriophage And Urinary Microbiome Lysogens, Michael Bochert

Master's Theses

Bacteriophages (phages) are viruses that infect bacteria. They are the most abundant life forms on earth. It is estimated that there are approximately 10³⁰ phages on the planet (Chibani-Chennoufi et al. 2004). They outnumber their bacterial hosts approximately 10 to 1. Bacteria inhabit most niches on the planet including the human body, where they play critical roles in health and disease. Since phages shape bacterial populations the same way that viruses, such as smallpox and measles, shaped human populations, it is important to understand how they interact with their bacterial hosts. The majority of the phages that we know of …

Modulation Of Coxsackievirus Protease Activity By Polyamines, Courtney Noelle Dial

Master's Theses

Coxsackievirus type B (CVB3) is one of the six serotypes of the Coxsackievirus family of non-enveloped, linear, and positive-sense single-stranded RNA viruses. It is a pathogenic enterovirus that belongs to the same genus as the notable pathogen poliovirus. CVB3 can cause a range of illnesses from a fever to gastrointestinal distress but is most noteworthy for the ability to cause viral myocarditis, a swelling of the heart muscle. Coxsackievirus, like all RNA viruses, tends to develop mutations rapidly due to its error prone polymerase and lack of proofreading activity. These mutations can be advantageous for the virus, allowing it to …

A Possible Role Of Clostridium Difficile Association With The Gi Tract In Relapsing Clostridium Difficile Infection, Asiyya Ashraf

Master's Theses

Clostridium difficile infection (CDI), caused by the anaerobic spore-former C. difficile, is a major cause of health-care associated morbidity. CDI occurs in individuals that have a disrupted GI tract microbiota, typically in patients who are on antibiotic therapy. After recovery from an episode of CDI, approximately, 15-30% of patients experience relapse of disease. The mechanism of relapse is not well understood. It is plausible for relapse to occur if C. difficile cells and spores associate with the GI tract mucosa during infection and persist after infection. However, little is known about the association of cells and spores with the GI …

The Role Of Polyamines In Enteroviral Attachment, Thomas Mckenzie Kicmal

Master's Theses

Polyamines are small polycationic molecules with flexible carbon chains that are found in all eukaryotic cells. Polyamines are involved in the regulation of many host processes and have been shown to be implicated in viral replication. Depletion of polyamine pools in cells with FDA approved drugs restricts replication of diverse RNA viruses. Viruses can exploit host polyamines to facilitate packaging, transcription, translation, and protease activity but other mechanisms remain largely unknown. Picornaviruses, including Coxsackievirus B3 (CVB3), are sensitive to depletion of polyamines and remain a significant public health threat. We employed CVB3 as a model system to investigate a potential …

Dissecting A Role For Polyamines In Rift Valley Fever Virus Infection, Vincent Mastrodomenico

Master's Theses

Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. Rift Valley fever virus is a particularly devastating bunyavirus, infecting both humans and livestock with significant morbidity and mortality. By coordinating several host and viral processes Rift Valley fever virus is able to produce infectious virions. Polyamines are small, positively-charged host-derived molecules that play diverse roles in human cells and in infection. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated polyamines' role in the replication of the Rift Valley fever virus (vaccine strain MP-12). …

Longitudinal Urinary Microbiome Studies: A Need To Transition To Voided Urine, Baylie Hochstedler

Master's Theses

It is now established that the bladder is not sterile; it contains communities of microbes (microbiota). While the healthy bladder microbiota have been defined using expanded quantitative urine culture (EQUC) and urines obtained by transurethral catheterization (TUC), longitudinal and population studies have not previously been possible. These studies cannot be done using TUC urines, as it would be impractical to catheterize participants daily. Instead, we must transition to using voided urines.In order to make this switch, we have addressed three main issues raised by using voided urine. First, we showed that EQUC out performs standard urine culture on voided urines …

Characterizing Immune Response To Hiv-1 Infection In Bicd2-Knockout Cells, Omar Abdel-Rahim

Master's Theses

An important part of the HIV-1 infection cycle is the attachment of the intracellular viral core to the host microtubule network, facilitated by attachment of the viral capsid to cargo adaptor proteins. One such cargo adaptor is Bicaudal D Homolog Protein 2 (BICD2). BICD2 can attach to both the HIV-1 capsid and the dynein/dynactin complex and facilitate the trafficking of the viral core towards the host nucleus. Removal of BICD2 can disrupt this viral translocation, resulting in an elevated immune response that impairs productive HIV-1 infection. In my research, we investigated what viral particles are detected in the absence of …

Characterizing The Roles Of Staphylococcus Aureus Secreted Factors In Virulence And Modulation Of Innate Immune Cell Activity, Cameron Harvey

Master's Theses

Staphylococcus aureus is a prominent human pathogen that is responsible for a massive burden on healthcare. This thesis takes two approaches to characterize how S. aureus secreted factors subvert the innate immune response. First, characterization of the integral membrane protease 1984 showed that it regulates secretion of a putative lytic transglycosylase, IsaA, which is implicated in virulence. My results suggest IsaA and 1984 are capable of modulating immune responses that result in enhanced heart colonization in vivo. In addition, recent studies have uncovered TLR-independent pathways that induce inflammatory responses after S. aureus insult. Therefore, I hypothesized the bacterium secretes factors …

Understanding The Dynamics Of Protein Lipoylation In Staphylococcus Aureus, Sarah C. Flury

Master's Theses

Staphylococcus aureus is a commensal bacterium that also acts as an opportunistic pathogen. The pathogenicity of S. aureus has often been attributed to the wide range of virulence factors that the bacterium produces. While virulence factors do contribute a great deal, there is a growing field of research that aims to investigate the role of metabolism in bacterial virulence.

My project focuses on the necessity of a metabolic cofactor, lipoic acid. To ensure sufficient amounts of lipoic acid are available for enzyme activity, S. aureus has evolved two pathways to obtain the important nutrient. The lipoic acid salvage pathway is …

Immunobiology Of Adenovirus-Vector Vaccines For Mrsa, Emily Orvis

Master's Theses

Staphylococcus aureus is a gram-positive, extracellular bacterium that has emerged as an

important human pathogen. This bacterium is a leading cause of skin and soft tissue infections

(SSTIs) in humans, often leading to invasive and life-threatening infections. Treatment of S.

aureus infections is becoming more complicated due to the rise of methicillin-resistant S. aureus

(MRSA) strains, which are becoming increasingly resistant to a number of antibiotics. In the

United States, invasive MRSA infections result in more deaths annually than any other infectious

agent.

Despite a dire need, there is currently no vaccine against S. aureus infections. The failure

of past …

Surveying Host Innate Immune Responses To Interferon Antagonist-Deficient Murine Coronaviruses, Aaron Brian Volk

Master's Theses

Two coronaviruses (CoVs)—severe acute respiratory syndrome (SARS) virus and Middle East respiratory syndrome (MERS) virus—have emerged in the 21st century from animal reservoirs into the human population, each causing an epidemic associated with significant disease and mortality. CoV epidemics are currently only controllable by rigorous public health measures; no targeted therapeutics or vaccines exist to treat or prevent any human CoV infection. One method of generating attenuated CoV strains to be studied as vaccine candidates involves specifically disrupting CoV-encoded interferon (IFN) antagonists, thereby rendering the virus vulnerable to host innate antiviral immunity. Deubiquitinating (DUB) activity encoded within CoV nonstructural protein …

Utilizing Genetic Techniques To Identify Amino Acids Within The Putative Glycosyltransferase Sypq That Are Essential For Its Role In Biofilm Formation By Vibrio Fischeri, Mary Kathryn Flaherty

Master's Theses

The polysaccharide component of the V. fischeri biofilm is produced by proteins encoded by the symbiosis polysaccharide (syp) locus. The syp locus encodes 18 genes, six of which are putative glycosyltransferases (GTs). One of these GTs, sypQ, encodes a putative family 2 glycosyltransferase (GT-2). The mechanism by which SypQ facilitates biofilm formation is not yet understood. To uncover which specific residues are essential for the function of SypQ, I utilized Bioinformatics, site-directed mutagenesis, and random mutagenesis. The Bioinformatics identified putative GT-2 motifs in the SypQ primary sequence. The mutagenesis (both site-directed and random), identified five residues (D95, D151, S153, D236, …

Phenotyping Temperature-Sensitive Coronaviruses, Amani Eddins

Master's Theses

Coronaviruses (CoVs) can cause a range of symptoms; from a mild common cold to life threatening acute respiratory distress syndrome (ARDS) upon infection. These emerging viruses have the ability to be highly pathogenic and detrimental to the human population. Two prime examples of CoV emergence, SARS-CoV (Severe Acute Respiratory Syndrome) and MERS-CoV, (Middle Eastern Respiratory Syndrome), which exhibits the pandemic potential of emerging CoVs that gain human tropism. Given the increasing potential of an emerging CoV outbreak there is a state of urgency to develop vaccines that will help protect the human population against current and future circulating strains. The …

Optimizing Clean Catch Urine Collection And Its Applications In Urinary Microbiome Studies, Danielle Johansen

Master's Theses

Clean catch was developed before the advancement of more sensitive assays for culturing urinary bacteria. I reassessed clean catch, endeavoring to optimize this method. Periurethral swabs and voided urines were routinely collected, while analyzing different aspects of the method. I determined that midstream urine contained the least amount of vulvo-vaginal contamination and that the time of collection did not impact the results. I also determined that the use of antiseptic wipes prior to void increased the level of vulvo-vaginal contamination. A small cohort of females collected daily urines over a 17-day period and kept a strict alcohol diary. Females who …

Defining The Roles Of The Lipoic Acid Ligases In Promoting Staphylococcus Aureus Metabolic Homeostasis And Virulence, Irina Laczkovich

Master's Theses

Staphylococcus aureus is a major human pathogen known to cause disease in a wide range of tissues. In order to thrive in such diverse environments, S. aureus uses multiple adaptive traits such as trace metal/nutrient acquisition, shifts in metabolic activity, and expression of detoxification systems, all of which allow the bacterium to proliferate and survive in nutritionally deficient and inhospitable environments.

One essential metabolite used by S. aureus is lipoic acid, a cofactor of enzyme complexes used in aerobic metabolism, fatty acid biosynthesis, glycine detoxification, and maintenance of redox homeostasis. Prior studies in the lab used a genetic approach to …

Characterization Of Inhibitory Molecules Produced By Lactobacillus Crispatus, Giuseppe Anthony Pistone

Master's Theses

Lactobacillus species are widely accepted as beneficial bacteria of the human microbiota1-8. Lactobacilli spp. are well documented to inhibit pathogens by production and secretion of hydrogen peroxide (H2O2), organic acids, and/or proteinaceous bacteriocins into their environment. Most research attributes bactericidal activity of cell free supernatant (CFS) to H2O2 and/or lactic acid. Here, I demonstrate that CFS from a clinical isolate of Lactobacillus crispatus contains a molecule(s) that inhibits uropathogenic Escherichia coli (UPEC) colony formation independently of H2O2 and organic acids. Physiologic concentrations of H2O2 and organic acids produced by L. crispatus do not inhibit UPEC colony formation. Incubation of UPEC …

Lactobacillus Crispatus Produces A Bacteridical Molecule That Kills Uropathogenic E. Coli, Katherine Diebel

Master's Theses

As many as 1 in 2 women will have at least one urinary tract infection (UTI) in their lifetime. UTIs can cause complications in pregnancy and decrease quality of life, and their treatment and prevention are expensive. Uropathogenic E. coli (UPEC) is the primary cause of UTI. The probiotic and bactericidal capacities of gut and vaginal Lactobacillus isolates have been studied, but the same attention has not been paid to urinary strains. These urinary isolates of L. crispatus appear to have a greater killing capacity against UPEC and this bactericidal activity does not depend on the cells themselves, consistent with …