Microbiology Commons^™

Study Of Rickettsia Parkeri Colonization And Proliferation In The Tick Host Amblyomma Maculatum (Acari: Ixodidae), Khemraj Budachetri

Dissertations

Amblyomma maculatum (Gulf coast tick) ticks are prevalent across the Atlantic to Gulf Coast region of United States. These ticks are recognized vectors of Rickettsia parkeri, a spotted fever group of Rickettsia (SFGR) known to cause American boutonneuse fever associated with fever and eschar rashes localized to the site of bites. We hypothesized that Rickettsia parkeri colonization and proliferation in the tick vector involve pathogen-symbiont dynamics and tick-pathogen interactions, which influence rickettsial transmission to the victims after tick bites. The rickettsial infection is maintained across the tick life cycle for many generations due to transovarial and transstadial transmission of …

The Female Urogynecological Microbiome: Determining Niche Specificity, Krystal Thomas-White

Dissertations

Contrary to dogma, urine is not sterile. Recent discoveries show that the female urinary bladder contains a unique microbiome,with differences in healthy and disease states. This female urinary microbiome (FUM) is distinguishable from vulvo-vaginal contamination; yet, only the few centimeters of the urethra separate these two niches. Recent studies show that the most common bladder organisms are Lactobacillus, Gardnerella, Streptococcus, Corynebacterium, and a diverse group of anaerobes, the same genera commonly found in the vagina. Thus, we asked if the bladder and vaginal microbiomes are interconnected.

Previous work suggests a link between these two niches. The vaginal tract of reproductive …

Cellular Determinants Of Coronavirus Entry Routes, James Thomas Earnest

Dissertations

Coronaviruses (CoVs) represent some of the greatest modern threats to global health. CoVs are zoonotic viruses that generally cause respiratory or enteric infections. The ability of CoVs to move between species and into human populations ensures that CoVs will remain important and dangerous pathogens. Therefore, understanding how CoVs infect different hosts is vital to human health.

CoVs are enveloped and must undergo fusion of viral and host membranes to initiate infection. Viral glycoproteins, called Spike (S) proteins, are responsible for host cell binding and carrying out the membrane fusion reaction. S proteins store energy in their folded, pre-fusion conformation that …

Mutual Regulation Of Crp And N(Epsilon)-Lysine Acetylation In Escherichia Coli, Robert James Davis

Dissertations

Post-translational modifications, such as N(epsilon)-lysine acetylation, are known to alter the behavior of transcriptional regulators in eukaryotes, but very little is known about the consequences of acetylation on transcriptional regulation in bacteria. Here, I provide evidence that a global transcriptional regulator of carbon metabolism, cAMP Receptor Protein (CRP), promotes both enzymatic and non-enzymatic lysine acetylation in E. coli. Non-enzymatic lysine acetylation occurs when cells ferment acetate, such as during growth on high concentrations of glucose. Intriguingly, CRP can be non-enzymatically acetylated on several lysines, including lysine 100 (K100). I provide evidence that neutralization of the K100 positive charge, as would …

Epidemiology Of White Spot Syndrome Virus In The Daggerblade Grass Shrimp (Palaemonetes Pugio) And The Gulf Sand Fiddler Crab (Uca Panacea), Muhammad

Dissertations

Ever since the first outbreaks of White spot syndrome virus (WSSV), which causes White Spot Disease (WSD), in Asia in the early 1990s, the pathogen has been a major constraint to the profitability of the shrimp aquaculture industry across the globe. WSSV has a broad host range and is routinely detected in wild decapod crustaceans. In the present study, two common species in the tidal salt marsh along the northern coast of the Gulf of Mexico, the daggerblade grass shrimp (Palaemonetes pugio) and the Gulf sand fiddler crab (Uca panacea), were investigated for their role as …

A Microbiomic Approach To The Characterization Of The Impacts And Influences Of Viral, Bacterial, And Harmful Algal Bloom Toxins On The Bottlenose Dolphin, Corey David Russo

Dissertations

As apex predators that display high site fidelity Tursiops truncatus (bottlenose dolphin) are indicators of marine ecosystem health. Bottlenose dolphins, additionally, display pathogenesis and immune response similar to that of humans. Humans and coastal bottlenose dolphins, in particular, are constantly exposed to the same industrial, agricultural and domestic toxins and pathogens, contaminants and pollutants. Thus, studies on the bottlenose dolphin are also valuable in bridging the gap between ocean and human health. Bottlenose dolphins are susceptible to viral bacterial and toxin infection. Infection in the bottlenose dolphin manifests itself in the form of mass stranding events, unusual mortality events, chronic …

Lignocellulose As Carbon Source Promotes Bacterial Synergism And Reduces Antagonism, Yijie Deng

Dissertations

Lignocellulose decomposes slowly in nature because it consists of complex polymers resistant to enzymatic degradation by most organisms. Some bacteria are capable of producing cellulolytic enzymes but the way in which bacteria interact within a community to enhance degradation of the recalcitrant substrate is poorly understood. A better understanding of how bacterial interactions affect lignocellulose degradation would provide potential approaches to improve the efficiency of lignocellulose degradation for biofuel production.

To study whether bacterial interactions enhance lignocellulose degradation, I grew environmental bacterial isolates in mixed cultures and pure cultures. I found that bacterial synergism in mixed cultures was common in …

Adenovirus Evasion Of Cell-Intrinsic Immunity, Andrew Michael Burrage

Dissertations

Virus cell entry represents one of the earliest opportunities for a host to respond to infection. Understanding the processes of pathogen detection and restriction employed by the host, as well as strategies utilized by the virus itself to evade such processes, is critical in developing therapeutics to counter pathogenesis. Adenovirus (Ad) infections are self-limiting in healthy populations, but can be devastating to individuals with compromised immune systems. Currently, no specific antiviral treatments exist to combat Ad infections in susceptible populations. However, because Ad infections are not severe in healthy individuals, employing replication-defective Ads as vaccine vectors is generally regarded as …

Regulation Of Vancomycin Resistance And Stress Response By The Msaabcr Operon In Staphylococcus Aureus, Dhritiman Samanta

Dissertations

Staphylococcus aureus is the predominant cause of public health problems around the world. Vancomycin has been an important antibiotic against Methicillin Resistant Staphylococcus aureus (MRSA) infections. However, Vancomycin Intermediate S. aureus (VISA) strains have been reported. These strains are characterized by thick cell walls, reduced autolytic rate, reduced PBP4 activity, and increased amount of D-Ala-D-Ala termini in the cell wall. In this study, we show that the msaABCR operon regulates vancomycin resistance in two clinical VISA strains. Deletion of the msaABCR operon in strains Mu50 and HIP6297 resulted in a significant decrease in the minimum inhibitory concentration (MIC) for vancomycin. …

Investigations Of Filarial Nematode Motility, Response To Drug Treatment, And Pathology, Charles Nutting

Dissertations

More than a billion people live at risk of chronic diseases caused by parasitic filarial nematodes. These diseases: lymphatic filariasis, onchocerciasis, and loaisis cause significant morbidity, degrading the health, quality of life, and economic productivity of those who suffer from them. Though treatable, there is no cure to rid those infected of adult parasites. The parasites can modulate the immune system and live for 10-15 years. Testing of compounds against filarial nematodes is complicated due to a lack of an objective platform on which to analyze in vitro treatments. There is no published, immunocompetent laboratory model for lymphatic filariasis. This …

Signaling Between Two Sensor Kinases Controls Biofilms And Host Colonization In A Bacterial Symbiont, Allison N. Norsworthy

Dissertations

Organisms within all domains of life must acclimate to fluctuating environments to survive. To do this, cells utilize sensory circuits, which function to connect environmental stimuli to an intracellular response. One common sensory pathway utilized by bacteria is two-component signaling (TCS), composed of an environmental sensor (the sensor kinase, SK) and a cognate, intracellular effector (the response regulator, RR). The marine bacterium Vibrio fischeri uses an elaborate TCS phosphorelay containing a hybrid SK, RscS, and two RRs, SypE and SypG, to colonize its natural squid host, Euprymna scolopes. This TCS pathway regulates V. fischeri's ability to form a biofilm, or …

Characterization And Role Of Msaabcr In Biofilm Development And Virulence In Staphylococcus Aureus, Gyan Sundar Sahukhal

Dissertations

Community-acquired, methicillin-resistant Staphylococcus aureus strains often cause localized infections in immune-compromised hosts, but some strains show enhanced virulence leading to severe infections even among healthy individuals with no predisposing risk factors. The genetic basis for this enhanced virulence has yet to be determined. S. aureus possesses a wide variety of virulence factors, the expression of which is carefully coordinated by a variety of regulators. Several virulence regulators have been well characterized, but others have yet to be thoroughly investigated. Previously, the msa gene as a regulator of several virulence genes, biofilm development, and antibiotic …

Antiviral Responses In Mouse Embryonic Stem Cells: Differential Development Of Cellular Mechanisms In Type I Interferon Production And Response, Ruoxing Wang

Dissertations

Embryonic stem cells (ESCs) have been recognized as a promising cell source for regenerative medicine. Intensive research over the past decade has led to the possibility that ESC-derived cells will be used for the treatment of human diseases. However, increasing evidence indicates that ESC-derived cells generated by the current differentiation methods are not fully functional. It is recently recognized that ESC-derived cells lack innate immunity to a wide range of infectious agents and inflammatory cytokines. When used in patients, ESC-derived cells would be placed in wounded sites that are exposed to various pathogens and inflammatory cytokines; therefore, their viability and …

Relative Roles Of Host Exposure And Parasite Establishment In Producing Patterns Of Infection Using Eptesicus Fuscus (Chiroptera: Vespertilionidae) And Its Helminths As A Model System, Elizabeth Warburton

Dissertations

Parasite abundance and community structure could vary depending on conditions that are both extrinsic and intrinsic to the host. Variation in parasite burden, resulting from heterogeneous exposure to parasites or heterogeneous establishment of parasites in the host, often drives transmission dynamics. Changes in parasite community structure influence parasite species diversity and thus, diversity of infection within a single host or entire host population. I sought to quantify relative roles of exposure and establishment in producing patterns of infection using bats, Eptesicus fuscus, and their helminths. I utilized subsets of data from bats captured in 16 colonies from three states …

Coronavirus Proteases As Therapeutic Targets: Development Of Biosensors To Detect Inhibition Of Protease Activity And Separation Of The Multiple Functions Of Coronavirus Papain-Like Proteases, Andrew Kilianski

Dissertations

Coronaviruses are important human pathogens and have the potential to severely impact public health on an international scale. The emergence of SARS-CoV and MERS-CoV highlight the need for research to identify antivirals and vaccines against coronaviruses. To develop therapeutics against current and potentially emergent coronaviruses, I utilized two approaches targeting the proteases encoded within all coronaviruses. The papain-like protease and 3C-like protease of coronaviruses are responsible for cleaving viral polyproteins early during infection, and this step is required for viral replication. To quantitatively assess the inhibition by small-molecule compounds on MERS-CoV protease activity, I developed a luciferase-based biosensor to monitor …

Host Proteins Interact With The Hiv-1 Core To Facilitate And Restrict, Zana Lukic

Dissertations

Host cell proteins, termed restriction factors, which inhibit viral replication at various stages of the viral life cycle, determine the species-specific tropism of numerous retroviruses. Many members of the TRIM family of proteins act as viral restriction factors. One well-characterized example is the ability of TRIM5á from rhesus macaques (rhTRIM5á) to inhibit human immunodeficiency virus type-1 (HIV-1) soon after viral entry but prior to reverse transcription (RT). It is well established that the restriction requires an interaction between the viral capsid lattice and the B30.2/SPRY domain of TRIM5á. Following the binding of the viral core, TRIM5á mediates an event or …

Adenovirus Vectors As Potent Adjuvants In Vaccine Development, Kathleen Ann Mcguire

Dissertations

Due to their ability to activate the immune system, replication-defective Adenoviruses (Ad) are potential vaccine vectors for several pathogens. The proinflammatory response to Ad contributes to the response to vaccine antigens. We found that reactive oxygen species (ROS) are an important signal in the proinflammatory response to Ad. We identified that serotype 5 adenovirus (Ad5) elicits ROS by inducing mitochondrial membrane damage, a process that is dependent on endosomal membrane rupture and Cathepsin release. This mitochondrial dysfunction contributes to NLRP3 inflammasome- and NFκB-dependent innate immune activation. The ROS-dependent inflammatory response likely contributes to the adaptive immune response by supporting DC …

Host Entry Factors For Human Coronaviruses, Taylor Heald-Sargent

Dissertations

Coronaviruses infect a diverse range of animals from birds to pigs and cats to humans. Coronaviruses employ RNA-based replicative processes and as such are genetically adaptable to acquire novel host ranges. A coronavirus from one species can jump to another by shifting its entry requirements. As we learned from the Severe Acute Respiratory Syndrome (SARS) pandemic, this shift in species can have detrimental consequences. Thus, it is imperative that we understand the virus-host interaction during the entry process.

This dissertation focuses on host entry factors that influence human coronavirus entry. Recently, a new class of proteases, the type II transmembrane …

Identifying Novel Factors Involved In Biofilm Formation By Vibrio Fischeri, Valerie Ray

Dissertations

Biofilm formation by bacteria provides them with a means to survive harsh environmental conditions and promote colonization of a particular surface (biotic or abiotic). Due to the resistance properties of biofilms, biofilm-forming bacteria are difficult to treat in the context of host infections. Thus, investigating the processes that control biofilm formation will promote the development of novel therapeutics. To study biofilm formation in the context of a host, I utilized as a model system the marine bacterium Vibrio fischeri, which must be competent to form a biofilm to colonize its squid host Euprymna scolopes. Biofilm formation by V. fischeri requires …

Multifunctional Coronavirus Papain-Like Proteases As Targets For Antiviral Therapeutics And Vaccines, Anna Maria Mielech

Dissertations

Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle Ease Respiratory Syndrome coronavirus (MERS-CoV) pose a severe threat to humans because of high mortality. Despite the risk of coronavirus (CoV) emerging in the human population there are no antiviral drugs or vaccines to combat coronavirus infection. The focus of my dissertation was to study the multifunctionality of papain-like proteases (PLPs) encoded within coronavirus genomes to facilitate the development of antiviral drugs and vaccines. The viral PLPs are critical for processing the amino-terminal end of the replicase during virus replication and are attractive targets for antiviral therapies.

In my research, I analyzed …

Understanding The Posttranslational Regulation Of The Response Regulator Rcsb And Acetyl Phosphate As An Acetyl Group Donor In E. Coli, Linda Hu

Dissertations

The observation that Nepsilon-lysine acetylation occurs on a hundreds of proteins in bacteria is a recent discovery. To study the mechanisms that regulate acetylation and to determine if acetylation affects physiology, I studied the Escherichia coli response regulator and transcription factor RcsB, which was reported to be acetylated in vitro. To monitor RcsB activity, I measured transcription from the rprA promoter, which requires RcsB. I confirmed that RcsB is activated by phosphorylation through the Rcs phosphorelay and acetyl phosphate and showed that acetyl phosphate could phosphorylate RcsB. However, a mutant that accumulates acetyl phosphate (ackA) exhibited reduced rprA transcription instead …

The Role Of Linker 2 (L2) Region In Rhtrim5Α Assembly And Hiv-1 Restriction, Jayalaxmi Sastri

Dissertations

The cellular restriction factor TRIM5alpha inhibits infection by numerous retroviruses in a species specific manner. TRIM5alpha protein from rhesus macaques (rhTRIM5alpha) and a related protein TRIM-Cyp from Owl monkeys restrict infection by HIV-1 while human TRIM5α (huTRIM5alpha) restricts infection by N-tropic murine leukemia virus (N-MLV) but not HIV-1. Several models have been proposed for retroviral restriction by TRIM5 proteins (TRIM5alpha and TRIMCyp). These models collectively suggest that TRIM5 proteins mediate restriction by recognizing specific determinants in the viral capsid and directly binding the capsid. Following binding, the TRIM5 proteins self-associate into large assemblies around the viral capsid, which leads to …

Characterization Of The Novel Response Regulator Sype: A Dual Regulator Of Biofilm Formation And Colonization In Vibrio Fischeri, Andrew Robert Morris

Dissertations

Biofilms, or surface-associated microbial communities encased in a matrix, represent a common mode of growth for many bacterial species. Importantly, biofilms often play significant roles in the association of bacteria with their animal hosts, including promoting attachment to and colonization of host tissues. The symbiosis between the bacterium Vibrio fischeri and its animal host Euprymna scolopes provides a natural model system for investigating the impact of biofilms on host infection. In particular, V. fischeri produces a specific biofilm to successfully initiate host colonization.

Biofilm formation by V. fischeri is a tightly regulated process involving a complex network of regulatory genes. …

Evading Innate And Adaptive Immunity During Adenovirus Cell Entry, Shauna Marvin

Dissertations

Adenovirus (Ad), a non-enveloped, dsDNA virus, enters cells via clathrin-mediated endocytosis. For viral genome delivery to the nucleus, Ad must penetrate endosomal membranes to create defects sufficient for the passage of the 90 nm diameter capsid across cell membranes. Recent observations suggest that adenovirus type 5 (Ad5) capsid uncoating occurs at the cell surface upon binding to both the coxsackievirus and adenovirus receptor and α_v integrins. This uncoating event leads to the exposure of the capsid membrane lytic protein VI. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture, we demonstrate that Ad5 membrane rupture occurs …

Going Beyond Phosphorylation: Acetylphosphate-Mediated Ran Polymerase Acetylation And Caper Phosphorylation Co-Regulate Transcription Of Escherichia Coli Stress-Responsive Gene Cpxp, Bruno P. Lima

Dissertations

The ability of bacteria to sense and adapt to environmental changes has al-lowed these organisms to thrive in all parts of the globe and to establish many complex interactions with the environments they live as well as other members of these environments. Crucial to monitoring extracellular conditions is a group of signaling pathways known as two-component signal transduction system (2CST). These systems relay information from the extracellular environment to the interior of the cell via a transfer of phosphoryl groups from the sensory protein know as sensor kinase (SK) to the output protein known as response regulator (RR).

In addition …

Mechanisms Of Adenovirus Membrane Permeabilization, Oana Maier

Dissertations

For a successful infection to occur, a virus must first penetrate host cell membranes to access intracellular sites of viral replication. Currently the mechanism through which adenovirus, a non-enveloped, dsDNA virus, disrupts the endosomal membrane during cell entry is not well characterized.

Recent studies suggest that adenovirus protein VI, which is released from the interior of the capsid during cell entry, has all of the in vitro membrane lytic activity of the virion. We found that protein VI binds membranes via an amino-terminal 80 residue α-helical domain. Critical to this interaction are conserved hydrophobic and basic lysine residues within this …

Cofactors In Coronavirus Entry, Ana Shulla

Dissertations

Viruses have evolved complex ways to penetrate host barriers and cause disease. One of the most important barriers the virus has to cross is the cellular membrane. Enveloped viruses accomplish this task by viral glycoprotein-mediated binding to host cells and fusion of virus and host cell membranes. For the coronaviruses, viral spike (S) proteins execute these cell entry functions. In my dissertation research I focused on understanding the coronavirus spike proteins as well as other cofactors required for S-mediated entry into cells.

The S proteins are set apart from other viral and cellular membrane fusion proteins by their extensively palmitoylated …

Hypervirulent Clostridium Difficile Strains: Adherence, Toxin Production And Sporulation, Michelle Marie Merrigan

Dissertations

Clostridium difficile is a leading cause of nosocomial infections, and recently emerged "hypervirulent" C. difficile strains have caused epidemics worldwide. We hypothesized that multiple factors were responsible for this phenotype, particularly the interaction of C. difficile with the epithelial cell, as well as toxin production and sporulation.

To test if host interaction varied in HV strains, we developed a quantitative host-cell adherence assay, and found that while C. difficile strains varied in adherence to human intestinal epithelial cells, hypervirulent strains were not significantly more adherent than other strains. The bacterial surface protein SlpA varied in both size and amount between …

Coronavirus Replicase Proteins: Multifunctional Mediators Of Replication And Innate Immunity Evasion, Mark Anthony Clementz

Dissertations

Coronaviruses are positive-sense, single-stranded RNA viruses. The majority of the RNA encodes non-structural proteins (nsps) that are translated as a large polyprotein, which is cleaved by the papain-like (PLP) and picornavirus 3C-like (3CLpro) proteases. The nsps modify host membranes to produce double membrane vesicles (DMVs) upon which the replicase-transcriptase assembles and synthesizes viral RNA. nsp3, nsp4, and nsp6 are integral membrane proteins believed to be involved in DMV formation. Work presented here demonstrates that nsp4 is subjected to N-linked glycosylation and mutation of N258 to threonine in nsp4 confers a temperature sensitive phenotype to MHV-A59 infectious clone virus. This virus …

Characterization Of Tram, A Required Bacteroides Fragilis Conjugal Apparatus Protein That Interacts With The B. Fragilis Putative Coupling Protein Bcta And The B. Fragilis Relaxase Protein Bmph, Mai Thi Thanh Nguyen

Dissertations

Bacteroides spp. organisms, the most prominent bacteria of the human colon, are reservoirs of antibiotic resistance genes which promiscuously transfer within and from the genus via conjugation. The B. fragilis clinical isolate LV23 harbors a 37kb conjugative transposon, BTF-37. Two important conjugal apparatus proteins encoded by BTF-37 are BctA, a predicted coupling protein (CP) that powers DNA translocation, and ORF7 (TraM). In this study, we demonstrated that TraM exhibits characteristics of a required conjugal apparatus protein including 1) upregulated expression under conjugation conditions; 2) localization to the bacterial inner membrane; 3) interaction with the putative coupling protein BctA and the …