Microbiology Commons^™

Characterization Of Transfer Of The Mobile Genomic Island Encoding Methicillin Resistance Among Staphylococci, Melissa D. Ray

Theses and Dissertations

The gene encoding methicillin resistance in Staphylococcus aureus (MRSA) is carried in the chromosome on a large genomic island called SCCmec and is always inserted at the att site within orfX. SCCmec has been designated a mobile genetic element but a mechanism by which it moves among different strains and species of staphylococci has never been demonstrated. This work shows that bacteriophage 80α is capable of transducing SCCmec into a recipient cell, after which it can integrate into the bacterial chromosome via homologous recombination. More importantly, this work characterizes a conjugative mechanism of SCCmec transfer. …

The Characterization Of A Putative Virulence Factor Expressed By Sneathia Amnii, Amy Sanford

Theses and Dissertations

Preterm birth, defined at birth before 37 weeks gestation, affects millions of newborns worldwide every year. Preterm birth is a leading cause of infant morbidity and mortality. One major cause of preterm birth is preterm premature rupture of membranes (PPROM), which can be triggered by bacterial infection and inflammation. A bacterial species that has been implicated in preterm birth and other obstetric complications is Sneathia amnii. The goals of this study were to observe cytopathogenic effects caused by S. amnii strain Sn35 and identify putative virulence factors causing those effects. Sn35 was able to adhere to, invade, and damage/kill …

A Novel Antimicrobial Drug Discovery Approach For The Periodontal Pathogen Porphyromonas Gingivalis, Victoria N. Stone

Theses and Dissertations

The human body is colonized by more than 100 trillion microbes which make up an essential part of the body and plays a significant role in health. We now know the over use and misuse of broad-spectrum antibiotics can disrupt this microbiome contributing to the onset of disease and runs the risk of promoting antibiotic resistance. With antibiotic research still on the decline, new strategies are greatly needed to combat emerging pathogens while maintaining a healthy microbiome. We therefore set out to present a novel species-selective antimicrobial drug discovery strategy.

Disruption of the homeostasis within the oral cavity can trigger …

Orientia Tsutsugamushi Ankyrin-Repeat Protein Family Targeting Of The Host Endoplasmic Reticulum, Lauren Viebrock

Theses and Dissertations

Abstract

ORIENTIA TSUTSUGAMUSHI ANKYRIN REPEAT-PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM

By Lauren VieBrock, B.S.

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University

Virginia Commonwealth University, 2015

Director: Jason A. Carlyon, Ph.D.

Professor

Microbiology and Immunology

Scrub typhus is an understudied, potentially fatal febrile illness, which poses threat to one billion people annually in the Asia-Pacific region. The host-pathogen interactions that facilitate the intracellular survival of the etiologic agent, Orientia tsutsugamushi, are not well understood. The Orientia tsutsugamushi genome encodes a large number of ankyrin repeat-containing proteins …

Integrated Nanoscale Imaging And Spatial Recognition Of Biomolecules On Surfaces, Congzhou Wang

Theses and Dissertations

Biomolecules on cell surfaces play critical roles in diverse biological and physiological processes. However, conventional bulk scale techniques are unable to clarify the density and distribution of specific biomolecules in situ on single, living cell surfaces at the micro or nanoscale. In this work, a single cell analysis technique based on Atomic Force Microscopy (AFM) is developed to spatially identify biomolecules and characterize nanomechanical properties on single cell surfaces. The unique advantage of these AFM-based techniques lies in the ability to operate in situ (in a non-destructive fashion) and in real time, under physiological conditions or controlled micro-environments.

First, AFM-based …

Development Of Novel Chimeric Epitope Based Diagnostic Antigens And Vaccines For Lyme Disease, Lee D. Oliver Jr.

Theses and Dissertations

Lyme disease (LD) is the leading arthropod-borne disease in North America with 300-600,000 cases each year. There are currently no approved human LD vaccines. Outer surface protein C (OspC) has emerged as a leading vaccine candidate and an attractive diagnostic marker due to its antigenicity and expression early in infection. Several chimeric, epitope based OspC derived proteins were generated. The constructs were found to be highly immunogenic in mice and vaccination induced complement-dependent bactericidal antibodies. These results suggest that a broadly protective polyvalent OspC epitope based vaccine can be produced. Currently, LD diagnostic approaches are unreliable and unable to differentiate …

Characterization Of Salmonella Enterica Isolates From Food And Clinical Samples: Functionality Of Cytolethal Distending Toxin (Cdt) From Non-Typhi Salmonella Enterica, Ezat Hussain Mezal

Theses and Dissertations

Salmonella enterica is recognized worldwide as a significant cause of both human and animal disease. Recently, many random outbreaks of infections associated with the pathogens have been documented. Thus, state of the art molecular tools are necessary to conduct effective surveillance that would aid monitoring and prevention of disease. In this study, 110 Salmonella enterica serovars (Enteritidis & Javiana) strains isolated from food, environmental and clinical samples were analyzed for genetic diversity, antibiotic resistance, presence of virulence genes, plasmids, and plasmid replicon types. To assess the genetic diversity, pulsed-field gel electrophoresis (PFGE) fingerprinting, multiple-locus variable-number tandem repeat analysis (MLVA) and …

Enhancing Protein And Enzyme Stability Through Rationally Engineered Site-Specific Immobilization Utilizing Non-Canonical Amino Acids, Jeffrey Chun Wu

Theses and Dissertations

The demand for economical, efficient protein production, reuse, and recovery has never been greater due to their versatility in a large variety of applications ranging from industrial chemical manufacturing to pharmaceutical drug production. The applications for naturally and artificially produced proteins include protein drugs and other pharmaceutical products, as biocatalysts in environmentally friendly chemical manufacturing, as enzymes for food processing purposes, and as an essential component in many biomedical devices. However, protein production suffers from many challenges, which include the cost of production, protein stability especially under harsh conditions, and recoverability and reusability of the proteins. The combination of two …

Studies Of Phou In Escherichia Coli: Metal Binding, Dimerization,Protein/Protein Interactions, And A Signaling Complex Model, Stewart G. Gardner

Theses and Dissertations

Phosphate is an essential nutrient for all forms of life. Escherichia coli has a PhoR/PhoB two component regulatory system that controls the expression of various genes whose products allow the cell to thrive in low phosphate environments. The signaling mechanism of the PhoR/PhoB system has been studied and the phosphorylation cascade that controls gene expression is well understood. What is still unknown is how PhoR senses the phosphate level of the environment. The PstS, PstC, PstA, PstB, and PhoU proteins play a role in this signal sensing. This work confirms the hypothesis that the PstSCAB complex senses the environmental phosphate …

Pharmacologic Immunomodulation Of Macrophage Activation By Caffeine, Ryan Perry Steck

Theses and Dissertations

Caffeine is one of the most widely used neurostimulants in the world and there is considerable debate on its effect in immune cells. One of its main targets is proposed to be adenosine receptors which mediate an anti-inflammatory switch in activated immune cells while another target is phosphodiesterase where it acts as an inhibitor. In macrophages, caffeine has been shown to cause both pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. If the primary effect of caffeine on macrophages were to antagonize adenosine receptors we would expect cells exposed to caffeine to have a prolonged M1 response. However, we show that caffeine …

Snf1 Mediated Phosphorylation And Activation Of Pas Kinase, Bryan D. Badal

Theses and Dissertations

Nutrient sensing kinases sense available nutrients and regulate cell activity accordingly. Three of these enzymes are AMP regulated kinase (AMPK, or Snf1 in yeast), PAS kinase, and target of rapamycin (TOR), are conserved from yeast to man and have overlapping function. AMPK and Snf1 are important in sensing when nutrient status in the cell is low and down regulating energy consuming pathways. PAS kinase is required for glucose homeostasis in the cell, and responds to glucose levels. TOR senses nutrients such as amino acids and upregulates cell growth pathways primarily through protein synthesis. Due to the varying nature of these …

Cell Wall And Autolytic Properties Of A Cold-Sensitive Listeria Monocytogenes Mutant Deficient In Branched-Chain Fatty Acid Biosynthesis, Anthony M. Martini

Theses and Dissertations

Cellular membranes modulate several critical functions of bacterial physiology, including solute diffusion and nutrient transport. Biophysical properties of phospholipids, as well as environmental pressures such as temperature, can have a profound effect on these membrane functions and the kinetics of associated enzymes. Listeria monocytogenes is a facultative intracellular pathogen capable of robust low temperature growth, a characteristic thought to be facilitated by a membrane highly enriched in branched-chain fatty acids (BCFA). Mutants deficient in the biosynthesis of these fatty acids exhibit severe growth defects under low temperature and extreme pH conditions. Morphological investigations have also revealed these mutants to undergo …

Analysis Of Nucleosome Mobility, Fragility, And Recovery: From Embryonic Stem Cells To Invitrosomes, Ashley Nicolle Wright

Theses and Dissertations

Several factors direct the placement of specific nucleosomes, which in turn have the ability to regulate DNA accessibility. These factors include, but are not limited to, nucleotide sequence preference, nucleotide modifications, the type of histones present within the nucleosome, and the presence of additional transcription factor or chromatin remodelers. A combination of these and other factors are responsible for tightly controlled efficient transcription within the eukaryotic cell. In order to contribute to the understanding of these complicated processes, three separate hypothesis-driven investigations were carried out. First, we looked into nucleosome positioning and phasing within closely related cells lines. Second, we …

Characterization Of The Effects Of Iron On Neisseria Gonorrhoeae Surface Protein Modulation And Host Cell Interactions, Aminat Oki

Theses and Dissertations

Iron is an essential nutrient that is sequestered by iron-binding proteins in the human host resulting in a hostile environment for microbes. Neisseria gonorrhoeae, however, can utilize numerous iron-binding proteins such as transferrin and lactoferrin to acquire this nutrient. During initial infection, gonococci have access to transferrin and lactoferrin present in semen and vaginal fluids, as well as to hemoglobin present in blood during menses or disseminated infections. Consequently, the gonococcus likely encounters conditions of high iron at some stages in the course of natural infection. Potential contributions of iron to gonococcal invasion have however been largely over looked in …

Anaplasma Phagocytophilum Nutritional Virulence Mechanisms Target The Host Cell Secretory Pathway, Hilary Kay Truchan

Theses and Dissertations

Obligate intracellular pathogens must acquire host cell-derived nutrients to facilitate their survival. One such bacterial pathogen, Anaplasma phagocytophilum, replicates within neutrophils and non-phagocytic cells in a bacterial-modified, host cell-derived vacuole. The bacterium exploits host cell vesicular trafficking pathways to route nutrients to its vacuole and utilizes Rab GTPases, guanine nucleotide-dependent, vesicular trafficking regulators, to do so. We previously discovered that the A. phagocytophilum vacuolar membrane is decorated with a specific subset of Rab GTPases - Rab1, Rab4A, Rab10, Rab11, Rab14, Rab22A and Rab35. Rab1 is exclusively found on the endoplasmic reticulum (ER) and thus its localization suggests that the …

Identification Of The Binding Partners For Hspb2 And Cryab Reveals Myofibril And Mitochondrial Protein Interactions And Non-Redundant Roles For Small Heat Shock Proteins, Kelsey Murphey Langston

Theses and Dissertations

Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity. As such, mutations in this family of proteins result in a wide variety of diseases from cancers to cardiomyopathies. The sHSPs Beta-2 (HspB2) and alpha-beta crystalline (CryAB) are two of the ten human sHSPs and are both expressed in cardiac and skeletal muscle cells. A heart that cannot properly recover or defend against stressors such as extreme heat or cold, oxidative/reductive stress, and heavy metal-induced stress will constantly struggle to maintain efficient function. Accordingly, CryAB is required …

The Effect Of Media Selection On The Membrane Characteristics And Fatty Acid Composition Of Staphylococcus Aureus, Seth Russell Johnson

Theses and Dissertations

The cellular membrane is a vital structure to a microorganism and impacts important aspects of environmental regulation and growth. The physical structure of cellular membranes plays a key role in bacterial growth at low temperatures, membrane permeability and susceptibility to membrane active molecules as well as broader aspects of bacterial physiology and pathogenesis. Mueller Hinton medium is the required medium for antimicrobial susceptibility testing in the US, but there are many other types of media used in staphylococcal research. Different media vary in their nutrient compositions, and the effect of media composition on bacterial physiology is incompletely understood. The aim …

A Quadruplex Real-Time Pcr Assay For The Rapid Detection And Differentiation Of The Burkholderia Pseudomallei Complex: B. Mallei, B. Pseudomallei, And B. Thailandensis, Chinn-Woan Lowe

Theses and Dissertations

Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, …

Nuclear Bmp2 And The Immune Response, Daniel S. Olsen

Theses and Dissertations

Nuclear bone morphogenetic protein 2 (nBMP2) is a nuclear variant of the secreted growth factor BMP2. Experiments in nBmp2NLStm mutant mice, which lack nBMP2 in the nucleus, have shown that nBMP2 affects intracellular calcium transport in skeletal muscle and hippocampal neurons. The objective of this study was to determine whether nBMP2 affects the immune system, since activation of lymphocytes and other immune cells depends on intracellular calcium transport. We found that spleens in nBmp2NLStm mutant mice were 24% smaller than in wild type mice. The white pulp of the spleen contains many immune cells, particularly B and T lymphocytes and …

The Role Of Nuclear Bmp2 In The Cell Cycle And Tumorigenesis, Brandt Alan Nichols

Theses and Dissertations

Bone morphogenetic protein 2 (BMP2) is a secreted growth factor that is essential for proper embryonic development and proliferation. Our laboratory discovered a nuclear variant of BMP2 (nBMP2) which is produced when translation is initiated at an alternative start codon within the BMP2 gene. When translation occurs at the downstream start codon, the resulting protein lacks the ER signal peptide, thereby allowing cytoplasmic translation and nuclear localization. Our aim is to distinguish the role of this nuclear localized variant from secreted BMP2. Overexpression of nBMP2 in HEK293 and HT29 cell lines resulted in a higher percentage of cells in proliferative …

Promoter Polymorphisms In Interferon Regulatory Factor 5, Daniel N. Clark

Theses and Dissertations

The promoter region of interferon regulatory factor 5 (IRF5) contains the rs2004640 T or G single nucleotide polymorphism (SNP) and a CGGGG indel. Both of these polymorphisms have been implicated as genetic risk factors for several autoimmune diseases, including systemic lupus erythematosus, whose pathology involves altered apoptosis and cytokine signaling. The polymorphisms' overall effect is to increase IRF5 levels. IRF5 is a transcription factor of several cytokines, including interferon, and is pro-apoptotic. Thus an alteration of cytokine levels and apoptosis signaling due to high IRF5 levels is the proposed source of autoimmune risk. Each of IRF5's four first exons (1A, …

Hypersaline Lake Environments Exhibit Reduced Microbial Dormancy, Joshua Christopher Vert

Theses and Dissertations

From acid seeps and deep-sea thermal vents to glacial ice and hypersaline lakes, extreme environments contain relatively simplified communities consisting of extremophiles that have evolved to survive and thrive under adverse abiotic conditions. In more neutral environments, microorganisms use dormancy as a common life history strategy to weather temporal fluctuations of resources or stresses until more 'optimal' conditions are present. It is unclear if dormancy is an essential survival mechanism for microorganisms in extreme environments; however, recent studies suggest that extreme environments may create stable conditions for extremophiles to the extent that dormancy is of less ecological importance. Using lake …

Thymidine Kinase 1: Diagnostic And Prognostic Significance In Malignancy, Melissa Marie Alegre

Theses and Dissertations

Thymidine kinase 1 (TK1) is a cancer biomarker which has diagnostic and prognostic potential in a variety of malignancies. TK1 is significantly elevated in the serum and tumor tissue of most malignancies. This increase in TK1 can be detected in the very early stages of malignancy, including in pre-malignant disease with an increased risk for progression. Several studies have demonstrated that elevated TK1 is found in serum months before any clinical symptoms of malignancy. It has also been demonstrated that TK1 is elevated months before clinical recurrence of malignancy. This work first sought to demonstrate the early nature of TK1 …

Modulators Of Symbiotic Outcome In Sinorhizobium Meliloti, Matthew B. Crook

Theses and Dissertations

Microorganisms interact frequently with each other and with higher organisms. This contact and communication takes place at the molecular level. Microbial interactions with eukaryotes can be pathogenic or mutualistic. One of the best-studied symbioses is the complex interaction between nitrogen-fixing soil bacteria, termed rhizobia, and legumes. This symbiosis culminates in the elaboration of a new organ, the root nodule. Many of the molecular signals exchanged between the host plant and the invading rhizobia have been deduced, but there is still much that remains to be discovered. The molecular determinant of host range at the genus level of the plant host …

Evidences For Protein-Protein Interactions Between Pstb And Phou In The Phosphate Signaling Complex Of Escherichia Coli, Kristine Dawn Johns

Theses and Dissertations

The PstSCAB2 complex serves the dual function of being a phosphate transporter as well as the primary sensor of phosphate for the Pho regulon. PhoU is an integral protein required for the signal from PstSCAB2 to be transmitted to PhoR. Our hypothesis is that conformational changes of PstSCAB2 during the phosphate transport process are the mechanism by which information about environmental phosphate levels are transduced to the cell. Additionally, we propose that direct protein-protein interactions between PhoU and the alternating conformations of PstSCAB2 mediate PhoU interactions with PhoR. By means of genetic and biochemical approaches, we have found substantial evidence …

Effects Of Chemical Stimulation And Tumor Co-Incubation On Macrophage Activation And Aggressiveness, Measured Through Phagocytosis And Respiratory Burst, Bo Marcus Gustafsson

Theses and Dissertations

Macrophages are a cornerstone in innate immunity, especially important in detecting and killing invading microorganisms. In tumor biology, the macrophages can contribute both to anti-tumor activity and tumor promotion depending on individual tumor microenvironment and therefore have a large impact on both tumor progression and prognosis. Two of the most important functions of macrophages are the ability to phagocytose microorganisms and then kill them through the respiratory burst. Phagocytosis activates the respiratory burst, but the more subtle interactions between these processes are less known. Since phagocytosis and reactive oxygen species production are two attributes that change between the classically and …

Loss Of The Lipopolysaccharide Core Biosynthesis Rfad Gene Increases Antimicrobial Chemokine Binding And Bacterial Susceptibility To Ccl28 And Polymyxin: A Model For Understanding The Interface Of Antimicrobial Chemokines And Bacterial Host Defense Avoidance Mechanisms, Cynthia S. Lew

Theses and Dissertations

In order to better understand the mechanism of antimicrobial chemokine activity, including binding to and killing of bacteria, random transposon mutagenesis was performed in Yersinia pseudotuberculosis. Resulting mutants were screened for increased binding to chemokine and high binding clones were selected for further study. One mutant, designated mutant 27, was found to have a single insertion mutation in the rfaD gene. The rfaD gene product is involved in heptose biosynthesis, one of the sugars of the inner core oligosaccharide of Gram- negative lipopolysaccharide (LPS). Mutant 27 was found to bind both CCL25 and CCL28, two antimicrobial chemokines, more efficiently than …

Characterizing The Role Of Hspb2 In Cardiac Metabolism And Muscle Structure Using Yeast And Mammalian Systems, Jonathan Paul Neubert

Theses and Dissertations

HspB2 is a small heat shock protein encoded on human chromosome 11. Less than 1000 base pairs away from HSPB2 and situated in a head-to-head orientation lies the gene encoding another small heat shock protein, CRYAB. Because they are uncommonly close to one another they share regulatory elements. In addition, they share protein homology as sHSPs, suggesting that they perhaps perform aimilar functions. SHSPs such as HspB2 and CryAB are traditionally thought to provide protective effects to cells in response to a variety of stress inducers. In response to stress they form complexes around misfolded proteins or proteins in danger …

Characterization Of The Cellular And Organellar Dynamics That Occur With A Partial Depletion Of Mitochondrial Dna When Arabidopsis Organellar Dna Polymerase Ib Is Mutated, John D. Cupp

Theses and Dissertations

Plant mitochondrial genomes are large and complex, and the mechanisms for maintaining mitochondrial DNA (mtDNA) remain unclear. Arabidopsis thaliana has two DNA polymerase genes, polIA and polIB, that have been shown to be dual localized to mitochondria and chloroplasts but are unequally expressed within primary plant tissues involved in cell division or cell expansion. PolIB expression is observed at higher levels in both shoot and root apexes, suggesting a possible role in organelle DNA replication in rapidly dividing or expanding cells. It is proposed that both polIA and polIB are required for mtDNA replication under wild type conditions. An Arabidopsis …

Transgene Delivery Via Microelectromechanical Systems, Aubrey Marie Mueller Wilson

Theses and Dissertations

The invention of pronuclear microinjection initiated the field of transgenic research. Over 30 years later microinjection remains the most straight-forward and most commonly used transgene delivery option. In this work we address the current progress of microelectromechanical systems (MEMS) used as transgenic delivery mechanisms. The nanoinjector is a specially designed MEMS device which uses electrostatic charge to manipulate transgene molecules. The process of nanoinjection was designed as an alternative to microinjection which causes less damage to developing embryos, improves embryo survival, birth rates, and overall efficiency of injections. In vivo testing of nanoinjection demonstrates it is both safe and effective. …