Browsing by Author "Schubot, Florian D."

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    Analysis of the Quorum Sensing Regulons of Vibrio parahaemolyticus BB22 and Pantoea stewartii subspecies stewartii
    Burke, Alison Kernell (Virginia Tech, 2015-12-07)
    Quorum sensing is utilized by many different proteobacteria, including the two studied for this dissertation work, Vibrio parahaemolyticus and Pantoea stewartii subsp. stewartii. V. parahaemolyticus causes acute gastroenteritis in people who eat contaminated raw or undercooked shellfish. It is found in warmer marine waters and in rare cases, causes systemic infections when bacteria enter the body through open wounds. P. stewartii, on the other hand, is a phytopathogen that causes Stewart's wilt in maize. It is found in soil or the mid-gut of the corn flea beetle, its insect vector. Both V. parahaemolyticus and P. stewartii utilize quorum sensing to control their pathogenicity. Quorum sensing enables coordinate gene expression across a bacterial population. The V. parahaemolyticus quorum-sensing system utilizes the master regulator OpaR, which is homologous to the V. harveyii LuxRVh and the P. stewartii system contains EsaR which is homologous to the V. fischeri LuxRVf regulator. While the two systems differ in the molecular details of their mechanistic control, they are both forms of cell density dependent regulation that are either directly or indirectly controlled by small signaling molecules. Three different signaling molecules are found in V. parahaemolyticus, and only one signal is used in P. stewartii. The focus of this dissertation has been on understanding the downstream targets of OpaR and EsaR in their respective quorum-sensing systems. Prior to this work, it was known that when OpaR is not present or is nonfunctional V. parahaemolyticus changes from an opaque to a translucent colony morphology phenotype and the cells also become swarm proficient and more pathogenic. The complete genome of the V. parahaemolyticus BB22OP strain was assembled and annotated (Chapter 2). RNA-Seq was then used to analyze the transcriptomes of OpaR-active and OpaR-deficient strains of V. parahaemolyticus and identify genes that were regulated via quorum sensing (Chapter 3). Similarly, P. stewartii was also analyzed using RNA-Seq to identify genes controlled by EsaR in the transcriptome that had not been detected through prior proteomic studies. The initial RNA-Seq work confirmed the control of some previously identified direct targets of EsaR and newly identified ten other genes also directly controlled by EsaR (Chapter 4). Two direct targets of EsaR, rcsA and lrhA, became the focus of additional studies to further define the hierarchy of gene control downstream of the quorum-sensing regulator EsaR. RcsA controls capsule production, while LrhA controls motility and adhesion in P. stewartii. The regulons of rcsA and lrhA were defined by RNA-Seq, which also revealed multi-level control of rcsA gene expression (Chapter 5). Tight coordinated and temporal control of virulence factors is important for successful disease progression by pathogens. This dissertation work aims to enable a better understanding of the quorum-sensing hierarchy of genetic control in V. parahaemolyticus and P. stewartii.
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    Backbone Interactions Between Transcriptional Activator ExsA and Anti-Activator ExsD Facilitate Regulation of the Type III Secretion System in Pseudomonas aeruginosa
    Shrestha, Manisha; Bernhards, Robert C.; Fu, Yichen; Ryan, Kylie; Schubot, Florian D. (Springer Nature, 2020)
    The type III secretion system (T3SS) is a pivotal virulence mechanism of many Gram-negative bacteria. During infection, the syringe-like T3SS injects cytotoxic proteins directly into the eukaryotic host cell cytoplasm. In Pseudomonas aeruginosa, expression of the T3SS is regulated by a signaling cascade involving the proteins ExsA, ExsC, ExsD, and ExsE. The AraC-type transcription factor ExsA activates transcription of all T3SS-associated genes. Prior to host cell contact, ExsA is inhibited through direct binding of the anti-activator protein ExsD. Host cell contact triggers secretion of ExsE and sequestration of ExsD by ExsC to cause the release of ExsA. ExsA does not bind ExsD through the canonical ligand binding pocket of AraC-type proteins. Using site-directed mutagenesis and a specific in vitro transcription assay, we have now discovered that backbone interactions between the amino terminus of ExsD and the ExsA beta barrel constitute a pivotal part of the ExsD-ExsA interface. Follow-up bacterial two-hybrid experiments suggest additional contacts create an even larger protein– protein interface. The discovered role of the amino terminus of ExsD in ExsA binding explains how ExsC might relieve the ExsD-mediated inhibition of T3SS gene expression, because the same region of ExsD interacts with ExsC following host cell contact.
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    Biochemical Characterization of Two Aminopeptidases Involved in Hemoglobin Catabolism in the Food Vacuole of Plasmodium falciparum
    Ragheb, Daniel Raafat Tadros (Virginia Tech, 2011-03-31)
    The parasite Plasmodium falciparum is the causative agent of the most severe form of human malaria. During its intraerythocytic life cycle, P. falciparum transports red blood cell contents to its acidic organelle, known as the food vacuole, where a series of proteases degrade a majority of the host hemoglobin. Two metalloaminopeptidases, PfAPP and PfA-M1, have been previously localized to the food vacuole (in addition to distinct secondary locations for each), implicating them in the final stages of hemoglobin catabolism. Prior genetic work has determined these enzymes are necessary for efficient parasite proliferation, highlighting them as potential anti-malarial drug targets. This study presents the biochemical basis for the catalytic roles of these two enzymes in the hemoglobin degradation pathway. PfAPP, an aminopeptidase P homolog, is specific for hydrolyzing the N-termini of peptides containing penultimate prolines. PfA-M1 is a member of the expansive M1 family of proteases and exhibits a broad specificity towards substrates. The two enzymes are ubiquitous, found in organisms across all kingdoms of life. Their presence in an acidic environment is unique for aminopeptidase P proteins and rare for M1 homologs. Our immunolocalization results have confirmed the dual distribution of these two enzymes in the parasite. Vacuolar targeting was found to be associated with the Plasmodium specific N-terminal extension found in the PfA-M1 sequence by yellow fluorescent protein fusion studies. Kinetic analysis of recombinant forms of PfAPP and PfA-M1 revealed both enzymes are stable and catalytically efficient in the substrate rich, acidic environment of the parasite food vacuole. In addition, mutagenic exploration of the PfA-M1 active site has determined a residue important in dictating substrate specificity among homologs of the same family. These results provide insight into the parasite's functional recruitment of these enzymes to deal with the final stages of hemoglobin catabolism and necessary considerations for inhibitor design.
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    Characterization of the Sinorhizobum Meliloti Chotaxis System
    Castaneda Saldana, Rafael (Virginia Tech, 2019-12-19)
    Increasing awareness to global climate change has drastically focused attention on finding solutions to reduce environmental impacts while still providing sufficient food for the increasing world population. Beneficial Nitrogen Fixing (BNF) microbes provide a possible solution by delivering biological nitrogen to plants resulting in reduced environmental impacts due to fertilizer runoff and eutrophication. One well studied model is that of Sinorhizobium meliloti and its legume host Medicago sativa (alfalfa), the fourth largest USA crop used for animal feed. Advancing research for this symbiosis model can provide solutions to enhance yield while minimizing environmental impacts. Chapter 2 focuses on the deviation of the S. meliloti chemotaxis system from the enteric paradigm. Quantitative immunoblots determined the cellular amounts of chemotaxis proteins. Overall, chemotaxis protein levels were approximately 10-fold lower in S. meliloti compared to B. subtilis and E. coli. Focusing on cellular stoichiometric ratios, S. meliloti generally exhibits drastically higher values for CheB, CheR, and CheY to the histidine kinase CheA monomer compared to E. coli and B. subtilis. Chapter 3 characterizes the role of McpX to quaternary ammonium compound (QAC) sensing. QACs are exuded by germinating alfalfa seeds. In vitro binding assays were performed to determine ligand binding characteristics. S. meliloti chemotaxis behavior to QACs was analyzed in in vivo capillary assays under real-time imaging. These studies strengthen our knowledge of the chemotaxis system in the symbiosis model of S. meliloti and alfalfa. The data can further be used to create a mathematical model of the dynamics of bacteria-host interaction. The results can be used to optimize chemotaxis to host plants to improve crop yield and protect watersheds.
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    Characterizing the Innate Immune Response of Human Airway Cells to the Unique Fungal Allergen Alt a 1
    Hayes, Tristan Alonzo (Virginia Tech, 2017-04-25)
    Allergic airway diseases such as rhinitis, asthma, and chronic rhinosinusitis are responsible for causing a huge economic burden on patients and society. Patients suffering from asthma often have allergies to pollen, dust mite, and mold. Interestingly, studies have shown that there is a correlation between severe asthma and sensitization to fungi including Aspergillus, Alternaria, Cladosporium, and Penicillium. This project has been focused on studying the innate immunomodulatory activities of the major allergen Alt a 1, from the ubiquitous airborne fungus, Alternaria alternata. In several studies, 90-100% of allergic patients who are sensitized to Alternaria, have Alt a 1 specific IgE antibodies indicating that it is a major and clinically relevant allergen. Although progress has been made over the past few decades regarding elucidating the mechanistic underpinnings of allergic inflammation, more research needs to be done, especially in regards to innate immunity and its role in the sensitization and exacerbation aspects of allergic diseases. Published studies have increasingly made it clear that Toll-like receptors (TLRs) are key players in innate immunity to several allergens. For example, the dust mite allergen, Der p 2, has been shown to mimic the activity of human and mouse MD2 in the presence of LPS to trigger a response through TLR4. Bet v 1, an allergen from Birch tree, has been shown to enter and be transported through lung epithelium in patient cells. It is hypothesized that transcytosis of allergens like Bet v 1 may contribute to sensitization and exacerbation in atopic individuals. This project was focused on two primary aims; (1) Characterize the innate immune response of Alt a 1 in human airway epithelial cells, and (2) Identify if and how Alt a 1 can enter human airway cells. We found that Alt a 1 was able to stimulate innate immune responses in bronchial epithelial cells and this was dependent upon TLR2, TLR4 and the downstream adaptor proteins MyD88 and TIRAP. We also found in our studies that Alt a 1 rapidly enters bronchial epithelial cells. Furthermore, our data suggests that endocytosis of Alt a 1 may be partially dependent upon interaction with phosphatidyl-inositol-3-phosphate (PI-3-P).
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    Circadian modulation of the estrogen receptor alpha transcription
    Villa, Linda Monique (Virginia Tech, 2012-07-12)
    The circadian clock is a molecular mechanism that synchronizes physiological changes with environmental variations. Disruption of the circadian clock has been linked to increased risk in diseases and a number of disorders (e.g. jet lag, insomnia, and cancer). Period 2 (Per2), a circadian protein, is at the center of the clock's function. The loss or deregulation of per2 has been shown to be common in several types of cancer including breast and ovarian [1, 2]. Epidemiological studies established a correlation between circadian disruption and the development of estrogen dependent tumors. The expression of estrogen receptor alpha (ERα) mRNA oscillates in a 24-hour period and, unlike Per2, ERα peaks during the light phase of the day. Because up regulation of ERα relates to tumor development, defining the mechanisms of ERα expression will contribute to our comprehension of cellular proliferation and regulation of normal developmental processes. The overall goal of this project is to investigate the molecular basis for circadian control of ERα transcription. Transcriptional activation of ERα was measured using a reporter system in Chinese hamster ovary (CHO) cell lines. Data show that Per2 influences ERα transcription through a non-canonical mechanism independent of its circadian counterparts. Breast cancer susceptibility protein 1 (BRCA1) was confirmed to be an interactor of Per2 via bacterial two-hybrid assays, in accordance with previous studies [2]. BRCA1 is a transcriptional activator of ERα promoter in the presence of octamer transcription factor-1 (OCT-1) [3]. Our results indicate that the DNA binding domain of OCT-1, POU, to directly interact with Per2 and BRCA1, in vitro. Pull-down assays were used to map direct interaction of various Per2 and BRCA1 recombinant proteins and POU. Chromatin immunoprecipitation assays confirmed the recruitment of PER2 and BRCA1 to the estrogen promoter by OCT-1 and the recruitment of Per2 to the ERα promoter decreases ERα mRNA expression levels in MCF-7 cells. Our work supports a circadian regulation of ERα through the repression of esr1 by Per2 in MCF-7 cells.
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    Development of Methods for Structural Characterization of Pantoea stewartii Quorum-Sensing Regulator EsaR
    Pennerman, Kayla Kara (Virginia Tech, 2014-02-04)
    The LuxR family of proteins serves as quorum-sensing transcriptional regulators in proteobacteria. At high population densities, a small acyl-homoserine lactone (AHL) molecule, produced by a LuxI homologue, accumulates in the environment. The LuxR proteins bind to their respective AHL when the ligand accumulates to sufficient levels. Once bound to AHL, the holoproteins usually become functional as transcriptional activators. However, there is a subset of LuxR homologues, the EsaR subfamily, which is active without the AHL ligand and becomes inactivated once bound to it. EsaR is the best understood member of this subfamily. It controls virulence in the corn pathogen Pantoea stewartii ssp. stewartii. Solubility issues have previously limited structural studies of LuxR homologues as the proteins could not be purified without the AHL ligand. A soluble recombinant EsaR protein, HMGE, is biologically active and can be purified in the absence and presence of AHL, unlike most other LuxR homologues. Using HMGE, amino acid substitutions and Förster resonance energy transfer (FRET), experimental methods were designed for determining the dimerization interface of EsaR and for testing the hypothesis that EsaR undergoes a conformational shift when presented with the AHL ligand. To identify residues of the dimerization interface, heterodimerization assays were designed, involving either coexpression or coincubation of wild-type EsaR and variant HMGE proteins. In this assay, the inability of the proteins to copurify by nickel affinity chromatography would indicate that the modified residue(s) are important for dimerization of EsaR. To determine the conformational change that EsaR undergoes when bound to the AHL ligand, a FRET assay was developed to estimate the distances between amino acid residues in the absence and presence of AHL. Future work will have to include a few modifications to the methods and/or control experiments. This study provides the basis upon which the present methods can be further developed and later used for structural studies of EsaR.
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    Differential Expression Analysis of Type II Toxin-Antitoxin Genes of Pseudomonas aeruginosa PAO1 under Different Environmental Conditions
    Haque, Anamul (Virginia Tech, 2018-07-02)
    Bacterial persistence is considered as one of the primary reason for antibiotic tolerance besides genetically acquired antibiotic resistance. Persisters are the subpopulation of a clonal bacterial population, which can survive environmental extremes and become invulnerable to stresses due to limited metabolic activities and physiological functions. Cognate toxin and antitoxin (TA) pairs, which are transcribed simultaneously from the same or different operons within the bacterial chromosomes or plasmids, play an important role for bacterial survival during stressful growth environments. Pseudomonas aeruginosa PAO1 is one of the most versatile microorganisms in the environment. Despite its ubiquitous presence, no studies have shown the differential expression pattern of its toxin-antitoxins, and persistence related genes. The purpose of the following study is to analyze differential expression of P. aeruginosa PAO1 type II toxin-antitoxins and persistence related genes under different growth conditions and to show how their stoichiometric ratio changes during different growth conditions. Differential expression analysis indicated that the toxins and antitoxin pairs behave differently under different growth conditions. In addition, the genes related to persistence presented relatively consistent differential expression pattern under different growth environment.
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    Elucidation of the Specificity of S. meliloti Chemoreceptors for Host Derived Attractants
    Webb, Benjamin A. (Virginia Tech, 2016-08-24)
    The bacterium Sinorhizobium (Ensifer) meliloti is a member of the Rhizobiaceae family and can enter a mutualistic, diazotrophic relationship with most plants of the genera Medicago, Melilotus, and Trigonella. Medicago sativa (alfalfa) is an agriculturally important legume that hosts S. meliloti and allows the bacterium to invade the plant root and begin fixing nitrogen. Prior to invasion, S. meliloti exists as a free living bacterium and must navigate through the soil to find alfalfa, using chemical signals secreted by the root. Alfalfa is the 4th most cultivated crop in the United States, therefore, identification of plant host signals that lure S. meliloti, and identification of the bacterium's chemoreceptors that perceive the signals can aid in propagating the symbiosis more efficiently, thus leading to greater crop yields. Investigations here focus on discovering alfalfa derived attractant signals and matching them to their respective chemoreceptors in S. meliloti. We have determined the chemotactic potency of alfalfa seed exudate and characterized and quantified two classes of attractant compounds exuded by germinating alfalfa seeds, namely, amino acids and quaternary ammonium compounds (QACs). At all points possible, we have compared alfalfa with the closely related non-host, spotted medic (Medicago arabica). The chemotactic potency of alfalfa seed exudate is the same as spotted medic seed exudate, however, the attractant compositions are chemically different. The amount of each proteinogenic amino acid (AA) exuded by spotted medic is slightly greater than the amounts exuded by alfalfa. In addition, the five QACs studied are exuded in various amounts between the two Medicago species. In comparison, the total amount of proteinogenic AAs exuded be alfalfa and spotted medic are 2.01 μg/seed and 1.94 μg/seed respectively, and the total amount of QACs exuded are 249 ng/seed and 221 ng/seed respectively. By performing a chemotaxis assay with synthetic AA mixtures mimicking the amounts exuded from the medics, it was found that the AA mixtures contribute to 23% and 37% of the responses to alfalfa and spotted medic exudates, respectively. The chemoreceptor McpU was found to be the most important chemoreceptor of the eight for chemotaxis to the whole exudates and the AA mixtures. Furthermore, McpU is shown to mediate chemotaxis to 19 of 20 AAs excluding aspartate. McpU directly interacts with 18 AAs and indirectly mediates chemotaxis to glutamate. Through single amino acid residue substitutions, it is determined that McpU directly binds to amino acids in the annotated region called the Cache_1 domain, likely utilizing residues D155 and D182 to interact with the amino group of AA ligands. In all, McpU is a direct sensor for AAs except for the acidic AAs aspartate and glutamate. Work is presented to show that the QACs betonicine, choline, glycine betaine, stachydrine, and trigonelline are potent attractants for S. meliloti, McpX is the most important chemoreceptor for chemotaxis to these QACs, and we demonstrate the binding strength of McpX to the QACs with dissociation constants ranging from low millimolar to low nanomolar, thus making McpX the first observed bacterial MCP that mediates chemotaxis to QACs. Overall, we match medic derived AAs with McpU and QACs with McpX. These results can aid in optimizing chemotaxis to the host derived attractants in order to propagate the symbiosis more efficiently resulting in greater crop yields. Chapter 2 characterizes the function of the S. meliloti Methyl accepting Chemotaxis Protein U (McpU) as receptor for the attractant, proline. A reduction in chemotaxis to proline is observed in an McpU deletion strain, but the defect is restored in an mcpU complemented strain. Single amino acid substitution mutant strains were created, each harboring a mutant mcpU gene. The behavioral experiments with the mutants display a reduction in chemotaxis to proline when aspartate 155 and aspartate 182 are changed to glutamates. The periplasmic region of wild type McpU was purified and demonstrated to directly bind proline with a dissociation constant (Kd) of 104 μM. The variant McpU proteins show a reduction in binding affinity confirming McpU as a direct proline sensor. Chapter 3, describes the development of a high-throughput technique that is able to observe chemotaxis responses in ten separate chemotaxis chambers all at once. This procedure also allows for real time observations at intervals of two minutes for however long the experiment is scheduled. Using this new method it was found that McpU and the Internal Chemotaxis Protein A (IcpA) are the most involved with chemotaxis to seed exudates followed by McpV, W, X, and Y. The amounts of each proteinogenic amino acid (AA) in host and non-host seed exudates are quantified, which reveals that similar amounts are exuded from each species. It is shown that McpU is the most important receptor for chemotaxis toward synthetic mixtures that mimic the amounts seen in the exudates. Chapter 4 further investigates the role of McpU in sensing amino acids using the high-throughput technique developed in Chapter 3. It is shown that McpU is important for chemotaxis to all individual proteinogenic amino acids except the acidic AA, aspartate. In vitro binding experiments confirm that McpU directly interacts with all AAs except the acidic AAs aspartate and glutamate. Binding parameters are determined for aspartate, glutamate, phenylalanine and proline. In Chapter 5, five quaternary ammonium compounds (QACs) are quantified from the host and non-host seed exudates, which reveals distinctive QAC profiles. S. meliloti is found to display strong chemotaxis to all QACs, which is further shown to be mediated mostly by McpX. McpX is then established as a direct binder to all QACs as well as proline, with dissociation constants ranging from nanomolar to millimolar. These studies have increased our knowledge of how chemoreceptors sense attractants, and they have contributed to the bank of known attractant molecules for bacteria. Our new understandings of chemotaxis and how it relates to the Sinorhizobium-alfalfa model can allow for manipulations of the system to enhance chemotaxis to the host, thus propagating the symbiosis more efficiently, ultimately leading to greater crop yields.
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    Exploring protein interactions and intracellular localization in regulating flavonoid metabolism
    Bowerman, Peter A. (Virginia Tech, 2010-08-02)
    The organization of biological processes via protein-protein interactions and the subcellular localization of enzymes is believed to be fundamental to many aspects of metabolism. Although this organization has been demonstrated in several systems, the mechanisms by which it is established and regulated are still not well understood. The flavonoid biosynthetic pathway offers a unique system in which to study several important aspects of metabolism. Here we describe a novel toolset of mutant alleles within the flavonoid biosynthetic pathway. In addition, we discuss the use of several of these alleles together with a number of emerging technologies to probe the role of subcellular localization of chalcone synthase, the first committed flavonoid biosynthetic enzyme, on metabolic flux, and to characterize a novel chalcone synthase-interacting protein. The over-expression of this interacting protein induces novel phenotypes that are likely associated with the production or distribution of auxin. Further, interaction analyses between recombinant flavonoid biosynthetic enzymes point to the possibility that post-translational modifications play an important role in promoting interactions.
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    Global and targeted proteomics in Arabidopsis thaliana: A study of secondary metabolism and phytohormone signaling
    Slade, William O. (Virginia Tech, 2013-09-20)
    Proteomics is defined as a tool to explore how proteins control and regulate important molecular and physiological processes. Further, peptide-centric approaches, or bottom-up methods, provide more comprehensive coverage of a proteome compared to whole-protein approaches. This body of work assesses the technical feasibility of several bottom-up proteomics technologies applied to Arabidopsis thaliana, including gel-based methods, those that require peptide derivitization, and those that do not. Selected-reaction monitoring (SRM) for targeted proteomics, and data-independent acquisition (MSE) was also evaluated. In addition to assessing the capabilities of these technologies, we then applied them to the context of uncovering new insights into the flavonoid biosynthetic pathway and the auxin and ethylene signaling pathways. Chapter one provides background information related to secondary metabolism, phytohormone signaling, and the status of proteomics in plants. In Chapter 2 and Appendix A, we establish the methodology to apply traditional and DiGE-based 2D-GE strategies to global proteomics in Arabidopsis. Our results suggest that while 2D-GE is applicable to Arabidopsis, there are practical and conceptual limitations that must be understood. Further, our results suggest that pertubations in the flavonoid pathway do not affect the abundance of proteins in Arabidopsis seedlings, roots, or flowers that can be studied using 2D-GE and DiGE. Additionally, we demonstrated the first parallel comparison of the effects of auxin and ethylene on the Arabidopsis root proteome and observed no overlap among the proteins regulated by the two phytohormones, at least for the most abundant proteins observed by 2D-GE. Chapter 3 explores the efficacy of selected reaction monitoring for relative peptide quantification in Arabidopsis roots. Our results suggest that while the technology parallels application in yeast and humans, there are substantial analytical challenges that much be addressed. In Chapter 4 we explore the MSE data acquisition scheme for global proteomics in Arabidopsis. We observe that treatment with exogenous auxin affects the abundance of many proteins representing diverse biological processes. Interestingly, we observe minimal overlap among genes and proteins regulated by exogenous auxin. Appendix B explores the efficacy of iTRAQ labeling for relative peptide quantification in Arabidopsis roots.
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    Identification and Analysis of Germination-Active Proteins in Bacillus Spores
    Sayer, Cameron Vincent (Virginia Tech, 2019-07-02)
    Many spore forming bacteria are the causative agents of severe disease, such as Bacillus anthracis and anthrax. In these cases, the spore often acts as the infectious agent. Spores boast extreme resistance to chemical and UV damage among other bactericidal conditions. This is problematic due to the difficulty and economic costs of decontaminating exposure sites. The present work focuses on identifying and characterizing proteins active within spore germination, with a focus towards understanding the triggering of the major stages of germination. Understanding how each stage is initiated could allow for development of methods that induce these processes to efficiently germinate spores, thus facilitating cheap and effective decontamination. Sequencing of a spore transposon insertion library after exposure to germinants led to the identification of 42 genes with previously uncharacterized roles in spore germination. Fourteen of the genes, encoding proteins associated with the inner spore membrane, were further characterized. Mutants lacking these genes portrayed phenotypes consistent with failure of a GerA receptor-mediated germination response, and these genes affect the earliest stages of germination. Chemical cross-linking was used to characterize protein interactions important for stage II of spore germination. Site-directed in vivo crosslinking indicated that YpeB may exist as a multimer within the dormant spore. Further investigation of individual protein domains using bacterial two-hybrid analysis suggested that both N- and C-terminal domains of YpeB contribute to the formation of a multimer. In addition, the uncharacterized YpeB N-terminal domain was demonstrated to have strong self-association and may mediate self-association within the dormant spore. Additional genes that contribute to efficient initiation of spore germination in a GerA-dependent manner were identified via TnSeq. Chemical cross-linking of dormant spores was implemented to characterize protein interactions leading to stabilization and activation of an important enzyme that contributes to cortex degradation in stage II of germination. The presented studies employed a variety of techniques to provide additional insight into both stages of spore germination with a goal of furthering understanding of specific events that contribute to a loss of spore dormancy.
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    Identification and Characterization of the Enzymes Involved in Biosynthesis of FAD and Tetrahydromethanopterin in Methanocaldococcus jannaschii
    Mashhadi, Zahra (Virginia Tech, 2010-06-30)
    Methanogens belong to the archaeal domain, are anaerobes and produce methane from CO2 or other simple carbon compounds. Methanogenesis is a key process of the global carbon cycle and methanogens produce about 75-85% of all methane emissions. Besides the universally occurring coenzymes that are needed in normal metabolic pathways, such as biotin, coenzyme A, thiamine, FAD, PLP, etc.; methanogens need six additional coenzymes that are involved in the methane production pathway: methanofuran, tetrahydromethanopterin, coenzyme F₄₂₀, coenzyme M, coenzyme B, and coenzyme F₄₃₀. Although now it is known that some non-methanogenic archaea and bacteria have several of these coenzymes, they are named methanogenic coenzymes since these six coenzymes were first isolated and identified from methanogens. We are using Methanocaldococcus jannaschii as a model organism of methanogens to understand and investigate pathways of coenzymes biosynthesis. Our laboratory is involved in establishing the chemical functions of hypothetical proteins that function in targeted biochemical pathways leading to coenzyme production within the euryarchaeon M. jannaschii and identifying their corresponding genes. While there are many coenzymes present in this organism, my focus is on the biosynthetic pathways of tetrahydromethanopterin and FAD. 7,8-Dihydro-D-neopterin 2',3'-cyclic phosphate (H₂N-cP) is the first intermediate in the biosynthesis of the pterin portion of tetrahydromethanopterin (H₄MPT), a C₁ carrier coenzyme. This intermediate is produced from GTP by MptA (MJ0775 gene product), a new class of GTP cyclohydrolase I. An Fe(II)-dependent cyclic phosphodiesterase (MptB, MJ0837 gene product) hydrolyzes the cyclic phosphate of H₂N-cP to a mixture of 7,8-dihydro-D-neopterin 2'-monophosphate and 7,8-dihydro-D-neopterin 3'-monophosphate. MptB requires Fe²⁺ for activity, the same as observed for MptA. Thus the first two enzymes involved in H4MPT biosynthesis in the Archaea are Fe²⁺ dependent. In the FAD biosynthetic pathway, the conversion of riboflavin first into FMN and then to FAD is catalyzed by a bifunctional enzyme (RibF) that first acts as a kinase converting riboflavin to FMN in the presence of ATP and then acts as a nucleotidyl transferase using a second ATP to convert the FMN to FAD. Identification of the archaeal CTP-dependent riboflavin kinase, RibK (MJ0056 gene product) led us to identify a archaeal monofunctional FAD synthetase, RibL (MJ1179 gene product). RibL is the only air-sensitive FAD synthetase identified.
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    Insights into Mechanisms of Amyloid Toxicity:  Molecular Dynamics Simulations of the Amyloid andbeta-peptide (Aandbeta) and Islet Amyloid Polypeptide (IAPP)
    Brown, Anne M. (Virginia Tech, 2016-04-07)
    Aggregation of proteins into amyloid deposits is a common feature among dozens of diseases. Two such diseases that feature amyloid deposits are Alzheimer's disease (AD) and type 2 diabetes (T2D). AD toxicity has been associated with the aggregation and accumulation of the amyloid β-peptide (Aβ); Aβ exerts its toxic effects through interactions with neuronal cell membranes. A characteristic feature of T2D is the deposition of the islet amyloid polypeptide (IAPP) in the pancreatic islets of Langerhans. It is currently unknown if IAPP aggregation is a cause or consequence of T2D, but it does lead to β-cell dysfunction and death, exacerbating the effects of diabetes. Characterizing the fundamental interactions between both Aβ and IAPP with lipid membranes and in solution will give greater insight into mechanisms of toxicity exhibited by amyloid proteins. In this work, molecular dynamics (MD) simulations were used to study the secondary, tertiary, and quatnary structure of Aβ and IAPP, in addition to peptide-membrane interactions and membrane perturbation as independently caused by both peptides. Studies were conducted to address the following questions: (1) what influence do solution conditions and oxidation state have on monomeric Aβ] (2) how and in what way does monomeric Aβ interact with model lipid membranes and what role does sequence play on these peptide-membrane interactions; (3) can MD simulations be utilized to understand Aβ tetramer formation, rearrangement, and tetramer-membrane interactions; (4) how does IAP interact with model membranes and how does that vary from non-toxic (rat) IAPP peptide-membrane interactions. These studies led to conclusions that showed variance in lipid affinity and degree of perturbation as based on peptide sequence, in addition to insight into the type of perturbation caused to membranes by these amyloid peptides. Understanding the differences in peptide-membrane interactions of amyloidogenic and non-amyloidogenic (rat) peptides gave insight into the overall mechanism of amyloidogenicity, leading to the detection of specific amino acids essential in peptide-membrane perturbation. These residues can then be targeted for novel therapeutic design to attenuate the perturbation and potential cell death as caused by these peptides.
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    Investigating viral subversion of intercellular communication
    Calhoun II, Patrick James (Virginia Tech, 2020-06-19)
    Adenoviruses are non-enveloped, dsDNA tumor viruses responsible for a breadth of pathogenesis including acute respiratory disease and viral myocarditis. Gap junctions, which are formed by connexin proteins, directly couple the cytoplasms of apposed cells enabling immunological, metabolic, and electrical intercellular communication. The gap junction protein connexin43 (Cx43; gene name – GJA1) is the most widely expressed human connexin protein and is the predominant connexin in the working myocardium. Given the immunological role for Cx43 gap junctions, we hypothesized that gap junctions would be targeted during adenoviral infection. We find reduced Cx43 protein due to suppression of GJA1 transcription dependent upon β-catenin during adenoviral infection, with viral protein E4orf1 sufficient to induce β-catenin phosphorylation. Loss of gap junction function occurs prior to reduced Cx43 protein levels with Ad5 infection rapidly inducing Cx43 phosphorylation at residues previously demonstrated to alter gap junction conductance. Direct Cx43 interaction with ZO-1 plays a critical role in gap junction regulation. We find loss of Cx43/ZO-1 complexing during Ad5 infection by co-immunoprecipitation, with complementary studies in human induced pluripotent stem cell derived-cardiomyocytes revealing Cx43 gap junction remodeling concomitant with reduced ZO-1 complexing. These findings demonstrate specific targeting of gap junction function by Ad5 leading to disruptions in intercellular communication which would contribute to dangerous pathological states including arrhythmias in infected hearts. Intercellular junction proteins belonging to classically defined unique junctions exhibit extensive cross-talk and interdependency for expression and localization. We find reduced connexin43 (Cx43) phosphorylation at a known internalization motif, leading us to hypothesize that gap junctions are maintained during adenoviral infection in order to stabilize intercellular junctions and adenoviral receptors therein. Utilizing immunofluorescence confocal microscopy, we demonstrate that Cx43 reductions are primarily cytosolic with Cx43 preservation at the plasma membrane. Click-IT chemistry, a non-radioactive pulse-chase technique, reveals that Cx43 ½ life is extended during adenoviral infection. In order to test if remaining Cx43 exists in de facto gap junctions (i.e. not undocked or cytosolic connexons) we utilized 1 % Triton X-100 solubility fractionation and find Cx43 is indeed primarily junctional during adenoviral infection. Having demonstrated increases in junctional Cx43, we next asked how tightly coupled cells were during adenoviral infection and by ECIS measurements of electrical resistance we demonstrate a transient increase in mechanical coupling during infection. Our future aims are to uncover changes in Coxsackievirus and adenovirus receptor (CAR) protein localization to determine if adenoviral-induced changes to subcellular architecture predisposes neighboring cells to infection and enhances viral spread. These findings will add to the existing model of adenoviral infection and more broadly, contribute to the therapeutic design of adenoviral vectors for cancer and gene therapy.
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    Investigation of Pantoea stewartii Quorum-Sensing Controlled Regulators and Genes Important for Infection of Corn
    Duong, An Duy (Virginia Tech, 2018-02-27)
    Bacteria interact with their eukaryotic hosts using a variety of mechanisms that range from being beneficial to detrimental. This dissertation focuses on Pantoea stewartii subspecies stewartii (P. stewartii), an endosymbiont in the corn flea beetle gut that causes Stewart's wilt disease in corn. Gaining insights into the interactions occurring between this bacterial pathogen and its plant host may lead to informed intervention strategies. This phytopathogen uses quorum sensing (QS) to coordinate cell density-dependent gene expression and successfully colonize corn leading to wilt disease. Prior to the research presented in this dissertation, the QS master regulator EsaR was shown to regulate two major virulence factors of P. stewartii, capsule production and surface motility. However, the function and integration of EsaR downstream targets in P. stewartii were still largely undefined. Moreover, only a draft genome of a reference strain of P. stewartii was publicly available for researchers, limiting bioinformatics and genome-scale genetic approaches with the organism. The work described in this dissertation has now addressed these important issues. The function of two EsaR direct targets, LrhA and RcsA, was explored (Chapter Two) and the existence of integration in the regulation between them was discovered (Chapters Two and Four). RcsA and LrhA are transcription factors controlling capsule production and surface motility in P. stewartii, respectively. In Chapter Two, the RcsA and LrhA regulons were investigated using RNA-Seq. This led to the discovery of a potential regulatory interaction between them that was confirmed by qRT-PCR and transcriptional gene fusion assays. The involvement of LrhA in surface motility and virulence was also established in this project. A direct interaction between LrhA and promoter of rcsA was defined in Chapter Four. Additional direct regulatory targets of LrhA were also identified. A project to generate a complete assembly of the P. stewartii genome (Chapter Three) enabled more thorough genome-wide analysis and revealed the existence of a previous unknown 66-kb region in the P. stewartii genome believed to contain genes important for motility and virulence. In addition, completion of the genome sequence permitted genes for two distinctive Type III secretion systems, used for interactions with corn or the corn flea beetle, to be placed on two mega-plasmids. Furthermore, the complete genome sequence facilitated a Tn-Seq approach (Chapter Five). Tn-Seq is a potent tool used to identify bacterial genes required for certain environmental test conditions. This project is a pioneering utilization of a Tn-Seq analysis in planta to investigate genes important for colonization and survival of P. stewartii within its corn host. It was discovered that OmpC and Lon are important to in planta growth and OmpA plays a role in plant virulence. In conclusion, these studies have broadened our understanding about the role of the QS regulon and other genes important for the pathogenesis of this phytopathogen. This knowledge may now be applied toward the development of future disease intervention strategies against P. stewartii and other wilt-disease causing plant pathogens.
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    Investigations into the molecular evolution of plant terpene, alkaloid, and urushiol biosynthetic enzymes
    Weisberg, Alexandra Jamie (Virginia Tech, 2014-07-09)
    Plants produce a vast number of low-molecular-weight chemicals (so called secondary or specialized metabolites) that confer a selective advantage to the plant, such as defense against herbivory or protection from changing environmental conditions. Many of these specialized metabolites are used for their medicinal properties, as lead compounds in drug discovery, or to impart our food with different tastes and scents. These chemicals are produced by various pathways of enzyme-mediated reactions in plant cells. It is suspected that enzymes in plant specialized metabolism evolved from those in primary metabolism. Understanding how plants evolved to produce these diverse metabolites is of primary interest, as it can lead to the engineering of plants to be more resistant to both biotic and abiotic stress, or to produce more complex small molecule compounds that are difficult to derive. To that end, the first objective was to develop a schema for rational protein engineering using meta-analyses of a well-characterized sesquiterpene synthase family encoding two closely-related but different types of enzymes, using quantitative measures of natural selection on amino-acid positions previously demonstrated as important for neofunctionalization between two terpene synthase gene families. The change in the nonsynonymous to synonymous mutation rate ratio (dN/dS) between these two gene families was large at the sites known to be responsible for interconversion. This led to a metric (delta dN/dS) that might have some predictive power. This natural selection-oriented approach was tested on two related enzyme families involved in either nicotine/tropane alkaloid biosynthesis (putrescine N-methyltransferase) or primary metabolism (spermidine synthase) by attempting to interconvert a spermidine synthase to encode putrescine N-methyltransferase activity based upon past patterns of natural selection. In contrast to the HPS/TEAS system, using delta dN/dS metrics between SPDS and PMT and site directed mutagenesis of SPDS did not result in the desired neofunctionalization to PMT activity. Phylogenetic analyses were performed to investigate the molecular evolution of plant N-methyltransferases involved in three alkaloid biosynthetic pathways. The results from these studies indicated that unlike O-MTs that show monophyletic origins, plant N-MTs showed patterns indicating polyphyletic origins. To provide the foundation for future molecular-oriented studies of urushiol production in poison ivy, the complete poison ivy root and leaf transcriptomes were sequenced, assembled, and analyzed.
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    Mechanistic Studies of the Roles of the Transcriptional Activator ExsA and Anti-activator Protein ExsD in the Regulation of the Type Three Secretion System in Pseudomonas aeruginosa
    Shrestha, Manisha (Virginia Tech, 2018-06-19)
    Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that is a substantial threat, particularly in hospital settings, causing severe infections in immunocompromised patients that may lead to death. Pseudomonas aeruginosa harbors a multitude of virulence factors that enable this pathogen to establish both acute and chronic infections in humans. A key determinant of acute infections is a hollow molecular needle structure used for injecting toxins into a host cell, called the type three secretion system (T3SS). The secretion machinery itself is highly complex and, together with the specific secreted factors, requires expression of more than 30 genes. Due to the high energy cost of its synthesis to the organism this system is highly regulated to finely time gene expression to coincide with host contact. ExsA, a member of the AraC-type transcription factor family, is the main transcriptional activator of all the genes necessary for expression of the T3SS. Members of the AraC family are characterized by the presence of two helix-turn-helix (HTH) motifs, which bind to the promoter DNA and activate transcription. ExsA uses its HTH containing C-terminal domain (CTD) to regulate gene expression from 10 different promoters. The N-terminal domain (NTD) of ExsA mediates dimerization and regulation of ExsA-activity. While most AraC-type activators are regulated by a small molecule ligands, ExsA is regulated by another protein, ExsD. As part of a four-protein signaling cascade, ExsD interacts directly with ExsA to prevent transcription of T3SS-associated genes under non-inducing conditions prior to host cell contact. The entire regulatory cascade includes of two additional proteins, ExsC and ExsE. ExsA, ExsC, ExsD, and ExsE follow a partner-switching mechanism to link expression of the secretion system with host cell contact. Our laboratory is working to understand this unique signaling mechanism by determining the molecular basis for the regulation of this important virulence factor. Previous studies in the laboratory have solved the structures of ExsE, ExsC and ExsD, and shed light on how these proteins interact and compete for overlapping binding sites. However, it is still unclear as to how the ExsA and ExsD interact and thus how regulation is mediated at the molecular level. In the presented study, we sought to map the molecular interface between ExsA and ExsD. First, the crystal structure of ExsA-NTD is presented wherein the dimerization interface of the protein was identified. Two of the well-studied AraC-type proteins, AraC and ToxT crystal structures have been solved by others in the presence of their respective ligands. Residues that were involved in ligand binding in AraC and ToxT were aligned with the residues in ExsA and analyzed for interaction with ExsD. However, this canonical binding pocket appeared to be not involved in the interaction between ExsA and ExsD. Structure directed site-specific mutagenesis was carried out to construct many different variants of ExsD and ExsA. Thus constructed variants were purified and analyzed in a functional assay. Using this approach, we were able to identify regions on ExsD and ExsA that are crucial for the interaction and for the regulation of ExsA-dependent transcription. It turns out that backbone interactions between the amino-terminal residues of ExsD and the beta-barrel region of the ExsA-NTD are pivotal. This result explains how ExsA and ExsC compete for ExsD binding, since both target the same regions on ExsD.
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    Microfluidic Approaches for Probing Protein Phosphorylation in Cells
    Deng, Jingren (Virginia Tech, 2018-07-31)
    Protein phosphorylation plays critical roles in diverse cellular functions, including cell cycle, growth, differentiation, and apoptosis. Deregulated phospho-signaling is often associated with many human diseases and cancers. Despite tremendous efforts to investigate the molecular mechanisms that control the functionality of phospho-signaling pathways, only limited progress has been made on describing the temporal and spatial profiles of cellular protein phosphorylation. The main challenges associated with the study of phospho-signaling processes in cells are related to the short time-scale of certain phosphorylation and dephosphorylation events, the low abundance of the phosphorylated protein forms as compared to their non-phosphorylated counterparts, the complicated and time-consuming sample preparation methods that are accompanying such type of work, and, last, the performance of the detection methods that are suitable for assessing protein phosphorylation. To tackle the challenges associated with the investigation of protein phosphorylation in cells, our objective was to develop a combined mass spectrometry (MS) and microfluidics strategy that enables fast sampling and sensitive detection of key signaling phosphoproteins in complex biological samples. MS is the most widely used analytical tool in the field of proteomics due to its high sensitivity, specificity, and throughput. Microfluidics has been proven as a suitable platform for handling small volumes of scarce samples, being also amenable to automation, integration, and multiplexing. To achieve our objective, this study was conducted in multiple steps: (1) We performed a comprehensive analysis of the factors that affect the performance of mass spectrometry detection (i.e., sensitivity, reproducibility, ability to accurately identify a large number of proteins from complex samples), when used in conjunction with technologies that are conducted in a non-standard fashion, on short time-scales; (2) We developed and evaluated a miniaturized strategy for rapid proteolytic digestion and phosphopeptide enrichment; (3) We demonstrated sensitive detection and quantification of phosphopeptides from complex biological samples using multiple reaction monitoring mass spectrometry (MRM-MS) and microfluidic sample processing; and (4) We developed a microfluidic platform for handling and processing cells that enables the investigation of biological processes that occur on short time-scales, and that can be integrated with the devices developed for the analysis of phospho-proteins. SKBR3 cells were used as a model system for developing and demonstrating the microfluidic chips. The detection and quantification of phospho-proteins involved in MAPK (mitogen activated protein kinase) signaling pathways was achieved at the low nM level. Overall, this study demonstrates proof-of-concept applicability of a microfluidics-MS strategy for monitoring phosphorylation processes in signaling networks.
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    Molecular Analysis of Type IV Pilus Assembly in Clostridium perfringens
    Hendrick, William Anthony (Virginia Tech, 2016-07-19)
    Clostridium perfringens is a Gram-positive anaerobe capable of causing disease in humans and many animals. C. perfringens is able to move across surfaces in a manner that is dependent on growth and type IV pili. Type IV pili are filaments that can be extended away from the cell by rapid polymerization, and retracted by depolymerization. Furthering the understanding of the initial and final energetic states of the pilins will reveal insights into possible mechanisms of type IV pilus assembly. Toward that end, a pilin was purified from the Gram-negative pathogen Pseudomonas aeruginosa and incorporated into an artificial membrane. The pilin was probed by a solid state nuclear magnetic resonance (ssNMR) technique that can determine the angle and depth of insertion of a helical peptide, as well as fluorescent and electron microscopy. All type IV pilus systems involve the action of an assembly ATPase to provide energy to polymerize the pilus. One proposed mechanism involves two primary proteins: an ATPase and an integral membrane core protein (IMCP). Other type IV pilus proteins are thought to play supportive roles in aiding the traversal of the cell envelope. In order to evaluate this model, the assembly ATPase PilB2 and IMCP PilC2 from C. perfringens were purified and examined for interactions. The evidence presented here suggest that PilB2 and PilC2 do not interact directly, and cannot function as a core assembly apparatus. The carbonic anhydrase (Cpb) from C. perfringens strain 13 was characterized both biochemically and physiologically. Cpb belongs to the type I subclass of the β class and is the first β class enzyme investigated from a strictly anaerobic bacteria. Kinetic analyses revealed a two-step, pingpong, zinc-hydroxide mechanism of catalysis. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semi-defined medium and an atmosphere without CO₂. The grew well in nutrient-rich media with or without CO₂ in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO₂ fixation reactions by supplying bicarbonate to carboxylases.