Browsing by Subject "Chemotaxis"
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Item Characterization of QSEA and QSED in the Quorum Sensing Cascade of Enterohemorrhagic Escherichia Coli(2005-08-11) Sharp, Faith Christine; Sperandio, VanessaEnterohemorrhagic E. coli O157:H7 (EHEC) is an enteric pathogen that has been implicated in many outbreaks of bloody diarrhea worldwide. EHEC senses its environment through quorum sensing, a mechanism by which bacteria use chemical signals, termed autoinducers, to regulate key genes. In the gastrointestinal tract, EHEC responds to AI-3 produced by the endogenous gastrointestinal microbial flora and epinephrine/norepinephrine produced by the host to regulate expression of virulence genes. In particular, EHEC utilizes quorum sensing to regulate virulence processes, including motility and chemotaxis and the production of attaching and effacing lesions. Motility and chemotaxis processes are controlled under the complex flagella regulon in EHEC. The expression of genes within the locus of enterocyte effacement (LEE) results in the production of the characteristic attaching and effacing lesions created as a result of production of a type III secretion apparatus. The LEE1 operon encodes for a transcriptional activator, Ler, which is responsible for the activation of other genes within the pathogenicity island. The virulence mechanisms that enable EHEC to circumvent the host defenses and compete for essential nutrients for survival are controlled by several transcriptional regulators, many of which are controlled in response to quorum sensing in EHEC. Quorum sensing E. coli regulator A, QseA, recently was described as a transcription factor that is activated via quorum sensing in EHEC. QseA, which belongs to the family of LysR transcription factors, activates the transcription of LEE1/ler directly; therefore, QseA indirectly activates the expression of other genes within the LEE pathogenicity island. The work in the first specific aim of this thesis examines the specific regulation of the LEE1/ler promoter by QseA through the use of genetic and biochemical methods. Quorum sensing E. coli regulator D, QseD, is a previously uncharacterized transcription factor that is repressed through quorum sensing in EHEC. QseD appears to play a significant role in the overall quorum sensing cascade, as it is involved in the modulation of both motility and type III secretion in EHEC. The second aim of this thesis is to study the role of QseD modulation in quorum sensing signaling in EHEC.Item Comparison of the mechanism of transmembrane signaling in bacterial chemoreceptors and sensor kinases(Texas A&M University, 2006-10-30) Ward, Scott MichaelMembrane-bound receptors transmit information from the cell exterior to the cell interior. Bacterial receptors capable of transmitting this information include sensor kinases, which control gene expression via response regulators, and methyl-accepting chemotaxis proteins (MCPs), which control rotation of the flagellar motor. These receptors, which have a similar general architecture and function, are predicted to share similar mechanisms of transmembrane signaling. The majority of such work has been conducted on MCPs. Our goal is to extend this work to the closely related sensor kinases by creating functional hybrid transducers. I show that a chimeric protein (Nart) that joins the periplasmic, ligandbinding domain of the sensor kinase NarX (nitrate/nitrite sensor) to the cytoplasmic signaling domain of the chemoreceptor Tar is capable of modulating flagellar rotation in response to both nitrate and nitrite. Consistent with the properties of NarX, our Nart elicits a stronger response to nitrate than to nitrite. Introduction of mutations into a highly conserved periplasmic region affects Nart signaling in a fashion that is consistent with the effects seen in NarX. I also present the first example of a substitution in a presumed ligand-binding domain that confers a reverse-signal phenotype for both nitrate and nitrite in Nart. These results support the hypothesis that the key aspects of transmembrane signaling are closely similar in homodimeric bacterial chemoreceptors and sensor kinases.Item Design of a Website and Signaling Map Template Acting as a Database Interface and Providing a Visual Explanation of Cellular Signaling Networks an Their Operative Components(2004-12-30) Wable, Lisa Jo; Calver, LewisThe intention of this thesis is to document and describe the design of a website and signaling map template wherein the maps are standardized, expandable in detail, and act as a database interface while providing a visual explanation of cellular signaling networks and their operative components. This template was designed to provide scientific researchers in the cell signaling community with a unique method of presenting cell signaling networks (maps) within the web-based environment that can be used by persons with little web design experience. It was further designed to offer the scientific community a new way to access cell signaling network (map) information which provides the viewer/researcher multiple levels of map detail, consistent map formats, and a map interface that taps into a large database with information on specific molecules and isoforms, narratives related to biochemical and dynamic processes, references, illustrations, animations, or other information which the webpage author may feel relevant to the specific network. For this thesis, development of the website and a set of sample pages with maps on chemotaxis were constructed for the Alliance for Cellular Signaling (AfCS). A manual was written and assembled for use by key AfCS members to use the template and thus expand the Signaling Maps pages of the AfCS website, and the sample pages from this thesis project were posted online for members to view and evaluate. This thesis explores the needs of the cell signaling community and the AfCS, discusses the unique environment of the internet as a medium for displaying and sharing cell signaling pathway information, and documents the research, creative process, and discovery that went into the creation of the template and sample web pages.Item Functional analysis of Abp1 in Dictyostelium(2006-08) Wang, Yanqin, 1974-; O'Halloran, TheresaThis work identified an ortholog of Abp1 (actin binding protein 1) in Dictyostelium (Dabp1). In order to analyze the functions of Dabp1 in Dictyostelium, loss-of–function studies and gain-of-function studies were performed by generating cells that either deleted the Dabp1 gene from the genome or overexpressed the Dabp1 protein. In these mutants, most actin-based processes were intact. However, cell motility was altered during early development. During chemotactic streaming, more than 90% of wild type cells had a single leading pseudopodium and a single uropod, whereas more than 27% of Dabp1 null cells projected multiple pseudopodia. Similarly, ~ 90% of cells that overexpressed Dabp1 projected multiple pseudopodia during chemotactic streaming, and displayed reduced rates of cell movement. Expression of the SH3 domain of Dabp1 showed this domain to be an important determinant in regulating pseudopodium number. These results suggest that Abp1 controls pseudopodium number and motility in early stages of chemotactic aggregation in Dictyostelium. This work also revealed an interplay between Dabp1 and MyoB, one of the Myosin I proteins, in controlling pseudopodia formation in Dictyostelium. These two proteins colocalize partially at the cortex in growing cells. The peripheral localization of MyoB was dependent on Dabp1. Depletion of both Dabp1 and MyoB caused defects in organization of the actin cytoskeleton and actin related activities such as formation of small F-actin filled spikes on the cell cortex of growing cells, a higher percentage of multinucleated cells, and an increased number of pseudopodia branching extensively. When MyoB was overexpressed in Dabp1 null mutants, cells had similar phenotypes as Dabp1/MyoB double null mutants, and displayed an increased number of pseudopodia with many branches. Overexpression of Dabp1 in MyoB null mutants rescued the defects in pseudopodia formation. The SH3 of Dabp1 was shown to be important for the rescue of defects caused by depletion of MyoB. Collectively, these data suggest that MyoB and Dabp1 work cooperatively to regulate the uniformity and integrity of the actin extensions during chemotaxis. MyoB requires Dabp1 to function in this process. Dabp1 may function as a scaffold to recruit MyoB to the proper localization. These studies of Dabp1 in Dictyostelium raise broad question about functions of actinassociated proteins in pseudopodia formation and the importance of uniformity and integrity for actin structures in chemotaxis.Item The Role of the PIP5 Kinase Gamma 87 Isoform in the Regulation of the Actin Cytoskeleton(2010-01-12T18:49:50Z) Corgan, Anne Marie; Yin, Helen L.Phosphatidylinositol-4,5-bisphosphate (PIP2) is an important regulator of the actin cytoskeleton and plasma membrane functions. It is primarily synthesized by the type 1 phosphatidylinositol 4 phosphate 5 kinases (PIP5Ks). Mammals have three PIP5K genes (PIP5K alpha, PIP5K beta, and PIP5K gamma), and the gamma isoform has two ubiquitous 90 kDa and an 87 kDa splice variants. We found that the depletion of each PIP5K isoform individually by RNA interference (RNAi) or gene knockout by homologous recombination generated distinct changes in the actin cytoskeleton and signaling responses. The actin phenotype of the PIP5K gamma depletion (using pan siRNA, which is directed against a common sequence shared by the 90 and 87kDa isoforms) in HeLa cells is particularly striking: it results in increased actin stress fibers, decreased chemotaxis, and increased adhesion to fibronectin-coated substrates. There is also a striking increase in prominent focal adhesions (FA). Using real-time IRM, we found that the turnover of FA is 48% slower in the PIP5K gamma depleted cells. Likewise, there is a large decrease in the dynamic turnover of green fluorescent protein (GFP)-labeled vinculin and paxillin in FA, as monitored by fluorescence recovery after photobleaching. Since PIP5K gamma 90 has already been implicated in FA assembly, we depleted it specifically without depletion of the much more abundant PIP5K gamma 87 by using a PIP5K gamma 90 specific targeting sequence not found in PIP5K gamma 87. This fails to produce robust stress fibers. Overexpression of PIP5K gamma 87, but not the kinase dead enzyme, is able to rescue the pan PIP5K gamma knockdown actin phenotype in HeLa cells. Thus, PIP5K gamma 87 is the major contributor to the pan PIP5K gamma depletion/knockout robust actin and FA phenotype. Similar results were obtained in mouse embryonic fibroblasts (MEFs) from PIP5K gamma -/- mice. We sought to identify the molecular mechanisms of the PIP5K gamma depleted actin phenotype. Inhibitors of myosin, Rho-associated coiled-coil-containing protein kinase (ROCK), and RhoA GTPase all decreased the amount of thick actin stress fibers in PIP5K gamma RNAi cells, suggesting that the phenotype is due to abnormal RhoA activation. This is confirmed by the finding that RhoA activity is elevated in PIP5K gamma depleted/knock out cells. We hypothesize that PIP5K gamma regulates the actin cytoskeleton by inhibiting Rho, and thus its downstream effectors ROCK and myosin.Item The C-Terminus of Transmembrane Helix 2 (TM2) of the Escherichia coli Tar Chemorecptor Determines Signal Output and Ligand Sensitivity(2012-11-20) Adase, Christopher A. 1981-Methyl-accepting chemotaxis proteins MCPs can bind one or more receptor- specific ligands. In the case of the Tar MCP of Escherichia coli (TarEc), a primary attractant ligand is aspartate. Its binding to the periplasmic domain of Tar generates a conformational change that is transmitted via helix 4 transmembrane helix 2 (TM2). An inward movement of TM2 initiates a transmembrane signal to the cytoplasmic HAMP (histidine kinases, adenyl cyclases, methyl-accepting proteins, phosphatases) domain. Baseline CheA kinase-stimulating activity and ligand-induced responses are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). The cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in TarEc, is of particular importance. These residues are not highly conserved among transmembrane receptors having a HAMP domain, although there are almost always some aromatic residues in this region. The question thus becomes what properties of this aromatic anchor are necessary for proper signal transduction. In this dissertation, I studied the effect on TarEc function by substituting all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. This library of TarEc variants allowed the direct assessment of the effect of the residue composition of the aromatic anchor and led to a model of how the wild-type anchor maintains the base-line signaling state in TarEc. Additional receptor variants containing double aromatic tandems and Ala substitutions for the periplasmic Trp residue were created, and the aromatic residues were also shifted in position within the six residues 207-212. Trp, Tyr, and Phe, in that order, had the greatest effect on function when they were moved to novel positions. It was also discovered that Gly-211 plays a critical role in maintaining receptor function. A model was generated that proposes that Gly-211 plays a role in maintaining the flexibility of the TM2-HAMP domain connector. The results suggest that the signaling properties of the transmembrane sensor kinases of two-component systems can be predicted by the nature of their TM2-HAMP connections. It may also be possible to modulate their activity in a controlled way by manipulating the amino acid sequences that comprise those connections.Item The Tsr chemoreceptor/CheW/CheA ternary complex as an allosteric enzyme(Texas A&M University, 2006-08-16) Fan, LinThe transmembrane serine receptor Tsr associates with a coupling protein, CheW, and a histidine kinase, CheA, to form a ternary complex that regulates the activity of CheA. CheA activity is inhibited by binding of L-serine to Tsr. This work aims to characterize the ligand-binding properties of Tsr and the inhibitory effect of L-serine on CheA activity. The periplasmic domain of Tsr (pTsr) was purified and characterized. Analytical gel filtration and analytical ultracentrifugation indicated that binding of Lserine promotes dimerization. The binding stoichiometry and dissociation constant for binding of L-serine to pTsr were determined by fluorescence spectroscopy. As protein concentration decreased, the dissociation constant increased. A working model was proposed to account for the interactions between L-serine and pTsr. The activity of CheA in a ternary complex with full-length Tsr and CheW was analyzed by measuring the production of [32P]-phospho-CheY. (Phospho-CheY is the product of CheA catalysis.) The results revealed that binding of L-serine decreased CheA activity without changing its affinity for ATP. These findings suggest that the allosteric effect of L-serine on CheA activity might occur through V-type inhibition. Optimization of an alternative, continuous, non-radioactive assay for CheA is underway.