Browsing by Subject "Escherichia coli Proteins"
Now showing 1 - 5 of 5
Results Per Page
Sort Options
Item The characterization of quorum sensing E. coli regulators E,F, and G (Qse EFG) and their role in pathogenesis(2008-09-18) Reading, Nicola Catherine; Sperandio, VanessaEscherichia coli O157:H7 (EHEC) causes hemorrhagic colitis and life-threatening hemolytic uremic syndrome (HUS) worldwide. EHEC colonizes the large intestine and adheres to intestinal epithelial cells by forming attaching and effacing lesions (AE). These lesions result in the rearrangement of the actin cytoskeleton to pedestal-like structures, which cup each bacterium. The genes necessary for formation of pedestals are encoded in the Locus of Enterocyte Effacement (LEE), including a type III secretion system, an effector protein, Tir, and the outer membrane protein, Intimin. Also, a prophage encoded effector protein, EspFu, is required. EHEC regulates many of its virulence genes including the AE lesion genes in response to environmental signals. Utilizing these signals allows EHEC to colonize the intestine efficiently and effectively. Environmental signals are often recognized by bacterial sensor kinases. In response to cognate signals, sensor kinases autophosphorylate and transfer the phosphate to a response regulator. The regulator then binds downstream genes to regulate transcription. This pathway for flagellation and motility, which allows EHEC to be motile, has been well-characterized and involves the two-component sytem QseBC. Less is known about the signaling towards EHEC's AE lesion formation capability. Here, we describe a unique signaling system important for EHEC pedestal formation. In contrast to conventional two-component signaling systems, this one consists of three-components. Quorum sensing E.coli regulators E (qseE), qseG, and qseF, encode a sensor kinase, membrane protein, and response regulator respectively. qseF and qseG mutant strains cannot form pedestals on epithelial cells. We have shown that QseF transcriptionally regulates espFu. When espFu is expressed on a plasmid, pedestal formation is restored to the qseF mutant. Microarray analysis comparing qseE, qseF, and qseG mutants to wild-type revealed that these genes may also play a role in metabolism and stress. The similar profiles of these mutants in the microarray indicate that these proteins may work together. QseE is able to autophosphorylate and this activity is stimulated by epinephrine, phosphate, and sulfate sources. These data indicate that QseEFG is a three-component system involved in regulation of virulence and metabolism in EHEC. The following study undertakes a genetic and functional analysis of these proteins.Item Determining Structural Transitions That Occur Upon Gating a Bacterial Mechanosensitive Channel(2006-05-15) Bartlett, Jessica Louise; Blount, PaulEssentially all bacteria, including pathogens, must be able to rapidly adapt to changing environments, specifically a rapid decrease in osmotic environment which can result in major trauma to the cell. The mechanosensitive channel of large conductance (MscL) is one of a handful of channels that responds to tension in the membrane and acts as a lifesaving mechanism for the cell in instances of osmotic down shock. Solutes are jettisoned before the cell explodes due to internal pressures. The goal of this project was to further define the mechanism of opening of the MscL channel. I used two approaches to solve this problem. The first utilized the Substituted-Cysteine Accessibility Method (SCAM). Specific amino acids are replaced with a cysteine residue and the ability of that cysteine to react with a substrate is assessed. A cysteine library of the transmembrane domains of E. coli MscL was created. I screened the mutants for their ability to react with MTSET before and after shock using whole-cell physiological assays. This in vivo SCAM study gave support for a clockwise rotation of TM1 predicted in another model, and defined a number of residues that appear to constitute the pore of the open E. coli MscL channel. Furthermore, the precise manner in which the channel activity was modified by the MTSET reagent was then determined by examining a select number of residues using electrophysiology. In this way, the transition from closed to open states could be examined. The data presented confirm many of our previous predictions as well as give new insight into the structural transitions that occur upon gating. The second approach to define opening utilized functional differences between homologues that have relatively similar sequences. For instance, the E. coli and M. tuberculosis MscL proteins are similar in sequence. However, they exhibit different sensitivities to pressure both in vivo and in vitro. Another homologue, found in S. aureus, exhibits faster kinetics and a different conductance. The chimeras constructed between E. coli and M. tuberculosis and E. coli and S. aureus MscLs have given insight into structural domains that can alter channel threshold tension, kinetics and conductance.Item The Functional Characterization of QSEC a Bacterial Adrenergic Receptor and the Luxr Homologue SDIA in EHEC(2009-09-04) Hughes, David T.; Sperandio, VanessaEnterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for outbreaks of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). The histidine sensor kinase QseC is an inner membrane adrenergic receptor which responds to the bacterial signal autoinducer-3 (AI-3) and the host signals epinephrine and norepinephrine. EHEC senses these signals in the gut in order to coordinate expression of multiple virulence factors. These factors include the locus of enterocyte effacement (LEE) genes which facilitate attachment and effacement (AE) of the gut epithelium, Shiga toxin (Stx) which causes HUS, and secreted effectors like NleA. We had previously reported that QseC is autoregulatory and regulates the flagellar genes through its cognate response regulator QseB. Here, we examined the global role of QseC in EHEC gene regulation. Microarray analysis of 뱳eC along with real time RT-PCR (qPCR) revealed QseC's regulation of Stx, NleA, and Ler, the master regulator of the LEE. Additionally, phosphotransfer studies between QseC and thirty two E. coli response regulators, revealed two new QseC phosphotransfer partners: QseF and KdpE. qPCR confirmed a role for QseC in QseF and KdpE genetic regulation. Additionally, QseC appears to regulate the LEE genes through KdpE and regulates Stx through QseF. Finally, 뱳eC and 뱳eB do not have the same phenotype. We examined this phenomenon by monitoring the flagellar response in 뱳eC and 뱳eB. It appears that, QseB plays a dual role in gene regulation based on its phosphorylation state. ?? We also studied the role of EHEC cell-cell signaling in cattle, the asymptomatic natural reservoirs of EHEC. We have shown that mutation of the LuxR homologue SdiA, decreases EHEC's ability to colonize the bovine intestine. The LuxR proteins are transcription factors that are activated or repressed by the quorum sensing molecules, autoinducer-1 (AI-1) which are N-acyl homoserine lactones (AHL). Generally, in these systems, LuxI synthesizes the AHL that LuxR senses. EHEC does not encode a LuxI homologue, indicating that it can respond to AHLs through SdiA, but cannot produce them. EHEC uses SdiA to sense its environment through other AHL-producing bacteria. ?? Microarray analysis and qPCR confirmed that, in response to AHL, SdiA represses the transcription of the LEE genes, which encode bovine colonization and human virulence factors. Additionally, electrophoretic mobility shift assays have indicated that SdiA binds the promoter of ler.?? Previous reports have indicated that glutamate-dependent acid resistance (AR2) is required for EHEC to survive in cattle. qPCR comparing WT EHEC to 볤iA showed a decreased expression of AR2 genes. When AHL was added to WT EHEC an increase was seen in AR2 gene expression. This effect was absent in 볤iA. Functional acid resistance tests have confirmed that SdiA is essential in facilitating acid resistance specifically through the AR2. ?? Finally, previous reports have indicated that sdiA is required for EHEC to survive in cattle. To this end, we have confirmed the presence of AHLs in the bovine rumen and have shown that hydrophobic rumen extracts containing AHLs can decrease LEE gene expression and increase AR2 gene expression. This effect is enhanced in the presence of SdiA. ?? These findings have led us to compose a more complete picture of adrenergic signaling in EHEC and given us a greater understanding of the role of cell-cell signaling in cattle, the natural reservoir of EHEC.??Item Regulation of WASP in Actin Signaling: From Angstroms To Microns(2011-08-26T17:34:28Z) Cheng, Hui-Chun; Rosen, Michael K.Wiskott-Aldrich syndrome protein (WASP) regulates membrane-attached force generation by promoting actin polymerization, which is key to cell motility and immune responses. Mutations in WASP lead to Wiskott-Aldrich syndrome and neutropenia. My graduate research was focused on allosteric regulation of WASP by an Enterohaemorrhagic E. coli (EHEC) effector EspFU. This study leads to novel mechanisms of WASP regulation and also sheds light on how cells conduct μm-scale assembly by orchestrating nm-scale proteins spatially and temporally. EHEC hijacks the host cytoskeleton during infection to form actin-rich ‘pedestals’ beneath the invading bacteria. The multiple repeat-containing effector EspFU stimulates pedestal formation by activating WASP, which is itself autoinhibited through interactions between a regulatory GTPase binding domain (GBD) and an activity bearing VCA region. First, I investigated how a single repeat fragment (1R) from EspFU relieves WASP autoinhibition. By determining the solution structure of a complex between the WASP GBD and 1R, I showed that EspFU binds to WASP by mimicking the C region of the VCA domain. 1R has a higher affinity toward the GBD than does VCA, allowing it to displace the VCA and activate WASP. Next, I examined the role of multiple repeats of EspFU. Surprisingly, a two-repeat fragment is much more potent in stimulating WASP-dependent actin polymerization than a single repeat at the same total repeat concentration. My colleagues and I showed that the inter-repeat cooperativity of EspFU originates from its ability to engage two active WASP molecules. Such dimers have much higher affinity (~100-fold) than monomers for the actin nucleation factor, the Arp2/3 complex. The combined mechanism enables EHEC to dominate the eukaryotic cytoskeletal machinery. These studies culminated in discovery of the hierarchical regulation of WASP proteins: allosteric activators release autoinhibition, and dimerization/oligomerization of active WASP molecules further enhances activity in promoting actin assembly by the Arp2/3 complex. Multivalency is widespread from extracellular sugar binding proteins to transmembrane receptors to cytoplasmic adapters to nuclear chromatin. Multivalent interactions play a fundamental but incompletely understood role in biology. To assess its roles in signal transduction, my colleagues and I utilized a battery of biochemical and biophysical tools to study a model system, where each multivalent molecule harbors identical modules, and a natural system, where each multivalent molecule consists of homologous modules. In the model system, I observed μm-scale droplet-like protein assembly (phase separation) upon mixing multivalent proteins above a critical concentration. The critical concentration correlates with the valency and individual module binding affinity. In the natural system, the multivalent signaling network consisting of WASP, Nck and Nephrin is necessary to maintain the structural and functional integrity of glomeruli in the kidney. Upon phosphorylation, three phosphotyrosine motifs in the cytoplasmic domain of a transmembrane scaffolding protein Nephrin recruit the C terminal SH2 domain of Nck, whose three SH3 domains interact with multiple proline-rich motifs of WASP. We also observed phase transitions in this system and found that the phase boundary position is highly dependent on the degree of nephrin phosphorylation, suggesting that kinases could induce phase transitions to remodel cellular structure. Furthermore, the phase transition here correlates with a sharp transition in the ability of WASP to stimulate actin assembly by the Arp2/3 complex. Together, this work suggests that cells may exploit multivalency as one of means of regulating the spatial organization and biochemical activity of signaling molecules in response to the environment.Item The WXXXE Effector Map Functions as a Potent and Specific Guanine Nucleotide Exchange Factor for CDC42(2009-09-04) Wallenfang, Adam James; Alto, NealMany gram negative bacterial pathogens utilize a type three secretion system (TTSS) in order to infect eukaryotic cells. A TTSS looks and acts like a molecular syringe, allowing bacteria to inject effector proteins directly into the host cell cytoplasm. These effectors commandeer and manipulate host signaling pathways to support the lifecycle of the particular pathogen. Common targets for these effectors are small GTPases, particularly those controlling the actin cytoskeletal architecture. GTPases are molecular switches found within a diverse consortium of eukaryotic signaling pathways. As their name denotes, the primary enzymatic activity of GTPases is the conversion of bound guanine triphosphate (GTP) to guanine diphosphate (GDP) via the hydrolysis of the gamma phosphate. The oscillation between the "off" or GDP-bound form and the "on" or GTP-bound form is facilitated by the action of GTPase activating factors (GAPs) and Guanine Nucleotide Exchange Factors (GEFs). GAPs accelerate the intrinsic catalytic activity of the GTPase to physiologically relevant levels and lead to the rapid inactivation of a GTPase. GEFs conformationally eject bound GDP from inactivated GTPases allowing the binding of GTP and the activation GTPases and downstream pathways. Lipid modifications also allow GTPase activity to be localized within a eukaryotic cell. Their switch-like function, preeminence in signaling, and localized activity make GTPases great targets for bacterial effectors. Several members of a multispecies group of effectors known as the WxxxE family appear to mimic certain Rho GTPases. The WxxxE family is defined by the motif of an invariant tryptophan and glutamic acid separated by three variable residues. Given the frequent incidence of horizontal gene transfer in bacterial evolution, it is not surprising that such a family might exist between TTSS pathogens, but this family does not contain a high level of homology besides the titular motif. Upon expression in eukaryotic cells, some WxxxE effectors have been shown to produce phenotypes identical to those seen when over-expressing certain GTPases. For example, the Shigella effectors IpgB1 and IpgB2 produce Rac1-characteristic lamellipodia and RhoA-characteristic actin stress fibers. The enteropathogenic and enterohaemorrhagic E. coli effector Map produces filipodia similar to Cdc42. This study demonstrates that the enzymatic activity of EHEC O157:H7 Map as a specific GEF for cellular Cdc42. Structure/function studies were used to identify key residues and protein regions of Shigella IpgB2, E. coli Map, and Salmonella SifA that suggests that the WxxxE family functions through a SopE-like GEF biochemical activity. We propose that the WxxxE family members physically couple GTPase activation to downstream signaling pathways via a highly evolved pathogenic mechanism. These findings also suggest the possibility of GAP or GAP-like bacterial effectors counter to the WxxxEs over the course of pathogenesis, which is briefly explored in the EHEC effector EspH.