Browsing by Subject "Escherichia coli O157"
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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 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 E. coli O157: H7 infections(1997-11-20) Cryer, ByronItem Post-Transcriptional Regulation of Virulence Genes by GlmY and GlmZ in Enterohemorrhagic E. Coli(2014-04-14) Gruber, Charley C.; Hooper, Lora V.; Sperandio, Vanessa; Gardner, Kevin H.; Conrad, NicholasEnterohemorrhagic E. coli O157:H7 (EHEC) is a major cause of foodborne illness and hemolytic uremic syndrome (HUS) throughout the world. One of the major virulence factors in this pathogen is a type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE). EHEC uses this proteinaceous needle to inject effector proteins into host cells to hijack various host cellular processes, as well as the translocation and insertion of the translocated intimin receptor (Tir) into the host cell membrane. The bacterial adhesin intimin then binds to Tir, allowing EHEC to tightly adhere to the host cell’s membrane (109). Tir also indirectly recruits another bacterial effector EspFu, which induces actin polymerization. This causes the formation of the characteristic pedestal that cups the bacterial cell. This process from the assembly of the needle apparatus to the formation of the pedestal must be tightly regulated both transcriptionally and post-transcriptionally. The two two-component systems QseEF and QseBC have been previously shown to regulate various virulence genes. We established that these systems both regulate the transcription of the small RNA (sRNA) glmY. GlmY is known to stabilize another sRNA, GlmZ, which activates the translation of glucosamine synthetase (GlmS) (72). Here we show that GlmY and GlmZ are also important players in the post-transcriptional regulation of virulence genes in EHEC. The transcription factor QseF is required for the expression of EspFu and thus pedestal formation. This defect can be complemented by overexpression of either GlmY or GlmZ and is not at the transcriptional level. Instead, the expression of espFu requires a processing event that is QseF dependent. We have shown that GlmZ also post-transcriptionally regulates two of the operons of the LEE, LEE4 and LEE5. Both of these operons are transcribed from a single promoter, but there is a processing event that separates the first gene of the operon from the rest that requires the endoribonuclease RNase E. Overexpression of either sRNA results in the downregulation of the latter fragment of both the LEE4 and LEE5 operons. In the case of LEE4, this is through direct binding of GlmZ to a region within the LEE4 mRNA. We also investigated the global role of GlmY and GlmZ in EHEC through microarrays and RNA sequencing of the knockout strains. Aside from the LEE, GlmZ also regulates curli which are used to facilitate bacteria attachment to host cells. These data show that GlmZ has been co-opted into being an important regulator of virulence genes in EHEC.