Browsing by Subject "Enterohemorrhagic Escherichia coli"
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Item Characterization of Host and Microbiota Derived Signals that Regulate the Locus of Enterocyte Effacement of EHEC(2020-05-01T05:00:00.000Z) Jimenez Lopez, Angel Giovanni; Hooper, Lora V.; Winter, Sebastian E.; Greenberg, David; Sperandio, VanessaHumans are populated by an extensive community of microorganisms, primarily in organs such as the skin, mucosal membranes in the mouth, reproductive organs, and the gut. This complex community, termed the microbiota, is in part comprised of bacteria, many of which have intimate associations with their hosts to promote physiological homeostasis. These organisms, commonly termed commensal bacteria, have a rich and long history with their human hosts and accomplish important functions such as providing the host with nutrients, developing the immune system, and preventing colonization by pathogenic organisms in a process known as colonization resistance. These functions are especially apparent within the gastrointestinal (GI) tract, which contains the richest and most densely populated community of microbes in the body. Healthy gut function relies on the proper structure and balance of this microbial community. Disruption of the community, termed dysbiosis, has been associated with a plethora of diseases such as increased susceptibility to GI infections, neurological disorders, intestinal inflammation, and cancer progression. Dysbiosis is most commonly caused by pharmacological interventions with antibiotics or infection with a GI pathogen. The microbiota is regarded as a barrier against intestinal pathogens, partly due to intense competition for a limited supply of nutrients and space. This suggests that GI pathogens have evolved mechanisms to overcome colonization resistance and outcompete the resident microbiota for resources within the GI tract. Microbiota and host-derived metabolites have a significant impact on the abilities of GI pathogens to successfully establish intestinal infection and the subsequent development of disease. However, the precise mechanism by which microbiota or host metabolites affect the pathogenesis of GI pathogens is not well understood. Many of these nutrients, whether host-, diet-, or microbiota-derived, serve as chemical cues for incoming pathogens. These signals are used by pathogens to gauge resource availability, microbiota composition, host physiology, and location within the intestines to properly deploy virulence strategies that allow for colonization. Microbiota-derived small molecules include toxins, antimicrobials, oligopeptides, hormones, and products of microbial metabolism of host-derived and dietary molecules. Pathogens can directly sense many of these host- and microbiota-derived small molecules, which in turn can regulate their virulence mechanisms. Taken together, developing therapeutics that target the signaling pathways that control virulence-associated functions in pathogens represent an attractive alternative or secondary strategy to tackle bacterial infections. In a previous study, our group conducted a candidate-based screen of 372 independent mutants to look for novel regulators of the T3SS [1]. The candidates of this screen consisted primarily of transcription factors, two-component regulatory systems, anti-terminators and anti-toxins. This work generated a great number of hits that potentially regulate the T3SS of EHEC. Our work sought to characterize novel signaling pathways that directly affect the virulence of enterohemorrhagic Escherichia coli (EHEC) through characterization of some of the hits of said screen in particular the transcriptional regulators ExuR and FadR. Understanding of these signaling pathway could lead us to develop novel strategies to drive down the virulence of enteric pathogens and improve colonization resistance as an alternative approach to control bacterial infections. Here, we found that EHEC senses and utilizes galacturonic-acid (GalA) as a nutrient during infection and moonlights as a signal to downregulate the expression of virulence associated genes. Furthermore, we demonstrated that a pectin-rich diet, which is a source of GalA, increased mice tolerance towards a Citrobacter rodentium infection, a surrogate mice model for EHEC infection. AE pathogens like EHEC and C. rodentium thrive in an inflamed environment. During the onset phase of inflammation, the host-derived polyunsaturated omega-6 long-chain fatty acid (LCFA), arachidonic acid (AA) becomes elevated to produce endogenous lipid signaling molecules like prostaglandins and leukotrienes that act as inducers of inflammation. EHEC can sense long-chain fatty acids through the FadR response regulator. We found that AA is processed by EHEC using canonical LCFA signaling pathways involving the FadL LCFA transporter, the FadD acyl-CoA synthase and the FadR transcriptional regulator. In conclusion, we characterized the signaling pathways that mediate the sensing of galacturonic-acid and arachidonic-acid. We demonstrated that a diet high in pectin can effectively be used to control an infection by C. rodentium by effectively modulating the levels of GalA and affecting virulence in an ExuR dependent manner. We also showed that EHEC is capable of sensing a host produced long chain fatty acid like arachidonic acid to regulate its virulence. These studies highlight the complexity that underlies regulation of the locus of enterocyte effacement and perhaps will serve as a starting point for the development of new strategies to control enteric infections.Item The Characterization of the Fucose Sensing Kinase (FUSK) and the Fucose Sensing Response Regulator (FUSR) and Their Role in Virulence Regulation in Enterohemorrhagic Escherichia Coli O157:H7(2013-01-17) Pacheco, Alline Roberta; Sperandio, VanessaEHEC causes outbreaks of bloody diarrhea worldwide, by colonizing the human large intestine, where it forms attaching and effacing (AE) lesions on the intestinal epithelium. AE lesion development requires the presence of the locus of enterocyte effacement (LEE) that encodes for a molecular syringe, a type three secretion system (T3SS), which translocates effectors to the host cell. Expression of the LEE is controlled by the AI-3/Epi/NE interkingdom signaling cascade. The two-component systems QseBC and QseEF are at the core of the AI-3/Epi/NE signaling, controlling expression of flagellar motility genes, the LEE and type 3 secreted effectors in response to AI-3 and the catecholamine hormones Epi and NE. The network of regulatory proteins that form the AI-3/Epi/NE continues to expand, as shown by recent studies from our laboratory. Microarray analyses indicate that a putative two-component system (TCS), herein named FusKR, is repressed by QseBC and QseEF. FusK is the histidine kinase and FusR is the response regulator. In this work, we started to unravel the role of FusKR in EHEC pathogenicity. We constructed isogenic knockouts of fusK and fusR, and investigated their participation in virulence gene regulation in EHEC. Microarray analysis shows that deletion of fusK and fusR alters transcription of virulence and metabolic genes. Phenotypic analyses show that fusK- and fusR- strains are hypervirulent in vitro, overexpress the LEE genes and produces higher amounts of the T3 secreted protein EspB. Nonetheless, the fusK mutant is attenuated for colonization of the mammalian intestine. Biochemical studies revealed that FusK senses fucose. Fucose is an important carbon source for commensal and pathogenic bacteria during intestinal colonization. Transcriptional analyses shows that FusKR signal transduction system regulates fucose utilization indirectly, through regulation of the predicted membrane transporter Z0461, involved in optimal fucose uptake. Gut commensal Bacteroides thetaiomicron (B.theta) degrades mucin, releasing free monosaccharides, including fucose, into the gut lumen. Co-culture of B.theta and EHEC on mucin indicates that this commensal supplies mucin-derived fucose to EHEC, reducing expression of the LEE. Our studies demonstrate that a novel TCS, FusKR, modulates intestinal colonization by EHEC, and it is involved in complex interactions with the microbiota during infection.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 ΔqseC 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, ΔqseC and ΔqseB do not have the same phenotype. We examined this phenomenon by monitoring the flagellar response in ΔqseC and ΔqseB. 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 ΔsdiA 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 ΔsdiA. 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 The Functional Characterization of the LysR-Type Transcriptional Regulator QseD and the SorC-Type Transcriptional Regulator LsrR in Enterohemorrhagic Escherichia coli(2010-05-14) Habdas, Benjamin J.; Sperandio, VanessaEnterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for numerous outbreaks of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) throughout the world. EHEC is able to sense and respond to biotic cues from its environment, such as the human host produced catecholamines epinephrine and norepinephrine, through two two-component systems QseBC and QseEF, and abiotic environmental cues, such as phosphate and sulfate levels through QseEF [1-2]. Additionally, quorum sensing (QS) signaling cascades have evolved to sense microbial population density and diversity through the recognition of bacterially produced autoinducers (AI) AI-2, and 3 by LsrR, and QseBC respectively [1, 3]. Through the interpretation and integration of these multiple regulatory signaling networks that often involve intracellular regulatory proteins, such as the lysine regulator (LysR) type transcriptional (LTTR) family member QseA, EHEC is able to coordinate the expression of its multiple virulence factors [4]. These factors include the production of flagella that confer bacterial motility, the locus of enterocyte effacement (LEE) encoded type three secretion system (TTSS) that facilitates formation of attaching and effacing (AE) lesions on gut epithelium, and is positively regulated by QseA, and Shiga toxin (Stx), which causes cellular damage and HUS. Here, we show that yjiE, renamed Quorum Sensing E. coli Regulator D (QseD), which was predicted to encode a transcriptional regulator of the LTTR family, functions in a QS-dependent manner to regulate gene expression in both pathogenic and commensal strains of E. coli. LTTRs, the largest known family of prokaryotic DNA binding proteins, contain two functional domains, an N-terminal helix-turn-helix (HTH) and a C-terminal co-factor binding domain which allows for oligomerization [5]. We have demonstrated that QseD indirectly represses transcription of the LEE in EHEC and represses the flagella regulon expression in K-12 E. coli. Additionally QseD regulates the expression of iraD, which has recently been demonstrated to prevent degradation of RpoS by RssB sequestration, leading to an altered bacterial stress-response [6-7]. However, what is most intriguing is that while qseD is prevalent in many enterobacteria it seemingly exists almost exclusively in EHEC O157:H7 isolates as a helix-turn-helix truncated "short" isoform (sQseD). Due to the inability of the sQseD to bind to DNA and the predicted in silico ability of LTTR family members to form hetero-dimers in order to bind DNA, a targeted yeast-two-hybrid (Y2H) approach was used to exclude the known LTTR regulators of LEE transcription QseA and LrhA, as QseD interaction partners. Taken together, these results show that QseD regulates alternate targets in EHEC and K-12 E. coli, and that EHEC O157:H7 has evolved to encode a truncated form of this protein. We also studied the role of the LsrR regulon in EHEC pathogenesis and environmental persistence through biofilm formation. LsrR, a negative regulator of lsrK and of the lsrACDBFG operon, has been shown to regulate the uptake and removal of AI-2, the cell-to-cell signaling product of LuxS, from the environment through regulation of the LsrACDB AI-2 uptake pump [8-9]. LsrK, an AI-2 kinase, has been shown to alleviate lsrACDBFG operon repression by generating the inhibitory ligand of LsrR DNA binding, phospho-AI-2 [10]. In E. coli, LsrR has been implicated along with LsrK in AI-2 dependent regulation of biofilm architecture and small-RNA (sRNA) expression [11]. However, while it has been suggested that AI-2 signaling can affect pathogenesis in EHEC, the direct effects of LsrR and LsrK have never been examined [12]. Here we show that in EHEC both LsrR and LsrK regulate virulence expression, and that this regulation is altered in the absence of a functioning LuxS enzyme. In EHEC, while lsrR and lsrK both positively regulate motility in the presence of luxS, in its absence they both repress motility in a temperature dependent manner. Additionally, in the presence of luxS, lsrR increases biofilm formation. In microarray studies, LsrR was also shown to down-regulate the LEE, and differentially regulate non-LEE effectors (Nle's). Taken together, these results show that both LsrR and LsrK have regulatory roles in the pathogenesis of EHEC and that their effects are altered by the absence of luxS. These findings have given us a more complete and greater understanding of the genetic regulatory networks and their signaling and integration in EHEC.Item High-Throughput Identification of Regulators of the Locus of Enterocyte Effacement from Enterohemorrhagic E. coli(2017-06-12) Pifer, Reed Allen; Winter, Sebastian E.; Sperandio, Vanessa; Alto, Neal; Kliewer, Steven A.Identification of pathways involved in the regulation of the type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) of Enterohemorrhagic E. coli (EHEC) has been underway for more than twenty years, but significant knowledge gaps remain. We have created a generalizable, high-throughput method of identifying E. coli core genome components that facilitate T3S. Using this tool, we have generated a dataset of regulatory pathways that control the LEE under anaerobic conditions, a largely neglected but essential consideration for this enteric pathogen. As proof of principle as to the efficacy of this method, we have characterized two transcription factors, cutR and fadR, as previously unknown LEE regulators in EHEC and Citrobacter rodentium. We have determined that CutR functions as a transcriptional activator of the LEE in the presence of L-cysteine, in EHEC and C. rodentium. We have observed that CutR is capable of directly binding to the LEE1 regulatory region, suggesting a direct mechanism of action, while simultaneously controlling a network of LEE-governing genes. We have also determined that FadR functions as a transcriptional repressor of the LEE in EHEC and C. rodentium, directly binding to LEE1 of both organisms. Finally, we explore the broader dataset to begin assessing the potential of yet uncharacterized LEE regulators.Item Interplay Between Tryptophan Metabolites and the Virulence Factors of Enteric Pathogens(2020-08-01T05:00:00.000Z) Kumar, Aman; Winter, Sebastian E.; Sperandio, Vanessa; Conrad, Nicholas; Tu, BenjaminThe human gut consists of a complex milieu of several small molecules that helps in shaping its overall chemistry and biogeography. Trillions of bacteria, collectively known as the gut microbiota, colonize this landscape and occupy a specific niche. Small molecules derived from diet, gut microbial metabolism, and host metabolic activity have an impact on dictating the underlying gut microbiome composition. Gut bacterial populations effectively sense these molecular signatures and modulate gene expression to colonize this niche. An invading intestinal pathogen, to effectively colonize the gut, must sense and respond to the molecular signatures in the gut, which leads to effective colonization and infection. Diet is the principal source of energy by the intake of three main nutrients: carbohydrates, proteins, and fats. Tryptophan is an essential amino acid taken from the diet and plays an important role in protein biosynthesis. Tryptophan is also metabolized to different small molecules and its metabolic products are known to be present in high concentrations in the body. Upon ingestion, tryptophan is readily available in the luminal environment of the gut. Abundant levels of tryptophan are also absorbed by intestinal epithelial cells and is made available in different cellular compartments for normal physiological processes. Tryptophan present in the gut lumen can be further metabolized to numerous small molecules by the action of the gut microbial metabolic activity. Indole is one of the most abundant tryptophan-derived metabolites present in the gut and is absorbed by the host epithelial cells. The host can also metabolize tryptophan to various small molecules including serotonin that is made available in the gut lumen upon release from the host cells. Bacteria in the gut sense these bacterial and host-derived small molecules to colonize and maintain their niche. Similarly, an invading food-borne pathogen such as Enterohemorrhagic Escherichia coli (EHEC), which causes gastroenteritis by primarily colonizing the human colon, sense these small molecules and respond in a way that is conducive for its colonization and virulence gene expression. The role of these highly abundant tryptophan-derived small molecules in dictating the infectivity of an enteric pathogen remains unknown. Because the concentrations of these small molecules naturally present in the body are in the range of the drug concentrations that are used to treat certain diseases, it is possible to repurpose the information gained from these studies to treat intestinal infections. In the present study, we focused on two highly abundant tryptophan-derived small molecules present in the gut. Indole is derived from bacterial metabolism while serotonin is present via the action of host metabolism of tryptophan. Indole and serotonin are structurally similar and therefore may have similar effects against a pathogen in vivo. Indeed, we identified that both indole and serotonin decrease the virulence of the human pathogen Enterohemorrhagic E. coli (EHEC) and the mouse pathogen Citrobacter rodentium. We used multiple strategies including genetic manipulation, pharmacological inhibitors, and knock-out murine models to show that both indole and serotonin are inhibitory signals for virulence gene expression in EHEC and C. rodentium. We further investigated the mechanism used by these pathogens to sense these signals. We identified the first bacterial receptor for both indole and serotonin, and showed that these signals are sensed by a bacterial membrane bound histidine kinase CpxA. Upon sensing indole or serotonin, CpxA dephosphorylates itself and the transcription factor CpxR. In its phosphorylated state, CpxR directly activates expression of the virulence genes, its dephosphorylation prevents its action, decreasing expression of these genes. Together, our studies highlight the importance of sensing small molecules and understanding the gut biogeography by invading pathogens to successfully colonize the gut.Item Regulation of the EHEC Lee Pathogenicity Island by Bacterial and Host Signaling(2006-08-11) Walters, Matthew S.; Sperandio, VanessaEnterohemorrhagic E. coli O157:H7 (EHEC) causes outbreaks of bloody diarrhea and hemolytic-uremic syndrome throughout the world. The locus of enterocyte effacement (LEE) consists of five major operons (LEE1 - LEE5) and is required for formation of attaching and effacing (AE) lesions that disrupt intestinal epithelial microvilli. We have previously reported that expression of EHEC LEE genes is regulated by the luxS quorum sensing system. The luxS gene in EHEC affects the production of autoinducer-3 (AI-3), which activates the LEE. Epinephrine and norepinephrine also activate the LEE in a manner similar to AI-3. The luxS mutant had diminished transcription from the LEE promoters during mid-exponential growth phase, decreased levels of the LEE-encoded proteins EscJ, Tir, and EspA, and reduced secretion of EspA and EspB, encoded by LEE4. Epinephrine enhanced LEE expression in both wildtype (WT) and the luxS mutant, but WT still exhibited greater LEE activation. The results suggest a possible synergistic relationship between AI-3 and epinephrine. The combined effects of these two signaling molecules may lead to greater LEE expression and a more efficient infection. Given the virulence defects resulting from the luxS mutation, we next examined pathways which may be affected that lead to reduced AI-3 synthesis. We show that several species of bacteria synthesize AI-3, suggesting a possible role for AI-3 in inter-species bacterial communication. The LuxS enzyme produces the autoinducer-2 (AI-2) precursor 4,5-dihydroxy-2,3-pentanedione (DPD) and homocysteine. Homocysteine is required for the de novo synthesis of methionine in the cell. The luxS mutation leaves the cell with only one pathway for the synthesis of homocysteine, involving the use of oxaloacetate and Lglutamate. The exclusive use of this pathway appears to alter metabolism in the luxS mutant, leading to decreased production of AI-3. Addition of aspartate and increasing the cellular concentration of aromatic amino acids, such as tyrosine, restored AI-3-dependent phenotypes in a luxS mutant. The defect in AI-3 production, but not in AI-2 production, was also restored by expressing the P. aeruginosa S-adenosylhomocysteine hydrolase, which produces homocysteine directly from S-adenosylhomocysteine, in the luxS mutant. Furthermore, Phenotype MicroArrays (Biolog) revealed that the luxS mutation caused numerous metabolic deficiencies, while AI-3 signaling had little effect on metabolism. These studies examine the effects of the luxS mutation on LEE expression, how AI-3 production is affected by mutation of luxS, and explores the roles of the LuxS / AI-2 system in metabolism and QS.Item The Role of Host Hormones and Metabolites in the Regulation of Virulence in Enterohemorrhagic Escherichia coli (EHEC)(2012-07-17) Njoroge, Jacqueline W.; Sperandio, VanessaGastrointestinal bacteria, including the enteric pathogen enterohemorrhagic Escherichia coli O157:H7 (EHEC) that causes hemorrhagic colitis, sense diverse environmental signals, and use them as cues for differential gene regulation and niche adaptation. This allows for a temporal and energy efficient up-regulation of EHEC virulence factors that is essential for successful colonization and infection of the host. These virulence factors include motility genes, Shiga toxin, and attaching and effacing (AE) lesion formation on colonic epithelial cells. AE lesion formation is primarily regulated by a pathogenicity island (PI) known as the locus of enterocyte effacement (LEE). One of the signals sensed by EHEC to activate virulence is the mammalian hormone epinephrine. We investigated the extent of epinephrine regulation in EHEC through transcriptome studies. The bacterial adrenergic kinases QseC and QseE both respond to epinephrine to regulate the LEE PI positively and negatively respectively. We also demonstrated for the first time that co-incubation with epinephrine increases the formation of AE lesions, and that QseC and QseE are the only sensors of epinephrine in EHEC. Epinephrine is not the only host hormone sensed by EHEC. We showed that another human hormone, serotonin is sensed by EHEC, Citrobacter rodentium and uropathogenic E.coli. In EHEC and C.rodentium we showed that serotonin inhibits the transcription of the LEE PI. We also determined that the mechanism of LEE PI inhibition by serotonin is through the reduction of autophosphorylation of the bacterial sensor kinase CpxA, which is itself an activator of the LEE PI. In addition to chemical signaling, nutrient availability plays an important role in bacterial gene regulation. We investigated the role that carbon nutrients play in the regulation of EHEC virulence. We showed that the LEE PI is activated under gluconeogenic conditions, which has been shown to be important for the maintenance of colonization in vivo, and inhibited under glycolytic conditions. We also identified a novel glucose concentration dependent regulator of the LEE PI, Cra. These findings enhanced our understanding of the role that epinephrine plays in virulence, and introduced two other signals, serotonin and glucose which are both important for the regulation of EHEC virulence genes.Item Structural and Mechanistic Roles of Novel Chemical Ligands on Quorum Sensing Transcription Regulator SdiA in Enterohemorrhagic Escherichia coli(2014-11-18) Nguyen, Y. Nhu; Alto, Neal; Sperandio, Vanessa; Hooper, Lora V.; Winter, Sebastian E.Microorganisms and their eukaryotic hosts have co-evolved for millions of years. How bacteria sense and adapt to different environments is still unclear. Most Gram-negative bacteria use the LuxR family of transcription factors to regulate gene expression to coordinate population behavior by sensing endogenously produced chemical signaling molecules, acyl-homoserine lactones (AHLs) [1]. However, some bacteria such as Escherichia coli (E. coli) do not produce AHLs and, therefore, their quorum sensing LuxR-type proteins are thought to be regulated by AHLs from other bacteria [2-4]. These sub-family of LuxR proteins known as LuxR solos can also regulate and detect non-AHL signals to regulate gene expression independently of AHLs [5,6]. This AHL-dependent and -independent regulation of transcription is still unknown. Here we present several structures of one such solo LuxR-type protein, SdiA, from E. coli, in the presence and absence of AHL. Our study demonstrated that without AHL, SdiA is actually not in an apo-state, but regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL), which is ubiquitously found throughout the tree of life, and serve as energy sources, signaling molecules, and substrates for membrane biogenesis. While exogenous AHL renders SdiA much higher stability and DNA binding affinity, we propose that OCL may function as a chemical chaperone placeholder in the absence of AHL and stabilizes SdiA as a dimer, allowing for some basal activity. Structural comparison between SdiA-AHL and SdiA-OCL complexes provides some crucial mechanistic insights into the ligand regulation of SdiA transcription activity. Understanding the role of ligand binding on the function SdiA is important for elucidating how SdiA regulates expression of virulence genes in the human pathogen enterohemorrhagic E. coli (EHEC) O157:H7. Although EHEC causes foodborne infections worldwide that result in bloody diarrhea and hemolytic uremic syndrome (HUS), cattle is the major reservoir of EHEC. In cattle, EHEC colonizes predominately at the recto-anal junction (RAJ). Colonization at the RAJ poses a serious risk for fecal shedding and contamination of the environment. We previously demonstrated that EHEC senses AHLs produced by the microbiota in the rumen to activate the gad acid resistance genes necessary for survival through the acidic stomachs in cattle and to repress the locus of enterocyte effacement (LEE) genes important for colonization of the RAJ, but unnecessary in the rumen. Devoid of AHLs, the RAJ is the prominent site of colonization of EHEC in cattle. To determine whether the presence of AHLs in the RAJ could repress colonization at this site, we engineered EHEC to express the Yersinia enterocolitica (Y. enterocolitica) AHL synthase gene yenI, which constitutively produces AHLs, to mimic a constant exposure of AHLs in the environment. The yenI⁺ EHEC produces endogenous AHLs, and has a significant reduction in LEE expression, effector protein secretion, and attaching and effacing (A/E) lesion formation in vitro compared to the wild type (WT). The yenI⁺ EHEC also activated expression of the gad genes. To assess whether AHL production, which decreases LEE expression, would decrease RAJ colonization by EHEC, cattle were challenged at the RAJ with WT or yenI⁺ EHEC. Although the yenI⁺ EHEC colonized the RAJ with equal efficiency to that of the WT, there was a trend for the cattle to shed the WT strain longer than the yenI⁺ EHEC. The findings demonstrate that the regulation of EHEC in cattle is complex. Other factors such as fimbriae [161] may also contribute the colonization of EHEC in cattle. Identifying new factors and mechanisms of EHEC regulation is crucial for developing a better preventive approach against EHEC survival and colonization in cattle and subsequent EHEC contamination in the environment.