Structural and Mechanistic Roles of Novel Chemical Ligands on Quorum Sensing Transcription Regulator SdiA in Enterohemorrhagic Escherichia coli

dc.contributor.advisorAlto, Nealen
dc.contributor.committeeMemberSperandio, Vanessaen
dc.contributor.committeeMemberHooper, Lora V.en
dc.contributor.committeeMemberWinter, Sebastian E.en
dc.creatorNguyen, Y. Nhuen 2014
dc.description.abstractMicroorganisms 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.en
dc.subjectEnterohemorrhagic Escherichia colien
dc.subjectEscherichia coli Proteinsen
dc.subjectGene Expression Regulation, Bacterialen
dc.subjectQuorum Sensingen
dc.titleStructural and Mechanistic Roles of Novel Chemical Ligands on Quorum Sensing Transcription Regulator SdiA in Enterohemorrhagic Escherichia colien
dc.type.materialtexten School of Biomedical Sciencesen Microbiologyen Southwestern Medical Centeren of Philosophyen


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