The Functional Characterization of the LysR-Type Transcriptional Regulator QseD and the SorC-Type Transcriptional Regulator LsrR in Enterohemorrhagic Escherichia coli

Enterohemorrhagic 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.