Multiple functions of l0036 in the regulation of the pathogenicity island of enterohaemorrhagic Escherichia coli O157:H7

Abstract

Diarrhoeagenic enterohaemorrhagic Escherichia coli and enteropathogenic E. coli attach to human intestinal epithelium and efface brush-border microvilli, forming an A/E (attaching and effacing) lesion. These human pathogens are phenotypically similar to the mouse pathogen Citrobacter rodentium. Genetically, they all have a homologous set of virulent genes involved in the A/E lesion, and these genes are organized on a LEE (locus of enterocyte effacement), a pathogenicity island. This island comprises 41 specific open reading frames, of which most are organized at five operons, LEE1, LEE2, LEE3, LEE4 and tir (LEE5). The expression of the LEE genes is regulated in a complicated manner, and current knowledge is that there are at least two positive regulators, Ler (LEE-encoded regulator) and GrlA (global regulator of LEE activator), and one negative regulator, called GrlR (global regulator of LEE repressor). In enterohaemorrhagic E. coli, GrlA is encoded by l0043, whereas GrlR is encoded by l0044. Here we report a fourth regulatory gene located in LEE3, namely l0036. Its expression is tightly controlled. When overexpressed, this factor, named Mpc (multiple point controller), interacts with Ler and suppresses the expression of the LEE proteins. When the translation is not initiated or terminated before maturation, the type III secretion of effectors is completely abolished. Therefore, together with the fact that several cis elements reside in the region that l0036 spans, l0036 appeared to have multiple functions in the regulation of LEE expression.

Gene l0017 encodes a second chaperone for EspA of enterohaemorrhagic Escherichia coli O157 : H7

“…To delete l0017 from the EHEC chromosome, a previously used method of homologous recombination without inserting a selectable marker was employed (Link et al, 1997;Tsai et al, 2006). First, a 59 fragment flanking l0017 was amplified from the chromosomal DNA of the parental WT strain (WT) using primers L17-33909F (59-GCTGA-AGATCTTGCAGAC-39) and L17-34978R(XbaI) (59-TGCTCTAG-ACCGCCCACACCAGTATCTTATT-39).…”

“…Anti-L0017 was prepared by immunizing mice with purified recombinant proteins from E. coli JM109. Anti-EspA, anti-EspB and anti-Tir have been described previously (Tsai et al, 2006). Immunoblots were developed with Renaissance Western Blot Chemiluminescence Reagent Plus (NEN) and the images were captured using X-ray film (Fuji).…”

“…CesAB, CesD, CesD2, CesF and CesT) (Creasey et al, 2003a, c;Wainwright & Kaper, 1998), regulators (Ler, GrlR, GrlA and Mpc) (Deng et al, 2004;Mellies et al, 1999;Tsai et al, 2006) and adhesin (intimin) (Donnenberg et al, 1993;Jerse & Kaper, 1991;Kenny et al, 1997b). While some of them have been thoroughly studied, others remain less well characterized, and gene l0017 of EHEC is one of the latter.…”

“…The system (Stratagene) was used as described previously (Tsai et al, 2006). In essence, pBT, which encodes the lcI protein, was used to produce a bait protein fused with lcI, and pTRG was used to generate a target protein fused to the C terminus of an N-terminal domain of the RNA polymerase a subunit.…”

Netrin-1 Signaling RegulatesDe NovoProtein Synthesis of κ Opioid Receptor by Facilitating Polysomal Partition of Its mRNA

“…Equal amount of proteins were added into the gel loading buffer and resolved on an 8% SDS-PAGE. Western blots of various antigens were performed as described previously (Tsai et al, 2006). Immunohistochemistry was conducted as described (Bi et al, 2003) with anti-KOR (1:200) or anti-Tau (1:500) antibodies.…”