Characterization of SepL of Enterohemorrhagic Escherichia coli

Abstract

The sepL gene is expressed in the locus of enterocyte effacement and therefore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up-and downstream of this gene. In this study, the sepL gene of the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that sepL is transcribed monocistronically and independently from the esp operon located downstream, which codes for the secreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of the sepL start codon. The analysis of the upstream regions led to the identification of canonical promoter sequences between positions ؊5 and ؊36. Translational fusions using lacZ as a reporter gene demonstrated that sepL is activated in the exponential growth phase by stimuli that are characteristic for the intestinal niche, e.g., a temperature of 37°C, a nutrient-rich environment, high osmolarity, and the presence of Mn 2؉ . Protein localization studies showed that SepL was present in the cytoplasm and associated with the bacterial membrane fraction. To analyze the functional role of the SepL protein during infection of eukaryotic cells, an in-frame deletion mutant was generated. This sepL mutant was strongly impaired in its ability to attach to HeLa cells and induce a local accumulation of actin. These defects were partially restored by providing the sepL gene in trans. The EDL933⌬sepL mutant also exhibited an impaired secretion but not biosynthesis of Esp proteins, which was fully complemented by providing sepL in trans. These results demonstrate the crucial role played by SepL in the biological cycle of EHEC.Enterohemorrhagic Escherichia coli (EHEC) strains are the major cause of bloody diarrhea and acute renal failure (10, 18). EHEC interacts with the gut mucosa, leading to histopathological changes which are collectively called attaching and effacing (A/E) lesions (18). While the production of Shiga toxins is a distinctive feature of Shigella and EHEC, the capacity to cause A/E lesions of EHEC is shared by many other enteric pathogens like enteropathogenic E. coli (EPEC), diffusely adhering E. coli, Hafnia alvei, and Citrobacter freundii (2,37,44). These bacteria contain a pathogenicity island called the locus of enterocyte effacement (LEE), which codes for bacterial products sufficient for triggering the A/E lesions (33, 34). The LEE sequences of EPEC and EHEC have been published (15,42), and most of the open reading frames (ORFs) are highly conserved, in particular the identified components of the type III secretion apparatus (98 to 100% identity), except for the sepZ gene. The secreted structural and putative effector proteins EspA, EspB, EspD, and Tir are more diverse (84.63, 74.01, 80.36, and 66.48% identity, respectively).Despite the overall sequence similarities of the ORFs within the LEE in EPEC and EHEC, Esp proteins appear to be involved to different...

Adhesion and Colonization of Enterohemorrhagic Escherichia coli O157:H7 in Cecum of Mice

“…EspA and SepL, products of espA and sepL genes located on the LEE, respectively, are known to be essential to compose a type III secretion apparatus, and the mutants showed poorly adherent activity to HeLa cells (Fig. 1) in agreement with the other's reports (5,9). The needle structure composed of EspA may contribute to initial diffuse adherence to epithelial cells (21) as described below.…”

“…The needle structure composed of EspA may contribute to initial diffuse adherence to epithelial cells (21) as described below. Mutation of the sepL gene causes an impaired secretion of Esp proteins including EspA (9). However, the role of SepL in type III secretion system was not known precisely.…”

Modulation of chemokine gene expression by Shiga-toxin producing Escherichia coli belonging to various origins and serotypes

“…EspD protein is essential for the formation of surface appendages and is integrated in the cytoplasmic membranes of eukaryotic cells [8]. Two additional proteins, SepL and EscD, also called Pas, are involved in the translocation of Tir and secretion of Esp proteins [9,10]. TTSS-secreted proteins interact with the host signal transduction, leading to actin polymerization and microvilli effacement [1].…”

Using Integrated Bioinformatics Strategy to Identify Critical Factors for the Structural Integrity of Salmonella T3SS

“…More specific examples of host cell manipulation include: inducing the host cell to engulf the bacterium; inducing apoptosis [24] by the interaction between Shigella flexneri effector IpaB and caspase 1 of host cell [25] ; and entering the nucleus of host cell and then activating the expression of genes that are beneficial for bacterial infection as shown in the case of Xanthomonas effector TAL [26] . Deletion of proteins in the export apparatus significantly influenced the secretion of other proteins [17,19,[27][28][29][30] , and hence may alter the virulence and ability of affection of the bacteria.…”