Simon J. Elliott

The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69

Enteropathogenic Escherichia coli (EPEC) are an important aetiological agent in infant diarrhoea and the prototype for a family of pathogens exhibiting the unique virulence mechanism known as attaching and effacing (AE) (Nataro and Kaper, 1998). All genes necessary for AE are encoded on a 35 kb chromosomal pathogenicity island called the locus of enterocyte effacement (LEE), which contains genes encoding a type III secretion system, secreted proteins (Esp) and the adhesin intimin (McDaniel et al., 1995; McDaniel and Kaper, 1997). Study of the LEE will illuminate our understanding of the pathogenesis of EPEC and other AE pathogens and contribute to the growing body of knowledge about type III secretion systems and pathogenicity islands. We have recently sequenced the entire LEE of EPEC strain E2348/69 and describe below our initial analysis. Further details can be found in GenBank (accession number AF022236) and on the Molecular Microbiology Web site (http://www.blackwellscience.com/products/journals/mole.htm). The complete region was 35 624 bp with an average G þ C content of 38.36%, which is far below that of the E. coli chromosome (50.8%; Blattner et al., 1997), a pattern in keeping with many other pathogenicity islands (Hacker et al., 1997). The LEE contains 41 predicted open reading frames (ORFs) (of > 50 amino acids) arranged in at least five polycistronic operons, as predicted by the close spacing of co-directional genes. The LEE may be divided into at least three functional domains (Fig. 1): the central eae (encoding intimin), the region encoding the secreted Esp proteins and a large region encoding the type III secretion apparatus. Several LEE genes have been reported previously, and our final LEE sequence entry contains corrections to some of these previously reported genes and predicted proteins. Additionally, we have decided to adopt a standardized nomenclature (Bogdanove et al., 1996a; Yahr et al., 1997), which changes the name of several previously described genes comprising the type III secretion system of EPEC (Jarvis et al., 1995). Those genes homologous to Yersinia type III secretion (ysc) genes are referred to as esc (E. coli secretion) genes with the same suffix as the Yersinia homologue (e.g. sepA becomes escV, homologous with yscV; Table 1). Within the family of type III secretory genes, the LEE shares the highest level of predicted amino acid similarity and genetic organization with ssa genes from the SPI-2 pathogenicity island of Salmonella typhimurium (Shea et al., 1996). Genes that are not ysc homologues but are involved in type III secretion are named sep (secretion of E. coli proteins). The chaperone for the secretion of EspD is named cesD for chaperone for E. coli secreted protein D (Wainwright and Kaper, 1998). The remaining named genes, esp (E. coli secreted protein), eae (E. coli attaching and effacing) and orfU will retain their designations, and remaining ORFs are designated orf or rorf depending on the direction of transcription relative to eae. A brief description of selected LEE ORFs follows. More details can be found in Table 1, Fig. 1 and on the Molecular Microbiology home page (http://www.blackwell-science. com/products/journals/mole.htm. rOrf1 is similar to a protein of unknown function from E. coli K-12 and to a predicted lipoprotein that is encoded on the S. typhimurium virulence plasmid adjacent to rck (Heffernan et al., 1992), which has been shown to be important for virulence (Cirillo et al., 1996). rOrf2 is similar to the VirA protein of Shigella flexneri, a type III secreted protein that is involved in invasion and intercellular spreading (Uchiya et al., 1995). Secretion of rOrf2 has not been observed, and it is unclear what functions rOrf2 may have in EPEC, in which the role of invasion remains undefined. Molecular Microbiology (1998) 28(1), 1–4

The Per regulon of enteropathogenic Escherichia coli : identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)‐encoded regulator (Ler)

Enteropathogenic Escherichia coli (EPEC) is the prototype organism of a group of pathogenic Gram‐negative bacteria that cause attaching and effacing (AE) intestinal lesions. All EPEC genes necessary for the AE phenotype are encoded within a 35.6 kb pathogenicity island termed the locus of enterocyte effacement (LEE). The LEE encodes 41 predicted open reading frames (ORFs), including components of a type III secretion apparatus and secreted molecules involved in the disruption of the host cell cytoskeleton. To initiate our studies on regulation of genes within the LEE, we determined the genetic organization of the LEE, defining transcriptional units and mapping transcriptional start points. We found that components of the type III secretion system are transcribed from three polycistronic operons designated LEE1, LEE2 and LEE3. The secreted Esp molecules are part of a fourth polycistronic operon designated LEE4. Using reporter gene fusion assays, we found that the previously described plasmid‐encoded regulator (Per) activated operons LEE1, LEE2 and LEE3, and modestly increased the expression of LEE4 in EPEC. Using single‐copy lacZ fusions in K‐12‐derived strains, we determined that Per only directly activated the LEE1::lacZ fusion, and did not directly activate the other operons. Orf1 of the LEE1 operon activated the expression of single‐copy LEE2::lacZ and LEE3::lacZ fusions in trans and modestly increased the expression of LEE4::lacZ in K‐12 strains. Orf1 was therefore designated Ler, for LEE‐encoded regulator. Thus, the four polycistronic operons of the LEE that encode type III secretion components and secreted molecules are now included in the Per regulon, where Ler participates in this novel regulatory cascade in EPEC.

The Locus of Enterocyte Effacement (LEE)-Encoded Regulator Controls Expression of Both LEE- and Non-LEE-Encoded Virulence Factors in Enteropathogenic and Enterohemorrhagic Escherichia coli

ABSTRACT Regulation of virulence gene expression in enteropathogenicEscherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is incompletely understood. In EPEC, the plasmid-encoded regulator Per is required for maximal expression of proteins encoded on the locus of enterocyte effacement (LEE), and a LEE-encoded regulator (Ler) is part of the Per-mediated regulatory cascade upregulating the LEE2, LEE3, andLEE4 promoters. We now report that Ler is essential for the expression of multiple LEE-located genes in both EPEC and EHEC, including those encoding the type III secretion pathway, the secreted Esp proteins, Tir, and intimin. Ler is therefore central to the process of attaching and effacing (AE) lesion formation. Ler also regulates the expression of LEE-located genes not required for AE-lesion formation, including rorf2, orf10,rorf10, orf19, and espF, indicating that Ler regulates additional virulence properties. In addition, Ler regulates the expression of proteins encoded outside the LEE that are not essential for AE lesion formation, including TagA in EHEC and EspC in EPEC. Δler mutants of both EPEC and EHEC show altered adherence to epithelial cells and express novel fimbriae. Ler is therefore a global regulator of virulence gene expression in EPEC and EHEC.

Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli.

The locus of enterocyte effacement of enteropathogenic Escherichia coli encodes a type III secretion system, an outer membrane protein adhesin (intimin, the product of eae ) and Tir, a translocated protein that becomes a host cell receptor for intimin. Many type III secreted proteins require chaperones, which function to stabilize proteins, prevent inappropriate protein–protein interactions and aid in secretion. An open reading frame located between tir and eae, previously named orfU, was predicted to encode a protein with partial similarity to the Yersinia SycH chaperone. We examined the potential of the orfU gene product to serve as a chaperone for Tir. The orfU gene encoded a 15 kDa cytoplasmic protein that specifically interacted with Tir as demonstrated by the yeast two‐hybrid assay, column binding and coimmunoprecipitation experiments. An orfU mutant was defective in attaching–effacing lesion formation and Tir secretion, but was unaffected in expression of other virulence factors. OrfU appeared to stabilize Tir levels in the cytoplasm, but was not absolutely necessary for secretion of Tir. Based upon the physical similarities, phenotypic characteristics and the demonstrated interaction with Tir, orfU is redesignated as cesT for the chaperone for E. coli secretion of T ir.

EspG, a Novel Type III System-Secreted Protein from Enteropathogenic Escherichia coli with Similarities to VirA of Shigella flexneri

ABSTRACT The function of the rorf2 gene located on the locus of enterocyte effacement (LEE) pathogenicity island of enteropathogenicEscherichia coli (EPEC) has not been described. We report that rorf2 encodes a novel protein, named EspG, which is secreted by the type III secretory system and which is translocated into host epithelial cells. EspG is homologous withShigella flexneri protein VirA, and the clonedespG (rorf2) gene can rescue invasion in a Shigella virA mutant, indicating that these proteins are functionally equivalent in Shigella. An EPECespG mutant had no apparent defects in in vitro assays of virulence phenotypes, but a rabbit diarrheagenic E. coli strain carrying a mutant espG showed diminished intestinal colonization and yet diarrheal attack rates similar to those of the wild type. A second EspG homolog, Orf3, is encoded on the EspC pathogenicity islet. The clonedorf3 gene could also rescue invasion in aShigella virA mutant, but an EPEC espG orf3 double mutant was not diminished in any tested in vitro assays for EPEC virulence factors. Our results indicate that EspG plays an accessory but as yet undefined role in EPEC virulence that may involve intestinal colonization.

Complete Nucleotide Sequence and Analysis of the Locus of Enterocyte Effacement from Rabbit Diarrheagenic Escherichia coli RDEC-1

ABSTRACT The pathogenicity island termed the locus of enterocyte effacement (LEE) is found in diverse attaching and effacing pathogens associated with diarrhea in humans and other animal species. To explore the relation of variation in LEE sequences to host specificity and genetic lineage, we determined the nucleotide sequence of the LEE region from a rabbit diarrheagenic Escherichia coli strain RDEC-1 (O15:H−) and compared it with those from human enteropathogenicE. coli (EPEC, O127:H6) and enterohemorrhagic E. coli (EHEC, O157:H7) strains. Differing from EPEC and EHEC LEEs, the RDEC-1 LEE is not inserted at selC and is flanked by an IS2 element and the lifA toxin gene. The RDEC-1 LEE contains a core region of 40 open reading frames, all of which are shared with the LEE of EPEC and EHEC. orf3 and the ERIC (enteric repetitive intergenic consensus) sequence present in the LEEs of EHEC and EPEC are absent from the RDEC-1 LEE. The predicted promoters of LEE1, LEE2, LEE3, tir, andLEE4 operons are highly conserved among the LEEs, although the upstream regions varied considerably fortir and the crucial LEE1 promoter, suggesting differences in regulation. Among the shared genes, high homology (>95% identity) between the RDEC-1 and the EPEC and EHEC LEEs at the predicted amino acid level was observed for the components of the type III secretion apparatus, the Ces chaperones, and the Ler regulator. In contrast, more divergence (66 to 88% identity) was observed in genes encoding proteins involved in host interaction, such as intimin (Eae) and the secreted proteins (Tir and Esps). A comparison of the highly variable genes from RDEC-1 with those from a number of attaching and effacing pathogens infecting different species and of different evolutionary lineages was performed. Although RDEC-1 diverges from some human-infecting EPEC and EHEC, most of the variation observed appeared to be due to evolutionary lineage rather than host specificity. Therefore, much of the observed hypervariability in genes involved in pathogenesis may not represent specific adaptation to different host species.

SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD

Enteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface. This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE). The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function. Among this last group is sepL. To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non‐polar sepL mutant. We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG). Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments. Fractionation studies revealed that SepL is a soluble cytoplasmic protein. Yeast two‐hybrid and affinity purification studies indicated that SepL interacts with the LEE‐encoded protein SepD. In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins. Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery.

A gene from the locus of enterocyte effacement that is required for enteropathogenic Escherichia coli to increase tight-junction permeability encodes a chaperone for EspF.

ABSTRACT Disruption of the barrier properties of the enterocyte tight junction is believed to be important in the pathogenesis of diarrhea caused by enteropathogenic Escherichia coli (EPEC). This phenotype can be measured in vitro as the ability of EPEC to reduce transepithelial resistance (TER) across enterocyte monolayers and requires the products of the locus of enterocyte effacement (LEE) and, in particular, the type III secreted effector protein EspF. We report a second LEE-encoded gene that is also necessary for EPEC to fully reduce TER. rorf10 is not necessary for EPEC adherence, EspADB secretion, or formation of attaching and effacing lesions. However, rorf10 mutants have a diminished TER phenotype, reduced intracellular levels of EspF, and a reduced ability to translocate EspF into epithelial cells. The product of rorf10 is a 14-kDa intracellular protein rich in α-helices that specifically interacts with EspF but not with Tir or other EPEC secreted proteins. These properties are consistent with the hypothesis that rorf10 encodes a type III secretion chaperone for EspF, and we rename this protein CesF, the chaperone for EPEC secreted protein F.

Antibiotic-resistant cell-detaching Escherichia coli strains from Nigerian children.

ABSTRACT The properties of 23 cell-detaching Escherichia coli strains that were isolated from stool specimens in Nigeria are described. Common properties of the strains included the presence of genes encoding α-hemolysin (100%), pyelonephritis-associated pili (100%), and cytotoxic necrotizing factor 1 (70%) as well as lactose negativity (70%) and multiple antibiotic resistance (74%). Antibiotic resistance was shown in most cases to be transferable and associated with the presence of class 1 integrons. Phenotypic properties and pulsed-field gel electrophoresis analysis demonstrated that the majority of the strains, particularly multiply resistant, lactose-negative O4:H40 strains, were closely related. Multiply-resistant cell-detaching E. coli strains may represent an important reservoir for antibiotic resistance genes.

Functional analysis of the enteropathogenic Escherichia coli type III secretion system chaperone CesT identifies domains that mediate substrate interactions

In many Gram‐negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion‐competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone–chaperone and chaperone–substrate associations, were determined. The CesT N‐terminal is implicated in chaperone dimerization, whereas the amphipathic α‐helical region of the C‐terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT–Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.