Enterohemorrhagic Escherichia coli O157:H7 and enteropathogenic E. coli cause a characteristic histopathology in intestinal cells known as attaching and effacing. The attaching and effacing lesion is encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, which encodes a type III secretion system, the intimin intestinal colonization factor, and the translocated intimin receptor protein that is translocated from the bacterium to the host epithelial cells. Using lacZ reporter gene fusions, we show that expression of the LEE operons encoding the type III secretion system, translocated intimin receptor, and intimin is regulated by quorum sensing in both enterohemorrhagic E. coli and enteropathogenic E. coli. The luxS gene recently shown to be responsible for production of autoinducer in the Vibrio harveyi and E. coli quorum-sensing systems is responsible for regulation of the LEE operons, as shown by the mutation and complementation of the luxS gene. Regulation of intestinal colonization factors by quorum sensing could play an important role in the pathogenesis of disease caused by these organisms. These results suggest that intestinal colonization by E. coli O157:H7, which has an unusually low infectious dose, could be induced by quorum sensing of signals produced by nonpathogenic E. coli of the normal intestinal flora.

Regulation of virulence gene expression in enteropathogenic Escherichia 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, and LEE4 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. ⌬ler 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.Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are important enteric pathogens for humans. EPEC is the most common bacterial cause of diarrhea in infants (35), while EHEC, especially those of serotype O157: H7, are important emerging pathogens causing diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (35). Central to the pathogenesis of both EPEC and EHEC infections is the formation of attaching and effacing (AE) lesions on infected host intestinal epithelial cells. The AE lesion is characterized by the loss of microvilli (effacement) and the induction of a pedestal of polymerized actin and other cytoskeletal elements that forms underneath and around the infecting bacterium (13,24,35). In EPEC strain E2348/69, the AE phenotype is encoded by a 35.6-kb pathogenicity island, the locus of enterocyte effacement (LEE) (31, 32). The LEE contains genes encoding an outer membrane protein (intimin), a type III secretion system (Esc, Sep, and Ces proteins), secreted proteins (Esp), and the translocated intimin receptor (Tir), as well as a number of open reading frames of undetermined function (8). These genes are also found in the same organization on the LEE of EHEC (36) and are necessary but not sufficient for AE lesion formation by EHEC in vitro (11).In addition to the LEE pathogenicity island and the AE phenotype, other parts of the genome in both EPEC and EHEC encode additional virulence factors and pathogenic mechanisms. The EPEC virulence plasmid encodes the regulator Per (18) and the type IV bundle-forming pili (BFP) (16), which are necessary both for in vitro EPEC adherence to HEp-2 cells in the characteristic localized-adherence pattern and for full virulence in humans (2). The EHEC virulence plasmid encodes a large...

During the search for unknown factors involved in motility, we have found that expression of the flagellar master operon flhDC is affected by mutations of the pta and ackA genes, encoding phosphotransacetylase and acetate kinase, respectively (S. Shin, J. Sheen, and C. Park, Korean J. Microbiol. 31:504-511, 1993). Here we describe results showing that this effect is modulated by externally added acetate, except when both pta and ackA are mutated, suggesting the role of acetyl phosphate, an intermediate of acetate metabolism, as a regulatory effector. Furthermore, the following evidence indicates that the phosphorylation of OmpR, a trans factor for osmoregulation, regulates flagellar expression. First, in a strain lacking ompR, the expression of flhDC is no longer responsive to a change in the level of acetyl phosphate. Second, an increase in medium osmolarity does not decrease flhDC expression in an ompR mutant. It is known that such an increase normally enhances OmpR phosphorylation. Third, OmpR protein binds to the DNA fragment containing the flhDC promoter, and its affinity is increased with phosphorylation by acetyl phosphate. DNase I footprinting revealed the regions of the flhDC promoter protected by OmpR in the presence or absence of phosphorylation. Therefore, we propose that the phosphorylated OmpR, generated by either osmolarity change or the internal level of acetyl phosphate, negatively regulates the expression of flagella.The flagellar and chemotaxis regulon of Escherichia coli contains more than 40 genes, organized into at least 13 operons, that specify functions for motility and chemosensory signal processing (for a review, see reference 27). These genes are transcriptionally regulated in a cascade fashion in order to be coordinated with flagellar assembly (16,22,25). The flagellar synthesis is inhibited when the bacteria are grown under a catabolite-repressive condition (1, 45) or stressful conditions (1, 42) such as high concentrations of salts, sugars, or alcohols, high temperatures, or conditions of blocked DNA replication. The expression of flhDC, the master operon, is required for the expression of the whole flagellar regulon. It is reported that the heterotetramer consisting of two FlhD and two FlhC proteins binds directly to the upstream region of three class II operons (26). The mutation called cfs (constitutive flagellar synthesis), which is insensitive to catabolite repression, was mapped to the flhDC operon (45). In addition, mutations circumventing the high-temperature inhibition of flagellation were mapped in both the flhDC and fliA operons (46). The heat shock proteins, DnaK, DnaJ, and GroEL, were shown to be required for the expression of the flhDC operon (43), while mutations which result in defective in phospholipid biosynthesis, such as those in pss, psd (41), and pgsA (34) genes, and the mutations in galU (23) and mdoA (12) genes inhibit motility.E. coli not only produces acetate as part of the mixed-acid fermentation process but also utilizes it as a carbon source (20). The fe...

Escherichia coli K1 is a major gram-negative organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMEC) are a prerequisite for E. coli penetration into the central nervous system in vivo. In the present study, we showed using DNA microarray analysis that E. coli K1 associated with HBMEC expressed significantly higher levels of the fim genes compared to nonassociated bacteria. We also showed that E. coli K1 binding to and invasion of HBMEC were significantly decreased with its fimH deletion mutant and type 1 fimbria locked-off mutant, while they were significantly increased with its type 1 fimbria locked-on mutant. E. coli K1 strains associated with HBMEC were predominantly type 1 fimbria phase-on (i.e., fimbriated) bacteria. Taken together, we showed for the first time that type 1 fimbriae play an important role in E. coli K1 binding to and invasion of HBMEC and that type 1 fimbria phase-on E. coli is the major population interacting with HBMEC.The mortality and morbidity associated with neonatal gramnegative bacterial meningitis have remained significant despite advances in antimicrobial chemotherapy. This is mainly attributed to inadequate knowledge of the pathogenesis and pathophysiology of this disease. Escherichia coli K1 is the most common gram-negative bacterium that causes meningitis during the neonatal period (26).E. coli meningitis develops as a result of hematogenous spread, but it is not clear how circulating bacteria cross the blood-brain barrier. Our laboratory has successfully isolated and cultivated human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier (9, 10). We showed that E. coli invasion of HBMEC is a prerequisite for E. coli penetration into the central nervous system in vivo. However, the basis of E. coli-HBMEC interactions involved in binding to HBMEC is incompletely understood.Adherence of bacteria to their host cells is considered the initial step of the pathogenesis of infectious diseases including meningitis. To date OmpA is the only identified determinant that is involved in E. coli K 1 binding to HBMEC (8, 10). Previous reports have implied that S fimbriae might be another potential E. coli K1 factor involved in adherence to HBMEC (3,16,24,32). However, according to our recent data, S fimbriae do not play a significant role in binding of E. coli K1 to HBMEC (35).Type 1 fimbriae are filamentous surface organelles produced by E. coli and mediate mannose-sensitive adhesion of E. coli to various eukaryotic cells. In E. coli K1, type 1 fimbriae have been shown to be important for oropharyngeal colonization in a neonatal rat model (4). Type 1 fimbriae are encoded by a fim gene cluster, including at least nine genes required for their biosynthesis (20). The fimbriae are composed primarily of the major FimA protein and a small tip structure containing FimF, FimG, and FimH (12). The lectin-like adhesin, FimH, located at the tip of the fimbrial shaft is responsible for the mannosesens...