is a well established cause of human diarrhea, particularly in young children. Although outbreaks were still frequent in developed countries until the 1940s and 1950s (8), the incidence of EPEC infection in the United States and United Kingdom has since declined. However, EPEC is still responsible for occasional outbreaks in daycare centers and pediatric wards (9). EPEC has remained an important cause of infant mortality in developing countries, with recent outbreaks reporting a mortality rate of 30% (10). Thus, EPEC infection is estimated to cause the deaths of several hundred thousand children per year (2). The hallmark of EPEC infection is the A/E histopathology often observed in small bowel biopsy specimens from infected patients, and seen after the infection of epithelial cells in tissue culture (2, 3). Infection generally causes acute diarrhea, but severe cases can lead to a protracted disease (3). Aside from profuse watery diarrhea, both vomiting and the development of fever are common symptoms of EPEC infection (3). Based on the morbidity and mortality associated with this microbe, EPEC strains remain a significant health threat to children worldwide.
Unlike the nonpathogenic strains of E. coli found within the human intestine, EPEC and other pathogenic E. coli strains contain pathogenicity islands within their genome. All of the genes necessary for the formation of A/E lesions and pedestals are contained within a 35-kbp pathogenicity island termed the locus of enterocyte effacement (LEE) (2, 5). The G + C content of the LEE is 38.4%, significantly lower than the 50% composition of the nonpathogenic E. coli K-12 chromosome. This discrepancy suggests that the LEE was originally acquired from a foreign source and was subsequently inserted into EPEC's chromosome. The insertion site for the LEE region in the E. coli K-12 genome is at the site encoding the tRNA for selenocysteine. Interestingly, this location appears to be a hot spot for the insertion of several virulence factor genes, including a large but different pathogenicity island found in uropathogenic E. coli (11). The complete LEE region has been sequenced and contains a type III secretion system (2), as well as other genes necessary for pedestal formation. These include several genes coding for type III-secreted proteins, termed Esps (EPEC-secreted proteins), including EspA, EspB, EspD, and EspF, as well as an adhesin, intimin, and its translocated receptor, Tir. Mutation of any of these bacterial factors, with the exception of EspF (12), prevents A/E lesion formation in epithelial cell culture models (3). As with other type III secretion systems, cytosolic chaperone proteins have been shown to be required for the translocation of secreted effector proteins. Two chaperones have been identified in the LEE, CesD for EspB and EspD (13), and CesT that chaperones Tir (14). DNA sequences with a high degree of homology to the EPEC LEE have been found in the other A/E lesion-causing bacteria, including EHEC, as well as the mouse pathogen C. rodentium, suggesting a common pathway underlying A/E lesion formation (2). This pathway is also self contained because the introduction of the cloned LEE of EPEC into a previously nonpathogenic E. coli strain conferred the ability to form A/E lesions (15).
Interactions between EPEC and host cells entail several distinct steps and have classically been viewed as a three-stage process. The first stage in EPEC pathogenesis involves the initial adherence of the bacterium to the host's intestinal epithelium. In this stage, EPEC form dense microcolonies on the surface of tissue culture cells in a pattern known as localized adherence (3). The bacterium is thought to initially attach to the host cell through a plasmid-encoded bundle forming pilus (BFP). Although mutants lacking this plasmid still attach to host cells, they do not form microcolonies and produce fewer A/E lesions than wild-type EPEC ( 5, 16). Even so, BFP remains an important virulence factor because BFP mutant strains show severe impairment in their ability to cause diarrhea in human volunteers (17). This loss of virulence probably indicates that both initial adherence to host cells as well as microcolony formation are critically involved in the ability of EPEC to successfully infect its host. Curiously, the mediators of initial attachment appear to vary among the family of A/E pathogens. The related pathogen EHEC lacks BFP and, unlike EPEC, infects the human colon rather the ileum (3). Therefore, whether bacterial colonization occurs preferentially in the small or the large bowel may be influenced by the expression of BFP and other adhesins as well as by environmental factors regulating the expression of other virulence factors (18).
The second stage of EPEC pathogenesis involves the production of bacterial proteins including EspA, EspB, and EspD. These proteins are translocated from the bacterial cytoplasm to the external environment by a type III secretion system (Fig. 1), encoded by the esc and sep genes, also found within the LEE pathogenicity island. The type III secretion machinery is thought to generate a pore permitting this translocation to occur (2, 5). Type III secretion systems also play an important role during infection by other Gram-negative pathogenic bacteria such as Yersinia and Salmonella, enabling virulence factors to be translocated directly from the bacterial cytoplasm to the host-cell membrane or cytoplasm. Although the majority of the Esps produced by EPEC are necessary for A/E lesion formation, their precise role in EPEC pathogenesis is not well defined. EspA makes filamentous appendages surrounding the bacterium that are transiently present on the bacterial surface (19). These filaments interact with the host cell, possibly forming a translocation tube reaching into the host cell. In support of this theory, EspB is translocated into the host cytosol and membrane by a process dependent on EspA (19). EspD is known to be inserted into the host cell membrane (20). Although the exact functions of EspB and EspD are unknown, their sequence homology to the