The enteropathogenic Escherichia coli (EPEC) locus of enterocyte effacement (LEE)-encoded effectors EspF and Map are multifunctional and have an impact on the tight junction barrier while the non-LEE-encoded proteins NleH1 and NleH2 possess significant anti-inflammatory activity. In order to address the temporal expression of these important genes in vivo, their promoters were cloned upstream of the luxCDABE operon, and luciferase expression was measured in EPEC-infected mice by bioluminescence using an in vivo imaging system (IVIS). Bioluminescent images of living mice, of excised whole intestines, and of whole intestines longitudinally opened and washed were assessed. The majority of bioluminescent bacteria localized in the cecum by 3 h postinfection, indicating that the cecum is not only a major colonization site of EPEC but also a site of EPEC effector gene expression in mice. espF, nleH1, and nleH2 were abundantly expressed over the course of infection. In contrast, map expression was suppressed at 2 days postinfection, and at 4 days postinfection it was totally abolished. After 2 to 4 days postinfection, when map is suppressed, EPEC colonization is significantly reduced, indicating that map may be one of the factors required to maintain EPEC colonization. This was confirmed in a competitive colonization study and in two models of chronic infection, repeated exposure to ketamine and Citrobacter rodentium infection. Our data suggest that map expression contributes to the maintenance of EPEC colonization. E nteropathogenic Escherichia coli (EPEC) is a human pathogen that causes infantile diarrhea in developing countries (1-7). EPEC adheres to host intestinal epithelium and induces attaching and effacing (A/E) lesions, which are characterized by intimate attachment of the organisms to host epithelial cells and effacement of the surrounding enterocyte microvilli (8). A pathogenicity island known as the locus of enterocyte effacement (LEE) encodes the proteins that promote the attachment and effacement phenotype, including the type III secretion system (T3SS) and T3SS effector proteins (9, 10). The T3SS translocates bacterial effector proteins into host cells, thus mediating host cell responses and alterations. Effector proteins secreted through the T3SS are comprised of not only LEE-encoded proteins but also non-LEEencoded proteins. The temporal hierarchy of translocated effectors through the T3SS has been defined using in vitro models. Tir is the first effector to be translocated from EPEC into cultured cells, followed by EspZ, EspF, EspH, EspG, and Map (11). Therefore, it is likely that the expression of effector proteins in vivo also varies in accordance with time and localization of infection. A recent report described the expression of virulence genes in Citrobacter rodentium, a natural mouse pathogen, by studying the expression of Ler, a regulator for most LEE genes (12). This study showed that virulence genes were expressed and required for pathogen growth during early infection but that they were downre...