SummaryEnteropathogenic Escherichia coli (EPEC) elicit changes in host cell morphology and cause actin rearrangement, a phenotype that has commonly been referred to as attaching/effacing (AE) lesions. The ability of EPEC to induce AE lesions is dependent upon a type III protein secretion/translocation system that is encoded by genes clustered in a 35.6 kb DNA segment, named the locus of enterocyte effacement (LEE). We used transcriptional fusions between the green fluorescent protein (gfp) reporter gene and LEE genes rorf2, orf3, orf5, escJ, escV and eae, together with immunoblot analysis with antibodies against Tir, intimin, EspB and EspF, to analyse the genetic regulation of the LEE. The expression of all these LEE genes was strictly dependent upon the presence of a functional integration host factor (IHF). IHF binds specifically upstream from the ler (orf1) promoter and appears to activate expression of ler, orf3, orf5 and rorf2 directly. The ler-encoded Ler protein was involved in activating the expression of escJ, escV, tir, eae, espB and espF. Expression of both IHF and Ler was needed to elicit actin rearrangement associated with AE lesions. In conclusion, IHF directly activates the expression of the ler and rorf2 transcriptional units, and Ler in turn mediates the expression of the other LEE genes.

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhoea in young children. The locus of enterocyte effacement (LEE) pathogenicity island comprises a cluster of operons encoding a type III secretion system and related proteins that are associated with EPEC virulence. The LEE1 operon encodes Ler that positively regulates the LEE2, LEE3, LEE4, LEE5 and espG transcriptional units. The LEE operons are repressed at 27 SC and expressed at 37 SC. This paper describes a regulatory cascade of the thermoregulation of LEE operons. LEE1 including ler is repressed by H-NS at 27 SC but not at 37 SC. In contrast, the expression of the LEE2, LEE3, LEE4, LEE5 and espG transcriptional units is repressed by H-NS at both 27 SC and 37 SC. Upon shifting the culture temperature from 27 SC to 37 SC, Ler is synthesized and in turn activates the expression of LEE2, LEE3, LEE4 and espG by releasing the H-NS mediated repression. In the case of LEE5, Ler acts both by alleviating the H-NS mediated repression and by an additional mechanism, as yet to be defined.

Enteropathogenic Escherichia coli (EPEC) are extracellular pathogens that colonize mucosal surfaces of the intestine via formation of attaching and effacing (A/E) lesions. The genes responsible for induction of the A/E lesions are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes the adhesin intimin and the type III secretion system needle complex, translocator and effector proteins. One of the major EPEC translocator proteins, EspA, forms a filamentous conduit along which secreted proteins travel before they arrive at the translocation pore in the plasma membrane of the host cell, which is composed of EspB and EspD. Prior to secretion, many type III proteins, including translocators, are maintained in the bacterial cytoplasm by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins Tir, Map and EspF, and the translocator proteins EspD and EspB. In this study, CesAB (Orf3 of the LEE) was identified as a chaperone for EspA and EspB. Specific CesAB–EspA and CesAB–EspB protein interactions are demonstrated. CesAB was essential for stability of EspA within the bacterial cell prior to secretion. Furthermore, a cesAB mutant failed to secrete EspA, as well as EspB, to assemble EspA filaments, to induce A/E lesion following infection of HEp-2 cells and to adhere to, or cause haemolysis of, erythrocytes.