137 ATTACHING AND EFFACING BACTERIA INDUCE AN INCREASE IN TISSUE PERMEABILITY THAT IS SPECIES-SPECIFIC

Abstract

Introduction. A three-steps of Enteropathogenie Escherichia coli (EPEC) pathogenicity include a fimbrial-mediated loose adherence to the enterocyte apical membrane (step. 1), followed by intimate adherence with formation of characteristic attaching and affacing (A/E) lesions (step 2). The genetic elements necessary for step 2 are clustered in a 35kb region called Locus for Enterocyte Effacement (LEE). Finally, step 3 involves an intracellular signalling cascade that seems to mediate ion secretion. Other data shows that EPEC decrease tissue resistance and increase transepithelial flux of mannitol, which may be a complementary mechanism of diarrhea. Aim. We investigated whether the decrease in tissue resistance is species-specific and whether intimate adherence is necessary for the resistance drop to occur. Methods. Stripped rabbit ileal mucosa was mounted in Ussing chambers, batheds with Ringer's solution, gassed with 95%O2-5%CO2 and potential difference (PD) and short-cirtcuit current (Isc) were monitored after challenging the tissue with PBS-resuspended cultures of wild type A/E forming bacteria and genetically derived strains. Tissue resistance (Rt) was calculated from PD and Isc according to Ohm's law and its variation (ΔRt) was expressed as percentage of the basal Rt value obtained ca 10 min after mounting the tissue. Results and conclusions. Rabbit specific A/E strain RDEC-1 induced a ΔRt of -17% (±3) while negative control (i.e. no fimbria, no A/E formation) strain DH5α induced a decrease of -7% (±2). Wild type, human specific EPEC strain E2348/69 did not induce a significant Δrt while transforming it with the plasmid (pM5) containing RDEC-1 fimbria gene evokes a ΔRt of -27% (±4). No significant ΔRt were noted transforming pM5 into a negative control strain. Finally, cloning RDEC-1 LEE and pM5, but not the LEE alone, into DH5α induced a ΔRt comparable to wild type RDEC-1. In summary, our data show that the ability of decreasing Rt is dependent on species-specific step 1 adherence. However, fimbrial adherence per se is not sufficient to give this effect, since elements contained in the LEE, most likely involved in A/E lesion formation, are required. Using optical and electron microscopy we are currently looking for a morphologic correlation between A/E lesions and decreased Rt. We are also engineering more selective genetic constructs to find out which gene(s) in the LEE is (are) involved in the process.