A comparison of enteropathogenic and enterohaemorrhagic Escherichia coli pathogenesis.

This review covers enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infections, focusing on differences in their virulence factors and regulation. While Shiga-toxin expression from integrated bacteriophages sets EHEC apart from EPEC, EHEC infections often originate from asymptomatic carriage in ruminants whereas human EPEC are considered to be overt pathogens and more host-restricted. In part, these differences reflect variation in adhesin repertoire, type III-secreted effectors and the way in which these factors are regulated.

Directional trans-epithelial transport of organic anions in porcine LLC-PK1 cells that co-express human OATP1B1 (OATP-C) and MRP2.

The transcellular transport of many compounds, which cannot readily cross the lipid bilayer, is mediated by drug uptake and efflux transporters. Human OATP1B1 and MRP2 have been implicated in the hepato-biliary transport of many endogenous and exogenous compounds. Here, we have established epithelial porcine kidney LLC-PK1 derived cell lines, that express both transporters in a polarized fashion, as a model to predict hepato-biliary transport. Immunological identification of OATP1B1 in the recombinant cell lines was greatly facilitated by its C-terminal tagging with a peptide sequence derived from hemagglutinin (HA) avoiding the generation of OATP1B1 specific antibodies. Importantly, the tag did not interfere with the functionality of the transporter. Compared to LLC-PK1 cells and cells which expressed only OATP1B1, the cell line that co-expressed MRP2 and OATP1B1 displayed high directional basolateral-to-apical transport of 17 beta-estradiol-17 beta-glucuronide and estrone-3-sulfate. Dehydroepiandrosterone sulfate already displayed a significant basolateral-to-apical transport in the parental cell line, which was further stimulated upon expression of both transporters. Transcellular flux of all steroid conjugates in the opposite direction (apical-to-basolateral) was much lower. By employing this cellular model we were able to demonstrate for the first time that OATP1B1 together with MRP2 mediates the trans-cellular transport of rifampicin. It is anticipated that the models established herein will greatly facilitate the identification of transporters involved in the disposition of novel drug candidates.

Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7.

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.

Endogenous drug transporters in in vitro and in vivo models for the prediction of drug disposition in man.

The epithelial canine and porcine kidney cell lines MDCK, MDCKII and LLC-PK1, respectively are employed to establish recombinant models of drug transport. Endogenous drug carriers in these cells may contribute to the activities of recombinant drug transporters, thus making it difficult to assess their properties. We analysed the expression of endogenous transporters in these cell lines by RT-PCR and by determining drug transporter activities. Concerning drug efflux, multidrug resistance protein 1 (MDR1) and MRP1 mRNAs were found in all lines. MRP2 mRNA was expressed in all cell lines except MDCK. Transepithelial transport of vinblastine and its modulation by a MDR1-specific inhibitor or by the MDR1- and MRP-inhibitor verapamil, indicated that MDCKII cells have, in comparisons to the other cell lines, relatively high levels of functional MDR1 while vinblastine transport in MDCK cells is likely to be mediated more by MRP1. Notably, LLC-PK1 cells displayed little activity attributable to either MDR1 and MRP1, thus making them suitable for the expression of these efflux pumps. Of the drug uptake carriers, OATP-A mRNA was only expressed in MDCK cells. OATP-C mRNA was barely detectable in MDCK cells and absent in MDCKII and LLC-PK1 cells. In agreement with transcriptional profiling, the OATP-mediated uptake of either estradiol-glucuronide or estrone-sulfate was either absent or barely detectable in all cell lines thus implying that they are suitable to establish recombinant models for human OATP's. Transcriptional profiling was also performed on porcine and canine tissues and revealed that MRP1 was expressed in canine but not in human or porcine liver, whereas surprisingly OATP-C was expressed in canine kidney but only in human and porcine liver. The findings presented are relevant to the use of porcine and canine models for drug disposition.