Intestinal fatty acid-binding protein (I-FABP) is a small protein that binds long-chain dietary fatty acids in the cytosol of the columnar absorptive epithelial cells (enterocytes) of the intestine. The binding cavity of I-FABP is much larger than is necessary to bind a fatty acid molecule, which suggests that the protein may be able to bind other hydrophobic and amphipathic ligands such as lipophilic drugs. Herein we describe the binding of three structurally diverse lipophilic drugs, bezafibrate, ibuprofen (both R- and S-isomers) and nitrazepam to I-FABP. The rank order of affinity for I-FABP determined for these compounds was found to be R-ibuprofen approximately bezafibrate > S-ibuprofen >> nitrazepam. The binding affinities were not directly related to aqueous solubility or partition coefficient of the compounds; however, the freely water-soluble drug diltiazem showed no affinity for I-FABP. Drug-I-FABP interaction interfaces were defined by analysis of chemical shift perturbations in NMR spectra, which revealed that the drugs bound within the central fatty acid binding cavity. Each drug participated in a different set of interactions within the cavity; however, a number of common contacts were observed with residues also involved in fatty acid binding. These data suggest that the binding of non-fatty acid lipophilic drugs to I-FABP may increase the cytosolic solubility of these compounds and thereby facilitate drug transport from the intestinal lumen across the enterocyte to sites of distribution and metabolism.

SummaryThe she pathogenicity island (PAI) is a chromosomal, laterally acquired, integrative element of Shigella flexneri that carries genes with established or putative roles in virulence. We demonstrate that spontaneous, precise excision of the element from its integration site in the 3 ¢ ¢ ¢ ¢ terminus of the pheV tRNA gene is mediated by an integrase gene ( int ) and a gene designated rox (regulator of excision), both of which are carried on the she PAI. Integrase-mediated excision occurs via recombination between a 22 bp sequence at the 3 ¢ ¢ ¢ ¢ terminus of pheV and an imperfect direct repeat at the pheV -distal boundary of the PAI. Excision leads to the formation of a circular episomal form of the PAI, reminiscent of circular excision intermediates of other mobile elements that are substrates for lateral transfer processes such as conjugation, packaging into phage particles and recombinase-mediated integration into the chromosome. The circle junction consists of the pheV -proximal and pheV -distal boundaries of the PAI converging on a sequence identical to 22 bp at the 3 ¢ ¢ ¢ ¢ terminus of pheV . The isolated circle was transferred to Escherichia coli where it integrated specifically into phe tRNA genes, as it does in S. flexneri , independently of recA . We also demonstrate that Rox stimulates, but is not essential for, excision of the she PAI in an integrase-dependent manner. However, Rox does not stimulate excision by activating the transcription of the she PAI integrase gene, suggesting that it has an excisionase function similar to that of a related protein from the P4 satellite element of phage P2.

No abstract

Pathogenicity islands are capable of excision and insertion within bacterial chromosomes. We describe a protein, Rox, that stimulates excision of the Shigella resistance locus pathogenicity island in Shigella flexneri. Sequence analysis suggests that Rox belongs to a new subfamily of recombination directionality factors, which includes proteins from P4, enterohemorrhagic Escherichia coli, and Yersinia pestis.

The Shigella resistance locus (SRL) pathogenicity island (PAI) in Shigella spp. mediates resistance to streptomycin, ampicillin, chloramphenicol, and tetracycline. It can be excised from the chromosome via site-specific recombination mediated by the P4-related int gene. Here, we show that SRL PAI attP is capable of RecA-independent, site-specific, int-mediated integration into two bacterial tRNA attB sites.Shigella spp. are the causative agents of bacillary dysentery, a disease responsible for the deaths of over 1.1 million people annually (10). The infection, which is spread via the fecal-oral route, is commonly treated with a combination of rehydration and antimicrobial therapy. However, over the past few decades, treatment has become increasingly difficult as resistance to most of the widely used therapeutic antibiotics has emerged (9, 16).Multiple-antibiotic resistance to Shigella spp. was first reported as early as the 1950s (21), and since that time, multiresistance gene clusters have been identified on R plasmids, transposons, and integrons. Recently, a 16-kb cluster of genes known as the Shigella resistance locus (SRL) was identified on the chromosome of Shigella flexneri 2a strain YSH6000. In this strain, the SRL, which confers resistance to the antibiotics streptomycin, ampicillin, chloramphenicol, and tetracycline, is present on a 66-kb pathogenicity island (PAI) designated the SRL PAI (11). Recent findings show that the SRL PAI is present in numerous Shigella strains, and it is hypothesized that the SRL PAI may be involved in the spread of multiple-antibiotic resistance in Shigella spp. (17).PAIs, which are believed to be acquired by horizontal gene transfer, frequently carry phage-related integrase genes, are often integrated adjacent to tRNA genes, and are flanked by short direct repeats (DRs) which resemble phage attachment (att) sites (1). It is thought that the genetic similarities of PAIs and phages may extend to related mechanisms of integration and excision (7). However, genetic instability of PAIs has been observed in only a small number of cases, with only two PAIs having been shown to be mobilizable, and very little is known about mechanisms of PAI transfer in general (5). In contrast, the mechanism by which the SRL PAI is excised from the serX tRNA gene in S. flexneri 2a YSH6000 is known to be integrase mediated and site specific. These data, in conjunction with the presence of the SRL PAI in other Shigella strains (17), suggest that the SRL PAI, and therefore the antibiotic resistance genes it carries, may still be mobile. However, nothing is known of the excised form of the SRL PAI or its ability to integrate into a new host.Circular form of the SRL PAI. The SRL PAI of strain YSH6000 is flanked by 14-bp sequences that correspond to the terminal 3Ј 14 bp of the serX tRNA gene at the right boundary and a directly repeated sequence at the left boundary. Integrase-mediated excision results in the loss of the PAI and one copy of the DR, while an intact serX gene and a single copy of the DR r...