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ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.

MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gram-negative Escherichia coli and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive Lactococcus lacks, our recent work in E. coli suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in L. lactis. MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with [K.sub.i] values of 16 and 11 [micro]LM, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a [K.sub.i] of 57 [micro]M, in a similar range as the [K.sub.i] for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on Msb-Amediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary E. coli proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium (C. L. Reyes and G. Chang, Science 308:1028-1031, 2005).

Author(s): Henrietta Venter; Richard A. Shilling; Saroj Velamakanni; Lekshmy Balakrishnan; Hendrik W. van Veen (corresponding author) Multidrug resistance, by which cells become resistant to multiple unrelated pharmaceuticals, is due to [...]

Cholic acid (CA) transport was investigated in nine intestinal Bifidobacterium strains. Upon energization with glucose, all of the bifidobacteria accumulated CA. The driving force behind CA accumulation was found to be the transmembrane proton gradient ([DELTA]pH, alkaline interior). The levels of accumulated CA generally coincided with the theoretical values, which were calculated by the Henderson-Hasselbalch equation using the measured internal pH values of the bifidobacteria, and a p[k.sub.a] value of 6.4 for CA. These results suggest that the mechanism of CA accumulation is based on the diffusion of a hydrophobic weak acid across the bacterial cell membrane, and its dissociation according to the [DELTA]pH value. A mixture of short-chain fatty acids (acetate, propionate and butyrate) at the appropriate colonic concentration (117 mM in total) reduced CA accumulation in Bifidobacterium breve JCM [1192.sup.T]. These short-chain fatty acids, which are weak acids, reduced the [DELTA]pH, thereby decreasing CA accumulation in a dose-dependent manner. The bifidobacteria did not alter or modify the CA molecule. The probiotic potential of CA accumulation in vivo is discussed in relation to human bile acid metabolism.

Research describes identification of a new phosphate transporter protein, PitB, in Escherichia coli. Data indicate that pitB gene, encoding the new protein, is formed in a double mutant revertant of strains containing the conventional phosphate transporters, PitA and Pst.

Multidrug transporters are a major impediment in the care and treatment of patients as they confer resistance to several of the antibiotics that are in use now. This review discusses the genetic basis and physiological and molecular mechanisms governing the microbial antibiotic resistance are discussed.

Research demonstrates that intestinal Lactobacillus accumulates bile acids as shown by the accumulation of cholic acid. Data indicate that the acid uptake is mediated by the transmembrane proton gradient.

The mutated form of ATP-binding cassette transporter MsbA homologue ABCB1 in humans shows alterations in transmembrane helices (TMHs) that affect its drug-specificity. The mutations in TMH 6 in MsbA of Gram negative bacteria are found to be significant.

The overexpression, purification, and functional reconstitution of Sav1866, an ATP-binding cassette (ABC) protein from the pathogen Staphylococcus aureus is performed to gain insight into multidrug transport by Sav1866. The results suggest that Sav1866 could serve as a well-defined model for studies on the molecular bases of drug-protein interactions in ABC transporters.

Deuterium solid-state nuclear magnetic resonance is used to investigate the molecular dynamics of the 64 kDa ATP-binding cassette (ABC) multidrug efflux pump LmrA from Lactococcus lactis within lipid membranes. The results have helped in understanding the molecular dynamics of a full-length ABC transporter during the catalytic cycle.