rifampin and fluorexon

Microorganisms are often studied as populations but the behaviour of single, individual cells can have important consequences. For example, tuberculosis, caused by the bacterial pathogen Mycobacterium tuberculosis, requires months of antibiotic therapy even though the bulk of the bacterial population dies rapidly. Shorter courses lead to high rates of relapse because subpopulations of bacilli can survive despite being genetically identical to those that are easily killed. In fact, mycobacteria create variability each time a cell divides, producing daughter cells with different sizes and growth rates. The mechanism(s) that underlie this high-frequency variation and how variability relates to survival of the population are unknown. Here we show that mycobacteria actively create heterogeneity. Using a fluorescent reporter and a fluorescence-activated cell sorting (FACS)-based transposon screen, we find that deletion of lamA, a gene of previously unknown function, decreases heterogeneity in the population by decreasing asymmetric polar growth. LamA has no known homologues in other organisms, but is highly conserved across mycobacterial species. We find that LamA is a member of the mycobacterial division complex (the 'divisome'). It inhibits growth at nascent new poles, creating asymmetry in polar growth. The kinetics of killing individual cells that lack lamA are more uniform and more rapid with rifampicin and drugs that target the cell wall. Our results show that mycobacteria encode a non-conserved protein that controls the pattern of cell growth, resulting in a population that is both heterogeneous and better able to survive antibiotic pressure.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Cell Division; Cell Polarity; Cell Wall; DNA Mutational Analysis; Flow Cytometry; Fluoresceins; Gene Deletion; Microbial Viability; Mycobacterium smegmatis; Phenotype; Reproducibility of Results; Rifampin; Single-Cell Analysis

Pharmacotherapy of central nervous system (CNS) disorders is impaired by the drug efflux transporter, P-glycoprotein, which limits drug penetration across the blood-brain barrier into the CNS. One strategy to increase brain drug levels is to modulate P-glycoprotein regulation. This approach requires understanding of the mechanisms that control transporter expression and function. One mechanism through which P-glycoprotein is regulated is the nuclear receptor, pregnane X receptor (PXR). Xenobiotics including drugs activate PXR and induce P-glycoprotein, which potentially affects pharmacokinetics/pharmacodynamics of coadministered drugs. Because rodent models are not suitable to predict xenobiotic interactions with human PXR, in a porcine model, we studied functional similarities between pig and human PXR. We used brain capillary endothelial cells from pig to study the effect of PXR activation on P-glycoprotein. To activate PXR, we used the PXR ligands, rifampicin, hyperforin, and pregnenolone-16alpha-carbonitrile (PCN), and measured abcb1 mRNA with quantitative polymerase chain reaction, P-glycoprotein expression with Western blotting, and P-glycoprotein transport activity with a calcein assay. We provide first proof of principle that the human PXR ligands, rifampicin and hyperforin, but not the rodent PXR ligand, PCN, activate pig PXR at the blood-brain barrier and induce mRNA, protein expression, and transport activity of P-glycoprotein. Our data indicate functional similarities between human and pig PXR that suggest the pig model could be useful for predicting xenobiotic-PXR interactions in humans. Because PXR is crucial in controlling drug efflux transporters, our findings will contribute to a better understanding of the regulation of blood-brain barrier function, which could potentially have important clinical implications for the treatment of CNS disorders.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blood-Brain Barrier; Blotting, Western; Bridged Bicyclo Compounds; Cell Separation; Cells, Cultured; Endothelial Cells; Fluoresceins; Humans; Immunohistochemistry; Myocytes, Smooth Muscle; Phloroglucinol; Pregnane X Receptor; Receptors, Steroid; Reverse Transcriptase Polymerase Chain Reaction; Rifampin; RNA, Messenger; Swine; Terpenes; Up-Regulation; Xenobiotics

The multidrug resistance-associated protein (MRP) is a drug efflux membrane pump conferring multidrug resistance on tumor cells. In order to look for compounds that can lead to reversal of such a resistance, the antituberculosis compound rifampicin, belonging to the chemical class of rifamycins, was examined for its effect on MRP activity in human multidrug resistant lung cancer GLC4/ADR cells. Rifampicin was shown to increase accumulation of the MRP substrate calcein in GLC4/ADR cells in a dose-dependent manner by inhibiting its MRP-mediated efflux from the cells; it also enhanced intracellular retention of another substrate of MRP such as the anticancer drug vincristine in the resistant cells. By contrast, the antituberculosis drug did not alter cellular levels of accumulation of either calcein or vincristine in parental drug-sensitive GLC4 cells. Other rifamycins such as rifamycin B and rifamycin SV were also demonstrated to increase intracellular accumulation of calcein in GLC4/ADR cells. These results therefore indicate that rifamycins, including rifampicin, probably constitute a new chemical class of modulators down-regulating MRP-mediated drug transport.

Topics: Antibiotics, Antitubercular; ATP-Binding Cassette Transporters; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Fluoresceins; Humans; Lung Neoplasms; Models, Chemical; Multidrug Resistance-Associated Proteins; Rifampin; Rifamycins; Tumor Cells, Cultured