Assessment of Plasmodium falciparum PfMDR1 transport rates using Fluo-4.

Mutations in the multidrug resistance transporter of Plasmodium falciparum PfMDR1 have been implicated to play a significant role in the emergence of worldwide drug resistance, yet the molecular and biochemical mechanisms of this transporter are not well understood. Although it is generally accepted that drug resistance in P. falciparum is partly associated with PfMDR1 transport activity situated in the membrane of the digestive vacuole, direct estimates of the pump rate of this transport process in the natural environment of the intact host-parasite system have never been analysed. The fluorochrome Fluo-4 is a well-documented surrogate substrate of PfMDR1 and has been found to accumulate by actively being transported into the digestive vacuole of several parasitic strains. In the present study, we designed an approach to use Fluo-4 fluorescence uptake as a measure of compartmental Fluo-4 concentration accumulation in the different compartments of the host-parasite system. We performed a 'reverse Fluo-4 imaging' approach to relate fluorescence intensity to changes in dye concentration rather than Ca(2+) fluctuations and were able to calculate the overall rate of transport for PfMDR1 in Dd2 parasites. With this assay, we provide a powerful method to selectively measure the effect of PfMDR1 mutations on substrate transport kinetics. This will be of high significance for future compound screening to test for new drugs in resistant P. falciparum strains.

Assessment and modulation of forsythiaside absorption with MDCKII cells and validation with in situ intestinal experiment.

Forsythiaside was characterized by low intestinal absorption by in situ rat experiment and Caco-2 cells. The mechanisms behind this low absorption had not yet been elucidated. The purpose of this study was to investigate the role of efflux transporters in the intestinal absorption of forsythiaside as a potential mechanism for its low small-intestinal absorption following oral administration. Polarized MDCKII cell lines stably transfected with human or murine complementary DNA encoding for various efflux transporters (P-gp/MDR1, MRP2 and Bcrp1) were used to study transepithelial transport of forsythiaside and compare results with the MDCKII-Wild type cells. The transportation inhibitors GF120918, MK571 and Ko143 were used to investigate the transport mechanism. The active transport of forsythiaside was found in MDCKII-WT cells. The MDCKII-MRP2 and MDCKII-Bcrp1 cells significantly increased forsythiaside efflux ratio compared with the parental cells due to the apically directed transport by MRP2 and Bcrp1, respectively. The efflux ratios in MRP2 and Bcrp1 transfected cell lines were greatly decreased in the presence of MK-571 and Ko143, respectively, which indicated that forsythiaside efflux by MRP2 and Bcrp1 were significantly inhibited by their selective inhibitors. MDCKII-MDR1 cells did not exhibit a significant reduction in the forsythiaside efflux compared with the parental cells, indicating that it was not a good substrate for MDR1. And the results were then validated by the in situ experiment. This study presents direct evidence that forsythiaside is effluxed by both MRP2 and Bcrp1, which may contribute to its poor oral bioavailability.

The role of RamA on the development of ciprofloxacin resistance in Salmonella enterica serovar Typhimurium.

Active efflux pump is a primary fluoroquinolone resistant mechanism of clinical isolates of Salmonella enterica serovar Typhimurium. RamA is an essential element in producing multidrug resistant (MDR) S. enterica serovar Typhimurium. The aim of the present study was to elucidate the roles of RamA on the development of ciprofloxacin, the first choice for the treatment of salmonellosis, resistance in S. enterica serovar Typhimurium. Spontaneous mutants were selected via several passages of S. enterica serovar Typhimurium CVCC541 susceptible strain (ST) on M-H agar with increasing concentrations of ciprofloxacin (CIP). Accumulation of ciprofloxacin was tested by the modified fluorometric method. The expression levels of MDR efflux pumps were determined by real time RT-PCR. In ST and its spontaneous mutants, the ramA gene was inactivated by insertion of the kan gene and compensated on a recombinant plasmid pGEXΦ(gst-ramA). The mutant prevention concentration (MPC) and mutant frequencies of ciprofloxacin against ST and a spontaneous mutant in the presence, absence and overexpression of RamA were tested. Four spontaneous mutants (SI1-SI4) were obtained. The SI1 (CIP MICs, 0.1 mg/L) without any target site mutation in its quinolone resistant determining regions (QRDRs) and SI3 (CIP MICs, 16 mg/L) harboring the Ser83→Phe mutation in its QRDR of GyrA strains exhibited reduced susceptibility and resistance to multidrugs, respectively. In SI1, RamA was the main factor that controlled the susceptibility to ciprofloxacin by activating MdtK as well as increasing the expression level of acrAB. In SI3, RamA played predominant role in ciprofloxacin resistance via increasing the expression level of acrAB. Likewise, the deficiency of RamA decreased the MPCs and mutant frequencies of ST and SI2 to ciprofloxacin. In conclusion, the expression of RamA promoted the development of ciprofloxacin resistant mutants of S. enterica serovar Typhimurium. The inhibition of RamA could decrease the appearance of the ciprofloxacin resistant mutants.