Study On The Mechanism Of Loss-of-Function Mutations Next To The Boundaries Of TMH6and TMH7in Pdr5p

Microbial resistance is one of the serious challenges in the antimicrobial treatment. In recent years, increasing multi-drug resistance phenomenon in pathogenic fungi lead to treat immunodeficiency and organ transplants patients on anti-infective more difficult and expensive, and a higher rate of clinical death. ABC transporter-mediated multi-drug resistance is widespread in a variety of organisms. As one of most important ABC transporter in Saccharomyces cerevisiae, Pdr5p are highly similar with ABC transporter in many pathogenic yeast strains in structure and function. And as a model, Pdr5p mediated multidrug-resistant molecular mechanism has become a research hotspot in recent years.In this thesis, we screened two single site mutations (C793F and S1230F) which caused serious damage to Pdr5p efflux function in fluconazole agar plate by PCR-mediated random mutagenesis. C793is located in the predicted transmembrane helix6(TMH6) intracellular boundary of Pdr5p, while S1230in transmembrane helix7(TMH7) extracellular boundary. Studying the drug resistance of these strains revealed that they had same phenotype with pdr5Δ strain on agar plate containing cycloheximide, rhodamine6G or red tetrazolium. Relative growth rate in liquid medium containing different concentrations of these drugs showed that capacity of C793F and S1230F efflux decreased significantly. Pdr5p specific ATP activity experiments and Western blot showed that the793and1230mutants did not result in Pdr5p-specific ATPase activity change, misallocation or protein expression level. All of these results indicated that differences of amino acid character were the main reason that damaged Pdr5p efflux.To further study the mechanism of damaging Pdr5p efflux, we constructed C793S, C793M, C793Y, S1230A, S1230N, S1230Y strains by site-directed mutagenesis. Studying the drug sensitivity of these mutant strains, Pdr5p-specific ATP activity and protein localization, expression suggested that mutants have similar ATP activity and membrane localization with WT strains. But the drug sensitivity were related with the nature of the amino acid. The experimental results showed that the amino acid space volume change at793caused pdr5p function losing; the polarity and space volume of the amino acids at1230play complicated role in Pdr5p efflux. In this paper, we screened two novel mutations that severely damaged pdr5p efflux function by PCR-mediated random mutagenesis on fluconazole drug plate. Drug resistance and biochemical evidence confirmed that sites at boundary of transmembranes are essential for Pdr5p function. These results further clarify new evidence on the molecular mechanism of ABC transporter...