Study On Biology Functional Sites Of Drug-resistance-related Protein Rta2p In Candida Albicans

In recent years, with the use of immunosuppressants after organ transplantation, and broad-spectrum antibiotic, the incidence of systemic fungal infections increased rapidly, especially the infections of Candida albicans are most serious. Carrying out research to find new antifungal drug targets of the new model for deep fungal infection resistance and mechanisms of the major pathogen Candida albicans is important for the designment of creation antifungal drugs and development of new antifungal agents, in order to overcome fungal infection and the growing problem of prevention and treatment of deep fungal infection.A new resistance gene RTA2was found in our preliminary studies, and we have proved that Ca2+increased the expression of RTA2by the calcineurin phosphatase pathway. The encoded protein Rta2p, located in cell membrane lipid rafts, was the ectopic enzyme of sphingolipid composition of Candida albicans, and could transport membrane sphingolipid skeleton composition-long chain base from inside to outside, which could regulate cell membrane on both sides of the sphingolipid composition distribution and enhance the stability of lipid rafts. Therefore, if we can study Rta2p protein structure-function relationship furthermore to find the biological active sites related with the calcineurin pathway and the long-chain base binding cassette transporter, we will lay the theoretical foundation of molecular mechanism in Candida albicans resistance, finding new antifungal drug targets, designing new anti-fungal inhibitors.We found four homologous proteins (Rsblp, Yer185w, Rtalp, Ylr046c) through analysis of Candida albicans genome database and the Saccharomyces cerevisiae genome database. By Blast comparison to find conserved sequences in transmembrane region, we designed pointed mutations in the structural differences and contrary nature of amino acids, a total of16mutations. First, based on the mismatch primers, we amplified the mutant plasmids by PCR, transformed into ultra-competent cells, extracted plasmids sequenced and got14pointed mutant plasmids. Then, according to the principle of homologous recombination, we used LiAC transfected method to transfect linear mutant plasmids to the RTA2absence strain JXM101, by using RTA2specific primers and nested PCR to identify positive transformed clone. All of the Rta2p mutant strains were proved by sensitivity experiments. We found that compared with JXM201, the difference of mutant strain M10-1(G234S) was obvious, and was similar with the empty vector strain P-B. The MICso showed no significant changes in high calcium conditions, and M10-1couldn't induce the formation of fungal resistance in the low calcium conditions and showed more sensitive to fluconazole. While the mutant strain M3-1(G158E) didn't showed obvious differences in the calcium compared with JXM201, and could not fully induce the formation of fungal resistance. Other mutant strains showed no significant difference with JXM201. In addition, the growth curves of each mutant to fluconazole and time of sterilization curve experiments also proved that the mutant strain M10-1(G234S) to fluconazole was changed. Besides, we found that the mutant strain M10-1(G234S) affected the experiments by the outake and intake of fluorescent substrate (NBD-DHS), and metabolism pathways downstream inhibitor sensitivity in the substrate transport and sensitivity to inhibitors of the sphingolipid metabolic pathway downstream with the difference of JXM201.So, through the study of resistant strains and the transport function on the transmembrane domain of resistance-associated protein Rta2p, we found a highly conserved region of the transmembrane region is an important function domain of RTA2. By studying different sites mutations of Rta2p transmembrane region we found that the difference of mutant strain M10-1(G234S) and wild strains JXM201was more obvious, and it was similar with the empty vector strain P-B in the high Ca2-conditions, which couldn't induce the formation of fungal resistance. While in the low Ca2+conditions it showed more sensitive to fluconazole. Besides we indicated that the G234is an important active site of transporting long-chain base.