Plasma Membrane Proteolipid 3 Participates In Fungal Resistances Of Polyene Class Amphotericin B

Deep fungal infection is a kind of fungus infectious diseases caused by pathogenic fungal invasion of the subcutaneous tissue and organs.The mortality rate of the disease is very high and the treatment is difficult.Amphotericin B(Amphotericin B,Am B)is an effective clinical drug to cure deep infection.However,with the increased use of Am B,drug-resistance became serious.The molecular mechanisms of drug-resistant strains are not clear.In our laboratory,previous studies have shown that overexpression plasma membrane protein 3(PMP3p)increased cell resistance of Am B,while the relationship between PMP3 and Am B is unkown.In this paper,we found that plasma membrane protein 3 is involved in Am B resistance in Saccharomyces cerevisiae and Canidia albicans.Previously,several investigations showed that there are two main types of the mechanism of action of Am B: the first one is to make the cell membrane form holes through the combination with sterol,so that cytoplasmic components flow out,resulting in cell death;the second one is that Am B can increase intracellular oxidative damage,leading to cell death.Hence,firstly by using PI chromatin integrity test and Rhodamine 123 absorption and efflux assay,we found that PMP3 can increase cell membrane stability.PMP3,is the earliest found gene in plants,which plays an important role in the balance of intracellular ions.Therefore,we explored the relationship between PMP3 regulation of Am B resistance and ions in yeast.The result showed that PMP3 plays an important role in the balance of Na +,and has no correlation with K + and Ca2+.We also found that PMP3-mediated resistance to Am B was not associated with the NHA1 and VCX1 pathways.Secondly,overexpression of PMP3 gene in sterol biosynthesis pathways blocked mutants(erg2△,erg6△,erg24△,hmg1△,hmg2△)could also enhance the resistance of cells to Am B.It suggests that blocking sterol biosynthesis pathway did not affect PMP3 regulation of Am B resistance.Further studies showed that overexpression of PMP3 did not significantly change the sterol contents in the wild type(BY4741)and different genetic mutation strains.It demonstrates that the effect of PMP3 on cell resistance of Am B is not associated with sterol synthesis.Finally,we investigated the relationship between PMP3 and reactive oxygen species induced by Am B.The results showed that Am B can increase intracellular reactive oxygen species,which elevated the contents of intracellular lipid peroxidation products TBRAS and protein carbonyl..And activities of antioxidative enzymes such as superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(Gpx)are also increased.Overexpression of PMP3 can make higher antioxidant enzyme synthesis in the cells,thus reducing the intracellular reactive oxygen content,and protecting cells against oxidative damage.the results indicate that overexpression of PMP3 can increase intracellular antioxidant enzymes to reduce reactive oxygen species,therefore enhances the resistance of cells to Am B.Meanwhile,the previous studies have shown that intracellular ROS reduction is not only related to antioxidant enzymes,but also has a close relationship with MAPK signaling pathway.In the absence of the key genes SPC1 and ATF1(atf1△ and spc1△)in the MAPK pathway,overexpression of PMP3 did not increase the resistance of Am B,and reduce intracellular reactive oxygen species by using RT-PCR and genetic analysis Altogether,the regulation of PMP3 on Am B resistance depends on the integrity of the MAPK pathway.