The Role Of PMT4 Gene In Formation And Drug Resistance Of Cryptococcus Noeformans Biofilms

BackgroundOne of the thorniest problem in research of Cryptococcosis is that we know little about how this susceptible pathogen sometimes becomes resistant to Antifungal drugs.In the past decade, the role of biofilms get attention of scientists. Some studies show that the susceptibility of fungi cells in biofilms to antifungal drugs is 30-2000 times lower that planktonic cells. But the mechanism is not clear yet.In this study, we chose C. neoformans H99 (serotype A, MATα) and PMT4 gene as the subjects. PMT4 gene encodes an enzyme which modificates proteins. A published study showed that PMT4 gene is necessary for resistance of Candida Albican against antifugal drugs.PMT4 gene of fungi cells is different from their analogous genes in mammal cells, so this gene and it's products can be a possible target of new therapeutics. Otherwise, we created a new animal model of C. neoformans biofilms and used an in vitro cryptococcus biofilm model described before.Objects1. To build a PMT4 mutant of C. neoformans H99.2. To build an new animal model of C. neoformans biofilms.3. To investigate the difference between H99 and PMT4 mutant in formation of biofilms.4. To investigate the difference between H99 biofilms and PMT4 mutant's in the resistance against antifugal drugs.Methods1. Build a PMT4 mutant of H99Long Flanking Homology (LFH-)PCR was used to get this work done. Transformants were preliminary screened on YPD plates with G418. The positive ones were verificated by PCR and gene sequencing.2. Build a new animal model of C. neoformansIn vitro model was built using basic culture media and 96-well plate. Animal model was built by setting a catheter (with polystyrene in lumens) into rabbit's central venous.3. Investigation on the differenc of the formation of biofilms between H99 and PMT4 mutantInverted phase contrast microscope and CLSM were used to observe the figure and construction of biofilmsCompare the amount of CFU in biofilms of H99 and PMT4 mutant4. To investigate the difference between H99 biofilms and PMT4 mutant's on the resistance against antifugal drugs.M27A2 program was used to test MIC of planktonic cells of H99 and PMT4 mutant and of their biofilmsMTT test was used to investigate the metabolic activity of H99 biofilm cells and PMT4 mutant biofilm cells, this method can show the susceptibility of both H99 and PMT4 mutant cells in both in vitro model and in vivo model.The concentration of drug in in vivo model was determinated by earlier in vitro experiment done before.Results1. Build a PMT4 mutant of C. neoformans H99PCR and gene sequencing showed that the PMT4 gene was knocked out from transformant No.12.2. Build an new animal model of C. neoformans.H99 and PMT4 mutant is able to form biofilms in the in vitro model and the animal model. So this new model can be used in the study on Cryptococcus neoformans biofilms.3. the differenc between H99 and PMT4 mutant in the formation of biofilms.The biofilms of H99 and PMT4 mutant were similar, but the latter had less amount of cells than the former in same phrase.There appeared construction like hypha in biofilms of PMT4 mutant 24h after inoculation.Biofilms of PMT4 mutant has less amount of CFU4. the difference between H99 biofilms and PMT4 mutant on the resistance against antifugal drugs. M27A2 test showed that FCZ,AmB,5-FC,ITR have MICs(μg/ml) against planktonic cells of H99 PMT4 mutant as 4,1,4,≤0.125 and 1,0.25,0.5,≤0.125 respectively; and against biofilm cells as 4,2,8,≤0.125 and 2,0.125,4,≤0.125。MTT test showed that the concentration of these four drugs to make the turbidity 80% off in H99 biofilms and PMT4 mutant biofilms were≥64,32,≥64,16 and 2,0.5,4,1 respectively.Treated with 64μg/ml FCZ, MTT test showed that the inhibition ratio of metabolic activity of H99 biofilm cells and PMT4 mutant biofilm cells were 8.7% and 96.7%, respectively.DiscussionLong Flanking Homology (LFH-)PCR was used in this study, and We get a verified PMT4 mutant of C. neoformans H99 in this study.We built successfully a new animal model and it is useful in research on biofilms of C. neoformans. In this animal model, the polystyrene film can be unloaded, so CLSM becomes nonessential sometimes in observation of biofilms.PMT4 mutant biofilms has less amount of CFU than H99's. That means PMT4 mutant biofilms delivers less fungal cell to the environment.There are construction like hypha in mature biofilms of PMT4 mutant. Maybe the defect of cell wall of PMT4 mutant results in this. But why in mature biofilms? we don't know yet.Drug sensitive test showed that planktonic cells of PMT4 mutant has a little higher susceptility. Maybe it also involves defect of cell wall. M27A2 test showed that biofilm cells of C. neoformans have similar susceptibility compared with planktonic cells. A possibility is, after separated from biofilms, the biofilm cells get again the biological characteristics of planktonic cells. Obviously, this program is not appropriate for biofilm cells.MTT test in both in vitro and in vivo model showed that H99 biofilm cells are significantly more resistant against antifugal drugs than PMT4 mutant biofilm cells are.These results suggest that PMT4 gene plays an essential role in the resistance of C. neoformans against antifungal agents. And because of the remarkable difference in properties and function of products between fungal PMT gene and it's analogous gene in mammals, PMT4 gene and it's products can be reasonably regarded as potential targets of new therapeutics.