| Candida is the most frequent human opportunistic fungus, which can parasitic on the mucosa and proliferation,inducing stomatitis, esophagitis and vaginitis and so on. Disseminated candidiasis often on lower immunity person, which often threats life. With the increase of radiation and chemotherapy, transplantation and AIDS patient, candidiasis has become the main threaten, which is one of the major fatal complications. In recent years, with the rapid increase of deep fungus infections’,80 percent of which is Candida albicans. In 2010, an survey of hospital infection in United States found that the infection of Candida in the fourth and causing the extremely high fatality rate. Candida albicans are the most strongest virulence and the most common clinical pathogenic fungi in Candida. With the rising of clinical morbidity and fatality rate on Candida albicans year by year, some reasons are attribute this condition, one of which is related to the long-term abuse of antibiotic, at the same tome, because of the powerful morbidity and drug resistance of Candida albicans biofilm, which is gradually come to attention by scientist. Biofilm are clinically significant because of their association with infection due to the presence of an implanted medical device. Use of these devices, such as venous catheters, pacemakers, and artificial joint, is routine in the United States, with well over 10 million recipients per year. These devices provide a very substantial health benefit but nonetheless are associated with infection rates as high as 30%. Based on the great clinical significance of Candida albicans biofilm, the study of the biofilms formation mechanism is helpful to further understanding pathogenicity and drug resistance.The vitro study of Candida albicans biofilm indicate that there are four stages on the formation of biofilm.(A) In the first step, yeast form cells adhere to the substrate.(B) In the second step, the cells multiply and accumulate. Germ tubes begin to form, and extracellular matrix production begins.(C) In the third step, the biofilm enlarges, enveloping the biofilm.(D) In the final step, yeast cells are dispersed to further colonize the surrounding environment. We used the medium of 10%serium+spider and on 37℃,200 rpm culture condition which induced the formation of Candida albicans in vitro. Using this method we screened the 300 mutant strains which used gene recombination and then found some genes which influence the formation of biofilm, so as to find some new genes about the formation of Candida albicans biofilm. We used the method of macroscopic observation screening 4genes in 300 mutant Candida aibicans strains. They needed. They are YPD(YEP+2%glucose), YEP+2%mannitol, Spider, Spider except mannitol+2%glucose. Under 37℃,200 rpm culture condition,we find that mitochondrial respiratory chain related deficiency strain M6(ORF19.287) is always on the condition of yeast on YEP+2%mannitol and Spider medium,however the mutant is always on the state of hyphae on YPD(YEP+2%glucose) and Spider except medium+2%glucose. Candida albicans standard stain SN250 is always on the condition of hyphae on four kinds of medium. Also can make use of measuring growth curve and spot assay, we find that the mutant growth deficiency low-grade in control with Candida albicans standard strain on YPD medium, however,the mutant grew obviously limited on Spider medium. We utilize four kinds of medium which they are YPD(YEP+2%glucose), YEP+2%mannitol, Spider, Spider except mannitol+2%glucose culturing biofilm in vitro. We find that SN250 can form intact biofilm,however, the mutant can form intact biofilm only on YPD(YEP+2%glucose) and Spider except mannitol+2%glucose. We can not find biofilm formation on YEP+2%mannitol and Spider medium. According to these results we find that the mitochondrial respiratory chain related gene deficiency influence Candida albicans using non-fermentation carbon source, which will affect its growth, hyphae formation and biofilm formation.We find that the mitochondrial respiratory chain related gene deficiency influence Candida albicans using mannitol(non-fermentation carbon source), which will affect its hyphae formation. We further confirmed by the method of RT-q PCR. The mutant M6(ORF19.287) is cultured on spider medium under the condition of 37℃.We identify that the hyphae related gene ALS3,ECE1,HWP1,HYR1 express decreased. We further identify mitochondrial respiratory chain related gene deficiency influence Candida albicans using mannitol(non-fermentation carbon source), which will affect its hyphae formation from gene level.Our study identify that mitochondrial respiratory chain related gene deficiency in Candida albicans utilize difference on between glucose(fermentation carbon source) and mannitol(non-fermentation carbon source) about growth,hyphae formation and biofilm formation from phenotype and gene level. are M6( ORF19.287),M36( ORF19.1625), M58( ORF19.2570), M136(ORF19.6607).We further identify the defect of the formation of biofilm used the method of XTT and biomass. We found the four mutant strains have the same function through the research of Candida albicans about mitochondrial respiratory chain and having the function of NADPH. Because they have no definite gene names, so we called the 4 screened mutant strains are mitochondrial respiratory chain related deficiency strains.Fungi can be gained energy by glucolysis and aerobic respiration through synthetic metabolism. The main energy will get from electronically respiratory chain transmit. When the gene of mitochondrial respiratory chain defected, the aerobic respiration will be restricted. Then glucolysis will become the main methods of providing energy. Mitochondrial respiratory chain related deficiency strains are different from growth, hyphae formation and biofilm formation by using fermentation carbon source and non-fermentation carbon source, which is the focus in our study. Firstly,we measured the difference of growth between mutant strains and standard using the method of Growth curve and Spot assay in fermentation carbon source(glucose) and non- fermentation carbon source(mannitol). Secondly, we observe the difference of formation of hyphae in the culture condition of 37℃,200 rpm using different carbon source providing energy. Thirdly, we compare the formation of Candida albicans in different carbon souce providing energy.The typical structure of Candida albicans biofilm is many yeast adhere to the object on the basal and lots of matrix is covered on the yeast and also we can see many hypha on the middle layer. The thickness of biofilm can be up to 50-350μm。The rich extracellular matrix is key to medicate the formation of Candida albicans biofilm, except that the morphologial transformation which is from yeast to pseudohyphae is also an important factor of Candida albicans formation, and at the same time the Candida albicans morphological changes is relevant to pathogenicity. In order to further identify mitochondrial respiratory chain related deficiency strains indeed influence the Candida albicans’ morphologial transformation. We use two classical inducing hyphae formation medium, which they are YPD and Spider medium. In order to further identify mitochondrial respiratory chain related deficiency strains on the utilization of different carbon source. We are based on two classical inducing hyphae formation medium collocating four kinds of different carbon source culture medium which is our experiment... |
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