A Study On Preliminary Analysis Of Exocrine Protein Of Crytococcus Neoformans Biofllms

Biofilms are composed of an extracellular matrix of polymeric substances. In otherand interdependent, commonly attached to biological or non-biological surfaces. Incomparison with planktonic cells, it is a living way that is adapted to the survivalwords, biofilms are structured microbial communities which are encapsulated byECM generated by themselves. Those microbial communities are unique, complicatedenvironment during the formation process of bacteria and funga. The feature ofbiofilms is their resistance to drug. At present,about65%human infections are relatedto the biofilm formation, which is considered more than80%by some reseachers. Inthe recent30years, infection of Cryptococcus neoformans is on the rise with theheavy use of broad-spectrum antibiotics and immunosuppressant, the increasing ofcancer patients and organ transplanters, and the widespread use of medicine materials,among which many are closely related to the formation of Cryptococcus neoformans.Once biofilms form on infection sites or the biological materials implanted in patients,the diseases caused by Cryptococcus neoformans are mostly chronic and refractorydue to the resistance to most antifungal drugs. In recent years the basic and clinicalresearches on c. neoformans biofilm have caused great attention.To deeply understand the relevant characteristics of the C. neoformans biofilm,firstly, we should observe the morphology of the biofilms. Researches have revealedthat there are several apparent phases in the formation process. MTT reduction assayis the most commonly used method in quantitative research of fungus, which canindirectly reflect the metabolic activity of the biofilms in order to analysis theformation processes of the biofilms. Isobaric tags for relative and absolutequantification in separating the protein mixed samples and comparing the differencesamong the samples have the advantages that cannot be replaced. It’s a newly methodthat can separate thousands of protein samples. Compared with traditionaltwo-dimensional electrophoresis, it has an advantage on evaluating species, molecular weight and sensitivity of the comparative proteomic. The objective of this study wasto analyze the dynamical processes of C.neoformans biofilm growth phases, which isunder the inverted phase contrast microscope and MTT reduction assay. From theresults, we can know better of characteristics of the C. neoformans biofilm andprovide the theory basis of resistance mechanism; meanwhile, with the method ofadding cryptococcal suspension to individual wells of polystyrene96-well plates inminimal medium and to tissue culture flask with low-speed rotary shaker to constructthe model of experiment group and control group in vitro, then, collect exocrineprotein from the supernatant as samples, which is needed to purify, at last, we need toanalyze samples by isobaric tags for relative and absolute quantitiation, from theresult,we can find the differentially expressed proteins, which is related with biofilmformation, of course the study is also helpful for us to know about the quorum sensingIn conclusion, Cryptococcus neoformans can form typical and mature biofilmstructures, which is supposed to contain three phases. The metabolic activity ofCryptococcus neoformans biofilms increases as time extends, and it becomesrelatively stable after48hours. Results: Cryptococcus neoformans biofilm isconsidered as three dimension configuration, which is variant, inhomogeneous andexoteric. And from the analysis of isobaric tags for relative and absolute quantitiation,we can discover the differential proteins.