Research Of Resistant Mechanism In Candida And Fusarium

In recent years, due to various reasons, fungal infection rate is rising. With the large number of antifungal medicine applications in the clinic and the extension of time, these result in increasing drug resistant strains, and multi-drug resistance, make fungal treatment more difficult. Fungal drug resistance is due to a number of factors, which works together as regulation and control. And studies of fungal resistant mechanisms in-depth to overcome and delay the development of drug resistance have become a hot research at home and abroad.Previous studies have found that a number of fungi-resistant mechanisms, mainly in the search for genes on the level of resistance genes. With the genome project researches in-depth, recent years proteomics method has gradually been used in pathogenic mechanisms of bacterial resistance in (including mycobacterium tuberculosis, pathogenic Escherichia coli and so on), and made some breakthrough progress. Comparing with pathogenic bacteria, pathogenic fungi related research has just started. There is no mechanism to study that proteomics methods are used for Fusarium resistance at home and abroad, so it is a very meaningful and worthy of exploration of new area.This study collected statistics and analyzed the character of regional distribution of clinical fungal infection from 2004 to 2006, isolated total strains of 246 fungi, and more than 10 strains of pathogenic fungi. Among these, Candia albicans is still the main pathogenic fungi. So it's the same with previous research. However, Candida tropicalis infection rate has increased, occupies the second place. While Candida kruse infection rate has turned down. Fusarium is still the main pathogens of eye disease, followed by Aspergillus and Candida. According to NCCLs M27-A and M38-P programs, Micro-dilution sensitivity test found that the clinical resistance of Candida to commonly used antifungal drugs has increased, while the resistance of Fusarium is very high, commonly used clinical antifungal drugs effects are not ideal.The preparations of protoplast are different between different species, or even different strains of the same species. This study observed a variety of factors which affect the yield of protoplast, established and optimized the preparation conditions of Fusarium protoplast. At last, we determined the preferred plan that PDB culture 36h. Under 32℃, 70r/min and 5h, mycelium is digested by the cell wall degrading enzyme, which is the mixture with 0.3mol/l MgSO4, 0.3mol/l NaCl buffer solution dissolved cellulose, snails enzyme and lysozyme (1:2:4).Efflux pump inhibitor FK506 can be used as the substrate of ABCT, thereby inhibiting ABCT as the intermediary of drug transit. After using FK506, this study found that different concentrations (20~0.3125μg/ml) of FK506 would the MIC value of Candida, Fusarium protoplast on FCZ and ICZ resistances, decreased several concentration gradients, and from resistance strain into sensitive strain. These proved an important mechanism by which fungi is resistant to triazole drug, that led to increasing drug efflux would lead to reduce concentration of drug in cells. And FK506 on AmB and 5-FC resistances have no reversal role, the relationship of ABCT and AmB, 5-FC resistance mechanism needs to be further explored.In addition, the use of Real Time RT-PCR gene expression of ABCT analysis shows that expression of drug resistant strains was significantly higher than sensitive strains, the gene expression of ABCT in Candida-drug resistant strain is 11 times of sensitive strain, the gene expression of ABCT in Fusarium solani-drug resistant strain is 13 times of sensitive strain, indicated that the differences of ABCT expression occur in the level of transcription.Optimization of protein extraction methods of the yeast (Candia albicans), filamentous fungi (Fusarium), obtaints adequate and stable high-quality protein, electrophoresis is clear, uniform and higher resolution. Using the comparative proteomics technology, respectively analyzed the protein expression pattern of sensitive and resistant strains, found composes of proteins exist differences. In Candia albicans, there are 35 different proteins, compared with the sensitive strain, 29 protein points of resistant strain express upward, 6 protein points express downward; in Fusarium, there are 25 different proteins, compared with the sensitive strain, 16 protein points of resistant strain express upward, 9 protein points express downward.This study firstly used proteomics methods combining with molecular biology tools in the study of fungal resistance mechanism; compared proteomics differences between different resistant strains; observed the proteome differences of changes of fungal resistance under certain conditions; comprehensive, dynamic clarified fungal drug resistance mechanisms. The search for new drug targets, development and selection of new antifungal is of great significance. The laboratory will improve the conditions to isolate and identify hydrophobic, high and low molecular weight proteins, while further identify by mass spectrometry protein and sequence, for showing the nature of and function different proteins.