The Study Of KpsF Gene Which Contains PxIxIT Motif In Aspergillus Fumigatus

Background Aspergillus fumigatus is a ubiquitous and widely distributed saprophytic fungus and it is an opportunistic pathogen which causes invasive aspergillosis.In recent years,the infection of immunodeficiency patients has increased markedly and the fatality rate was extremely high.Thus,the treatment has been becoming the common problem in clinical currently.Calcineurin has played an important role in the morphogenesis of pathogenic fungi and toxicity,which made it an attractive antifungal target.In study of aspergillus fumigatus,Ca N located on the tip of the hyphae and separation,had significant effects on pathogenic.Studies had shown that its functions were related to motif Px Ix IT.We found that similar motif existed in Kps F gene.Although the application of calcineurin inhibitor FK506 and cyclosporine A in modern medical has been very successful.But the side effects associated with calcineurin made the use of antifungal drug limited greatly.Therefore,study Kps F gene function and discover new endogenous calcineurin inhibitor,to provide a new method for the treatment of invasive aspergillosis is very important.Objective 1.To construct the Kps F Gene defect strains ?Kps F and Kps F-overexpress strains to explore the effect of radial and phenotypic growth in Aspergillus fumigatus.2.To study the susceptibility of Kps F gene to common antifungal drugs in vitro.3.Preliminary study the relationship between Kps F gene and calcineurin and calciumhomeostasis.4.Preliminary research the location of the Kps F Gene in the hyphae by constructing Kps F-EGFP strains.Methods 1.Used the protoplast technique to construct the ?Kps F and then verification of ?Kps F strains by Southern blot.2.Used p UCGH plasmid as a genetic vector,hygromycin as selection markers,and constructed a gene expression strains by protoplast method.3.Configured different concentration of three kinds of strains,inoculated a certain concentration on the GMM medium,observed the colony growth,recorded colony diameterin 24 h,48 h,72 h,96 h,120 h and made statistical analysis,added lactic acid phenol medan staining to observe strains morphology.4.Used Spot-assay to explore the sensitivity of antifungal drugs,added antifungal drugs in the GMM,recorded the colony diameter to observe the colony growth,respectively in 24 h,48 h,72 h,96 h,120 h and make statistical analysis.5.To compare the Cna A and Vcx A gene m RNA expression levels by RT-PCR under the condition of different concentrations of Ca2 +of three strains and statistical analysis.6.Used confocal microscopy to research the location of the Kps F Gene in the hyphae by constructing Kps F-EGFP strains.Results 1.Constructed the ?Kps F and Kps F-overexpres strains successfully,the laboratory has been built Kps F-EGFP strains.2.The radial growth results of ?Kps F and Ku80 had no significant difference(P > 0.05),Compared with Ku80,but Kps F-overexpression fell about 45% relatively.3.The phenotype of ?Kps F and Ku80 was normal and had no difference,Hyphae branch with 45°,Septum was clear.However,Kps F-overexpression strains had more hyphae,but the hyphae was unnormal,did't branched with 45°.the septum is relatively little;Conidium head is relatively short and cystic dysplasia.4.When the concentration from 1.0 to 5.0 ug/ml,? Kps F appear smaller paradoxical growth in response to caspofungin,the Ku80 and overexpression strains had no obvious change.For itraconazole,voriconazole,amphotericin B and FK506,four kinds of antifungal drugs,the drug sensitivity had no obvious difference of ? Kps F,Ku80 and Kps F-overexpression in vitro in different concentrations.5.Under the condition of 10 m M Ca Cl2,Cna A gene expression of ?Kps F is significantly higher than Ku80 and Kps F-overexpression.The Cna A gene expression of ?Kps F is 29.76 times higher than that of the Ku80(P < 0.01),Kps F-overexpression strain is 1.56 times of Ku80(P < 0.05).Under the condition of 10 m M Ca Cl2,Cna A gene expression of three strains is not obvious.As ordinary GMM medium,Cna A gene expression of ?Kps F is 2.81 times than that of Ku80,Kps F-overexpression has no change.6.Under the condition of 10 m M Ca Cl2,Vcx A gene expression of Kps F-overexpression is significantly higher than Ku80 and ?Kps F,Kps F-overexpression is 2.65 times higher than that of Ku80.The difference was significant statistically(P < 0.01),?Kps F compared with Ku80 has no obvious change.But under the condition of 100 m M Ca Cl2 and ordinary GMM medium,Vcx A gene expression of three strains were not obvious difference.7.Kps F-EGFP is widely and homogenously distributed within the cell membrane in Aspergillus fumigatus.Conclusions 1.The defection of Kps F gene has no effect on the radial growth.However,Kps F gene overexpression had limited radial growth in Aspergillus fumigatus.2.The defection of Kps F gene had no obvious change in phenotype,but Kps F gene overexpression influenced hyphal arrangement,branched and the formation of the septum,it also effected the growth of the Conidium.3.The defection of Kps F gene and Kps F-overexpression had no obvious effect on fungal drugs sensitivity in vitro in aspergillus fumigatus.4.Under low concentration Ca2+,Kps F genes may participate in the negative feedback regulation of calcineurin,and under the condition of 10 m M Ca2+,the negative feedback regulating functions was obvious,under the high concentration of Ca2+ the negative feedback regulation is suppressed.Kps F gene participated in calcium homeostasis in Aspergillus fumigatus.5.Kps F Gene may be associated with the formation of the cell membrane in Aspergillus fumigatus.