| Antifungal Susceptibility Test and Multi-locus Sequence Typing of Clinical Candida albicansObjective: To monitor the sensitivity of Candida albicans that caused infections in Jiangxi to the commonly used anifungal agents,and to provide the basis for the rational use of antifungal agents in Candida albicans infections.To find out the main epidemic strains of clinical Candida albicans in this region,the evolutionary relationships were analyzed by genotyping.Methods: 1.Isolated and purified the clinical strains,and identified the Candida albicans using CHROMagar Medium and multiple PCR.2.Susceptibility testing of Candida albicans to the five commonly used antifungal agents,amphotericin B(AMP B),5-flucytosine(5-FC),fluconazole(FCZ),itraconazole(ICZ)and ketoconazole(KETO),was performed according to a broth microdilution method,"Method for Extracting Micro-Dilute Antifungal Sensitivity of Yeast"(M27-A3),recommended by the Clinical and Laboratory Standards Institute(CLSI)for broth antifungal susceptibility testing of yeasts.3.The Multi-locus sequence typing(MLST)based on DNA sequencing was used for genotyping of Candida albicans.4.Using the MEGA6.0 to analyze the nucleotide polymorphism(SNP)existing in the MLST fragments.The minimal spanning tree was generated by using UPGMA,and the evolutionary relationship of Candida albicans isolates was analyzed with a p-distance 0.04 as the cutoff values.Results: 1.In this study,185 Candida albicans isolates were obtained by isolation,purification and identification.2.The 185 Candida albicans isolates were mainly isolated from sputum(103,55.7%),vaginal or cervical secretions(66,35.7%)and urine(10,5.4%).3.The resistance rates of Candida albicans against AMP B,5-FC,FCZ,ICZ and KETO were 0.0%,2.7%,7.0%,5.4% and 2.7%,respectively.4.A total of 122 double sequence type(DST)were obtained according to MLST genotyping,among which 99(DST2889~DST2899 and DST2945~DST3032)were newly discovered and recorded by Pub MLST database.5.The totoal sequence length of the seven housekeeping genes of MLST were 2883 bp,with 86(3.1%)SNP.The number of SNP of AAT1 a,AAC1,ADP1,MPIb,SYA1,VPS13 and ZWF1 b was 7(7/373,1.9%),10(10/407,2.5%),16(16/443,3.6%),16(16/375,4.3%),9(9/391,2.3%),16(16/403,4.0%)and 16(16/491,3.3%).6.The phylogenic cluster analysis showed that,with a p-distances 0.04,Candida albicans isolates were divided into 12 evolutionary branches(n≥5),and Clade 1 had the highest number of strains(56,30.3%),followed by Clade 2(17 strains,9.2%),Clade 3(16 strains,8.6%)and Clade 4(14 strains,7.6%).Conclusions: 1.The antifungal sensitivity of the 185 Candida albicans isolates was basically consistent with these studies in other regions of China.2.There were significant genetic variations among the clinical isolates of Candida albicans in the region.Of the 122 diploid sequences,99 were newly discovered,and these newly discovered DSTs were submitted into the Candida albicans MLST database.3.Phylogenetic analysis found that Candida albicans was divided into 12 evolutionary branches.The Clade 1 had the largest isolates number of and was the most predominant group of the Candida albicans.Preliminary Biological Function Study of The Serine Protease Ca Htr Ap of Candida albicansObjective: To investigate the precise function of serine protease high-temperature requirement A(Ca Htr A)protein of Candida albicans and its key catalytic serine residues.To screening the Candida albicans protein interacting with Ca Htr Ap to provide a basis for further study of Ca Htr Ap function and its role in the pathogenesis.Methods: 1.Using the NCBI Conserved domain prediction online tool to analyze the Ca Htr Ap conserved domain.2.Analyzing the leader sequence of Ca Htr Ap by using the Psort II online server and predicting its subcellular localization.3.The Ca Htr Ap domain and its homologous amino acid sequence were analyzed by software Clustal X1.86.4.The gene Ca Htr A1 encodes the functional Deg Q domain of Ca Htr Ap was amplified by PCR and its catalytic serine variants were constructed by over-lap PCR.The genes were then inverted into the prokaryotic expression plasmid p ET-28 c.The recombinant plasmid p ET28c-Htr A1,p ET28c-Htr A1S219 A,p ET28c-Htr A1S219 C,p ET28c-Htr A1S220 A,p ET28c-Htr A1S220 C and p ET28c-Htr A1S219/220 A were identified by PCR,restriction endonucleases digestion and DNA sequencing.5.The expression of recombinant plasmids was induced by using IPTG.Then the total protein was collected after breaking the cell wall by ultrasonic.And finally,the fusion proteins were purified using Ni+ affinity chromatography.6.The activity of Ca Htr A1 p serine protease and the effects of Ser219 and Ser220 were verified by the degradation of serine protease specific substrate β-casein in vitro.7.Immunizing Babl/c mice with fusion protein Ca Htr A1 p to prepare the anti-Ca Htr A1 p polyclonal antibody.8.The immunofluorescence technique was used to further verify the subcellularlocalization of Ca Htr Ap using the anti-Ca Htr A1 p antibody.9.Characterization of Candida albicans proteins interacting with Ca Htr Ap using in vitro co-immunoprecipitation techniques and mass spectrometry analysis.Results: 1.The conserved domain analysis showed that Ca Htr Ap was a double Deg Q domain protein consisting of two serine protease domains and four PDZ domains.The N-terminal Deg Q domain had a conserved tryptase-like serine protease motif ―catalytic triad‖(His105-Asp136-Ser220).2.Ca Htr Ap had no N-terminal leader sequence,as well as no nuclear localization signal,and thus the protein was predicted to be cytoplasmic.3.Amino acid sequence alignments showed that the N-terminal Deg Q domain of Ca Htr Ap had the ―catalytic triad‖ motif,and a conserved sequence GSSGS was among the predicted serine residue Ser220,with adjacent Ser219.While the C-terminal Deg Q domain function was non-functional.4.The results of PCR,restriction endonucleases digestion and DNA sequencing showed that the recombinant expression plasmids p ET28c-Htr A1,p ET28c-Htr A1S219 A,p ET28c-Htr A1S219 C,p ET28c-Htr A1S220 A,p ET28c-Htr A1S220 C and p ET28c-Htr A1S219 / 220 A were successfully constructed.5.The fusion proteins,Ca Htr A1 p,Ca Htr A1S219 Ap,Ca Htr A1S219 Cp,Ca Htr A1S220 Ap,Ca Htr A1S220 Cp and Ca Htr A1S219A/S220 Ap,were expressed,purified and concentrated,and the concentrations were 2.4 mg/m L,1.84 mg/m L,2.00 mg/m L,1.52 mg/m L,1.45 mg/m L and 1.9 mg/m L,respectively.6.In vitro protease activity test showed that Ca Htr A1 p could degrade the substrate β-casein.The degradation efficiency of fusion proteins Ca Htr A1S219 Cp,Ca Htr A1S220 Cp,Ca Htr A1S220 Ap,Ca Htr A1S219 A / S220 Ap and Ca Htr A1S219 Ap were respectively 0.84,0.74,0.66,0.46,0.42 and 0.35.7.Immunofluorescence verified the cytoplasmic subcellular localization of Ca Htr Ap,and the expression level of Ca Htr Ap in pseudohypha was significantly higher than that in yeast cells(unit area intensity value 0.23 vs.0.15,p=0.002)using a semi-quantitative analysis of fluorescence.8.A total of eight Candida albicans proteins,including three ribosomal subunit proteins(RPL3,RPS9 B and RPS3),and elongation factor 1-alpha(TEF1),actin(ACT1),ubiquinol-cytochrome c reductase core subunit 2(QCR2),Putative cytochrome P450 protein(TRI4)and dihydrolipoamide dehydrogenase 1(LPD1),were proved to interact with Ca Htr Ap by co-immunoprecipitation and mass spectrometry.Conclusions: 1.Ca Htr A1 p had the conserved trypsin-like serine protease ―catalytic triad‖(His105-Asp136-Ser220)motif and the typical serine protease activity.2.Both Ser219 and Ser220 had an important effect on Ca Htr A1 p protease activity,and there was a synergistic effect between these two serines.3.Ser219 and Ser220 had significant effects on Ca Htr A1 p protease activity,but the effect of Ser219 mutation on Ca Htr A1 p was significantly higher than that of predicted catalytic serine Ser220.4.Ca Htr Ap is a cytoplasmic protein that may be associated with the formation of pseudohypha;5.Eight Candida albicans proteins interacted with Ca Htr Ap,including RPL3,RPS9 B,RPS3,TEF1,ACT1,QCR2,TRI4 and LPD1,were successfully identified by co-immunoprecipitation and confirmed by mass spectrometry. |
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