Preliminary Purification And Identification Of The Novel Candida Albicans-secretory Factor Inducing Monocytes Apoptosis

Objective: To test the apoptosis-inducing activity of SIIF, a novel virulencefactor isolated from culture supernatants of Candida albicans, the proliferationand apoptosis effects of secretary IL-12inhibitory factor (SIIF) on humanmonocytes were observed. The position of the monocytes SIIF combines withwas tracked. The candidate proteins were screened and the protein sequencewas evaluated. These studies may contribute to mechanism of pathogenicity ofCandida albicans and new therapy strategies may be taken for local andsystemic infection of Candida albicans by interfering with SIIF activityspecifically. Methods: Fungal strain in this study was isolated from patientswith vulvovaginal candidiasis (VVC) in our previous study which wasidentified as Candida albicans by direct microscopic examination,morphological identification and extracting DNA blast with Genbank. Thehuman acute monocytic leukemia cell line (THP-1) was used as humanmonocyte cell. Candida albicans were inoculated in20ml of1640medium,shaking in37℃for24hours. After two times of passaging, the yeast wastransferred into500ml of culture medium for another24hours culturing. Thenthe supernatant was collected and allocated into centrifuge tubes, andcentrifuged to remove the yeast. Further, the supernatant was filtered through a0.22μm filter unit to remove the remaining fungi. Finally, the culturesupernatant was greatly concentrated by Centricon-70filters which also cut of protein less than30KDa. Protein concentration was measured by BCA proteinassay kit, and then stored at-80℃. According to whether SIIF was added intomonocytes culture medium, the experiment was divided into experimentalgroup and the control group. THP-1cells and400μg/ml of SIIF protein wereco-cultured24hours. The cell viability of each group was determined by XTTassay kit. Early apoptosis rate of THP-1cells was detected withAnnexinV-FITC/PI double staining by flow cytometry(FCM). The positionwhere SIIF bind to monocytes was observed under Fluorescence microscopy byConA-FITC/DAPI staining. SIIF was further separated and purified by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Afterelectrophoresed, bands in the gel were hydrolyzed by trypsin and analyzed withliquid chromatography–tandem mass spectrometric (LC-MS/MS). Then we usemascot software to search NCBInr database and get the relevant proteins.Finally, according to the protein molecular weight, the matching score and therelevant function, we screened out candidate proteins. The proliferation rate andearly apoptosis rate of the THP-1cells were evaluated by SPSS17.0softwarepackage for statistical analysis. Results:1. XTT assay showed that afterco-culturing with SIIF for24hours, the cell proliferation rate of the THP-1cells were significantly lower than that of control group (P=0.026).2. Flowcytometry assay with AnnexinV-FITC/PI double staining revealed that afterco-culturing with SIIF for24hours, the early apoptotic rate of the THP-1wassignificantly higher than that of the control group (11.867±1.604%vs5.300±0.265%, P=0.002).3. Fluorescence microscopy with ConA-FITC/DAPI double staining revealed that the green fluorescence where SIIF locatedoverlapped with the blue fluorescence which was the cell nucleus, indicatingthat SIIF got internalized into THP-1and combined with the nucleus.4. Afterseparated by SDS-PAGE,SIIF was digested with trypsin and analyzed throughLC-MS/MS. Based on the protein molecular weight, matching score andfunctional evaluation, secreted aspartic protease6(Sap6) and repressed byTUP1protein4(Rbt4p) were supposed as SIIF candidate proteins. Conclusions:1. SIIF can inhibit the proliferation of THP-1cells.2. SIIF may induce theapoptosis of THP-1cells.3. SIIF enters the THP-1and combines with thenucleus. Probably by mediating DNA transcription, thus blocking proteinsynthesis of the cells, SIIF induce apoptosis of THP-1.4. Sap6and Rbt4p areregarded as the candidate SIIF protein.