Phenotypic And Transcriptional Study Of Virulence Factors In Candida Tropicalis

Objectives The aim of this study is to determine virulent phenotypes of Candida tropicalis among different clonal complex using series of assays; to explore the expression profiles of virulence genes under three states with reverse transcriptase PCR; and to analyze the specific role of virulence factors in cell damage phenotypically and transcriptionally. The accomplishment of this research will help to select the dominant virulent complex, clarify the molecular basis for virulence disparity, and support the further research of pathogenicity in C. tropicalis, and will be quite important for further epidemiology monitoring, prevention and treatment of C. tropicalis.Methods Adhesion and biofilm formation were measured in 68 C. tropicalis isolates from different anatomic sits on polystyrene and human urinary bladder epithelial cell (TCC-SUP) by crystal violet (CV) assay and 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay. Filamentation of C. tropicalis was induced in serum and evaluated by inverted microscope. The activity of secreted hydrolytic enzymes including secreted aspartyl proteinases, phospholipases, esterase, lipase and hemolysins were determined on plates containing specific enzyme-substrate by observing precipitation or translucent halo. Based on the phenotypes of C. tropicalis in our previous assay, we selected nine representative strains with different activities of virulence factors (adhesion, biofilm formation, secreted aspartic proteinases and hemolysins), and ATCC750 was used as a reference strain. The expression levels of virulent genes (ALS1-3, LIP1, LIP4 and SAP1-4) were analyzed in planktonic, on polystyrene and TCC-SUP surface by reverse transcriptase PCR. The cell (TCC-SUP) damage induced by C. tropicalis was determined by lactate dehydrogenase assay (LDH). Furthermore, we will compare the activity of virulence factors among C. tropicalis from different clonal complex and anatomic sits, examine correlation between virulence genes expression and its phenotype, and analyze the specific role of virulence factors in cell damage phenotypically and transcriptionally.Results1. Most C. tropicalis could adhere and form biofilm on polystyrene and TCC-SUP surfaces, with specific strain-dependent feature. The adhesion ability grew stronger as time prolonged. Isolates demonstrated higher adhesion and BF capacity on PMP than TCC-SUP surface.2. All C. tropicalis isolates produced Saps (secreted aspartyl proteinases), esterase and hemolytic activity, but no phospholipases and lipase were detected. The activity of Saps achieved maximum at 48h, and esterase and hemolysins activity of C. tropicalis continued to grow within 72h.3. Adhesion and filamentation of C. tropicalis played an important role in biofilm formation (BF). However, the activity of Saps negatively correlated with adhesion and biomass of biofilm on polystyrene.4. Significant difference was found among diploid sequence types (DSTs) groups for adhesion and BF capacity on PMP and TCC-SUP surface. Most isolates in DSTs group5 and 6 were with high level of adhesion and BF, while group 1 contained isolates with low/medium adhesion and BF.5. Significant differences were observed for immediate adhesion (IA) and Saps among different anatomic sites. Blood isolates displayed weak adhesion on PMP, while the activity of Saps was strong.6. The expression of virulence genes (ALS1-3, LIP1, LIP4 and SAP1-4) was diversity and strain-specific among C. tropicalis strains. The gene of ALS3, LIP4 and SAPS were the highest expressed for most C. tropicalis in their corresponding gene family in three examining conditions.7. Virulence genes transcripts of C. tropicalis were all detected in planktonic and on polystyrene, whereas some virulent genes (ALS1, LIP], SAP1 and SAP4) in amount of isolates were rarely detected on TCC-SUP. The expression of virulence genes except ALS3 on polystyrene was up-regulate compared with the planktonic and TCC-SUP counterparts.8. Negative correlation was found between the expression of ALS1 on TCC-SUP (XTT) and adhesion; negative correlation existed between the expression of LIP7, LIP4 and SAP! on PMP and adhesion (LA); and positive correlation existed between the expression of SAPI and SAP2 in planktonic and the activity of Saps. Clustering analysis of virulence genes exhibited that isolates with high biofilm on polystyrene formed a group, while strains with low biofilm presented as a dispersion relation.9. Co-culture of TCC-SUP cell with each isolate separately, caused slight cell damage at 2,6 and 12h. The levels of LDH apparently increased at 24 and 48h. Statistical analysis showed that adhesion on epithelial cells and the expression of ALS2-3 encoding adhesions positively correlated with cell damage, which may suggested that adhesion on epithelial cells played an important role in cell damage.Conclusions We analyzed the virulence factors of C. tropicalis phenotypically and transcriptionally, simultaneously. The virulence phenotype including adhesion, biofilm formation, filamentation, Saps, esterase, lipase and hemolysins and the expression of virulence genes containing ALSI-3, LIP1&4 and SAP1-4 displayed strain-dependent. Significant difference was found among diploid sequence types (DSTs) groups for adhesion and BF capacity on PMP and TCC-SUP surface. Blood isolates displayed weak adhesion on PMP, while the activity of Saps was strong. The expression of virulence genes except ALS3 on polystyrene was up-regulate compared with the planktonic and TCC-SUP counterparts, which indicated that biofilm on PMP could promote the expression of virulence genes. Adhesion and filamentation of C. tropicalis played an important role in biofilm formation. However, the activity of Saps negatively correlated with adhesion and biomass of biofilm on polystyrene. Adhesion on epithelial cells and the expression of ALS2-3 encoding adhesions positively correlated with cell damage, which may suggested that adhesion on epithelial cells played an important role in cell damage. These data plays an important role in further research of pathogenicity, prevention, diagnosis and treatment of C. tropicalis.