الفهرس 
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Abstract
Yeast pathogenicity becomes an important public health challenge. Yeast is one of the most studied models for regeneration process. Cell progression of yeast cell is an important tool to identify the molecular pathways that switch yeast virulence.
Biofilm formation and secreted aspartic proteinase are two main important virulence factors associated with the pathogenicity of yeast. The transformation from planktonic cells to biofilm cells is contributed to a complex remodeling of morphological behavior supported by several change in several genes expression.
This study was conducted to quantify biofilm formation and secreted aspartic proteinase activity at optimum conditions for C. albicans and S. cerevisiae to follow up the expression level of some genes during biofilm development as EFG1, ZAP1, ALS3, HWP1, YWP1, SAP1 and SAP4 in C. albicans and FLO11 and YPS3 in S. cerevisiae at different time intervals to determine the real time required for genes activation and to investigate the relationship between morphological changes and switching time of their related virulence genes
To achieve these objectives, seven yeast strains were included in this study; five clinical identified C. albicans strains were obtained from Nephrology Department at Theodor Bilharz Research Institule Hospital, Giza, Egypt and two S. cerevisiae strains were obtained from Mersin Culture Collection, Faculty of Agriculture, Ain Shams University.
Biofilm formation was assessed qualitatively by tube adherence method and quantitatively by microplate method, and was evaluated and optimized at different pH values, incubation time and different media, and visualized by light microscope. The percentage of cell division and mitotic phases in yeast cells were determined. The production of secreted aspartic proteinase was assessed by bovine serum albumin agar plate method and evaluated at different pH values.
Seven different genes were detected for C. albicans (3), including transcription factors EFG1 and ZAP1, adhesion genes ALS3, YWP1 and HWP1 and aspartic proteinase SAP1 and SAP4 and two genes for S. cerevisiae 43 including adhesion gene FLO11 and aspartic proteinase YPS3. All these genes were quantified in relation to the concentration of the housekeeping genes CEF3 for C. albicans and ACT1 for S. cerevisiae, at different time intervals (90 min, 8 h, 24 h, and 48 h) by quantitative real time PCR (qRT- PCR).
Among the tested seven yeast strains, only four strains were positive for biofilm formation; C. albicans (1), C. albicans (2), C. albicans (3) and S. cerevisiae 43 and three strains were negative; C. albicans (4), C. albicans (5) and S. cerevisiae 44.
C. albicans (1) and C. albicans (3) showed strong biofilm formation and C. albicans (2) formed moderate biofilm. While S. cerevisiae 43 formed weak biofilm.
pH, incubation time and culture media have significant effect on biofilm formation.
Almost positive yeast strains showed marked significant difference in biofilm formation at a wide range of pH from 4 to 8 (0.5 interval). C. albicans (1), C. albicans (2), C. albicans (3) and S. cerevisiae 43 showed optimal biofilm formation at pH 4, 4, 7.5 and 6 respectively.
C. albicans (1), C. albicans (2), C. albicans (3) and S. cerevisiae 43 showed optimal and maximum biofilm formation at 48 h of incubation.
Optimal biofilm formation was observed using SDB broth with 8% glucose for C. albicans (1), C. albicans (2) and C. albicans (3). While S. cerevisiae 43 showed no significant difference in biofilm formation using SDB broth with 8% glucose and RPMI 1640 broth.
The morphological features of C. albicans (3) (strong biofilm formation) and S. cerevisiae 43 (the only S. cerevisiae strain that formed biofilm) grown in SDB at optimum conditions for 90 min, 8 h, 24 h and 48 h were microscopically examined. C. albicans (3) biofilm formation was characterized by yeast with germ tubes after 90 min, pseudohyphae and hyphae after 8 h, yeast and a lot of hyphae after 24 h and formation of a network of biofilm with yeast, pseudohyphae and hyphae with extracellular matrix (ECM) after 48 h. On the other hand, S. cerevisiae 43 formed ovoid budding yeast after 90 min, yeast and elongated yeast after 8 h, yeast and pseudohyphae after 24 h and the biofilm characterized by an increase in yeast and pseudohyphae after 48 h.
Staining of C. albicans (3) and S. cerevisiae 43 chromosomes indicated that cell division occurred in ordinary sequence and most of cells are accumulated at metaphase stage.
All yeast strains were positive for secreted aspartic proteinase (SAP) production. As well, pH has a significant effect on SAP activity of all the tested yeast strains. secreted aspartic proteinase was detected at acidic pH ranged from 4 to 6 with 0.05 interval and showed high SAP activity at pH 4. Surprisingly, S. cerevisiae 44 showed higher SAP activity than C. albicans strains.
In C. albicans (3), the gene expression level of EFG1 exhibited highly significant upregulation at 8 h and a significant increase at 48 h. Maximum expression level of ZAP1 gene was detected at 48 h and its expression level increased by increasing time.
For adhesion genes, 8 h showed the highest expression level for ALS3 and YWP1. ALS3 gene expression level was upregulated at 8 h and gradually decreased at other time intervals. While YWP1 gene expression level was significantly upregulated at 8 h and gradually downregulated by increasing time. However, HWP1 gene expression level showed gradual decrease and its expression level was less than the expression level of control.
For SAP genes; SAP1 and SAP4, 8 h showed the highest expression level. SAP1 gene expression level was significantly increased at 8 h and gradually decreased by increasing time. While the expression level of SAP4 was positively regulated at 8 and 48 h.
In S. cerevisiae 43, the expression level of adhesion gene FLO11 was upregulated at 8 h and a significant gradual downregulation at 24 and 48 h was observed. While the expression level of YPS3 was upregulated at 8 and 48 h.
These results indicate that there is a relationship between the transition from simple yeast budding form to a complex filament structure and gene expression time of their related genes. Moreover, time management of genetic control system is very important to control virulence factors at definite time. Furthermore, temporal gene expression level of commensal S. cerevisiae occurred in the same way that occurred in pathogenic C. albicans and it may take the pathogenesis line.