Comparative Study On ERG11 Sequence And The Activity Of Extracelluar Hydrolases Between The Hyphal Form And The Yeast Form Of Candida Albicans

1 ObjectivesTo compare the differences on ERG11 gene and the activity of extracelluar hydrolases between the hyphal form and yeast form of C. albicans, and to provide theoretical evidences for selecting the suitable form of C. albicans in medical mycology researches in vitro.2 MethodsBoth the hyphal form and yeast form of the sixteen C. albicans strains, CA-1, CA-2,……, CA-9, CA-11, CA-12,……, CA-17, were cultured and the genomic DNA were extracted. One set of primers was designed to amplify ERG11 gene, and the PCR products were digested after purification by AceⅠ, Mun I and AflⅡ, respectively, then the RFLP fingerprints between the two forms were compared after agarose gel electrophoresis. Furthermore, the PCR products were sequenced by using the downstream primer. Bovine serum albumin(BSA) and egg yolk agar medium(EYM) were used respectively to evaluate the secreted aspartyl proteinase activity(PA value) and phospholipase activity(PZ value) between the dimorphic C. albicans. The PA and PZ value of two forms, which determined as the ratio of colony diameter alone to the colony with the diameter of clearance zone on BSA or white precipitation zone on EYM, were analyzed statistically by paired t test.3 Results3.1 There were differences on ERG11 gene between the hyphal form and yeast form of C. albicans in strains CA-7, CA-8, CA-14, CA-16 and CA-17 on both PCR-RFLP fingerprints and DNA sequencing, and no differences were found between the two forms in the other strains (CA-1, CA-2,……, CA-6, CA-9, CA-11, CA-12, CA-13, CA-15).3.2 Differences on ERGll gene between the two forms of C. albicans were identified in strains CA-7, CA-8, CA-14, CA-16, CA-17 at position 1547, 1587 and 1617, which the position numbers were according to the ERG11 gene sequences in genebank (accession number: X13296).3.3 Differences on the activity of secreted aspartyl proteinase between the hyphal form and yeast form of C. albicans were statistically significant(t=4.980, P=0.000), and the activity in hyphal form was more powerful than that of yeast form(the former average PA value was 0.464 and the latter was 0.547).3.4 Differences on the activity ofphospholipase between the hyphal form and yeast form of C. albicans were statistically significant(t=5.867, P=0.000), and the activity in hyphal form was more powerful than that of yeast form(the former average PZ value was 0.543 and the latter was 0.606).4 Conclusion4.1 There are differences on ERG11 gene between hyphal form and yeast form of C. albicans.4.2 There are differences on the activity of secreted aspartyl proteinase and phospholipase between hyphal form and yeast form of C. albicans.4.3 To combine the former research which refer to the C. albicans hypha is more virulent and important than yeast in its pathogenity in vivo and the differences between the two forms in our study together, we think that hyphal form of C. albicans is more appropriate to reflect the real condition in vivo when studying on C. albicans in medical mycology researches in vitro.