Molecular Cloning And Characterization Of Candida Albicans CaBEM1, CaSRB9, CZF2 And CaFLO11 Genes

Candida albicans is the most frequently isolated fungal pathogen in humans. Thepathogenic fungus Candida albicans has a dimorphic transition in various environmentalconditions. Many regulatory factors and several transduction pathways have beenidentified in controlling filamentous growth. G1 cyclins Cln1 and Cln2 have been reportedas involved in the control of morphogenesis in Saccharomyces cerevisiae. Diploidcln1/cln1 cln2/cln2 strains completely lost the ability to form pseudohyphae. A C.albicans genomic DNA library was introduced into cln1/cln1 cln2/cln2 mutant to screengenes which could complement its filamentous growth defect. In this screening, a BEM1homolog, CaBEM1, was identified. The CaBEM1 gene has an ORF of 1899 bp, encodinga putative protein of 632 amino acids. The CaBem1 protein shares highest homology inamino acids with Bem1(38%) of S. cerevisiae and Scd2(32%) of Schizosaccharomycespombe. Sequence analysis showed that the CaBem1 contains two N-terminal SH3domains, a PX domain and a C-terminal PB1 domain. It is believed these domains arerequired for binding to proteins involved in polarized growth in S. cerevisiae and S. pombe.Ectopic expression of the CaBEM1 gene in diploid S. cerevisiae suppressed defects infilamentous growth of some mutants under nitrogen starvation conditions. Thissuppression bypassed MAPK pathway and cAMP/PKA pathway in filamentous growth.These results suggest that the CaBem1 protein may be a downstream component of thesetwo signal transduction pathways for filaments formation.Flo8 plays an important role in morphogenesis of Saccharomyces cerevisiae. In thiswork, a C. albicans genomic DNA library was introduced into an S. cerevisiae flo8/flo8mutant to screen genes which could complement its invasive growth defect. In thisscreening, 14 novel genes were isolated. One gene shares highest similarity in amino acids(38%) with Srb9 of S. cerevisiae and designated CaSRB9 (Candida albicans SRB9 gene).The deduced translation production of another gene is belong to a GAL4 fugaltranscription regulatory protein family, which was named CZF2 ( Candida albicans zincfinger protein). The third gene encoding a cell wall protein was designated CaFLO11.The CaSRB9 gene has an ORF of 4998 bp, encoding a predicted protein of 1665amino acids. Ectopic expression of the CaSRB9 gene in diploid S. cerevisiae suppresseddefect in filamentous growth of some mutants in filamentation MAPK pathway (ste7/ste7,ste12/ste12, and tec1/tec1) and flo8/flo8 mutant under nitrogen starvation conditions. Inhaploid S. cerevisiae, ectopic expressed CaSrb9 complemented the invasive growth defectof flo8 mutant but failed to complement the invasive growth defects of the mutants infilamentation MAPK pathway.The CZF2 gene has an ORF of 1875 bp, encoding a deduced protein of 624 aminoacids. Czf2 has a N-terminal conserved Zn2Cys6 zinc cluster motif. The region forms abinuclear zinc cluster, in which two Zn atoms are bound by six Cys residues . Ectopicexpression of the CZF2 gene in diploid S. cerevisiae partially suppressed defects infilamentous growth of the mutants in filamentation MAPK pathway and flo8/flo8 mutantunder nitrogen starvation conditions. In haploid S. cerevisiae, ectopic expressed Czf2complemented the invasive growth defects of the mutants in filamentation MAPKpathway and flo8 mutant. The deletion of CZF2 gene in C. albicans caused slightly defectin hyphal formation. czf2/czf2 mutant strain grown in YPD medium formed elongatedcells. These results suggested CZF2 contributed to C. albicans morphogenesis. In asystemic mouse model, czf2/czf2 mutant strains showed reduced virulence comparingwith wild type strains..The CaFLO11 gene has an ORF of 1962 bp, encoding a hypothetic protein of 653amino acids. CaFlo11 is similar in overall structure to the class of yeast serine/threoninerich GPI-anchored cell wall proteins. An N-terminal domain containing a signal sequence