Novel Regulatory Function for the CCAAT-Binding Factor in Candida albicans

ABSTRACT Candida albicans is an opportunistic human pathogen that can sense environmental changes and respond by altering its cell morphology and physiology. A number of environmental factors have been shown to influence this dimorphic transition, including pH, starvation, serum, and amino acids. In this report, we investigate the function of the C. albicans CCAAT-binding factor. In Saccharomyces cerevisiae, this heterooligomeric transcriptional activator stimulates the expression of genes that encode proteins involved in respiration. To examine the function of this transcription factor in C. albicans, we cloned CaHAP5 and generated a hap5Δ/hap5Δ mutant of C. albicans. Using mobility shift studies, we identified four separate complexes from C. albicans cell extracts whose DNA-binding activities were abolished in the hap5Δ/hap5Δ mutant, suggesting that they represented sequence-specific CCAAT-binding complexes. We found that the C. albicans hap5Δ homozygote was defective in hyphal development under a variety of conditions, and the mutant displayed a carbon source-dependent “hyperfilamentation” phenotype under certain growth conditions. In addition, the mRNA levels for two enzymes involved in respiration, encoded by COX5 and CYC1, were overexpressed in the hap5Δ/hap5Δ mutant when grown in medium containing amino acids as the sole carbon and nitrogen source. Thus, the C. albicans CCAAT-binding factor appeared to function as a repressor of genes encoding mitochondrial electron transport components, in contrast to its activator function in S. cerevisiae. These data provide the first evidence that the CCAAT-binding factor can act as a transcriptional repressor and raise new and interesting questions about how carbon metabolism is regulated in this opportunistic human pathogen.

[1]  T. Petit,et al.  Carbohydrate and energy-yielding metabolism in non-conventional yeasts. , 2000, FEMS microbiology reviews.

[2]  André Boorsma,et al.  Hap4p overexpression in glucose-grown Saccharomyces cerevisiae induces cells to enter a novel metabolic state , 2002, Genome Biology.

[3]  L. Alberghina,et al.  Evolutionary origin of nonuniversal CUGSer codon in some Candida species as inferred from a molecular phylogeny. , 1995, Genetics.

[4]  A. Rich,et al.  Subunit interaction in the CCAAT-binding heteromeric complex is mediated by a very short alpha-helix in HAP2. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[5]  L. Guarente,et al.  Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site , 1988, Molecular and cellular biology.

[6]  R. Tjian,et al.  Analysis of Sp1 in vivo reveals mutiple transcriptional domains, including a novel glutamine-rich activation motif , 1988, Cell.

[7]  P. Kozinn,et al.  Candida and candidiasis. , 1971, JAMA.

[8]  W. Fonzi,et al.  PHR2 of Candida albicans encodes a functional homolog of the pH-regulated gene PHR1 with an inverted pattern of pH-dependent expression , 1997, Molecular and cellular biology.

[9]  K. Struhl,et al.  The glutamine-rich activation domains of human Sp1 do not stimulate transcription in Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[10]  D. Landsman,et al.  A variety of DNA-binding and multimeric proteins contain the histone fold motif. , 1995, Nucleic acids research.

[11]  A. DeLuna,et al.  GDH1 expression is regulated by GLN3, GCN4, and HAP4 under respiratory growth. , 2002, Biochemical and biophysical research communications.

[12]  R. A. Butow,et al.  A Transcriptional Switch in the Expression of Yeast Tricarboxylic Acid Cycle Genes in Response to a Reduction or Loss of Respiratory Function , 1999, Molecular and Cellular Biology.

[13]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[14]  C. Lowry,et al.  Regulation of gene expression by oxygen in Saccharomyces cerevisiae. , 1992, Microbiological reviews.

[15]  D. McNabb,et al.  The Saccharomyces cerevisiae Hap5p homolog from fission yeast reveals two conserved domains that are essential for assembly of heterotetrameric CCAAT-binding factor , 1997, Molecular and cellular biology.

[16]  J. Boeke,et al.  Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications , 1998, Yeast.

[17]  W. Scheld,et al.  Brief Review of Fungal Infections , 1990, Pharmacotherapy.

[18]  A. Myers,et al.  Yeast/E. coli shuttle vectors with multiple unique restriction sites , 1986, Yeast.

[19]  P. Brown,et al.  Exploring the metabolic and genetic control of gene expression on a genomic scale. , 1997, Science.

[20]  A. Willems,et al.  Studies on the transformation of intact yeast cells by the LiAc/SS‐DNA/PEG procedure , 1995, Yeast.

[21]  C. Tonelli,et al.  Regulation of the CCAAT-Binding NF-Y subunits in Arabidopsis thaliana. , 2001, Gene.

[22]  Christophe Romier,et al.  The NF-YB/NF-YC Structure Gives Insight into DNA Binding and Transcription Regulation by CCAAT Factor NF-Y* , 2003, The Journal of Biological Chemistry.

[23]  L. Guarente,et al.  The HAP3 regulatory locus of Saccharomyces cerevisiae encodes divergent overlapping transcripts , 1988, Molecular and cellular biology.

[24]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[25]  C. Birse,et al.  PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis , 1995, Molecular and cellular biology.

[26]  L. Guarente,et al.  The HAP2 subunit of yeast CCAAT transcriptional activator contains adjacent domains for subunit association and DNA recognition: model for the HAP2/3/4 complex. , 1990, Genes & development.

[27]  D. McNabb,et al.  Cloning of yeast HAP5: a novel subunit of a heterotrimeric complex required for CCAAT binding. , 1995, Genes & development.

[28]  R. Mantovani,et al.  The molecular biology of the CCAAT-binding factor NF-Y. , 1999, Gene.

[29]  J. Fox.  Fungal infection rates are increasing , 1993 .

[30]  F. Odds,et al.  Candida and candidosis , 1979 .

[31]  M. Bolotin-Fukuhara,et al.  A new Hansenula polymorpha HAP4 homologue which contains only the N-terminal conserved domain of the protein is fully functional in Saccharomyces cerevisiae , 2005, Current Genetics.

[32]  L. Guarente,et al.  Sequence and nuclear localization of the Saccharomyces cerevisiae HAP2 protein, a transcriptional activator , 1987, Molecular and cellular biology.

[33]  L. Guarente,et al.  Communication between mitochondria and the nucleus in regulation of cytochrome genes in the yeast Saccharomyces cerevisiae. , 1989, Annual review of cell biology.

[34]  G. Fink,et al.  Life and Death in a Macrophage: Role of the Glyoxylate Cycle in Virulence , 2002, Eukaryotic Cell.

[35]  M. Bolotin-Fukuhara,et al.  HAP4, the glucose‐repressed regulated subunit of the HAP transcriptional complex involved in the fermentation–respiration shift, has a functional homologue in the respiratory yeast Kluyveromyces lactis , 1999, Molecular microbiology.

[36]  R. Stjernholm,et al.  Factors affecting filamentation in Candida albicans: changes in respiratory activity of Candida albicans during filamentation , 1975, Infection and immunity.

[37]  A. Mitchell,et al.  RIM101-Dependent and -Independent Pathways Govern pH Responses in Candida albicans , 2000, Molecular and Cellular Biology.

[38]  G. Fink,et al.  Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog. , 1994, Science.

[39]  L. Guarente,et al.  Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer. , 1989, Genes & development.

[40]  G. Braus,et al.  Three classes of mammalian transcription activation domain stimulate transcription in Schizosaccharomyces pombe , 1997, The EMBO journal.

[41]  F. Winston,et al.  A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. , 1987, Gene.

[42]  R. Mantovani,et al.  A survey of 178 NF-Y binding CCAAT boxes. , 1998, Nucleic acids research.

[43]  P. Bucher.  Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences. , 1990, Journal of molecular biology.

[44]  L. Guarente,et al.  Yeast HAP2 and HAP3: transcriptional activators in a heteromeric complex. , 1988, Science.

[45]  George Newport,et al.  Regulatory networks affected by iron availability in Candida albicans , 2004, Molecular microbiology.

[46]  K. L. Lee,et al.  An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida Albicans. , 1975, Sabouraudia.

[47]  M. Bolotin-Fukuhara,et al.  The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms , 1996, Journal of bacteriology.

[48]  M. Davis,et al.  HAP-Like CCAAT-binding complexes in filamentous fungi: implications for biotechnology. , 1999, Fungal genetics and biology : FG & B.

[49]  Janina Maier,et al.  Guide to yeast genetics and molecular biology. , 1991, Methods in enzymology.

[50]  Y. Xing,et al.  Mutations in yeast HAP2/HAP3 define a hybrid CCAAT box binding domain. , 1993, The EMBO journal.

[51]  P. Mukherjee,et al.  Purified arginine permease of Candida albicans is functionally active in a reconstituted system , 1998, Yeast.

[52]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[53]  W. Fonzi,et al.  Effect of Environmental pH on Morphological Development of Candida albicans Is Mediated via the PacC-Related Transcription Factor Encoded by PRR2 , 1999, Journal of bacteriology.

[54]  H. Chen,et al.  The Neurospora aab-1 gene encodes a CCAAT binding protein homologous to yeast HAP5. , 1998, Genetics.

[55]  A. Mitchell,et al.  Rapid Hypothesis Testing with Candida albicans through Gene Disruption with Short Homology Regions , 1999, Journal of bacteriology.