Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dignard, D. Articles by Whiteway, M. Search for Related Content PubMed PubMed Citation Articles by Dignard, D. Articles by Whiteway, M.

 Previous Article  |  Next Article 

Eukaryotic Cell, March 2007, p. 487-494, Vol. 6, No. 3
1535-9778/07/$08.00+0     doi:10.1128/EC.00387-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of MFA1, the Gene Encoding Candida albicans a-Factor Pheromone

Daniel Dignard,^1* Ahmed L. El-Naggar,² Mary E. Logue,³ Geraldine Butler,³ and Malcolm Whiteway¹

NRC Biotechnology Research Institute, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada,¹ Desert Research Center, 1 Mathaf El-Matariya Street, El-Matariya, P.O. Box 11753, Cairo, Egypt,² School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland³

Received 7 December 2006/ Accepted 18 December 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the opaque state, MTLa and MTL strains of Candida albicans are able to mate, and this mating is directed by a pheromone-mediated signaling process. We have used comparisons of genome sequences to identify a C. albicans gene encoding a candidate a-specific mating factor. This gene is conserved in Candida dubliniensis and is similar to a three-gene family in the related fungus Candida parapsilosis but has extremely limited similarity to the Saccharomyces cerevisiae MFA1 (ScMFA1) and ScMFA2 genes. All these genes encode C-terminal CAAX box motifs characteristic of prenylated proteins. The C. albicans gene, designated CaMFA1, is found on chromosome 2 between ORF19.2165 and ORF19.2219. MFA1 encodes an open reading frame of 42 amino acids that is predicted to be processed to a 14-amino-acid prenylated mature pheromone. Microarray analysis shows that MFA1 is poorly expressed in opaque MTLa cells but is induced when the cells are treated with -factor. Disruption of this C. albicans gene blocks the mating of MTLa cells but not MTL cells, while the reintegration of the gene suppresses this cell-type-specific mating defect.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recently, it has been established that opaque Candida albicans cells of opposite mating types mate efficiently (32). In other fungi, the mating process is coordinated through the action of cell-type-specific pheromones that are processed from precursor proteins (9, 34). The involvement of the Kex2 endopeptidase in the mating ability of MTL cells predicted a requirement for dibasic processing sites, which, in other systems, were involved in pheromone production (30). The observation that other Kex2-like protease-processed pheromones had multiple repeats of a short peptide sequence focused the search for genes encoding potential precursor proteins with C-terminal repetitive regions flanked by dibasic residues in C. albicans.

This approach (6, 26, 28, 38) established that C. albicans contains a single -factor gene, which encoded a candidate precursor protein containing three identical tridecapeptides, although the details of the processing have not been fully worked out. Overall, the mature pheromone is predicted to be generated in a fashion similar to that of -factor in Saccharomyces cerevisiae, through the combined activity of the Kex2 protease (22, 36) and the Ste13 dipeptidyl aminopeptidase A (2, 21). The requirement for a carboxypeptidase step (18) has not been confirmed in C. albicans. In the pathogen, MTL cells with a deletion of the MF1 gene are sterile, whereas the exposure of opaque a cells to synthetic -factor peptide results in the formation of shmoos and the induction of expression of genes involved in mating and pathogenesis (6, 28, 38).

In S. cerevisiae, two genes (S. cerevisiae MFA1 [ScMFA1] and ScMFA2) encode precursors that are acted on to make an a-cell-specific pheromone, termed a-factor. Similar 12-amino-acid mature peptides are generated through a series of processing events, including the modification of a C-terminal cysteine residue by the addition of a farnesyl moiety and a carboxyl methyl group, proteolytic removal of the three terminal amino acids, and three proteolysis steps at the N terminus (13). A final step in the production of the pheromone is export through the action of the Ste6p pump (31). Thus, the a- and -specific pheromones of yeast are generated through very different processing steps. This pattern of one pheromone being a prenylated peptide (a-factor) while the other pheromone is a simple peptide (-factor) is characteristic of ascomycetes (1); basidiomycetes typically have the pheromones from all the different mating types of the lipid-modified class (10).

C. albicans contains candidate genes for all the functions that are predicted to be essential for the processing of a lipid-modified peptide pheromone. These include the genes encoding the homologs of the prenylation enzymes Ram1p and Ram2p (ORF19.5046 and ORF19.4817), a carboxymethylase homolog encoded by ORF19.119, a candidate CAAX box protease encoded by ORF19.3825, homologs of other processing proteases encoded by ORF19.5654 and ORF19.7342, and, finally, an Ste6p-like transporter encoded by the HST6 gene, which has been shown to be essential for a-specific mating in C. albicans (30). These observations are therefore consistent with C. albicans producing a prenylated a-specific pheromone. However, the identification of the a-factor-encoding gene has been difficult. Many small open reading frames (ORFs) with the potential to be prenylated at the C terminus exist in the genome, and systematic analysis of these candidates would be a time-consuming process. We introduced oligonucleotides representing 80 candidate genes on microarrays to search for sequences that were induced by -factor, but none of the candidates were pheromone responsive. We have therefore made use of the recently established genome sequences of other Candida species to focus on candidate sequences that are common among the related organisms. This approach identified a potential a-factor precursor gene that we have designated C. albicans MFA1 (CaMFA1).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bioinformatics. The genome sequence of C. albicans (assembly 19) was downloaded from the Candida Genome Database (http://www.candidagenome.org/), while the Candida dubliniensis genome sequence (19 January 2006 release) was obtained from the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/sequencing/Candida/dubliniensis/). Each contig was translated in six frames, and all potential ORFs between 26 and 46 amino acids with a cysteine residue at the fourth position from the C terminus were identified. Sequences that were contained within larger ORFs were eliminated using TBLASTN searches against the C. albicans haploid coding sequences and a set of predicted C. dubliniensis coding sequences. The remaining short ORFs (1,004 in C. albicans and 1,211 in C. dubliniensis) were compared to find potential orthologs between the two species. One candidate (42 amino acids) was identical at the protein level in C. albicans and C. dubliniensis. The sequence contains a putative CAAX-like motif (CSVM) at the C terminus. Candidate MFA sequences in Candida tropicalis and Candida parapsilosis were identified with TBLASTN using the C. albicans sequence as a query and then, for C. tropicalis, using the first C. tropicalis sequence identified. Putative MFA sequences in Candida (Clavispora) lusitaniae and Candida (Pichia) guilliermondii were also determined by translating the genomic DNA in six frames. The K_a (number of nonsynonymous base changes) and K_s (number of synonymous base changes) values were determined by using the yn00 program, which is part of the PAML (44) package (http://abacus.gene.ucl.ac.uk/software/paml.html).

Strains. The strains used in this work are described in Table 1. The MFA1 gene was deleted from MTLa strain 3294 and from MTL strain 3740 as described below. For general propagation and maintenance conditions, the strains were cultured at 30°C in yeast-peptone-dextrose (YPD) medium supplemented with uridine (2% Bacto peptone, 1% yeast extract, 2% dextrose, and 50 µg/ml uridine, with the addition of 2% agar for solid medium). For transformation, the one-step lithium acetate protocol (12) was used with the modification that cells were incubated overnight at 30°C with the transforming DNA before plating. The opaque derivative of the strains was obtained after selection on phloxine B plates as described previously (17). For maintenance and experiments with opaque cells, the strains were grown at 24°C in supplemented synthetic dextrose (SD) medium as described previously (17). Cells from fresh opaque colonies were used in each experiment and were obtained after restreaking the opaque strain from a –80°C glycerol stock onto a phloxine B plate with incubation at 24°C; the quality was confirmed by microscopy for the typical elongated cell morphology of the opaque cells.

View this table: [in this window] [in a new window]

TABLE 1. Candida albicans strains used in this work

Plasmids and disruptions. Plasmid pBS-cHIS1 was used as the template to prepare the HIS1-PCR cassette, and plasmid pBS-cURA3 was used to prepare the URA3-PCR cassette as previously described (17). They both have a common DNA backbone, allowing the same oligonucleotide pair to be used for the amplification of the two PCR cassettes. The oligonucleotides used in this work are listed and described in Table 2. The MFA1 gene was deleted in two steps. In the first step, one allele was replaced by homologous recombination with a PCR cassette containing the HIS1 gene. Two chimeric oligonucleotides, AH1 and AH2, were composed of 80 nucleotides (nt) from the C. albicans genome at the MFA1 locus (contigs 19 to 10141, nt 5889 to 5968, for AH1 and contigs 19 to 10141, nt 6548 to 6628, for AH2) with a 20-nt addition corresponding to the vector sequence of the plasmid. Oligonucleotides AH1 and AH2 were used on plasmid pBS-cHIS1 to prepare the HIS1-PCR cassette, strain 3294 was transformed with the PCR fragment, and the cells were plated onto plates with SD medium minus histidine. Genomic DNA of the positive colonies was analyzed by PCR for the proper integration site of the cassette using oligonucleotides A plus H2 and B plus H1. The first allele deletion was confirmed with oligonucleotides A and B for the presence of both a 1.7-kb fragment corresponding to the integrated HIS1 and the 0.97-kb fragment for the wild-type allele. The strain deleted for a single MFA1 allele was named CA78. For the second allele disruption, oligonucleotides AH1 and AH2 were used to prepare the URA3-PCR cassette from vector pBS-cURA, strain CA78 was transformed with the PCR fragment, and the transformants were selected on plates with SD medium lacking histidine and uridine. The colonies were analyzed by PCR with oligonucleotides A and U2 and with oligonucleotides B and U1 to identify the proper integration site. The complete deletion of the MFA1 gene was confirmed by PCR, with oligonucleotides A and B generating only the larger 1.7-kb fragment (the HIS1 and URA3 cassettes used for the disruption are not resolved) and not the smaller 0.97-kb wild-type fragment. The absence of the wild-type 0.55-kb PCR fragment with oligonucleotides C and D, internal to the recombination sites, as well as the lack of PCR amplification with oligonucleotides A and D and B and C further confirmed the complete deletion of the MFA1 gene. The null mutant mfa1 strain was named CA83. This strain was also treated with 5-fluoroorotic acid, and the resulting mfa1 uridine-negative derivative strain was named CA84 and was used as the recipient strain for the reintegration of the MFA1 gene as described below. An identical disruption strategy was used to delete the MFA1 gene from MTL strain 3740. The strain deleted for a single MFA1 allele was named AE3. This strain was used for the second-step disruption, and the resulting null mutant mfa1 strain was named EL2. Strains CA83 and EL2 were also analyzed by PCR to confirm their corresponding mating types. Specific oligonucleotides for MTLa (a1-f with a1-r) and for MTL (alpha-f with alpha-r) were used for the PCR to confirm the mating types of the strains.

View this table: [in this window] [in a new window]

TABLE 2. Oligonucleotides used in this worka

Reintegration. For complementation experiments, the MFA1 gene was reintegrated into the null mutant mfa1 strain CA84. The wild-type MFA1 gene was amplified from genomic DNA from strain 3294 using oligonucleotides A and AH7 (Table 2) and Expand High Fidelity polymerase (Roche). The PCR fragment was digested with restriction enzymes BclI and XbaI. The BclI site is located about 12 nt downstream of oligonucleotide A, and the XbaI site is created from the tail of oligonucleotide AH7. The digested PCR fragment was purified on a column (QIAGEN) and cloned into the BamHI and XbaI sites of the CIp10 vector (33). The plasmid was sequenced to confirm the integrity of the MFA1 gene and was named CIp10MFA1. Plasmid CIp10MFA1 was digested with the StuI restriction enzyme and used to transform strain CA84. The uridine-positive colonies were analyzed by PCR with oligonucleotides C and D, and the presence of the 0.55-kb wild-type fragment confirmed the reintegration of the MFA1 gene. This reintegrated MFA1 strain was named CA96.

Mating. Mating was performed by auxotrophic marker complementation in patch mating experiments as described previously (17). In summary, opaque colonies from the assayed strains and from the tester strains were streaked separately across a YPD plate and incubated for 1 day at 24°C. The cells were transferred by replica plating and crossed together on a single plate for 1 day at 24°C before a last replica plating onto dropout plates containing SD medium minus five amino acids (uridine, lysine, histidine, arginine, tryptophan, and lysine) for the selection of the mating products and onto a YPD plate for control and reference. The plates were incubated at 24°C.

Microarray experiments. The Candida albicans microarrays were obtained from the BRI Microarray Facility (http://www.bri.nrc.gc.ca/services/microarray/scanning_e.html); they are printed with 70-mer oligonucleotide probes for each of the 6,354 ORFs of the annotated C. albicans genome to which a 70-mer oligonucleotide CaMFA probe (Table 2) corresponding to the MFA1 gene was added. Standard methods were used for the RNA isolation, cDNA construction, and DNA microarray hybridization and were described previously (17). In summary, the cDNAs were prepared from poly(A)⁺ mRNA enriched from total RNA extracted with the hot-phenol-plus-glass-beads method (17). The cells were grown in liquid SD medium at 24°C to an optical density at 600 nm (OD₆₀₀) of 0.8. For the induction with -factor peptide (38), cells were grown to an OD₆₀₀ of 0.5 before the addition of the peptide to the culture at a final concentration of 1 µg/ml for about 2 h to reach an OD₆₀₀ of 0.8.

Top Abstract Introduction Materials and Methods Results Discussion References

 
a-Factor sequences in yeasts are poorly conserved, but many, such as the Schizosaccharomyces pombe M-factor (16) and the a1 and a2 factors of Ustilago maydis (8), contain a terminal CAAX motif that acts as a recognition site for isoprenylation. Proteins modified at this motif tend to be localized to the membrane. However, even within recognized CAAX motifs, the internal A residues are not always aliphatic amino acids. We therefore searched the C. albicans genome for short open reading frames with a cysteine four residues from the C terminus. More than 1,000 putative ORFs were identified, most of which are likely to be spurious. We predicted that any true MFA gene would be conserved in Candida dubliniensis, a close relative of C. albicans with a functionally related pheromone, given that C. albicans and C. dubliniensis can mate (40). We therefore performed the same analysis using the C. dubliniensis genome sequence. One candidate predicted protein was identical in sequence and synteny in the two species. It is 42 amino acids long and contains a potential farnesylation site at the C terminus (CSVM) (Fig. 1). We named this gene CaMFA1 (in C. albicans) and CdMFA1 (in C. dubliniensis). In both organisms, the gene is found on chromosome 2; in Sc5314, it is between ORF19.2165 and an -factor-inducible reverse transcriptase gene encoded by ORF19.2219 that appears to be part of a transposon-like structure found in Sc5314 but that is not present in C. albicans strain WO-1 or in C. dubliniensis.

View larger version (7K): [in this window] [in a new window]

FIG. 1. Structure of predicted MFA genes from C. albicans and C. dubliniensis. The nucleotide sequence and predicted amino acid sequence of the a-factor precursors are shown. A putative CAAX motif is shown, and the predicted mature peptide is highlighted in gray. A potential cleavage site for the metalloprotease Axl1p is indicated.

In S. cerevisiae, the N terminus of the a-factor precursor is removed following three processing events carried out by the Ste24p and Axl1p proteases (13). These proteins are conserved in C. albicans, together with the prenyl-group-adding proteins Ram1p and Ram2p, the prenyl-dependent protease Rce1p, and the farnesyl cysteine-carboxyl methyltransferase Ste14p. The final N-terminal cleavage in S. cerevisiae follows an asparagine residue in the sequence KDN, generating the 12-amino-acid mature peptide. The putative Mfa proteins from C. albicans and C. dubliniensis contain the sequence KNN at a similar position, and, if processed in the same way as the S. cerevisiae pheromone, CaMFA1 would produce a mature peptide of 14 amino acids (Fig. 1).

While the S. cerevisiae genome contains two MFA genes, only one was identified in both C. albicans and C. dubliniensis. These species are closely related to two additional pathogenic Candida species, C. tropicalis and C. parapsilosis. C. tropicalis is likely to be asexual or parasexual, similarly to C. albicans. The mating locus in C. parapsilosis is defective, suggesting that it is unlikely to mate (29). In contrast, the related species Candida guilliermondii and Candida lusitaniae are likely to have fully functional mating pathways (45). The genome of C. parapsilosis is currently being sequenced by the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/sequencing/Candida/parapsilosis/), and the genomes of C. tropicalis, C. guilliermondii, and C. lusitaniae are being sequenced by the Broad Institute of the Massachusetts Institute of Technology and Harvard (http://www.broad.mit.edu/annotation/fgi/). We therefore looked for possible homologs of CaMFA1 in these species (Fig. 2). C. guilliermondii contains 1 putative gene, C. lusitaniae contains 2, C. parapsilosis contains 3, and, surprisingly, C. tropicalis has up to 12. The predicted mature peptides from C. parapsilosis are identical, and those from C. lusitaniae differ at one amino acid, whereas there are several differences among the predicted peptides from C. tropicalis, including substitutions in the terminal CAAX motif (Fig. 2).

View larger version (47K): [in this window] [in a new window]

FIG. 2. Alignment of putative MFA sequences from Candida species. The alignment of the precursor proteins was carried out using T-Coffee (37). Boxes were drawn using Boxshade (http://www.ch.embnet.org/software/BOX_form.html). Only three of the potential peptides from C. tropicalis are included.

It is very difficult to identify biologically relevant short open reading frames, particularly where the between-species conservation is low. We therefore determined the ratio between the K_a (number of nonsynonymous base changes per site) and the K_s (number of synonymous base changes per site) for several of the putative MFA peptides (Table 3). A low K_a/K_s ratio suggests that the sequence is functional and conserved during evolution. For known MFA peptides from S. cerevisiae, Saccharomyces paradoxus, Kluyveromyces lactis, and Ashbya gossypii, the K_a/K_s ratio is 0.5 or less. Most of the predicted Mfa peptides also have low levels of nonsynonymous nucleotide substitutions in pairwise comparisons. C. tropicalis MFA5 and MFA12 are poorly conserved, and it is difficult to obtain accurate K_a and K_s measurements (not shown). It is therefore possible that these are not translated in vivo.

View this table: [in this window] [in a new window]

TABLE 3. Ka/Ks ratios of the MFA peptides in several yeast species

The putative MFA genes in C. tropicalis are scattered throughout the genome, often on different contigs (not shown). However, the three genes in C. parapsilosis are close together, with a D-Tyr-tRNA^Tyr deacylase coding sequence inserted between MFA2 and MFA3. The protein sequences are identical, and the DNA sequences are very similar; it is very likely that these arose following a duplication event.

We tested directly whether the candidate MFA1 gene of C. albicans was involved in the mating process. Strain 3294, an MTLa/MTLa homozygous strain, was deleted for MFA1 in a two-step process. The first allele was replaced with HIS1, generating strain CA78, and the second allele was replaced with URA3, generating strain CA83, as described in Materials and Methods. As shown in Fig. 3, PCR analysis showed that MFA1 is completely deleted in strain CA83. Following the deletion of MFA1, opaque derivatives of strains 3294 and CA83 were isolated by screening on phloxine B plates. We analyzed the mating ability of the opaque version of strain CA83 using tester strain 3315; in contrast to the opaque versions of starting strain 3294, which was mating competent, the opaque version of the homozygous deletion strain was sterile (Fig. 4). To confirm that this sterility was a specific consequence of the mfa1 deletion, we reintroduced the MFA1 gene into the deleted strain. The MFA1 gene and surrounding sequences were cloned from the genomic DNA by PCR and introduced into plasmid CIp10, as described in Materials and Methods, to create plasmid CIp10MFA1. This plasmid was digested with restriction endonuclease StuI to target it to the RP10 (RPS1) locus (33). Strain CA96, containing the reintegrated MFA1 plasmid, was used to generate an opaque version through phloxine screening, and this opaque strain was able to mate in a manner similar to that of the wild-type strain (Fig. 4). This confirms that the mating defect of mfa1/mfa1 was due to the loss of MFA1 function in the cells.

View larger version (24K): [in this window] [in a new window]

FIG. 3. Disruption of the MFA1 gene. (A) Schematic representation of chromosome 2 at the MFA1 locus. The arrows represent the MFA1 ORF (identified in this study) and its closest flanking ORFs identified in the annotation of the Candida albicans genome. (B) Diagram of the strategy for the deletion of the MFA1 gene in two steps. The thick black bars represent the MFA1 locus, and the white rectangles represent the 80-bp segments used for the homologous recombination of the PCR cassettes containing the HIS1 gene and the URA3 gene, respectively. The small arrows represent the orientation and the relative position of the oligonucleotides used for the PCR analysis of the strains (the MTL locus-specific oligonucleotides are not shown). The diagram is not drawn to scale. (C) Confirmation of the disruption by PCR. Strain designations, labeled in white over the top part of the agarose gel, are as follows: wt, 3294, the parent MTLa (lanes 1, 6, 11, 14, 17, and 20); 1, CA78, single-allele disruption (lanes 2, 7, 12, 15, 18, and 21); , CA83, the complete mfa1 deletion (lanes 3, 8, 13, 16, 19, 22, 23, and 25); , EL2, deletion of mfa1 from the MTL strain (lanes 4, 9, 24, and 26); R, CA96, the reintegrated MFA1 gene (lanes 5 and 10); L, the molecular size reference (1-kb DNA ladder). The oligonucleotides used for PCR are identified in black by their respective designations above the gel. For details, see Materials and Methods.

View larger version (51K): [in this window] [in a new window]

FIG. 4. Mating of the mfa1 strains. Mating was assayed by auxotrophic marker complementation. Opaque colonies were streaked onto plates and then crossed for 24 h with tester strains 3315 and 3745 before replicating to a selection plate. The masterplate is shown at a reduced scale in the lower part of the figure. No colonies are detectable for mfa1 strains CA83 and CA84 after 4 days of incubation, while MFA1-reintegrated strain CA96 reverts the sterility. mfa1 MTL strain EL2 has mating similar to that of wild-type (wt) strain 3740, so the sterile phenotype of mfa1 is MTLa specific. The opaque cells from the mfa1 MTLa strains used in the experiment are similar to the cells of wild-type parent strain 3294, as shown by the microscopy pictures at the right side (magnification, x630), and are distinct from the white versions (data not shown).

To establish if the MFA1 gene was essential for mating in an MTL strain, we created strain EL2, a homozygous disruption of MFA1, in MTL/MTL strain 3740 (Fig. 3). An opaque version of strain EL2 was fully mating competent, showing that MFA1 was required for mating only in MTLa strains and not in MTL strains (Fig. 4).

We used microarray analysis to investigate the expression of the MFA1 gene. An oligonucleotide representing the gene was introduced onto our standard profiling arrays. Analysis of the transcription of the MFA1 gene from untreated MTLa cells showed a very low level of expression under standard growth conditions. However, the level of expression was greatly enhanced by the treatment of these cells with the -factor peptide (Fig. 5). This suggests that the production of the a-factor pheromone is enhanced by the treatment of cells with -factor; this type of positive feedback is typical of the pheromone response in other yeasts (41, 43). Using Clonemanager, we examined the upstream region of the MFA1 gene for regulatory sequences that may provide pheromone inducibility. Two repeats of the S. cerevisiae pheromone response element (TGAAACA) that may function in C. albicans as well (28) are found at positions –844 and –828, and complements of the motif directly predicted from C. albicans [TTGTTT(C/G)A] (5) are found at positions –1148 and –63 relative to the start of the gene.

View larger version (58K): [in this window] [in a new window]

FIG. 5. Induction of the MFA1 gene with -factor. DNA microarrays were used for the detection of MFA1 transcripts. cDNA probes were prepared from RNA from opaque cells and labeled with cyanine dyes before cohybridization. The subarrays (position 8.2 in the complete genome array) from two different chip experiments are presented: the array on the left shows a comparison between MTLa and MTL cells; the array on the right side shows a sample from MTLa cells after induction with the -factor peptide for 2 h compared to cells from the same strain without induction. The probes for the MFA1 gene, indicated by the white brackets (columns 11 and 12 in row 14, the second to the last row), are strongly induced when a-factor cells are treated with the -factor pheromone. The probes for the HST6 gene (columns 3 and 4 of the first row), the homolog of the STE6 gene in S. cerevisiae, the ABC transporter required to export the a-factor, has a pattern of induction similar to that of the MFA1 gene. FAV1 (17), another pheromone-inducible gene (columns 17 and 18 of row 4), is also present on this subarray. Independent comparisons of the RNA samples were used to confirm that the cells expressed opaque-specific and not white-specific genes. This slide is representative of two independent RNA preparations from two strains.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pheromone-mediated mating is a common process in fungi. Many Ascomycetes have two mating types that express structurally distinct pheromones: one a simple peptide and the other a prenylated peptide (7, 14, 39, 42). In Basidiomycetes, the structure of the mating locus can be much more complex, and the peptide pheromones are generally of the prenylated peptide class for all the mating types (10). The recent identification of mating in C. albicans has initiated a search for the mating pheromones of the MTLa and MTL cells; the MF1 gene has been predicted (26) and functionally characterized (6, 28, 38), while the a-factor pheromone gene has remained elusive. However, the involvement of a prenylated pheromone produced by MTLa cells was predicted because of the requirement for the Hst6p pump in the mating of MTLa cells (30); this pump is structurally similar to the transmembrane Ste6 protein that exports the prenylated a-factor in S. cerevisiae cells (31).

The recent sequencing of the genomes of other species related to C. albicans has allowed the use of comparative genomics to identify functional elements that are difficult to discern in a single genome. This approach has facilitated the identification of a single candidate for the C. albicans MFA1 gene; this gene predicts an identical processed lipopeptide in both C. albicans and C. dubliniensis (Fig. 2), which is consistent with the observation that these two organisms are able to mate (40). Disruption of this gene causes an MTLa-specific mating defect, while reintegration of the gene restores mating. MFA1 is induced by the treatment of MTLa cells by -factor, and the gene has potential pheromone response elements in the upstream region that are likely critical for this pheromone-mediated inducibility.

The involvement of two distinct processing and export pathways for generating the peptide and prenylated peptide pheromones is an interesting consistency among the Ascomycetes. Although the genes themselves show little similarity among the different organisms, the pattern of a prenylated and nonprenylated molecule is preserved. This distinction is somewhat surprising, since the cells must express unique and complex processing machinery for each pheromone, and thus suggests that the involvement of chemically distinct signaling molecules is optimum for the mating process. One obvious difference between a prenylated and a nonprenylated pheromone is solubility and range in an aqueous environment; the more hydrophilic nonprenylated pheromone will be able to diffuse over a greater distance than the prenylated molecule. If both pheromones were able to signal over long distances, cells might initiate the mating process and exit the mitotic cycle without being able to physically reach their partners, while if both pheromones were insoluble lipopeptides, the potential mating partners would have to be in very close proximity to be able to signal productively. Intriguingly, even in Basidiomycetes, there is an apparent relationship between pheromone solubility and the range of signaling, and both methylation and prenylation can serve as modulators of hydrophobicity (24). It is also interesting that the presence of one pheromone typically stimulates the expression of the other pheromone and that the pheromone-mediated induction of secreted proteases that degrade nonprenylated pheromones is also a typical component of these signaling systems (P. Cote, unpublished data) (5, 11, 25). Thus, a standard mating interaction can be seen to have one partner producing a long-range signaling molecule and the other partner ensuring a productive association by both replying with a short-range signal and demanding an increasing strength of the long-range signal through induced proteolysis.

The identification of the MFA1 gene of C. albicans extends the similarities of the mating systems of S. cerevisiae and the fungal pathogen. Although the process of mating and subsequent meiosis is a well-established component of the life cycle of the budding yeast, the function of mating in the life cycle of C. albicans is less clear. There is no currently identified meiotic process in the pathogen, and reduction from the tetraploid state generated by mating has been attributed to a parasexual process (3). Also, the need to undergo the apparently infrequent event of mitotic homozygosis of the mating locus, followed by the infrequent event of switching to the opaque state, suggests that mating potential in the natural environment could be very low (4, 20). Infrequent genetic exchange is also predicted from the population structure of the organism (19). Clearly, many of the components in the pathway function in other cellular processes such as the control of filamentation (27) and perhaps biofilm formation (15). However, C. albicans maintains the complete complex network of genes involved in the signaling process, suggesting that the mating machinery plays an important role in the biology of the fungus.

 
This work was supported in part by CIHR grant MOP-42516 to M.W., the Egyptian Government to A.L.E.-N., and the Science Foundation Ireland to G.B.

We thank Herve Hogues and Marco van het Hoog for bioinformatics support and Pete Magee and B. B. Magee for strains and interest in the early part of this study.

This is NRC publication number 47551.

 
* Corresponding author. Mailing address: NRC Biotechnology Research Institute, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Phone: (514) 496-6359. Fax: (514) 496-6213. E-mail: daniel.dignard{at}cnrc.gc.ca.

Published ahead of print on 5 January 2007.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Bardwell, L. 2005. A walk-through of the yeast mating pheromone response pathway. Peptides 26:339-350.[CrossRef][Medline]
2
2. Bautista-Munoz, C., C. Hernandez-Rodriguez, and L. Villa-Tanaca. 2005. Analysis and expression of STE13ca gene encoding a putative X-prolyl dipeptidyl aminopeptidase from Candida albicans. FEMS Immunol. Med. Microbiol. 45:459-469.[CrossRef][Medline]
3
3. Bennett, R. J., and A. D. Johnson. 2003. Completion of a parasexual cycle in Candida albicans by induced chromosome loss in tetraploid strains. EMBO J. 22:2505-2515.[CrossRef][Medline]
4
4. Bennett, R. J., and A. D. Johnson. 2005. Mating in Candida albicans and the search for a sexual cycle. Annu. Rev. Microbiol. 59:233-255.[CrossRef][Medline]
5
5. Bennett, R. J., and A. D. Johnson. 2006. The role of nutrient regulation and the Gpa2 protein in the mating pheromone response of C. albicans. Mol. Microbiol. 62:100-119.[CrossRef][Medline]
6
6. Bennett, R. J., M. A. Uhl, M. G. Miller, and A. D. Johnson. 2003. Identification and characterization of a Candida albicans mating pheromone. Mol. Cell. Biol. 23:8189-8201.[Abstract/Free Full Text]
7
7. Bobrowicz, P., R. Pawlak, A. Correa, D. Bell-Pedersen, and D. J. Ebbole. 2002. The Neurospora crassa pheromone precursor genes are regulated by the mating type locus and the circadian clock. Mol. Microbiol. 45:795-804.[CrossRef][Medline]
8
8. Bolker, M., M. Urban, and R. Kahmann. 1992. The a mating type locus of U. maydis specifies cell signaling components. Cell 68:441-450.[CrossRef][Medline]
9
9. Caldwell, G. A., F. Naider, and J. M. Becker. 1995. Fungal lipopeptide mating pheromones: a model system for the study of protein prenylation. Microbiol. Rev. 59:406-422.[Abstract/Free Full Text]
10
10. Casselton, L. A., and N. S. Olesnicky. 1998. Molecular genetics of mating recognition in basidiomycete fungi. Microbiol. Mol. Biol. Rev. 62:55-70.[Abstract/Free Full Text]
11
11. Chan, R. K., and C. A. Otte. 1982. Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G₁ arrest by a factor and factor pheromones. Mol. Cell. Biol. 2:21-29.[Abstract/Free Full Text]
12
12. Chen, D. C., B. C. Yang, and T. T. Kuo. 1992. One-step transformation of yeast in stationary phase. Curr. Genet. 21:83-84.[CrossRef][Medline]
13
13. Chen, P., S. K. Sapperstein, J. D. Choi, and S. Michaelis. 1997. Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor. J. Cell Biol. 136:251-269.[Abstract/Free Full Text]
14
14. Coppin, E., C. de Renty, and R. Debuchy. 2005. The function of the coding sequences for the putative pheromone precursors in Podospora anserina is restricted to fertilization. Eukaryot. Cell 4:407-420.[Abstract/Free Full Text]
15
15. Daniels, K. J., T. Srikantha, S. R. Lockhart, C. Pujol, and D. R. Soll. 2006. Opaque cells signal white cells to form biofilms in Candida albicans. EMBO J. 25:2240-2252.[CrossRef][Medline]
16
16. Davey, J. 1992. Mating pheromones of the fission yeast Schizosaccharomyces pombe: purification and structural characterization of M-factor and isolation and analysis of two genes encoding the pheromone. EMBO J. 11:951-960.[Medline]
17
17. Dignard, D., and M. Whiteway. 2006. SST2, a regulator of G-protein signaling for the Candida albicans mating response pathway. Eukaryot. Cell 5:192-202.[Abstract/Free Full Text]
18
18. Dmochowska, A., D. Dignard, D. Henning, D. Y. Thomas, and H. Bussey. 1987. Yeast KEX1 gene encodes a putative protease with a carboxypeptidase B-like function involved in killer toxin and alpha-factor precursor processing. Cell 50:573-584.[CrossRef][Medline]
19
19. Fundyga, R. E., T. J. Lott, and J. Arnold. 2002. Population structure of Candida albicans, a member of the human flora, as determined by microsatellite loci. Infect. Genet. Evol. 2:57-68.[CrossRef][Medline]
20
20. Johnson, A. 2003. The biology of mating in Candida albicans. Nat. Rev. Microbiol. 1:106-116.[CrossRef][Medline]
21
21. Julius, D., L. Blair, A. Brake, G. Sprague, and J. Thorner. 1983. Yeast alpha factor is processed from a larger precursor polypeptide: the essential role of a membrane-bound dipeptidyl aminopeptidase. Cell 32:839-852.[CrossRef][Medline]
22
22. Julius, D., A. Brake, L. Blair, R. Kunisawa, and J. Thorner. 1984. Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor. Cell 37:1075-1089.[CrossRef][Medline]
23
23. Kakar, S. N., R. M. Partridge, and P. T. Magee. 1983. A genetic analysis of Candida albicans: isolation of a wide variety of auxotrophs and demonstration of linkage and complementation. Genetics 104:241-255.[Abstract/Free Full Text]
24
24. Kosted, P. J., S. A. Gerhardt, C. M. Anderson, A. Stierle, and J. E. Sherwood. 2000. Structural requirements for activity of the pheromones of Ustilago hordei. Fungal Genet. Biol. 29:107-117.[CrossRef][Medline]
25
25. Ladds, G., and J. Davey. 2000. Sxa2 is a serine carboxypeptidase that degrades extracellular P-factor in the fission yeast Schizosaccharomyces pombe. Mol. Microbiol. 36:377-390.[CrossRef][Medline]
26
26. Lan, C. Y., G. Newport, L. A. Murillo, T. Jones, S. Scherer, R. W. Davis, and N. Agabian. 2002. Metabolic specialization associated with phenotypic switching in Candida albicans. Proc. Natl. Acad. Sci. USA 99:14907-14912.[Abstract/Free Full Text]
27
27. Leberer, E., D. Harcus, I. D. Broadbent, K. L. Clark, D. Dignard, K. Ziegelbauer, A. Schmidt, N. A. Gow, A. J. Brown, and D. Y. Thomas. 1996. Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans. Proc. Natl. Acad. Sci. USA 93:13217-13222.[Abstract/Free Full Text]
28
28. Lockhart, S. R., R. Zhao, K. J. Daniels, and D. R. Soll. 2003. -Pheromone-induced "shmooing" and gene regulation require white-opaque switching during Candida albicans mating. Eukaryot. Cell 2:847-855.[Abstract/Free Full Text]
29
29. Logue, M. E., S. Wong, K. H. Wolfe, and G. Butler. 2005. A genome sequence survey shows that the pathogenic yeast Candida parapsilosis has a defective MTLa1 allele at its mating type locus. Eukaryot. Cell 4:1009-1017.[Abstract/Free Full Text]
30
30. Magee, B. B., M. Legrand, A. M. Alarco, M. Raymond, and P. T. Magee. 2002. Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans. Mol. Microbiol. 46:1345-1351.[CrossRef][Medline]
31
31. Michaelis, S. 1993. STE6, the yeast a-factor transporter. Semin. Cell Biol. 4:17-27.[CrossRef][Medline]
32
32. Miller, M. G., and A. D. Johnson. 2002. White-opaque switching in Candida albicans is controlled by mating-type locus homeodomain proteins and allows efficient mating. Cell 110:293-302.[CrossRef][Medline]
33
33. Murad, A. M., P. R. Lee, I. D. Broadbent, C. J. Barelle, and A. J. Brown. 2000. CIp10, an efficient and convenient integrating vector for Candida albicans. Yeast 16:325-327.[CrossRef][Medline]
34
34. Naider, F., and J. M. Becker. 2004. The alpha-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors. Peptides 25:1441-1463.[CrossRef][Medline]
35
35. Negredo, A., L. Monteoliva, C. Gil, J. Pla, and C. Nombela. 1997. Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans. Microbiology 143:297-302.[Abstract/Free Full Text]
36
36. Newport, G., and N. Agabian. 1997. KEX2 influences Candida albicans proteinase secretion and hyphal formation. J. Biol. Chem. 272:28954-28961.[Abstract/Free Full Text]
37
37. Notredame, C., D. G. Higgins, and J. Heringa. 2000. T-Coffee: a novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302:205-217.[CrossRef][Medline]
38
38. Panwar, S. L., M. Legrand, D. Dignard, M. Whiteway, and P. T. Magee. 2003. MF1, the gene encoding the mating pheromone of Candida albicans. Eukaryot. Cell 2:1350-1360.[Abstract/Free Full Text]
39
39. Poggeler, S. 2000. Two pheromone precursor genes are transcriptionally expressed in the homothallic ascomycete Sordaria macrospora. Curr. Genet. 37:403-411.[CrossRef][Medline]
40
40. Pujol, C., K. J. Daniels, S. R. Lockhart, T. Srikantha, J. B. Radke, J. Geiger, and D. R. Soll. 2004. The closely related species Candida albicans and Candida dubliniensis can mate. Eukaryot. Cell 3:1015-1027.[Abstract/Free Full Text]
41
41. Roberts, C. J., B. Nelson, M. J. Marton, R. Stoughton, M. R. Meyer, H. A. Bennett, Y. D. He, H. Dai, W. L. Walker, T. R. Hughes, M. Tyers, C. Boone, and S. H. Friend. 2000. Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. Science 287:873-880.[Abstract/Free Full Text]
42
42. Shen, W. C., P. Bobrowicz, and D. J. Ebbole. 1999. Isolation of pheromone precursor genes of Magnaporthe grisea. Fungal Genet. Biol. 27:253-263.[CrossRef][Medline]
43
43. Xue-Franzen, Y., S. Kjaerulff, C. Holmberg, A. Wright, and O. Nielsen. 2006. Genomewide identification of pheromone-targeted transcription in fission yeast. BMC Genomics 7:303.[CrossRef][Medline]
44
44. Yang, Z. 1997. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput. Appl. Biosci. 13:555-556.[Free Full Text]
45
45. Young, L. Y., M. C. Lorenz, and J. Heitman. 2000. A STE12 homolog is required for mating but dispensable for filamentation in Candida lusitaniae. Genetics 155:17-29.[Abstract/Free Full Text]

---

Eukaryotic Cell, March 2007, p. 487-494, Vol. 6, No. 3
1535-9778/07/$08.00+0     doi:10.1128/EC.00387-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Dignard, D., Andre, D., Whiteway, M. (2008). Heterotrimeric G-Protein Subunit Function in Candida albicans: both the {alpha} and {beta} Subunits of the Pheromone Response G Protein Are Required for Mating. Eukaryot Cell 7: 1591-1599 [Abstract] [Full Text]  
• Yi, S., Sahni, N., Daniels, K. J., Pujol, C., Srikantha, T., Soll, D. R. (2008). The Same Receptor, G Protein, and Mitogen-activated Protein Kinase Pathway Activate Different Downstream Regulators in the Alternative White and Opaque Pheromone Responses of Candida albicans. Mol. Biol. Cell 19: 957-970 [Abstract] [Full Text]  
• Schaefer, D., Cote, P., Whiteway, M., Bennett, R. J. (2007). Barrier Activity in Candida albicans Mediates Pheromone Degradation and Promotes Mating. Eukaryot Cell 6: 907-918 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dignard, D. Articles by Whiteway, M. Search for Related Content PubMed PubMed Citation Articles by Dignard, D. Articles by Whiteway, M.