[Show abstract][Hide abstract]ABSTRACT: The ascomycetes Candida albicans, Saccharomyces cerevisiae and Scheffersomyces stipitis metabolize the pentose sugar xylose very differently. S. cerevisiae fails to grow on xylose, while C. albicans can grow, and S. stipitis can both grow and ferment xylose to ethanol. However, all three species contain highly similar genes that encode potential xylose reductases and xylitol dehydrogenases required to convert xylose to xylulose, and xylulose supports the growth of all three fungi. We have created C. albicans strains deleted for the xylose reductase gene GRE3, the xylitol dehydrogenase gene XYL2, as well as the gre3 xyl2 double mutant. As expected, all the mutant strains cannot grow on xylose, while the single gre3 mutant can grow on xylitol. The gre3 and xyl2 mutants are efficiently complemented by the XYL1 and XYL2 from S. stipitis. Intriguingly, the S. cerevisiae GRE3 gene can complement the Cagre3 mutant, while the ScSOR1 gene can complement the Caxyl2 mutant, showing that S. cerevisiae contains the enzymatic capacity for converting xylose to xylulose. In addition, the gre3 xyl2 double mutant of C. albicans is effectively rescued by the xylose isomerase (XI) gene of either Piromyces or Orpinomyces, suggesting that the XI provides an alternative to the missing oxido-reductase functions in the mutant required for the xylose-xylulose conversion. Overall this work suggests that C. albicans strains engineered to lack essential steps for xylose metabolism can provide a platform for the analysis of xylose metabolism enzymes from a variety of species, and confirms that S. cerevisiae has the genetic potential to convert xylose to xylulose, although non-engineered strains cannot proliferate on xylose as the sole carbon source.
[Show abstract][Hide abstract]ABSTRACT: Although gastrointestinal colonization by the opportunistic fungal pathogen Candida albicans is generally benign, severe systemic infections are thought to arise due to escape of commensal C. albicans from the gastrointestinal (GI) tract. The C. albicans transcription factor Efg1p is a major regulator of GI colonization, hyphal morphogenesis, and virulence. The goals of this
study were to identify the Efg1p regulon during GI tract colonization and to compare C. albicans gene expression during colonization of different organs of the GI tract. Our results identified significant differences in
gene expression between cells colonizing the cecum and ileum. During colonization, efg1− null mutant cells expressed higher levels of genes involved in lipid catabolism, carnitine biosynthesis, and carnitine utilization
than did colonizing wild-type (WT) cells. In addition, during laboratory growth, efg1− null mutant cells grew to a higher density than WT cells. The efg1− null mutant grew in depleted medium, while WT cells could grow only if the depleted medium was supplemented with carnitine,
a compound that promotes the metabolism of fatty acids. Altered gene expression and altered growth capability support the
ability of efg1− cells to hypercolonize naïve mice. Also, Efg1p was shown to be important for transcriptional responses to the stresses present
in the cecum environment. For example, during colonization, SOD5, encoding a superoxide dismutase, was highly upregulated in an Efg1p-dependent manner. Ectopic expression of SOD5 in an efg1− null mutant increased the fitness of the efg1− null mutant cells during colonization. These data show that EFG1 is an important regulator of GI colonization.
[Show abstract][Hide abstract]ABSTRACT: Scaffold proteins play central roles in the function of many signaling pathways. Among the best-studied examples are the Ste5 and Far1 proteins of the yeast Saccharomyces cerevisiae. These proteins contain three conserved modules, the RING and PH domains, characteristic of some ubiquitin-ligating enzymes, and a vWA domain implicated in protein-protein interactions. In yeast, Ste5p regulates the mating pathway kinases while Far1p coordinates the cellular polarity machinery. Within the fungal lineage, the Basidiomycetes and the Pezizomycetes contain a single Far1-like protein, while several Saccharomycotina species, belonging to the CTG (Candida) clade, contain both a classic Far1-like protein and a Ste5-like protein that lacks the vWA domain. We analyzed the function of C. albicans Ste5p (Cst5p), a member of this class of structurally distinct Ste5 proteins. CST5 is essential for mating and still coordinates the mitogen-activated protein (MAP) kinase (MAPK) cascade elements in the absence of the vWA domain; Cst5p interacts with the MEK kinase (MEKK) C. albicans Ste11p (CaSte11p) and the MAPK Cek1 as well as with the MEK Hst7 in a vWA domain-independent manner. Cst5p can homodimerize, similar to Ste5p, but can also heterodimerize with Far1p, potentially forming heteromeric signaling scaffolds. We found direct binding between the MEKK CaSte11p and the MEK Hst7p that depends on a mobile acidic loop absent from S. cerevisiae Ste11p but related to the Ste7-binding region within the vWA domain of Ste5p. Thus, the fungal lineage has restructured specific scaffolding modules to coordinate the proteins required to direct the gene expression, polarity, and cell cycle regulation essential for mating.
[Show abstract][Hide abstract]ABSTRACT: The yeast STE18 gene product has sequence and functional similarity to the gamma subunits of G proteins. The cloned STE18 gene was subjected to a saturation mutagenesis using doped oligonucleotides. The populations of mutant genes were screened for two classes of STE18 mutations, those that allowed for increased mating of a strain containing a defective STE4 gene (compensators) and those that inhibited mating even in the presence of a functional STE18 gene (dominant negatives). Three amino acid substitutions that enhanced mating in a specific STE4 (G beta) point mutant background were identified. These compensatory mutations were allele specific and had no detectable phenotype of their own; they may define residues that mediate an association between the G beta and G gamma subunits or in the association of the G beta gamma subunit with other components of the signalling pathway. Several dominant negative mutations were also identified, including two C terminal truncations. These mutant proteins were unable to function in signal transduction by themselves, but they prevented signal transduction mediated by pheromone, as well as the constitutive signalling which is present in cells defective in the GPA1 (G alpha) gene. These mutant proteins may sequester G beta or some other component of the signalling machinery in a nonfunctional complex.
No preview · Article · Jan 2011 · Biochemistry and Cell Biology
[Show abstract][Hide abstract]ABSTRACT: FIG S3 Detailed view of the protein-protein interaction identified by Y2H. An asterisk indicates interaction data that are explicitly indicated in Fig. S4 in the supplemental material. Download
[Show abstract][Hide abstract]ABSTRACT: FIG S2 Reverting a CST5 copy restores mating- and pheromone-induced shmoo and gene expression. Phenotypic characterization of a CST5 restored strain (CST5res; PCa65) in assays for determining mating- and pheromone-induced shmoo and gene expression is shown. The experimental design is identical to that presented in Fig. 2. Asterisks indicate genes that were considered induced in the CST5res strain following mating pheromone stimulation. Download
[Show abstract][Hide abstract]ABSTRACT: FIG S8 Multiple sequence alignment of the conserved spaced acidic region in the C terminus of Ste5 proteins lacking a vWA domain. Acidic residues are shown against a red background, and absolutely conserved residues are marked with asterisks. The MEME consensus motif sequence is displayed below the multiple sequence alignment. Download
[Show abstract][Hide abstract]ABSTRACT: FIG S5 Modeled conformational flap of the activation loop from the kinase domain of Hst11p. (A) The Ser719 residue is phosphorylated in the open-loop conformation (image on left) and unphosphorylated in the folded-back conformation (image on right). The side chains in the acidic insert in the middle of the activation loop (magenta tube) are shown in red, while basic residues from the activation and/or around the phosphorylation site are shown in blue. (B) MEK-binding acidic loop of S. cerevisiae Ste5-like proteins. Multiprotein sequence alignments of Ste5-like proteins showing overall amino acid conservation (blue gradient) and the acidic residues within their putative MEK-binding acidic loop (red gradient) are shown. Asterisks denote conserved putative phosphorylation sites. Download
[Show abstract][Hide abstract]ABSTRACT: FIG S7 Differential coordination of white and opaque C. albicans cell pheromone signaling cascades based on scaffold-scaffold interactions. In this model, Far1p function is implicated only in the opaque cell version of the mating MAPK signaling cascade. On the other hand, an additional putative protein, yet to be identified, might contribute specifically to the white cell version of the pathway, potentially helping to activate the Tec1p transcription factor instead of Cph1p. The biofilm and adhesion pictures representing the output response of the white cell signaling cascade were kindly provided by the David Soll laboratory. Download
[Show abstract][Hide abstract]ABSTRACT: FIG S6 The Ste11E707K mutant has a reduced mating capacity but is not sterile. Qualitative (petri plate) and quantitative (associated number) data are provided for the parental and wild-type reference strain 3294, the ste11/STE11 heterozygous strain, the Ste11E707K mutant, and the ste11/ste11 null mutant. For strain-complete genotype and parental connection, see Table S1 in the supplemental material. Download
[Show abstract][Hide abstract]ABSTRACT: FIG S4 Mapping of amino acid mutations crucial for Cst5p homo- or heterodimerization with Far1p. (A) Multiprotein sequence alignment of the RING domain found on Ste5p family members is shown. Identified mutations are indicated by stars and arrows: red, mutations allowing Far1p binding only; green, mutations allowing both Far1p and Cst5p binding. The shading represents the level of conservation, from high (dark blue) to low (light blue). The RING-H2 consensus sequence displayed further down includes cysteines and histidines/aspartic acids numbered according to their sequential order (C1 represents the first cysteine, etc.). C135R was identified multiple times in independent Y2H screenings (n = 3). (B) Schematic representation of Far1p structural organization featuring the minimal Cst5-binding region determined by Y2H (green lines) and point mutations identified that prevent this interaction (black arrows and stars). Download
[Show abstract][Hide abstract]ABSTRACT: FIG S1 Protein sequence homology and secondary folding of newly discovered vWA domains in Far1p proteins. Newly detected cryptic vWA domains of Far1-like proteins are represented. Sequence and secondary structure alignments between vWA domains predicted for representative Far1-like proteins (upper set) and selected structures of vWA domains are ranked by consensus fold recognition with the highest-scored protein from the fold recognition program (lower set; Protein Data Bank [PDB] accession numbers are in parentheses). The structurally aligned sequence of the S. cerevisiae Ste5p vWA domain of a known experimental structure is shown below the template alignment. Secondary structure elements: blue, β strand; red, α helix; and brown, G helix. Asterisks mark residues previously mutated in ScFar1p (21). Statistically significant 3D-Jury scores (>50) (39) for vWA fold detection are listed at the ends of the corresponding Far1 sequences. Download
[Show abstract][Hide abstract]ABSTRACT: Although the fungus Candida albicans is a commensal colonizer of humans, the organism is also an important opportunistic pathogen. Most infections caused by C. albicans arise from organisms that were previously colonizing the host as commensals, and therefore successful establishment of colonization is a prerequisite for pathogenicity. To elucidate fungal activities that promote colonization, an analysis of the transcription profile of C. albicans cells recovered from the intestinal tracts of mice was performed. The results showed that within the C. albicans colonizing population, cells expressed genes characteristic of the laboratory-grown exponential phase and genes characteristic of post-exponential-phase cells. Thus, gene expression both promoted the ability to grow rapidly (a characteristic of exponential-phase cells) and enhanced the ability to resist stresses (a characteristic of post-exponential-phase cells). Similarities in gene expression in commensal colonizing cells and cells invading host tissue during disease were found, showing that C. albicans cells adopt a particular cell surface when growing within a host in both situations. In addition, transcription factors Cph2p and Tec1p were shown to regulate C. albicans gene expression during intestinal colonization.
[Show abstract][Hide abstract]ABSTRACT: A pheromone-mediated signaling pathway that couples seven-transmembrane-domain (7-TMD) receptors to a mitogen-activated protein
kinase module controls Candida albicans mating. 7-TMD receptors are typically connected to heterotrimeric G proteins whose activation regulates downstream effectors.
Two Gα subunits in C. albicans have been identified previously, both of which have been implicated in aspects of pheromone response. Cag1p was found to
complement the mating pathway function of the pheromone receptor-coupled Gα subunit in Saccharomyces cerevisiae, and Gpa2p was shown to have a role in the regulation of cyclic AMP signaling in C. albicans and to repress pheromone-mediated arrest. Here, we show that the disruption of CAG1 prevented mating, inactivated pheromone-mediated arrest and morphological changes, and blocked pheromone-mediated gene expression
changes in opaque cells of C. albicans and that the overproduction of CAG1 suppressed the hyperactive cell cycle arrest exhibited by sst2 mutant cells. Because the disruption of the STE4 homolog constituting the only C. albicans gene for a heterotrimeric Gβ subunit also blocked mating and pheromone response, it appears that in this fungal pathogen
the Gα and Gβ subunits do not act antagonistically but, instead, are both required for the transmission of the mating signal.
[Show abstract][Hide abstract]ABSTRACT: Candida albicans cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent
opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at
37°C, block production of farnesol, and permit in vitro mating at 37°C at efficiencies of up to 84%. Aerobically, farnesol
prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned
off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized
gastrointestinal (GI) tract, could serve as niches for C. albicans mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells,
confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear,
and prototrophic, and they contained the heterologous hisG marker from one of the parental strains.
[Show abstract][Hide abstract]ABSTRACT: In the opaque state, MTLa and MTLα 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 α-factor. Disruption of this C. albicans gene blocks the mating of MTLa cells but not MTLα cells, while the reintegration of the gene suppresses this cell-type-specific mating defect.