Tec1 Mediates the Pheromone Response of the White Phenotype of Candida albicans: Insights into the Evolution of New Signal Transduction Pathways

The newly evolved pheromone response pathway of the white cell phenotype of the opportunistic human pathogen Candida albicans provides a unique view of how signal transduction pathways evolve

A Human-Curated Annotation of the Candida albicans Genome

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications

Systematic discovery of unannotated genes in 11 yeast species using a database of orthologous genomic segments

<p>Abstract</p> <p>Background</p> <p>In standard BLAST searches, no information other than the sequences of the query and the database entries is considered. However, in situations where two genes from different species have only borderline similarity in a BLAST search, the discovery that the genes are located within a region of conserved gene order (synteny) can provide additional evidence that they are orthologs. Thus, for interpreting borderline search results, it would be useful to know whether the syntenic context of a database hit is similar to that of the query. This principle has often been used in investigations of particular genes or genomic regions, but to our knowledge it has never been implemented systematically.</p> <p>Results</p> <p>We made use of the synteny information contained in the Yeast Gene Order Browser database for 11 yeast species to carry out a systematic search for protein-coding genes that were overlooked in the original annotations of one or more yeast genomes but which are syntenic with their orthologs. Such genes tend to have been overlooked because they are short, highly divergent, or contain introns. The key features of our software - called SearchDOGS - are that the database entries are classified into sets of genomic segments that are already known to be orthologous, and that very weak BLAST hits are retained for further analysis if their genomic location is similar to that of the query. Using SearchDOGS we identified 595 additional protein-coding genes among the 11 yeast species, including two new genes in <it>Saccharomyces cerevisiae</it>. We found additional genes for the mating pheromone a-factor in six species including <it>Kluyveromyces lactis</it>.</p> <p>Conclusions</p> <p>SearchDOGS has proven highly successful for identifying overlooked genes in the yeast genomes. We anticipate that our approach can be adapted for study of further groups of species, such as bacterial genomes. More generally, the concept of doing sequence similarity searches against databases to which external information has been added may prove useful in other settings.</p

The Transcriptomes of Two Heritable Cell Types Illuminate the Circuit Governing Their Differentiation

The differentiation of cells into distinct cell types, each of which is heritable for many generations, underlies many biological phenomena. White and opaque cells of the fungal pathogen Candida albicans are two such heritable cell types, each thought to be adapted to unique niches within their human host. To systematically investigate their differences, we performed strand-specific, massively-parallel sequencing of RNA from C. albicans white and opaque cells. With these data we first annotated the C. albicans transcriptome, finding hundreds of novel differentially-expressed transcripts. Using the new annotation, we compared differences in transcript abundance between the two cell types with the genomic regions bound by a master regulator of the white-opaque switch (Wor1). We found that the revised transcriptional landscape considerably alters our understanding of the circuit governing differentiation. In particular, we can now resolve the poor concordance between binding of a master regulator and the differential expression of adjacent genes, a discrepancy observed in several other studies of cell differentiation. More than one third of the Wor1-bound differentially-expressed transcripts were previously unannotated, which explains the formerly puzzling presence of Wor1 at these positions along the genome. Many of these newly identified Wor1-regulated genes are non-coding and transcribed antisense to coding transcripts. We also find that 5′ and 3′ UTRs of mRNAs in the circuit are unusually long and that 5′ UTRs often differ in length between cell-types, suggesting UTRs encode important regulatory information and that use of alternative promoters is widespread. Further analysis revealed that the revised Wor1 circuit bears several striking similarities to the Oct4 circuit that specifies the pluripotency of mammalian embryonic stem cells. Additional characteristics shared with the Oct4 circuit suggest a set of general hallmarks characteristic of heritable differentiation states in eukaryotes

Genes Selectively Up-Regulated by Pheromone in White Cells Are Involved in Biofilm Formation in Candida albicans

To mate, MTL-homozygous strains of the yeast pathogen Candida albicans must switch from the white to opaque phase. Mating-competent opaque cells then release pheromone that induces polarization, a G1 block and conjugation tube formation in opaque cells of opposite mating type. Pheromone also induces mating-incompetent white cells to become adhesive and cohesive, and form thicker biofilms that facilitate mating. The pheromone response pathway of white cells shares the upstream components of that of opaque cells, but targets a different transcription factor. Here we demonstrate that the genes up-regulated by the pheromone in white cells are activated through a common cis-acting sequence, WPRE, which is distinct from the cis-acting sequence, OPRE, responsible for up-regulation in opaque cells. Furthermore, we find that these white-specific genes play roles in white cell biofilm formation, and are essential for biofilm formation in the absence of an added source of pheromone, suggesting either an autocrine or pheromone-independent mechanism. These results suggest an intimate, complex and unique relationship between switching, mating and MTL-homozygous white cell biofilm formation, the latter a presumed virulence factor in C. albicans

Fungal G-protein-coupled receptors::mediators of pathogenesis and targets for disease control

G-protein signalling pathways are involved in sensing the environment, enabling fungi to coordinate cell function, metabolism and development with their surroundings, thereby promoting their survival, propagation and virulence. G-protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in fungi. Despite the apparent importance of GPCR signalling to fungal biology and virulence, relatively few GPCR–G-protein interactions, and even fewer receptor-binding ligands, have been identified. Approximately 40% of current pharmaceuticals target human GPCRs, due to their cell surface location and central role in cell signalling. Fungal GPCRs do not belong to any of the mammalian receptor classes, making them druggable targets for antifungal development. This Review Article evaluates developments in our understanding of fungal GPCR-mediated signalling, while substantiating the rationale for considering these receptors as potential antifungal targets. The need for insights into the structure–function relationship of receptor–ligand interactions is highlighted, which could facilitate the development of receptor-interfering compounds that could be used in disease control

The chromosome number of Saxifraga gaspensis Fernald

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Primary characterization of a panel of monoclonal antibodies raised against the fungal pathogen Candida albicans

Background: Candida albicans is an opportunistic pathogen responsible for non-life-threatening mucosal infections and is also a major cause of morbidity and mortality in immunosuppressed patients. While Candida infections can be treated with a variety of fungistatic antifungals, mortality rates from systemic candidiasis remain high. This fungi is capable of switching between various morphological forms including hyphae, associated with biofilm formation and tissue invasion, yeast, which is important for dissemination, the mating-competent opaque form and a commensal morphology that was recently observed in the gastro-intestinal tract. Methods: We immunized SJL and A/J mice with UV-killed hyphae, yeast or opaque C. albicans cells and used a ClonePixFL to select hybridoma clones that secrete immunoglobulins. These were then screened for anti-Candida monoclonal antibodies (mAbs) using an In-Cell ELISA assay. To date, we have produced 38 hybridoma cell lines and concentrated our characterization efforts on 23 mAbs that bind to live C. albicans cells. Results: Five mAbs recognize all morphological forms of C. albicans, 16 are hyphal-specific while 2 will only bind to opaque cells. In addition to C. albicans, 3 of the mAbs also recognize other fungal pathogens such as C. glabrata, C. tropicalis and C. dublininensis. All of the mAbs label the cell surface by immunofluorescence microscopy and 10 produce multiple bands or high-molecular weight smears in western blots. In order to narrow down the identity of mAb targets and to determine whether they recognize glycoproteins or glycans, we used the In-Cell ELISA assay to probe mannosylation ( 06och1, 06pmr1, 06mnt1 06mnt2) and chitin synthase ( 06chs2, 06chs3, 06chs8, 06chs2 06chs8) mutants. We have also produced and probed reverse-phase protein microarrays spotted with total protein extracts from 2,357 GRACE C. albicans strains in the hyphal or yeast morphologies. In these strains, one gene copy is inactivated by insertional mutagenesis while the other allele is under the control of a repressible Tet-promoter. Conclusions: These assays allowed us to cluster the members of this mAb panel according to their target specificities and to prioritize them for future investigations aimed at evaluating their potential for therapeutic or diagnostic purposes.NRC publication: Ye

Comparative xylose metabolism among the ascomycetes C. albicans, S. stipitis and S. cerevisiae

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. \ua9 2013 Boyles et al.Peer reviewed: YesNRC publication: Ye

Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3.

The cel-3 gene cloned from Fibrobacter succinogenes into Escherichia coli coded for the enzyme EG3, which exhibited both endoglucanase and cellobiosidase activities. The gene had an open reading frame of 1,974 base pairs, coding for a protein of 73.4 kilodaltons (kDa). However, the enzyme purified from the osmotic shock fluid of E. coli was 43 kDa. The amino terminus of the 43-kDa protein matched amino acid residue 266 of the protein coded for by the open reading frame, indicating proteolysis in E. coli. In addition to the 43-kDa protein, Western immunoblotting revealed a 94-kDa membranous form of the enzyme in E. coli and a single protein of 118 kDa in F. succinogenes. Thus, the purified protein appears to be a proteolytic degradation product of a native protein which was 94 kDa in E. coli and 118 kDa in F. succinogenes. The discrepancy between the molecular weight expected on the basis of the DNA sequence and the in vivo form may be due to anomalous migration during electrophoresis, to glycosylation of the native enzyme, or to fatty acyl substitution at the N terminus. One of two putative signal peptide cleavage sites bore a strong resemblance to known lipoprotein leader sequences. The purified 43-kDa peptide exhibited a high Km (53 mg/ml) for carboxymethyl cellulose but a low Km (3 to 4 mg/ml) for lichenan and barley beta-glucan. The enzyme hydrolyzed amorphous cellulose, and cellobiose and cellotriose were the major products of hydrolysis. Cellotriose, but not cellobiose, was cleaved by the enzyme. EG3 exhibited significant amino acid sequence homology with endoglucanase CelC from Clostridium thermocellum, and as with both CelA and CelC of C. thermocellum, it had a putative active site which could be aligned with the active site of hen egg white lysozyme at the highly conserved amino acid residues Asn-44 and Asp-52