Sequencing of a 4.3 kbp region of chromosome 2 of Candida albicans reveals the presence of homologues of SHE9 from Saccharomyces cerevisiae and of bacterial phosphatidylinositol-phospholipase C

The nucleotide sequence of a 4.3 kb fragment downstream of the LIG4 gene of Candida albicans has been determined. This fragment contains two entire ORFs (ORF1 and ORF2) and a truncated one (ORF3). ORF1 (1029 bp; EMBL databank, Accession No. AJ277539) encodes a putative protein of 343 amino acids with a high degree of similarity to phosphatidylinositol-specific phospholipases C (PI-PLC) of bacterial origin and, to a lesser degree, to similar proteins from trypanosome, fly and human. Isolated ORF1 confers PI-PLC activity to Escherichia coli transformants. ORF2 (1572 bp; EMBL databank, Accession No. AJ277538) predicts a protein of 524 amino acids with high similarity along most of the entire length to Ydr393w from Saccharomyces cerevisiae. This protein carries a domain with significant similarity to several cytoskeleton proteins of different origins. YDR393w (SHE9) is an orphan gene whose overexpression compromises cell growth. ORF3 appears to encode the homologue of the well-conserved proteasomal 26S regulatory subunit.     

A single-copy suppressor of the Saccharomyces cerevisae late-mitotic mutants cdc15 and dbf2 is encoded by the Candida albicans CDC14 gene.

The Saccharomyces cerevisiae CDC15, DBF2, TEM1 and CDC14 genes encode regulatory proteins that play a crucial role in the latest stages of the M phase of the cell cycle. By complementation of a S. cerevisiae cdc15-lyt1 mutant with a Candida albicans centromeric-based genomic library, we have isolated a homologue of the protein phosphatase-encoding gene CDC14. The sequence analysis of the C. albicans CDC14 gene reveals a putative open reading frame of 1626 base pairs interrupted by an intron located close to the 5' region. Analysis of C. albicans cDNA proved that the intron is processed in vivo. The CaCDC14 gene shares 49% of amino acid sequence identity with the S. cerevisiae CDC14 gene, 46% with Schizosaccharomyces pombe homologue, 35% with Caenorhabditis elegans and 37% and 38% with human CDC14A and CDC14B genes, respectively. As expected, the C. albicans CDC14 gene complemented a S. cerevisiae cdc14-1 mutant. We found that this gene was able to efficiently suppress not only a S.cerevisiae cdc15-lyt1 mutant but also a dbf2-2 mutant in a low number of copies and allowed growth, although very slightly, of a tem1 deletant. Overexpression of the human CDC14A and CDC14B genes complemented, although very poorly, S. cerevisiae cdc15-lyt1 and dbf2-2 mutants, suggesting a conserved function of these genes throughout phylogeny. The sequence of CaCDC14 was deposited in the EMBL database under Accession No. AJ243449.     

Comparative proteomic analysis of a Candida albicans DSE1 mutant under filamentous and non‐filamentous conditions

Candida albicans is a common opportunistic pathogen that causes a variety of diseases in immunocompromised hosts. In a pathogen, cell wall proteins are important virulence factors. We previously characterized Dse1 as a cell wall protein necessary for virulence and resistance to cell surface‐disrupting agents, such as Calcofluor white, chitin deposition, proper adhesion and biofilm formation. In the absence of decomplexation, our objectives were to investigate differential proteomic expression of a DSE1 mutant strain compared to the wild‐type strain. The strains were grown under filamentous and non‐filamentous conditions. The extracted cell proteome was subjected to tryptic digest, followed by generation of peptide profiles using MALDI–TOF MS. Generated peptide profiles were analysed and unique peaks for each strain and growth condition mined against a Candida database, allowing protein identification. The DSE1 mutant was shown to lack the chitin biosynthesis protein Chs5, explaining the previously observed decrease in chitin biosynthesis. The wild‐type strain expressed Pra1, involved in pH response and zinc acquisition, Atg15, a lipase involved in virulence, and Sod1, required for oxidative stress tolerance, in addition to proteins involved in protein biosynthesis, explaining the increase in total protein content observed compared to the mutants strain. The mutant, on the other hand, expressed glucoamylase 1, a cell wall glycoprotein involved in carbohydrate metabolism cell wall degradation and biofilm formation. As such, MALDI–TOF MS is a reliable technique in identifying mutant‐specific protein expression in C. albicans. Copyright © 2014 John Wiley & Sons, Ltd.     

Deletion of PDE2, the gene encoding the high-affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans

A role for the cAMP-dependent pathway in regulation of the cell wall in the model yeast Saccharomyces cerevisiae has recently been demonstrated. In this study we report the results of a phenotypic analysis of a Candida albicans mutant, characterized by a constitutive activation of the cAMP pathway due to deletion of PDE2, the gene encoding the high cAMP-affinity phosphodiesterase. Unlike wild-type strains, this mutant has an increased sensitivity to cell wall and membrane perturbing agents such as SDS and CFW, and antifungals such as amphotericin B and flucytosine. Moreover, the mutant is characterized by an altered sensitivity and a significantly reduced tolerance to fluconazole. The mutant's membrane has around 30% higher ergosterol content and the cell wall glucan was 22% lower than in the wild-type. These cell wall and membrane changes are manifested by a considerable reduction in the thickness of the cell wall, which in the mutant is on average 60-65 nm, compared to 80-85 nm in the wild-type strains as revealed by electron microscopy. These results suggest that constitutive activation of the cAMP pathway affects cell wall and membrane structure, and biosynthesis, not only in the model yeast S. cerevisiae but also in the human fungal pathogen C. albicans.     

Investigations into the polymorphisms at the ECM38 locus of two widely used Saccharomyces cerevisiae S288C strains,YPH499 and BY4742

The ECM38 gene encodes the gamma-glutamyl transpeptidase enzyme, an enzyme involved in glutathione turnover. The enzyme was found to be present in the S288C strain, BY4742, but absent in another widely used strain congenic to S288C, YPH499. Cloning and sequencing the genes from these yeasts indicated the presence of 11 single nucleotide polymorphisms in the coding region and eight single nucleotide polymorphisms in the promoter region of the ECM38 gene of YPH499 (but none in that of BY4742). One of the SNPs in the ECM38 ORF led to a G --> D conversion in a region conserved in all gamma-GT enzymes and was found to be responsible for the loss of activity in this strain. The presence of gamma-GT activity in other YPH strains led us to trace the origins of the polymorphisms in YPH499. Our results indicated that among the progenitor strains, YPH1 and YPH2, YPH1 carried the polymorphisms seen in YPH499 and also lacked the gamma-GT activity. The implications of these results for the use of these widely used S288C strains and the origin of these single nucleotide polymorphisms are presented.     

Physicochemical Properties,Fatty Acid Profiles,and Sensory Characteristics of Fermented Beef Sausage by Probiotics Lactobacillus plantarum IIA‐2C12 or Lactobacillus acidophilus IIA‐2B4

Probiotics may be used to enhance the functionality and nutritional values of fermented sausages. This study aims to evaluate the physicochemical and sensory properties of beef sausages fermented by lactic acid bacteria of Lactobacillus plantarum IIA‐2C12 and L. acidophilus IIA‐2B4. These strains were isolated from beef cattle and have shown to display probiotic features. While the nutrient contents were not affected by the probiotics, the pH, texture, and color varied among the sausages. Further analysis on fatty acids showed different profiles of saturated (C14:0, C17:0, and C20:0) and unsaturated (C14:1, C18:1n9c, C18:2n6c, and C22:6n3) fatty acids in sausages with probiotics. Gas chromatography–mass spectrometry further revealed some flavor development compounds including acid, alcohols, aldehydes, aromatic, ketones, sulfur, hydrocarbons and terpenes, varied among the sausages. Hedonic test showed no difference in the preference toward aroma, texture, and color for untrained panelists.