Morphogenesis beyond cytokinetic arrest in Saccharomyces cerevisiae.

The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37 degreesC, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother-daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.

Functional characterization of the MKC1 gene of Candida albicans, which encodes a mitogen-activated protein kinase homolog related to cell integrity.

Mitogen-activated protein (MAP) kinases represent a group of serine/threonine protein kinases playing a central role in signal transduction processes in eukaryotic cells. Using a strategy based on the complementation of the thermosensitive autolytic phenotype of slt2 null mutants, we have isolated a Candida albicans homolog of Saccharomyces cerevisiae MAP kinase gene SLT2 (MPK1), which is involved in the recently outlined PKC1-controlled signalling pathway. The isolated gene, named MKC1 (MAP kinase from C. albicans), coded for a putative protein, Mkc1p, of 58,320 Da that displayed all the characteristic domains of MAP kinases and was 55% identical to S. cerevisiae Slt2p (Mpk1p). The MKC1 gene was deleted in a diploid Candida strain, and heterozygous and homozygous strains, in both Ura+ and Ura- backgrounds, were obtained to facilitate the analysis of the function of the gene. Deletion of the two alleles of the MKC1 gene gave rise to viable cells that grew at 28 and 37 degrees C but, nevertheless, displayed a variety of phenotypic traits under more stringent conditions. These included a low growth yield and a loss of viability in cultures grown at 42 degrees C, a high sensitivity to thermal shocks at 55 degrees C, an enhanced susceptibility to caffeine that was osmotically remediable, and the formation of a weak cell wall with a very low resistance to complex lytic enzyme preparations. The analysis of the functions downstream of the MKC1 gene should contribute to understanding of the connection of growth and morphogenesis in pathogenic fungi.

A novel family of cell wall-related proteins regulated differently during the yeast life cycle.

The Saccharomyces cerevisiae Ygr189c, Yel040w, and Ylr213c gene products show significant homologies among themselves and with various bacterial beta-glucanases and eukaryotic endotransglycosidases. Deletion of the corresponding genes, either individually or in combination, did not produce a lethal phenotype. However, the removal of YGR189c and YEL040w, but not YLR213c, caused additive sensitivity to compounds that interfere with cell wall construction, such as Congo red and Calcofluor White, and overexpression of YEL040w led to resistance to these compounds. These genes were renamed CRH1 and CRH2, respectively, for Congo red hypersensitive. By site-directed mutagenesis we found that the putative glycosidase domain of CRH1 was critical for its function in complementing hypersensitivity to the inhibitors. The involvement of CRH1 and CRH2 in the development of cell wall architecture was clearly shown, since the alkali-soluble glucan fraction in the crh1Delta crh2Delta strain was almost twice the level in the wild-type. Interestingly, the three genes were subject to different patterns of transcriptional regulation. CRH1 and YLR213c (renamed CRR1, for CRH related) were found to be cell cycle regulated and also expressed under sporulation conditions, whereas CRH2 expression did not vary during the mitotic cycle. Crh1 and Crh2 are localized at the cell surface, particularly in chitin-rich areas. Consistent with the observed expression patterns, Crh1-green fluorescent protein was found at the incipient bud site, around the septum area in later stages of budding, and in ascospore envelopes. Crh2 was found to localize mainly at the bud neck throughout the whole budding cycle, in mating projections and zygotes, but not in ascospores. These data suggest that the members of this family of putative glycosidases might exert a common role in cell wall organization at different stages of the yeast life cycle.

Virulence genes in the pathogenic yeast Candida albicans.

In recent years, the incidence of fungal infections has been rising all over the world. Although the amount of research in the field of pathogenic fungi has also increased, there is still a need for the identification of reliable determinants of virulence. In this review, we focus on identified Candida albicans genes whose deletant strains have been tested in experimental virulence assays. We discuss the putative relationship of these genes to virulence and also outline the use of new different systems to examine the precise effect in virulence of different genes.

Cloning of the Candida albicans HIS1 gene by direct complementation of a C. albicans histidine auxotroph using an improved double-ARS shuttle vector.

ARS2 and ARS3 are two Candida albicans (Ca) DNA fragments with autonomous replicating activity that have been shown to promote non-integrative genetic transformation of both Ca and Saccharomyces cerevisiae (Sc). We have developed several shuttle vectors based on either ARS fragment, or the combination of both, and using the CaURA3 gene as a selection marker. The combination of ARS2 and ARS3 fragments in a single vector did not increase transformation frequencies but improved the stability of transformant plasmids in Ca cells, so that the degree of intracellular recombination was reduced. A Ca genomic DNA library was constructed on the double-ARS vector, pRM1, to be used for direct cloning in Ca by complementation of the histidine auxotrophy of strain CA9. By screening this library, we cloned CaHIS1, the Ca gene that encodes ATP phosphoribosyl transferase, one of the enzymes that participates in histidine biosynthesis. The deduced protein, CaHis1p, is 60.6% identical (73% similar) to ScHis1p (EC 2.4.2.17). The cloned gene is the first auxotrophic gene marker mapped to fragment I of chromosome 5 in the standard Ca genetic map. Our results represent the first demonstration of a direct cloning system in the opportunistic fungus Ca that does not require the use of an intermediate host such as Sc for plasmid rescue. This system could be used for the isolation of any gene affected in Ca mutants displaying a selectable or identifiable phenotype.

The role of HLA-DP beta residue 69 in the definition of antibody-binding epitopes.

Residue 69 of the DP beta chain has been previously identified as being involved in T-cell recognition as well as in the susceptibility to certain autoimmune diseases. The codon for Glu 69 in the DPB1*02012 allele was changed to the codon for Lys found in DPB1*0402, and transfectant L cells expressing wild-type or mutant HLA-DP molecule were obtained. The binding of a large panel of mAbs to these transfectants was tested by flow cytometry. Glu to Lys 69 substitution decreased the binding to the DPB1*02012 allele of some of the DP mAbs and completely eliminated the binding of four of the antibodies tested. These results clearly showed that this residue is involved in the formation of DP antibody-binding epitopes. Because this residue should be located in the alpha-helix of the DP beta polypeptide with the side chain pointing into the peptide-binding groove, its implication in the definition in some DP antibody-binding epitopes should be (a) defining conformational epitopes through effects on the conformation of adjacent regions of the molecule, and (b) determining the binding of peptides to the DP cleft which is directly or indirectly involved in these epitopes.

Protein localisation approaches for understanding yeast cell wall biogenesis.

Yeast cells are surrounded by the cell wall, a rigid but dynamic structure that is essential for their viability. The complexity and functionality of this structure suggest that a high number of proteins must be involved in the biogenesis of the cell wall architecture and, as a consequence, in the maintenance of cell integrity. Among them, a high percentage is assumed to be located at the cell surface, mostly as structural or enzymatic components of the cell wall. Therefore, the presence of a protein in the cell wall is suggestive of its cell wall-related function. Different techniques can be used to specifically detect the cell wall localisation of a given protein or to identify cell wall proteins in large-scale analyses. These include the detection of proteins in whole cells or specific cell wall fractions by immunological, biochemical, microscopic, or genetic approaches, as well as the emerging proteomic technology. The advantages, limitations, and usefulness of these techniques are discussed and illustrated with some examples.

The YGR194c (XKS1) gene encodes the xylulokinase from the budding yeast Saccharomyces cerevisiae.

We report the finding of a Saccharomyces cerevisiae gene necessary for growth in culture media with D-xylulose as the sole carbon source. This gene corresponds to the YGR194c open reading frame that we have previously described, and it is renamed now XKS1. Data bank comparisons of the protein encoded by the XKS1 gene showed significant homology with different xylulokinases, indicating a possible role in xylulose phosphorylation. The wild-type gene in a centromeric plasmid complemented defective growth of xks1 S. cerevisiae mutant strains in xylulose. By contrast, overexpression negatively influenced cell growth in this carbon source.

Candida albicans exoglucanase as a reporter gene in Schizosaccharomyces pombe.

The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cerevisiae and C. albicans, was tested in Schizosaccharomyces pombe. Unlike the budding yeast, S. pombe does not produce exoglucanase activity and hence this system would be applicable to any given strain of this organism. The XOG1 gene was located under the control of the nmt1 promoter and its functionality could be demonstrated even at high temperatures (37 degrees C). The exoglucanase activity can be measured both in vivo and in vitro by either a simple biochemical reaction (on cells or media) or by flow cytometry, because the cells remain viable after the assay.

Low virulence of a morphological Candida albicans mutant.

The virulence of Candida albicans 92', a morphological mutant unable to filament, was assayed in an experimental model of systemic candidiasis in three strains of mice with different susceptibilities to the infection. The mortality parameters studied pointed to the low virulence of this mutant strain. Study of the fungal load of C. albicans 92' in kidneys and brain revealed the presence of low numbers of CFUs and a high percentage of clearance, particularly in the brain. Adhesion studies demonstrated a reduced capability of the mutant to adhere to human epithelial cells. This strain can be considered a potential tool for cloning genes involved in virulence.

Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO red staining.

The fluorescent sensitive SYPRO Red dye was successfully employed to stain proteins in two-dimensional gels for protein identification by peptide mass fingerprinting. Proteins which are not chemically modified during the SYPRO Red staining process are well digested enzymatically in the gel and hence the resulting peptides can be efficiently eluted and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A SYPRO Red two-dimensional gel of a complex protein extract from Candida albicans was analysed by MALDI-TOF MS. The validity of SYPRO Red staining was demonstrated by identifying, via peptide mass fingerprinting, 10 different C. albicans proteins from a total of 31 selected protein spots. The peptide mass signal intensity, the number of matched peptides and the percentage of coverage of protein sequences from SYPRO Red-stained proteins were similar to or greater than those obtained in parallel with the modified silver protein gel staining. This work demonstrates that fluorescent SYPRO Red staining is compatible with the identification of proteins separated on polyacrylamide gel and that it can be used as an alternative to silver staining. As far as we know, this is the first report in which C. albicans proteins separated using 2-D gels have been identified by peptide mass fingerprinting. The improved technique described here should be very useful for carrying out proteomic studies.

A new system for the release of heterologous proteins from yeast based on mutant strains deficient in cell integrity.

A system has been developed for the release of heterologous proteins from Saccharomyces cerevisiae, based on the use of thermosensitive osmotic-remedial mutants, deficient in cell integrity, that lyse at the non-permissive temperature, thus releasing the bulk of intracellular proteins and leaving behind cell ghosts and debris. The strains developed combine the lyt2 mutation (which is allelic to gene SLT2/MPK1 coding for a MAP kinase homolog), with the disruption of genes PEP4 and PRB1 known to produce a protease-deficient background. Cells transformed with the appropriate bacterial gene, released about 70% of the heterologous protein chloramphenicol acetyl transferase (CAT) in bioreactor cultivation upon switching growth temperature to 37 degrees C, or by osmotic shock of the cells preincubated at 37 degrees C in the presence of 1 M sorbitol. It is suggested that our release system could be advantageous for obtaining large-scale protein preparations for downstream processing without any mechanical breakage of the cells, enzymatic treatment or chemical extraction.

[Molecular and genetic aspects of resistance to azoles in Candida albicans]. / Aspectos moleculares y genéticos de la resistencia a azoles en Candida albicans.

Fluconazole is one of the most useful drugs in the treatment of fungal systemic infections which frequently affect non immunocompetent individuals. However, the emergence of resistant strains in recent years may severely limit its usefulness in future. Although there are several described mechanisms involved in resistance to azoles, recent genetic studies demonstrate the role of specific genes in clinical resistance. Currently, the best characterized are the MDR1 and CDR1 genes, which code members of the MFS or ABC family of drug transporters, respectively. These proteins respond to the membrane potential (MFS) or hydrolyse ATP (ABC) thus promoting drug efflux and therefore reducing its intracellular accumulation. It has been shown that the mRNA from these genes is frequently increased in some Candida albicans resistant strains from patients receiving long term azole treatment. The development of molecular genetic tools in C. albicans is allowing characterization of their role in this and other important processes in the fungal cell.

The microbiome as a human organ.

The human organism is a complex structure composed of cells belonging to all three domains of life on Earth, Eukarya, Bacteria and Archaea, as well as their viruses. Bacterial cells of more than a thousand taxonomic units are condensed in a particular functional collective domain, the intestinal microbiome. The microbiome constitutes the last human organ under active research. Like other organs, and despite its intrinsic complexity, the microbiome is readily inherited, in a process probably involving 'small world' power law dynamics of construction in newborns. Like any other organ, the microbiome has physiology and pathology, and the individual (and collective?) health might be damaged when its collective population structure is altered. The diagnostic of microbiomic diseases involves metagenomic studies. The therapeutics of microbiome-induced pathology include microbiota transplantation, a technique increasingly available. Perhaps a new medical specialty, microbiomology, is being born.

Fungi sensing environmental stress.

Cells need to adapt to the external environment in order to survive. Signal transduction pathways are crucial mechanisms that allow cells to sense and respond to extracellular stimuli. Among the signal transduction pathways, we point out the cascades mediated by mitogen-activated protein kinases (MAPKs). The MAPKs are conserved from yeast to human and play relevant roles in the physiology of the cell. In pathogenic fungi these MAPK pathways control virulence factors. This review describes the MAPK cascades described in Candida albicans, the most frequently isolated fungus, from fungal systemic infections among individuals in developed countries.

The MAP kinase signal transduction network in Candida albicans.

MAP (mitogen-activated protein) kinase-mediated pathways are key elements in sensing and transmitting the response of cells to environmental conditions by the sequential action of phosphorylation events. In the fungal pathogen Candida albicans, different routes have been identified by genetic analysis, and especially by the phenotypic characterization of mutants altered in the Mkc1, Cek1/2 and Hog1 MAP kinases. The cell integrity (or MKC1-mediated) pathway is primarily involved in the biogenesis of the cell wall. The HOG pathway participates in the response to osmotic stress while the Cek1 pathway mediates mating and filamentation. Their actual functions are, however, much broader and Mkc1 senses several types of stress, while Hog1 is also responsive to other stress conditions and participates in two morphogenetic programmes: filamentation and chlamydospore formation. Furthermore, it has been recently shown that Cek1 participates in a putative pathway involved in the construction of the cell wall and which seems to be operative under basal conditions. As these stimuli are frequently encountered in the human host, they provide a reasonable explanation for the significant reduction in pathogenicity that several signal transduction mutants show in certain animal models of virulence. MAPK pathways therefore represent an attractive multienzymic system for which novel antifungal therapy could be designed.

The Cek1 and Hog1 mitogen-activated protein kinases play complementary roles in cell wall biogenesis and chlamydospore formation in the fungal pathogen Candida albicans.

The Hog1 mitogen-activated protein (MAP) kinase mediates an adaptive response to both osmotic and oxidative stress in the fungal pathogen Candida albicans. This protein also participates in two distinct morphogenetic processes, namely the yeast-to-hypha transition (as a repressor) and chlamydospore formation (as an inducer). We show here that repression of filamentous growth occurs both under serum limitation and under other partially inducing conditions, such as low temperature, low pH, or nitrogen starvation. To understand the relationship of the HOG pathway to other MAP kinase cascades that also play a role in morphological transitions, we have constructed and characterized a set of double mutants in which we deleted both the HOG1 gene and other signaling elements (the CST20, CLA4, and HST7 kinases, the CPH1 and EFG1 transcription factors, and the CPP1 protein phosphatase). We also show that Hog1 prevents the yeast-to-hypha switch independent of all the elements analyzed and that the inability of the hog1 mutants to form chlamydospores is suppressed when additional elements of the CEK1 pathway (CST20 or HST7) are altered. Finally, we report that Hog1 represses the activation of the Cek1 MAP kinase under basal conditions and that Cek1 activation correlates with resistance to certain cell wall inhibitors (such as Congo red), demonstrating a role for this pathway in cell wall biogenesis.

Identification of a nuclear export signal in the KSHV latent protein LANA2 mediating its export from the nucleus.

LANA2 is a latent protein detected in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected B cells that inhibits p53-dependent transcriptional transactivation and apoptosis and PKR-dependent apoptosis, suggesting an important role in the transforming activity of the virus. It has been reported that LANA2 localizes into the nucleus of both KSHV-infected B cells and transiently transfected HeLa cells. In this study, we show that LANA2 is a nucleocytoplasmic shuttling protein that requires a Rev-type nuclear export signal located in the C-terminus to direct the protein to the cytoplasm, through an association with the export receptor CRM1. In addition, a functional protein kinase B (PKB)/Akt phosphorylation motif partially overlapping with the nuclear export signal was identified. Nuclear exclusion of LANA2 was negatively regulated by the phosphorylation of threonine 564 by Akt. The ability of LANA2 to shuttle between nucleus and cytoplasm has implications for the function of this viral protein.