Human Pathogenic Yeast Research Articles

Dimorphism in Human Pathogenic and Apathogenic Yeasts. Contributions to Microbiology

Dimorphism in Human Pathogenic and Apathogenic Yeasts. Contributions to Microbiology

Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans.

CaMDR1 encodes a major facilitator superfamily (MFS) protein in Candida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p in Sf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance in C. albicans, were independently expressed in a common hypersensitive host JG436 of Saccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.

Antiviral and antimicrobial activities of Colombian medicinal plants

Strong antiviral and antimicrobial activities were detected in methanolic extracts of 24 plants used medicinally in the treatment of skin infections in four different regions of Colombia. Thirteen extracts displayed activity against herpes simplex virus (HSV) whereas none was active against poliovirus. The antiviral activity was indicated by a total inhibition of viral cytopathic effects (CPE) at a non-cytotoxic concentration of the extract. The most potent extract was obtained from Byrsonima verbascifolia (L.) HBK. which showed anti-HSV activity at a concentration as low as 2.5 μg/ml. Antimicrobial screening was conducted using the disc diffusion assay against Klebsiella pneumoniae, Escherichia coli, Streptococcus faecalis, Mycobacterium phlei, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium and the human pathogenic yeast, Candida albicans. Anti-Candida activity was observed for Piper lanceaefolium HBK. and Juglans neotropica Diels. Twenty-two extracts displayed activity against Gram-positive bacteria whereas none was active against the Gram-negative species. We concluded that these Colombian medicinal plants represent an untapped source of potentially useful antivirals and are worthy of further study.

Two telomerase reverse transcriptases (TERTs) expressed in Candida albicans

The human pathogenic yeast Candida albicans contains two telomerase reverse transcriptase (TERT) genes. CaTERT1 and CaTERT2 appear either to be two alleles of the same gene or two entirely different genes that encode 867-residue proteins that differ by five amino acids. Both TERTs have a calculated pI of 9.5 and a M(r) of 100.9 kDa and are the smallest TERTs identified to date. Both genes appear to be expressed. Based on sequence similarity between CaTERT1 and the Saccharomyces cerevisiae orthologue Est2p, we suggest a revised alignment for motif E of Est2p. The identification of these TERT genes provides the first opportunity to study telomerase in an important human pathogen.

The centromere-binding factor Cbf1p from Candida albicans complements the methionine auxotrophic phenotype of Saccharomyces cerevisiae.

A gene encoding the centromere binding factor 1 (Cbf1p) of the human pathogenic yeast Candida albicans was cloned and characterized. An open reading-frame was detected which encoded a 223 amino acid protein with a calculated molecular weight of 25.8 kDa and a relative isoelectric point of 5.55. It shares 39% overall amino acid sequence identity with Saccharomyces cerevisiae Cbf1p. We localized the CaCBF1 gene on chromosome 4. Southern analysis indicated that CaCBF1 is probably present as a single copy gene per haploid genome. The CaCBF1 gene under the control of its own promoter was able to complement the methionine auxotrophic growth, the increased mitotic instability of CEN plasmids and the slow growth of a Saccharomyces cerevisiae cbf1Delta mutant strain.

Genome sequencing suggests sexual reproduction in Candida albicans

Meiosis is the hallmark process of sexual reproduction. Haploid cells are produced by the reduction of homologous chromosomes in diploid parental cells. The recent identification of a mating-type-like (MTL) locus in the human pathogenic yeast Candida albicans strongly suggests that this fungus, which has been historically thought of as being asexual, does in fact have a sexual cycle. The lack of knowledge of a sexual cycle in C. albicans has hindered the ability to carry out conventional genetic analyses on this organism. Recently, however, shotgun sequencing of the diploid C. albicans genome has been completed to 10.4 haploid genome equivalents (http://sequence-www.stanford.edu/group/candida). With this sequence, as well as the sequence of Saccharomyces cerevisiae and higher eukaryotic organisms, genome comparisons should provide an insight into the possibility that C. albicans uses a complete sexual cycle.Tzung et al.1xGenomic evidence for a complete sexual cycle in Candida albicans. Tzung, K. et al. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3249–3253Crossref | PubMed | Scopus (134)See all References1 recently carried out such a genomic comparison between C. albicans, S. cerevisiae and some higher eukaryotes to search for homologues of genes known to be involved in meiosis. These investigators focused their search to genes crucial for mating and meiosis in budding yeast, as well as regulators known to be involved in cellular reproduction in higher eukaryotes. Using the BLASTP program, output was sorted and E-values of 1e−6 were used to assign homologues. Each sequence was also evaluated individually because there were some instances where biological data implied the presence of a particular homologue despite the lack of a gene with significant sequence homology. [Supplementary data summarizing the findings are available on the PNAS website (http://www.pnas.org)]. The genes identified were categorized according to their associated function: (1) mating differentiation; (2) nutritional control; (3) cell type control; (4) initiation of meiosis; (5) checkpoint control and progression through meiosis; (6) recombination and formation of synaptonemal complexes; and (7) spore wall morphogenesis and ascus formation. Five hundred genes analyzed by genome-wide transcriptional analysis of sporulation in S. cerevisiae were used as a reference point to begin the analysis.The presence of known S. cerevisiae homologues in C. albicans suggests similarities in the sexual cycle between these two fungi and the lack of known homologues of some genes highlights the interesting differences between them. With the identification of genes encoding homologues implicated in meiosis in C. albicans, a detailed functional characterization of each component should help to identify the role of these proteins and confirm the presence of a sexual cycle in this important human pathogen.

The deletion of CaVPS34 in the human pathogenic yeast Candida albicans causes defects in vesicle‐mediated protein sorting and nuclear segregation

The deletion of CaVPS34 in the human pathogenic yeast Candida albicans causes defects in vesicle‐mediated protein sorting and nuclear segregation

The in vitro Antimycotic Activity and Acute Toxicity of Third-Generation Bezylidenetetralones and Heteroarylidenetetralones

Objectives: A new series of E-2-arylmethylene-1-tetralones and E-2-heteroarylmethylene-1-tetralones (third generation) were designed as potential antimycotic agents against human pathogenic yeasts. Methods: The new compounds were obtained by aldol condensation of 1-tetralone with the appropriately substituted aldehydes in either acid- or base-catalysed conditions. These compounds were tested for their in vitro antimycotic activity against 24 strains of Cryptococcus neoformans, Candida spp. and Trichosporon cutaneum by a microtitre well technique in a liquid casitone medium, using a double dilution method. The toxicity of the new compounds was determined in vitro in cultured HeLa cells, in HeLa growth medium and in vivo in MFI mice. Results: Nineteen new E-2-benzylidine-1-tetralones were prepared, and 16 of the tested compounds showed superior antimycotic activity when compared to the first-generation E-2-benzylidine-1-tetralones synthesised previously. Twelve of these third-generation compounds were more active against different strains of yeasts than 6 commercial antimycotic agents similarly tested. An in vitro toxicity study and an in vivo acute toxicity study in MFI mice showed that these compounds were severalfold less toxic than most of the commerical antimycotics, with 4 being comparable to or less toxic than the least toxic of the commercial agents (amphotericin B) tested by the same procedures. Conclusions: Twelve members of this new E-2-benzylidine-1-tetralone class of compounds are promising candidates for antimycotic agents worthy of further investigation.

Dimorphism in human pathogenic and apathogenic yeast

Dimorphism in human pathogenic and apathogenic yeast

Sequencing and heterologous expression in Saccharomyces cerevisiae of a Cryptococcus neoformans cDNA encoding a plasma membrane H+-ATPase

A cDNA containing an open reading frame encoding a putative plasma membrane H+-ATPase in the human pathogenic basidiomycetous yeast Cryptococcus neoformans was cloned and sequenced by means of PCR and cDNA library hybridization. The cloned cDNA is 3475 bp in length, containing a 2994 bp open reading frame encoding a polypeptide of 997 amino acids. As in the case of another basidiomycetous fungus (Uromyces fabae), the deduced amino acid sequence of CnPMA1 was found to be more homologous to those of P-type H+-ATPases from higher plants than to those from ascomycetous fungi. In order to prove the sequenced cDNA corresponds to a H+-ATPase, it was expressed in Saccharomyces cerevisiae and found to functionally replace its own H+-ATPase. Kinetic studies of CnPMA1 compared to ScPMA1 show differences in Vmax values and H+-pumping in reconstituted vesicles. The pH optimum and Km values are similar in both enzymes.

Uniparental mitochondrial transmission in sexual crosses in Cryptococcus neoformans.

Restriction fragment length polymorphism (RFLP) in the large ribosomal RNA region of the mitochondrial DNA (mtDNA) was developed as a genetic marker for investigating mitochondrial transmission in sexual crosses of the human pathogenic basidiomycetous yeast Cryptococcus neoformans. Strain JEC20 of C. neoformans var. neoformans (mat a) was mated with six strains of C. neoformans var. grubii (mat alpha). Successful mating was indicated by the formation of hyphae and basidiospores. These basidiospores were examined for mtDNA RFLP genotypes. All 570 basidiospores examined from the six crosses showed the mtDNA genotype of strain JEC20. The failure to recover the C. neoformans var. grubii mtDNA in any cross indicates that the C. neoformans var. grubii mtDNA is either selectively eliminated in the newly formed dikaryon or selectively excluded in the immediate dikaryotic hyphae of the newly formed dikaryon.

Clonal and spontaneous origins of fluconazole resistance in Candida albicans.

The genotypes and susceptibilities to fluconazole of 78 strains of the human pathogenic yeast Candida albicans were compared. The strains comprised two sets of samples from Durham, N.C.: one from patients infected with the human immunodeficiency virus (HIV) and the other from healthy volunteers. For each strain, the MIC of fluconazole was determined by the standard National Committee for Clinical Laboratory Standards protocol. Genotypes were determined by PCR fingerprinting with five separate primers. The analysis revealed little evidence for genotypic clustering according to HIV status or body site. However, a small group of fluconazole-resistant strains isolated from patients infected with HIV formed a distinct cluster. In addition, two fluconazole-resistant strains were isolated from individuals who never took fluconazole, one from a patient infected with HIV and the other from a healthy person. The results suggest both clonal and spontaneous origins of fluconazole resistance in C. albicans.

Identification of a Candida albicans homologue of the PHO85 gene, a negative regulator of the PHO system in Saccharomyces cerevisiae.

In a screen for the protein kinase genes of the human pathogenic yeast Candida albicans, a putative homologue (CaPHO85) of PHO85, a negative regulator of the PHO system of Saccharomyces cerevisiae, which is one of the cyclin-dependent protein kinases (CDKs), was isolated. An open reading frame (ORF) of this gene was identified encoding a predicted protein of 326 amino acids with a calculated molecular weight of 37.6 kDa. The amino acid sequence is highly homologous to S. cerevisiae Pho85 (62% identity) and its Aspergillus nidulans homologue (70% identity), but less homologous to Cdc28 (50% identity) of S. cerevisiae and to its C. albicans homologue CaCdc28 (49% identity), both of which are also CDK. The coding region for the C. albicans gene was interrupted by an intron of 81 nucleotides near the sequence encoding the N-terminal region, similarly to the case of the S. cerevisiae PHO85 gene. Alignment of CaPho85 with various yeast CDKs revealed that most of the domains for ATP-binding and protein kinase activity are conserved among fungal species. Southern blot analysis indicated that CaPHO85 is most likely present as a single copy gene. This gene complemented the pho85 mutation of S. cerevisiae by transformation.

A phosphatidylinositol 3-kinase of Candida albicans: molecular cloning and characterization.

A phosphatidylinositol (PI) 3-kinase gene (CaVPS34) of the human pathogenic yeast Candida albicans was cloned by a PCR-based homology approach. The open reading frame encodes a 1020 amino acid protein with a calculated molecular weight of 118 kDa and a relative isoelectric point of 6.9. It shares 47% sequence identity with Saccharomyces cerevisiae Vps34p. Southern pattern indicated that CaVPS34 is probably present as a single copy gene per haploid genome in C. albicans. We localized the CaVPS34 gene on chromosome 1. Under all conditions tested a major CaVPS34 transcript of approximately 3. 5 kb could be detected. CaVPS34 mRNA levels increased during exponential growth up to 12-fold followed by a decline upon entry into stationary phase. The size of a 6xHis tag-CaVps34p fusion protein purified from Escherichia coli is in agreement with the calculated molecular mass of CaVps34p. It exhibits in vitro PI 3-kinase activity and produces only phosphatidylinositol 3-phosphate. The CaVPS34 gene under the control of its own promoter were not able to complement the temperature-sensitive growth of S. cerevisiae vps34. However, overexpression of CaVPS34 was sufficient to rescue the temperature-sensitive vps34 phenotype, suggesting a functional conservation in C. albicans.

A multicopper oxidase gene from Candida albicans: cloning, characterization and disruption.

A multicopper oxidase gene from the human pathogenic yeast Candida albicans was isolated and characterized. An open reading frame of 1872 bp, designated CaFET3, was identified, encoding a predicted protein of 624 amino acids and a molecular mass of 70.5 kDa. The identity between the deduced amino acid sequences of CaFET3 and the Saccharomyces cerevisiae FET3 gene is 55%. CaFET3 was localized on chromosome 6. A null mutant (fet3delta/fet3delta) was constructed by sequential gene disruption. Unlike the C. albicans SC5314 wild-type strain the fet3delta mutant was unable to grow in low-iron medium. The lack of growth of a S. cerevisiae fet3delta mutant in iron-limited medium was compensated by transformation with CaFET3. The null mutant strain showed no change in pathogenicity compared with the wild-type strain in the mouse model of systemic candidiasis.

DNA-dependent RNA polymerase II from Candida species is a multiple zinc-containing metalloenzyme.

We have purified DNA-dependent RNA polymerase II from Candida albicans, a human pathogenic yeast. The enzyme consists of 9 polypeptides that are unique to C. albicans, their mobility on SDS-PAGE being different from the mobility of the corresponding subunits of RNA polymerase II from Saccharomyces cerevisiae or C. utilis. In the present study we also demonstrate that RNA pol II from C. albican and C. utilis are metalloproteins containing approximately 5 mol of zinc per mole of enzyme. Although prolonged dialysis in 10 or 20 mM EDTA failed to remove Zn(II) from the C. albicans enzyme, in the C. utilis enzyme 3 Zn(II) ions could be removed and then reconstituted in the presence of excess Zn(II). o-Phenanthroline (5 mM) removed Zn(II) from C. albicans enzyme irreversibly in a time-dependent fashion with concomitant loss of enzyme activity. Circular dichroism studies revealed structural changes on removal of zinc, thus suggesting a role for Zn in maintenance of structural stability. Further, we demonstrate that the largest subunit of the C. utilis enzyme and the 3 large subunits of the C. albicans enzyme can bind radioactive zinc.

Antimicrobial activity of spices

Spices have been shown to possess medicinal value, in particular, antimicrobial activity. This study compares the sensitivity of some human pathogenic bacteria and yeasts to various spice extracts and commonly employed chemotherapeutic substances. Of the different spices tested only garlic and clove were found to possess antimicrobial activity. The bactericidal effect of garlic extract was apparent within 1 h of incubation and 93% killing of Staphylococcus epidermidis and Salmonella typhi was achieved within 3 h. Yeasts were totally killed in 1 h by garlic extract but in 5 h with clove. Some bacteria showing resistance to certain antibiotics were sensitive to extracts of both garlic and clove. Greater anti-candidal activity was shown by garlic than by nystatin. Spices might have a great potential to be used as antimicrobial agents.

Cloning of a centromere binding factor 3d (CBF3D) gene from Candida glabrata.

The gene encoding centromere binding factor 3d (CBF3D) of the human pathogenic yeast Candida glabrata has been isolated by hybridization of Saccharomyces cerevisiae CBF3D (ScCBF3D) DNA to a C. glabrata partial genomic library. Sequence analysis revealed a 540 bp open reading frame encoding a protein of 179 amino acids with a calculated molecular mass of 20.9 kDa. The amino acid sequence is highly homologous (78.6% identity) to ScCbf3d and 48.3% identical to the human homologue p19 (SKP1). Southern blot analysis indicates that CgCbf3d is encoded by an unique nuclear gene. The cloned CgCBF3D gene can functionally substitute the S. cerevisiae homologue in a S. cerevisiae CBF3D-deletion mutant. The GenBank Accession No. for this gene is AF 072472.

Asymmetric distribution of phosphatidylethanolamine in C. albicans: possible mediation by CDR1, a multidrug transporter belonging to ATP binding cassette (ABC) superfamily.

By using two molecular probes, we demonstrate that only 4% of total phosphatidylethanolamine (PtdEtn) in the plasma membrane (PM) of a human pathogenic yeast, Candida albicans, is present in its external half. Evidence is presented to show that the availability of PtdEtn could be related to the expression of a multidrug transporter CDR1 of C. albicans, and the process is energy-dependent. A homozygous CDR1 disruptant strain of C. albicans shows almost 23% reduction in the external labelling of PtdEtn. This report shows that, similar to human MDRs, yeast multidrug transporter could also be involved in aminophospholipid translocation.

Lack of consistent short sequence repeat polymorphisms in genetically homologous colonizing and invasive Candida albicans strains.

Short sequence repeats (SSRs), potentially representing variable numbers of tandem repeat (VNTR) loci, were identified for the human-pathogenic yeast species Candida albicans by computerized DNA sequence scanning. The individual SSR regions were investigated in different clinical isolates of C. albicans. Most of the C. albicans SSRs were identified as genuine VNTRs. They appeared to be present in multiple allelic variants and were demonstrated to be diverse in length among nonrelated strains. As such, these loci provide adequate targets for the molecular typing of C. albicans strains. VNTRs encountered in other microbial species sometimes participate in regulation of gene expression and function as molecular switches at the transcriptional or translational level. Interestingly, the VNTRs identified here often encode polyglutamine stretches and are frequently located within genes potentially involved in the regulation of transcription. DNA sequencing of these VNTRs demonstrated that the length variability was restricted to the CAA/CAG repeats encoding the polyglutamine stretches. For these reasons, paired C. albicans isolates of similar genotype, either found as noninvasive colonizers or encountered in an invasive state in the same individual, were studied with respect to potentially invasion-related alterations in the VNTR profiles. However, none of the VNTRs analyzed thus far varied systematically with the transition from colonization to invasion. In contrast to the situation described for some prokaryotic species, this finding suggests that VNTRs of C. albicans may not simply function as contingency loci related to straightforward on/off regulation of invasion-related gene expression.