Candida albicans ; genetics ; pathogenicity ; Extracellular Matrix ; metabolism

Humans ; Candida albicans/genetics* ; CARD Signaling Adaptor Proteins/genetics* ; Mutation ; Encephalomyelitis

The aim of this paper was to investigate the effect of berberine hydrochloride on the cell wall integrity of Candida albicans hypha. The minimal inhibitory concentration(MIC) of berberine hydrochloride against clinical and standard C. albicans strains was detected by micro liquid-based dilution method; the effect of berberine hydrochloride on the colony formation of C. albicans SC5314 was investigated by spot assay; the effect of berberine hydrochloride on the metabolism of C. albicans SC5314 hypha was checked by XTT reduction assay, and the viability of C. albicans SC5314 hypha was tested by fluorescent staining assay. The effect of berberine hydrochloride on the morphology of C. albicans SC5314 hypha was examined by scanning electron microscope. The changes in the cell wall of C. albicans SC5314 hypha after berberine hydrochloride treatment were detected by transmission electron microscopy. The effect of berberine hydrochloride on β-glucan from C. albicans SC5314 was detected by flow cytometry. The effect of berberine hydrochloride on hypha-specific gene ECE1 and β-glucan synthase genes FKS1 and FKS2 in C. albicans was examined by qRT-PCR. The results showed that berberine hydrochloride showed a strong inhibitory effect on both clinical and standard strains of C. albicans, and the MIC was 64-128 μg·mL~(-1). Spot assay, XTT redunction assay and fluorescent staining assay showed that with the increase of berberine hydrochloride concentration, the viability of C. albicans SC5314 gradually decreased. The transmission electron microscopy scanning assay showed that this compound could cause cell wall damage of C. albicans. The flow cytometry analysis showed the exposure degree of C. albicans β-glucan. The qRT-PCR further showed that berberine hydrochloride could significantly down-regulate hypha-specific gene ECE1 and β-glucan synthase-related gene FKS1 and FKS2. In conclusion, this compound can down-regulate C. albicans and β-glucan synthase-related gene expressions, so as to destroy the cell wall structure of C. albicans, expose β-glucan and damage the integrity of the wall.
Antifungal Agents/pharmacology* ; Berberine/pharmacology* ; Candida albicans/genetics* ; Cell Wall ; Hyphae ; Microbial Sensitivity Tests

Carotenoids have a range of diverse biological functions and actions, especially playing an important role in human health with provitamin A activity, anti-cancer activity, enhancing immune ability and so on. Human body can't synthesis carotenoids by itself and must absorb them from outside. However, carotenoid contents in many plant are very low, and many kinds of carotenoid are difficult to produce by chemical ways. With the elucidation of carotenoid biosynthetic pathway and cloning genes of relative enzymes from microorganisms and higher plants, it is possible to regulate carotenoid biosynthesis via genetic engineering. This article reviews gene cloning of carotenoid biosynthetic enzymes in microorganisms and higher plants, and advances in the studies of carotenoid production in heterologous microorganisms and crop plants using gene-manipulated carotenoid biosynthesis.
Candida albicans ; genetics ; Carotenoids ; biosynthesis ; Cloning, Molecular ; Escherichia coli ; genetics ; Genetic Engineering ; methods ; Plants ; genetics ; Saccharomyces cerevisiae ; genetics

BACKGROUNDCandida albicans (C. albicans) can become a pathogen causing superficial as well as life-threatening systemic infections, especially in immunocompromised patients. Many phenotypic attributes contribute to its capacity to colonize human organs. In our study, 93 C. albicans isolates from patients of various candidiasis in a hospital of China were surveyed. We aimed to investigate the white-opaque (WO) switching competence, drug sensitivity, and virulence of mating type-like (MTL) a/α isolates.

METHODSInternal transcribed spacer (ITS) gene and the MTL configuration were detected in all the isolates by reverse transcription-polymerase chain reaction. White/opaque phenotype and doubling time of cell growth were determined. The minimum inhibitory concentrations of antifungal agent were measured using broth microdilution method.

RESULTSSixty-four isolates (69.6%) were classified to serotype A, 19 (20.6%) to serotype B, and 9 (9.8%) to serotype C. Moreover, phylogenetic analysis showed that these isolates were divided into four different subgroups of ITS genotypes. Most of our clinical isolates were MTL a/α type, while 6.8% remained MTL a or MTLα type. The frequency of opaque phenotype was 71.0% (66 isolates). Following the guidelines of Clinical and Laboratory Standards Institute M27-A3, all isolates were susceptible to caspofungin and a few (0.6-3.2%) of them showed resistance against amphotericin B, flucytosine, fluconazole, itraconazole, and voriconazole.

CONCLUSIONSFrom these analyses, there were comparatively more C. albicans strains classified into serotype B, and the frequency of opaque phase strains was significant in the clinical isolates from China. Genetic, phenotypic, or drug susceptibility patterns were not significantly different from previous studies. MTL a/α isolates could also undergo WO switching which facilitates their survival.

Amphotericin B ; pharmacology ; Antifungal Agents ; pharmacology ; Candida albicans ; classification ; drug effects ; genetics ; Fluconazole ; pharmacology ; Genotype ; Humans ; Microbial Sensitivity Tests ; Phylogeny

Microarray analysis revealed that the expression of ferric reductase (FRP1) can be regulated by the Riml01 protein. In order to find new transcriptional regulatory element in the promoter of FRP1, we analyzed the 1000 bp sequence upstream of ATG to find 2 potential Riml01p binding sites. We generated site-specific mutations in each of the two sites and fused these mutated promoters to LacZ. Then the promoter-LacZ fusion construct was recombinant into wild type and riml01-/- strains for beta-galactosidase assay. The results revealed that the FRP1 was up-regulated in alkaline pH and this was caused by iron starvation. The -650 site, not the -160 site, had an important role in FRP1 Riml01p-dependent expression. We conclude that Riml01p may interact with the -650 binding site of the promoter to regulate the FRP1 expression.
Candida albicans ; enzymology ; genetics ; DNA-Binding Proteins ; genetics ; FMN Reductase ; genetics ; Fungal Proteins ; genetics ; Mutagenesis, Site-Directed ; Promoter Regions, Genetic ; genetics

OBJECTIVETo investigate the application of differential display-2PCR(DD-PCR) in research on gene expression of Candida.

METHODSResistance to fluconazole was induced in a Candida albicans isolate 435 from vagina by culturing in YEPD broth with increasing fluconazole concentration in vitro, and the resistant isolate 435-2 (MIC=128 microg/ml ) was obtained after 80 days of incubation. Comparisons between 435 and 435-2 either in fluconazole-containing medium or in drug-free medium were performed with the modified DD-PCR including amplification with long primers, silver staining, reverse dot blot and non-radiographic labeling techniques.

RESULTSThree differential displayed bands were found which showed high homology to alcohol dehydrogenase 1 (ADH1), TOP2 and CDR1, respectively. The up-regulating expression of ADH1 and CDR1 associated with fluconazole resistance was further identified by RT-PCR.

CONCLUSIONThe up-regulating expression of ADH1 and CDR1 was associated with fluconazole resistance in Candida albicans, ADH1 might be a candidate of novel fluconazole resistant gene.

Antifungal Agents ; pharmacology ; Candida albicans ; drug effects ; genetics ; Drug Resistance, Fungal ; genetics ; Fluconazole ; pharmacology ; Fungal Proteins ; genetics ; Membrane Transport Proteins ; genetics ; Oxidoreductases ; genetics ; Polymerase Chain Reaction ; methods

Candida albicans exhibits the ability to grow in either a yeast or a mycelia form in response to different environmental factors. The mycelia form, found in infected tissues, is important as a virulence factor in the adherence of the organism to the host epithelium. In vitro, the morphological transition can be induced by environmental shifts in the growing conditions, or by a variety of exogenous factors, including ambient pH, nutritional status and temperature. The differential-display reverse transcription polymerase chain reaction (DDRT-PCR) is a powerful technique for comparing gene expression between cell types, stages of development or differentiation. Hyphae related genes were identified and characterized using a PCR-based differential display. Candida albicans formed a germ tube when cultured in rabbit serum, RPMI 1640 medium or 39degrees C-YPD medium. We gained 21 cDNA bands showing a different expression pattern from that of the uninduced culture. DNA was extracted from the same location of the isolated bands, and PCR was performed under the same conditions, which reamplified the PCR product, showing the specific expression patterns according to the culture conditions. We cloned 18 germ tube-related cDNA clones (inserts average size is 80 - 700 bp) and sequenced them. The nucleotide sequences of the 18 clones were identified through in the present study from GenBank, and were found to have the accession number (AF405213-AF405230). We could not find any nucleotide sequence having a high homology with these clones. This study could form a part of the projects in the search for genes related to the germ tube formation of C. albicans.
Animals ; Base Sequence/genetics ; Candida albicans/genetics/*physiology ; Cloning, Molecular ; DNA, Complementary/genetics ; Molecular Sequence Data ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; Support, Non-U.S. Gov't

BACKGROUNDCandida albicans is the most frequently seen opportunistic human fungal pathogen. Terbinafine is an allylamine antifungal agent that has been proven to have high clinical efficacy in the therapy of fungal infections, the mechanism of action of terbinafine involves the specific inhibition of fungal squalene epoxidase, resulting in ergosterol deficiency and accumulation of intracellular squalene. We used cDNA microarray analysis technology to monitor global expression profile changes of Candida albicans genes in response to terbinafine treatment, and we anticipated a panoramic view of the responses of Candida albicans cells to the representatives of allylamine antifungal agents at the molecular level in an effort to identify drug class-specific and mechanism-independent changes in gene expression.

METHODSCandida albicans strain ATCC 90028 was exposed to either medium alone or terbinafine at a concentration equivalent to the 1/2 minimal inhibitory concentrations (MICs, 4 mg/L) for 90 minutes. RNA was isolated and gene expression profiles were compared to identify the changes in the gene expression profile using a cDNA microarray analysis. Differential expression of 10 select genes detected by cDNA microarray analysis was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSA total of 222 genes were found to be responsive to terbinafine, including 121 up-regulated genes and 101 down-regulated genes. These included genes encoding membrane transport proteins belonging to the members of the ATP-binding cassette (ABC) or major facilitator superfamily (MFS; CDR1, AGP2, GAP6, PHO84, HOL3, FCY23, VCX1), genes involved in stress response and detoxification (CDR1, AGP2, HOL3), and gene involved in the ergosterol biosynthesis pathway (ERG12). The results of semi-quantitative RT-PCR were consistent with that of the cDNA microarray analysis.

CONCLUSIONSThe up-regulation of the gene encoding the multidrug resistance efflux pump CDR1 may contribute to the terbinafine resistance in Candida albicans. However, the precise roles of other affected genes remain unclear, further studies of these genes and their respective products that play roles in the context of antifungal resistance are warranted.

Antifungal Agents ; pharmacology ; Candida albicans ; drug effects ; genetics ; Ergosterol ; biosynthesis ; Fungal Proteins ; genetics ; Gene Expression Profiling ; Genome, Fungal ; Membrane Transport Proteins ; genetics ; Naphthalenes ; pharmacology ; Oligonucleotide Array Sequence Analysis

BACKGROUNDThe cytochrome P450 lanosterol 14alpha-demethylase (Erg11p) encoded by ERG11 gene is the primary target for azole antifungals. Changes in azole affinity of this enzyme caused by amino acid substitutions have been reported as a mechanism of azole antifungal resistance. This study aimed to investigate the relationship between amino acid substitutions in Erg11p from fluconazole resistant Candida albicans (C. albicans) isolates and their cross-resistance to azoles.

METHODSMutations in ERG11 gene were screened in 10 clinical isolates of fluconazole resistant C. albicans strains. DNA sequence of ERG11 was determined by PCR based DNA sequencing.

RESULTSIn the 10 isolates, 19 types of amino acid substitutions were found, of which 10 substitutions (F72S, F103L, F145I, F198L, G206D, G227D, N349S, F416S, F422L and T482A) have not been reported previously. Mutations in ERG11 gene were detected in 9 isolates of fluconazole resistant C. albicans, but were not detected in 1 isolate.

CONCLUSIONSAlthough no definite correlation was found between the type of amino acid substitutions in Erg11p and the phenotype of cross-resistance to azoles, the substitutions F72S, F145I and G227D in our study may be highly associated with resistance to azoles because of their special location in Erg11p.

Antifungal Agents ; pharmacology ; Candida albicans ; drug effects ; genetics ; Cytochrome P-450 Enzyme System ; genetics ; Drug Resistance, Fungal ; Fluconazole ; pharmacology ; Fungal Proteins ; genetics ; Microbial Sensitivity Tests ; Mutation