Biofilm formation by fluconazole-resistant Candida albicans strains is inhibited by fluconazole.

OBJECTIVES: The fungal pathogen Candida albicans forms biofilms on implanted medical devices, resulting in infections with high mortality. Fully developed biofilms, which are adherent communities of microorganisms, characteristically exhibit high resistance to antimicrobial drugs, making treatment of device-associated infection problematic. The aim of this study was to determine the effect of the addition of the azole antifungal fluconazole on the initiation of biofilm formation by both drug-susceptible and drug-resistant C. albicans strains. RESULTS: Our data reported here show that biofilm formation by both fluconazole-susceptible and fluconazole-resistant C. albicans strains was inhibited when fluconazole was present. For the fluconazole-susceptible strains, inhibition of growth due to the presence of the antifungal drug probably prevented the acquisition of high-level fluconazole resistance. However, for fluconazole-resistant strains, the inhibition of biofilm development was unexpected. CONCLUSIONS: Unexpectedly, fluconazole inhibited biofilm formation by a variety of laboratory isolated and clinically isolated fluconazole-resistant strains.

Transcriptional regulation of MDR1, encoding a drug efflux determinant, in fluconazole-resistant Candida albicans strains through an Mcm1p binding site.

Constitutive, high-level transcription of the gene encoding the drug efflux facilitator Mdr1p is commonly observed in laboratory and clinical strains of Candida albicans that are resistant to the antifungal drug fluconazole (FLC). In five independently isolated FLC(R) laboratory strains, introduction of a wild-type MDR1 promoter fragment fused to the yeast enhanced green fluorescent protein (yEGFP) reporter gene resulted in high-level expression of GFP, demonstrating that overexpression of MDR1 is dependent on a trans-acting factor. This study identified a 35-bp MDR1 promoter element, termed the MDRE, that mediates high-level MDR1 transcription. When inserted into a heterologous promoter, the MDRE was sufficient to mediate high-level expression of the yEGFP reporter gene specifically in MDR1 trans-activation strains. The MDRE promoted transcription in an orientation-independent and dosage-dependent manner. Deletion of the MDRE in the full-length promoter did not abolish MDR1 trans-activation, indicating that elements upstream of the MDRE also contribute to transcription of MDR1 in these overexpression strains. Analysis of the MDRE sequence indicated that it contains an Mcm1p binding site very similar in organization to the site seen upstream of the Saccharomyces cerevisiae MFA1 and STE2 genes. Electrophoretic mobility shift analysis demonstrated that both wild-type, FLC-sensitive and MDR1 trans-activated, FLC-resistant strains contain a factor that binds the MDRE. Depletion of Mcm1p, by use of a strain in which MCM1 expression is under the control of a regulated promoter (44), resulted in a loss of MDRE binding activity. Thus, the general transcription factor Mcm1p participates in the regulation of MDR1 expression.

Genetic suppression analysis of non-antibiotic-producing mutants of the Streptomyces coelicolor absA locus.

The absA locus in Streptomyces coelicolor A3(2) was identified because mutations in it uncoupled sporulation from antibiotic synthesis: absA mutants failed to produce any of the four antibiotics characteristic of S. coelicolor. These mutants are now shown to contain point mutations in the absA1 gene which encodes the histidine kinase sensor-transmitter protein of a two-component signalling system. The absA1 non-antibiotic-producing mutants, which are unpigmented, spontaneously acquire pigmented colony sectors. Genetic analysis established that the pigmented sectors contain second-site suppressive mutations, sab (for suppressor of abs). Phenotypic characterization showed that sab strains produce all four antibiotics; some overproduce antibiotics and are designated Pha, for precocious hyperproduction of antibiotics. A set of sab mutations responsible for suppression was localized by plasmid-mediated and protoplast fusion mapping techniques to the vicinity of the absA locus. DNA cloned from this region was used to construct phage that could transduce sab mutations. Sequence analysis of sab strains defined sab mutations in both the absA1 gene and the absA2 gene; the latter encodes the two-component system's response regulator.

Two transcripts, differing at their 3' ends, are produced from the Candida albicans SEC14 gene.

A search for Candida albicans mutants defective in filamentous growth led to the isolation of a mutant strain with an insertion mutation in the SEC14 gene. SEC14 encodes the phosphatidylinositol/phosphatidylcholine transfer protein, an essential protein in the yeast Saccharomyces cerevisiae. In the dimorphic yeast Yarrowia lipolytica, SEC14 is needed for growth only in the hyphal form and is not required for growth in the yeast form. However, unlike Y. lipolytica SEC14, C. albicans SEC14 is probably essential for growth. Northern blot analysis and PCR amplification of transcripts produced from the SEC14 gene demonstrated that two transcripts differing at their 3' ends were produced. The two transcripts may regulate the activity of SEC14 so that Sec14p can perform two functions in C. albicans. One function may be an essential function analogous to the function of Sec14p in S. cerevisiae and the second function may be important during filamentous growth, analogous to the function of Sec14p in Y. lipolytica.