[show abstract][hide abstract] ABSTRACT: MUC7 12-mer is a cationic antimicrobial peptide derived from the N-terminal region of human low-molecular-weight salivary mucin. In order to gain new insights into the modes of action of the 12-mer against opportunistic fungal pathogen Candida albicans, we examined changes in the gene expression profile of C. albicans upon exposure to this peptide. Cells at an early logarithmic phase were exposed to 6 muM peptide and grown until an OD(600 nm) of approximately 0.4 was reached. Changes in gene expression were determined by microarray analysis and showed that 19 out of the total of 531 genes, whose expression was elevated in response to the peptide, are regulated by the calcium/calcineurin signalling pathway. Inactivation of this pathway by deletions, or by FK506, caused hypersensitivity to the peptide, demonstrating the importance of this pathway to the defense of C. albicans against the MUC7 peptide. Other differentially expressed genes that were detected include those encoding subunits of proteasome, and genes involved in cell stress, iron metabolism, cell wall maintenance and small-molecule transport. The presented results suggest that the calcium/calcineurin signalling pathway plays a role in the adaptation of C. albicans to the MUC7 antimicrobial peptide.
FEMS Yeast Research 08/2010; 10(5):579-86. · 2.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cap1p, a transcription factor of the basic region leucine zipper family, regulates the oxidative stress response (OSR) in Candida albicans. Alteration of its C-terminal cysteine-rich domain (CRD) results in Cap1p nuclear retention and transcriptional activation. To better understand the function of Cap1p in C. albicans, we used genome-wide location profiling (chromatin immunoprecipitation-on-chip) to identify its transcriptional targets in vivo. A triple-hemagglutinin (HA(3)) epitope was introduced at the C terminus of wild-type Cap1p (Cap1p-HA(3)) or hyperactive Cap1p with an altered CRD (Cap1p-CSE-HA(3)). Location profiling using whole-genome oligonucleotide tiling microarrays identified 89 targets bound by Cap1p-HA(3) or Cap1p-CSE-HA(3) (the binding ratio was at least twofold; P < or = 0.01). Strikingly, Cap1p binding was detected not only at the promoter region of its target genes but also at their 3' ends and within their open reading frames, suggesting that Cap1p may associate with the transcriptional or chromatin remodeling machinery to exert its activity. Overrepresented functional groups of the Cap1p targets (P < or = 0.02) included 11 genes involved in the OSR (CAP1, GLR1, TRX1, SOD1, CAT1, and others), 13 genes involved in response to drugs (PDR16, MDR1, FLU1, YCF1, FCR1, and others), 4 genes involved in phospholipid transport (PDR16, GIT1, RTA2, and orf19.932), and 3 genes involved in the regulation of nitrogen utilization (GST3, orf19.2693, and orf19.3121), suggesting that Cap1p has other cellular functions in addition to the OSR. Bioinformatic analyses of the bound sequences suggest that Cap1p recognizes the DNA motif 5'-MTKASTMA. Finally, transcriptome analyses showed that increased expression generally accompanies Cap1p binding at its targets, indicating that Cap1p functions as a transcriptional activator.
[show abstract][hide abstract] ABSTRACT: In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate the expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Overexpression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Overexpression of ERG11 leads to the increased production of lanosterol demethylase, which contributes to azole resistance in clinical isolates of C. albicans, but the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately upregulated with ERG11 in a fluconazole-resistant clinical isolate compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single-nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug-susceptible strain resulted in constitutive upregulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole-resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to the increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans.
[show abstract][hide abstract] ABSTRACT: Constitutive overexpression of the MDR1 (multidrug resistance) gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to fluconazole and other toxic compounds in clinical Candida albicans strains, but the mechanism of MDR1 upregulation has not been resolved. By genome-wide gene expression analysis we have identified a zinc cluster transcription factor, designated as MRR1 (multidrug resistance regulator), that was coordinately upregulated with MDR1 in drug-resistant, clinical C. albicans isolates. Inactivation of MRR1 in two such drug-resistant isolates abolished both MDR1 expression and multidrug resistance. Sequence analysis of the MRR1 alleles of two matched drug-sensitive and drug-resistant C. albicans isolate pairs showed that the resistant isolates had become homozygous for MRR1 alleles that contained single nucleotide substitutions, resulting in a P683S exchange in one isolate and a G997V substitution in the other isolate. Introduction of these mutated alleles into a drug-susceptible C. albicans strain resulted in constitutive MDR1 overexpression and multidrug resistance. By comparing the transcriptional profiles of drug-resistant C. albicans isolates and mrr1Delta mutants derived from them and of C. albicans strains carrying wild-type and mutated MRR1 alleles, we defined the target genes that are controlled by Mrr1p. Many of the Mrr1p target genes encode oxidoreductases, whose upregulation in fluconazole-resistant isolates may help to prevent cell damage resulting from the generation of toxic molecules in the presence of fluconazole and thereby contribute to drug resistance. The identification of MRR1 as the central regulator of the MDR1 efflux pump and the elucidation of the mutations that have occurred in fluconazole-resistant, clinical C. albicans isolates and result in constitutive activity of this trancription factor provide detailed insights into the molecular basis of multidrug resistance in this important human fungal pathogen.
[show abstract][hide abstract] ABSTRACT: A major mechanism of azole resistance in Candida albicans is overexpression of the genes encoding the ATP binding cassette transporters Cdr1p and Cdr2p due to gain-of-function mutations in Tac1p, a transcription factor of the zinc cluster family. To identify the Tac1p regulon, we analyzed four matched sets of clinical isolates representing the development of CDR1- and CDR2-mediated azole resistance by using gene expression profiling. We identified 31 genes that were consistently up-regulated with CDR1 and CDR2, including TAC1 itself, and 12 consistently down-regulated genes. When a resistant strain deleted for TAC1 was examined similarly, expression of almost all of these genes returned to levels similar to those in the matched azole-susceptible isolate. Using genome-wide location (ChIP-chip) analysis (a procedure combining chromatin immunoprecipitation with hybridization to DNA intergenic microarrays), we found 37 genes whose promoters were bound by Tac1p in vivo, including CDR1 and CDR2. Sequence analysis identified nine new genes whose promoters contain the previously reported Tac1p drug-responsive element (CGGN(4)CGG), including TAC1. In total, there were eight genes whose expression was modulated in the four azole-resistant clinical isolates in a TAC1-dependent manner and whose promoters were bound by Tac1p, qualifying them as direct Tac1p targets: CDR1, CDR2, GPX1 (putative glutathione peroxidase), LCB4 (putative sphingosine kinase), RTA3 (putative phospholipid flippase), and orf19.1887 (putative lipase), as well as IFU5 and orf19.4898 of unknown function. Our results show that Tac1p binds under nonactivating conditions to the promoters of its targets, including to its own promoter. They also suggest roles for Tac1p in regulating lipid metabolism (mobilization and trafficking) and oxidative stress response in C. albicans.
[show abstract][hide abstract] ABSTRACT: The aim of this study was to identify changes in the gene expression profile of Streptococcus pneumoniae in response to a subinhibitory concentration of penicillin in an effort to better understand mechanisms by which this organism copes with this stress.
S. pneumoniae serotype 2 strain D39 was grown for 1 h in the presence or absence of penicillin at a concentration equivalent to half the MIC (0.03 mg/L). RNA was isolated and gene expression profiles were compared using DNA microarrays. Differential expression of select genes was confirmed by real-time RT-PCR.
A total of 386 genes were found to be responsive to penicillin. Up-regulated genes included those of the ciaR-ciaH operon, luxS, genes encoding cell envelope proteins and genes of the pst locus. Down-regulated genes included genes involved in competence, genes encoding capsular polysaccharide biosynthesis proteins, genes involved in fatty acid chain elongation and genes of the polyamine transporter operon.
Altered expression of these genes reflects a protective response to perturbation of the bacterial cell wall by penicillin. Such genes may represent potential therapeutic targets for enhancing the activity of penicillin against this organism and provide insight into novel mechanisms of penicillin resistance.
Journal of Antimicrobial Chemotherapy 05/2007; 59(4):616-26. · 5.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: Antifungal agents exert their activity through a variety of mechanisms, some of which are poorly understood. We examined changes in the gene expression profile of Candida albicans following exposure to representatives of the four currently available classes of antifungal agents used in the treatment of systemic fungal infections. Ketoconazole exposure increased expression of genes involved in lipid, fatty acid, and sterol metabolism, including NCP1, MCR1, CYB5, ERG2, ERG3, ERG10, ERG25, ERG251, and that encoding the azole target, ERG11. Ketoconazole also increased expression of several genes associated with azole resistance, including CDR1, CDR2, IFD4, DDR48, and RTA3. Amphotericin B produced changes in the expression of genes involved in small-molecule transport (ENA21), and in cell stress (YHB1, CTA1, AOX1, and SOD2). Also observed was decreased expression of genes involved in ergosterol biosynthesis, including ERG3 and ERG11. Caspofungin produced changes in expression of genes encoding cell wall maintenance proteins, including the beta-1,3-glucan synthase subunit GSL22, as well as PHR1, ECM21, ECM33, and FEN12. Flucytosine increased the expression of proteins involved in purine and pyrimidine biosynthesis, including YNK1, FUR1, and that encoding its target, CDC21. Real-time reverse transcription-PCR was used to confirm microarray results. Genes responding similarly to two or more drugs were also identified. These data shed new light on the effects of these classes of antifungal agents on C. albicans.
Antimicrobial Agents and Chemotherapy 07/2005; 49(6):2226-36. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this study was to identify changes in the gene expression profile of Candida albicans upon exposure to the hydroxypyridone anti-infective agent ciclopirox olamine in an effort to better understand its mechanism of action.
C. albicans SC5314 was exposed to either medium alone or ciclopirox olamine at a concentration equivalent to the IC50 (0.24 mg/L) for 3 h. RNA was isolated and gene expression profiles were compared using DNA microarrays. Differential expression of select genes was confirmed by real-time reverse transcription (RT)-PCR. Mutants disrupted for CDR2 and both CDR1 and CDR2, as well as a clinical isolate overexpressing CDR1 and CDR2, were examined for changes in susceptibility to ciclopirox olamine.
A total of 49 genes were found to be responsive to ciclopirox olamine, including 36 up-regulated genes and 13 down-regulated genes. These included genes involved in small molecule transport (HGT11, HXT5, ENA22, PHO84, CDR4), iron uptake (FRE30, FET34, FTR1, FTR2, SIT1) and cell stress (SOD1, SOD22, CDR1, DDR48). Mutants disrupted for CDR2 and both CDR1 and CDR2, as well as a clinical isolate overexpressing CDR1 and CDR2, showed no change in susceptibility to ciclopirox olamine compared with the respective parent.
Consistent with the hypothesis that ciclopirox olamine acts as an iron chelator, it induced changes in expression of many genes involved in iron uptake. Despite induction of the multidrug efflux pump genes CDR1 and, to a lesser extent, CDR2 by ciclopirox olamine, these genes do not affect susceptibility to this agent.
Journal of Antimicrobial Chemotherapy 06/2005; 55(5):655-62. · 5.34 Impact Factor