A signaling pathway involving the extracellular protein Reelin and the intracellular adaptor protein Disabled-1 (Dab1) controls cell positioning during mammalian brain development. Here, we demonstrate that Reelin binds directly to lipoprotein receptors, preferably the very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Binding requires calcium, and it is inhibited in the presence of apoE. Furthermore, the CR-50 monoclonal antibody, which inhibits Reelin function, blocks the association of Reelin with VLDLR. After binding to VLDLR on the cell surface, Reelin is internalized into vesicles. In dissociated neurons, apoE reduces the level of Reelin-induced tyrosine phosphorylation of Dab1. These data suggest that Reelin directs neuronal migration by binding to VLDLR and ApoER2.

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 mrr1Δ 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.

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 ␤-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.Candida albicans is the most common human fungal pathogen and is the fourth leading cause of bloodstream infections in the United States (6,14,24). Currently only four antifungal drug classes are available for the management of systemic infections due to Candida species. Recently we examined changes in the genome-wide expression profile of Saccharomyces cerevisiae in response to representatives of the polyene, pyrimidine, azole, and echinocandin antifungal agents in an effort to identify class-specific and mechanism-independent changes in gene expression (1). In the present study, we extend this analysis to the pathogenic fungus C. albicans. By using the same representative drugs and similar growth conditions as in our previous study, we are able to show similarities and differences in the responses to these antifungal agents between S. cerevisiae and C. albicans. Gene expression profiling experiments revealed drug-specific responses consistent with their mechanisms of action, responses indicative of other pathways that may be affected by these agents, and responses that reflect known and potential mechanisms of resistance to these antifungal drugs. MATERIALS AND METHODSAntifungal agents. Ketoconazole (KTZ) and flucytosine (5-FC) were obtained from Sigma (St. Louis, MO). Amphotericin B (AMB) was obtained from ICN Biomedicals (Aurora, OH). The commercially available preparation of caspofungin (CPF) acetate for injection (Cancidas) was used. Stock solutions of various concentrations were made in dimethyl sulfoxide (DMS...

SummaryCandida glabrata emerged in the last decade as a common cause of mucosal and invasive fungal infection, in large part due to its intrinsic or acquired resistance to azole antifungals such as fluconazole. In C. glabrata clinical isolates, the predominant mechanism behind azole resistance is upregulated expression of multidrug transporter genes CDR1 and PDH1. We previously reported that azole-resistant mutants (MIC Ն 64 mg ml ) to both F15 and 66032 and eliminated both constitutive and fluconazole-induced CDR1-PDH1 expression. Reintroduction of wild-type or F15 PDR1 fully reversed these effects; together these results demonstrate a role for this gene in both acquired and intrinsic azole resistance. CDR1 disruption had a partial effect, reducing fluconazole trailing in both strains while restoring wild-type susceptibility (MIC = 16 mg ml -1 ) to F15. In an azole-resistant clinical isolate, PDR1 disruption reduced azole MICs eight-to 64-fold with no effect on sensitivity to other antifungals. To extend this analysis, C. glabrata microarrays were generated and used to analyse genome-wide expression in F15 relative to its parent. Homologues of 10 S. cerevisiae genes previously shown to be Pdr1-Pdr3 targets were upregulated (YOR1, RTA1, RSB1, RPN4, YLR346c and YMR102c along with CDR1, PDH1 and PDR1 itself) or downregulated (PDR12); roles for these genes include small molecule transport and transcriptional regulation. However, expression of 99 additional genes was specifically altered in C. glabrata F15; their roles include transport (e.g. QDR2, YBT1), lipid metabolism (ATF2, ARE1), cell stress (HSP12, CTA1), DNA repair (YIM1, MEC3) and cell wall function (MKC7, MNT3). These azole resistance-associated changes could affect C. glabrata tissue-specific virulence; in support of this, we detected differences in F15 oxidant, alcohol and weak acid sensitivities. C. glabrata provides a promising model for studying the genetic basis of multidrug resistance and its impact on virulence.

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.