RESUMEN
Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFkslp in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech[r]) or fully sensitive (Ech[s]) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech[s] allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.
Asunto(s)
Candida albicans/genética , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/genética , Glucosiltransferasas/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Secuencia de Aminoácidos , Animales , Antifúngicos , Secuencia de Bases , Farmacorresistencia Microbiana/genética , Equinocandinas , Proteínas Fúngicas/efectos de los fármacos , Genotipo , Proteínas de la Membrana/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , FenotipoRESUMEN
We previously isolated a clone from a human rhabdomyosarcoma (RH) cDNA library coding for a collagen chain different from those constituting the 18 reported types (Myers, J. C., Sun, M. J., D'Ippolito, J. A., Jabs, E. W., Neilson, E. G., and Dion, A. S. (1993) Gene (Amst.) 123, 211-217). The sequence translated to a 186-amino acid noncollagenous region, a 524-residue three-subdomain collagenous region, and a presumed 8-amino acid COOH-terminal peptide. To further elucidate the primary structure of this collagen, we have now determined the sequence of additional cDNA clones. Overlapping 3' clones, found to diverge exactly where the noncollagenous 8-residue COOH sequence began, encode two additional collagenous subdomains of 168 and 70 residues and a 19-residue COOH-terminal peptide. Analysis of genomic DNA spanning the region in question revealed several 45- and 51-base pair exons linked by 4 introns totaling over 5 kilobases (kb). A 2-kb intron, absent from the clones coding for the extended collagenous region, was used in Northern blot hybridization to detect an apparently prevalent splicing intermediate 2 kb larger than the major 12.4-kb RH transcript. Therefore, the triple-helical region of this collagen chain is likely to be composed of 832 amino acids divided into five collagenous subdomains separated by 20-44 residue interruptions. Two interruptions are similar in sequence and position to those located in the type XVI chain. Furthermore, the arrangement of 2 cysteines near the COOH terminus and two imperfections in collagenous subdomain 1 are conserved in the related subclass composed of type IX, XII, XIV, and XVI collagens. However, in contrast to the COOH-terminal interchain bridging in this latter collagen group, molecular modeling strongly predicts that the cysteines in RH collagen participate in intrachain disulfide bonds. Taken together, the data clearly show that RH collagen does not represent another chain of one of the known collagen types. We propose that it be designated the alpha 1 chain of type XIX collagen.
Asunto(s)
Colágeno/química , Colágeno/genética , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Cartilla de ADN , ADN Complementario/metabolismo , Exones , Biblioteca de Genes , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Conformación Proteica , Mapeo Restrictivo , Rabdomiosarcoma , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
Human collagen (COL) cDNA clones were isolated from a library representing transcripts synthesized by an established rhabdomyosarcoma (RH) cell line. The 0.6-kb insert of the first isolate encodes a discontinuous collagenous sequence not homologous to type I-XVI COL chains. Sequencing of a second clone with a 4-kb insert revealed an open reading frame (ORF) of 2154 nucleotides. The deduced amino acid (aa) sequence begins with an 186-aa noncollagenous region containing seven cysteines (Cys). Several of the Cys and surrounding aa residues can be aligned with those in type XVI, XII and IX COL. Due to the presence of two long interruptions, the 524-aa collagenous region is separated into three subdomains that each have smaller interruptions of 1-6 aa. The protein terminates with an 8-aa noncollagenous peptide including an unusual single Cys which would be expected to form an interchain disulfide bond. Results of Northern blot hybridization suggest that the new COL chain may be uncommonly large since the clone identified a low-abundance RNA at least 12.4 kb in size. The gene coding for RH COL is located on human chromosome 6. It is now important to elucidate the role of this unusual COL in the infrastructure of extracellular matrix.