We have compared the activities of posaconazole and other currently available antifungal agents against a collection of 3,378 clinical isolates of yeasts and filamentous fungi. A total of 1,997 clinical isolates of Candida spp., 359 of other yeast species, 697 strains of Aspergillus spp., and 325 nondermatophyte non-Aspergillus spp. were included. The average geometric means of the MICs of agents that were tested against Candida spp. were 0.23 g/ml for amphotericin B, 0.29 g/ml for flucytosine, 0.97 g/ml for fluconazole, 0.07 g/ml for itraconazole, 0.04 g/ml for voriconazole, 0.15 g/ml for caspofungin, and 0.03 g/ml for posaconazole. Voriconazole and posaconazole were active in vitro against the majority of isolates, with resistance to fluconazole and itraconazole, and against Cryptococcus neoformans and other Basidiomycota yeasts. Posaconazole was the most active of antifungal agents tested against Aspergillus spp., with an average geometric mean of 0.10 g/ml. It was active against Paecilomyces spp., Penicillium spp., Scedosporium apiospermum, and some black fungi, such as Alternaria spp. Multiresistant filamentous fungi, such as Scedosporium prolificans, Scopulariopsis brevicaulis, and Fusarium solani, were also resistant to voriconazole, caspofungin, and posaconazole. Amphotericin B and posaconazole were found to be active against most of the Mucorales strains tested. Posaconazole and currently available antifungal agents exhibit a potent activity in vitro against the majority of pathogenic fungal species.Posaconazole is a new triazole agent with an extended spectrum of in vitro activity. It is active against opportunistic, endemic, and dermatophytic fungi (10, 13, 14, 15) as well as Candida, Cryptococcus, and other yeast species (1,3,17,22,23), including isolates that exhibit resistance to fluconazole and itraconazole (3,10,23). It also has a potent activity in vitro against Aspergillus species; posaconazole appears to be more active in vitro against Aspergillus than are amphotericin B, itraconazole, voriconazole, and ravuconazole, inhibiting 95% of isolates at a MIC of Յ1 g/ml (2, 9, 24). In addition, the new triazoles have been shown to have antifungal activity against other species of filamentous fungi, such as Penicillium spp., Paecilomyces spp., and Acremonium spp., and Sporothrix schenckii as well as some isolates of Fusarium spp., Scedosporium apiospermum, and black fungi (9,10,13,18,24,30). Most notably, posaconazole is more active than the other triazole compounds against zygomycetes, with MICs around or below 1 g/ml. Within the class of Zygomycetes, posaconazole appears to be more active against Rhizopus spp. and Absidia corymbifera than against Mucor spp., which is consistent with the prior observation that the zygomycetes appear to be a heterogeneous group with regard to their susceptibilities to antifungal agents (7,9, 16,24,29).We have analyzed the in vitro activity of posaconazole and other currently available antifungal agents against a collection of 3,378 clinical isolates of yeasts and fila...

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

The glucan synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for ␤(1-3) glucans, showing that AfFks1p colocalized with the newly synthesized ␤(1-3) glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H ؉ -ATPase, and a 160-kDa protein which was labeled by an anti-␤(1-3) glucan antibody and was homologous to ABC bacterial ␤(1-2) glucan transporters.The fungal cell wall, which is specific and essential to fungal life, is mainly constituted of polysaccharides. Among all polysaccharides identified to date in the cell wall, ␤(1-3) glucans are the most prevalent, and they are present in all yeast and filamentous fungi investigated to date (14). Although ␤(1-3) glucan biosynthesis has been the subject of intensive research efforts for the last 30 years, the ␤(1-3) glucan biosynthetic pathway is not fully understood. It has been known since the early studies of Cabib and coworkers (22,31,35,36) that ␤(1-3) glucans are synthesized from UDP glucose by a membrane protein complex, ␤(1-3) glucan synthase (EC 2.4.1.34; UDP-glucose/liter 1,3-␤-D-glucan-3-␤-D-glucosyltransferase). Synthesis occurs on the cytoplasmic side of the plasma membrane, and ␤(1-3) glucan chains are extruded towards the periplasmic space (15,35). The glucan synthase complex has been characterized at the molecular level almost exclusively in the yeast Saccharomyces cerevisiae (5,7,12,19,29) and has been shown to be composed of two proteins: (i) the putative catalytic subunit Fksp, a large-molecular-size (Ͼ200 kDa) polypeptide with 16 transmembrane domains (12,29,30), and (ii) the regulatory subunit Rho1p, a small-molecular-size GTPase, which stimulates ␤(1-3) glucan synthase activity in its prenylated form (1,11,17,18,24,28,33).If the ␤(1-3) glucan synthase has been extensively analyzed in yeast, then this enzymatic complex has been poorly studied in filamentous fungi. Only one FKS gene had been cloned and sequenced to date in Aspergillus nidulans (23), and neither has a regulatory partner been identified nor has the cellular localization of the glucan synthase complex been investigated.This study was centered on the char...

The role of Aspergillus fumigatus 14␣-sterol demethylase (Cyp51A) in azole drug susceptibility was assessed. Targeted disruption of cyp51A in azole-susceptible and -resistant strains decreased MICs from 2-to 40-fold. The cyp51A mutants were morphologically indistinguishable from the wild-type strain, retaining the ability to cause pulmonary disease in neutropenic mice.In Aspergillus fumigatus, there are two distinct but related Cyp51 proteins encoded by cyp51A and cyp51B (7). Erg11 activity has been shown not to be essential in yeast (6, 16) but to date, there are not reports on Cyp51 functional studies in any filamentous fungi. Functional analysis of A. fumigatus Cyp51A by targeted disruption of the cyp51A gene in three clinical strains was performed.Strains. The strains used in this study were A. fumigatus strain CM-237, which was used for describing the sequence of cyp51A and cyp51B (7), and two clinical A. fumigatus strains, CNM-CM-1252 (AF-90) and CNM-CM-796 (filamentous fungus collection of the Spanish National Center for Microbiology), with elevated MICs to azole drugs (Table 1) and different Cyp51A amino acid substitutions (5, 8).Molecular cloning and DNA sequencing. The full coding sequence of cyp51A of A. fumigatus was PCR amplified as previously described (7) and cloned into the pGEM-T vector system (Promega, Madrid, Spain) to obtain plasmid pUM100. Restriction digestion of plasmid pID621 (kindly provided by D. W. Holden) was used to obtain the 1.4-kb SalI fragment of a hygromycin B (hph) resistance cassette (4) for construction of the disruption vector. The 1.4-kb hph cassette was inserted into the XhoI restriction site of pUM100 to create pUM102. A linear 3.0-kb DNA fragment obtained by SacI/SacII double digestion of pUM102 was used for A. fumigatus strain transformations (Fig. 1A).Aspergillus transformations. A. fumigatus transformation experiments were achieved by electroporation using a protocol previously described (15) with subsequent modifications (5, 18). Hygromycin B (130 g/ml; Sigma) was used for transformants selection. Mutants were named by a letter (e.g., A) followed by a number. Genomic DNAs from hygromycin-resistant transformants and the parental strain were digested with two different restriction enzymes (SalI and EcoRV; Amersham Biosciences, Madrid, Spain). Southern analysis was performed as previously described (7,14).Antifungal susceptibility testing. Broth microdilution susceptibility testing was performed as described in NCCLS document M38-A (10), with modifications (3, 11, 13). Itraconazole (ITC), ketoconazole (KTC) (both from Janssen Pharmaceutical S.A., Madrid, Spain), voriconazole, fluconazole (FLC) (both from Pfizer S.A., Madrid, Spain), ravuconazole (BristolMyers Squibb, Madrid, Spain), and amphotericin B (AmB; Sigma Aldrich Quimica, S.A., Madrid, Spain) were tested. Susceptibility tests were performed at least three times with each strain on different days.RNA extraction and LightCycler PCR. RNA extraction from the A. fumigatus CM-237 strain and the derived CM-A8 mutant st...