An association between reduced susceptibility to echinocandins and changes in the 1,3-␤-D-glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Cafks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei.

The lipopeptide antifungal agents, echinocandins, papulacandins, and pneumocandins, kill Candida albicans by inhibiting glucan synthesis. For this fungus, there is a good correlation of in vitro enzyme inhibition with in vitro assays of MICs. Semisynthetic lipopeptides such as cilofungin, LY303366,989,560 have activity in vivo against Aspergillus infections but appear to be inactive in broth dilution in vitro tests (MICs, > 128 ,ug/ml). To understand how compounds which lack activity in vitro can have good in vivo activity, we monitored the effect of pneumocandins on the morphology ofAspergillusf migatus and A. flavus strains by light microscopy and electron microscopy and related the changes in growth to inhibition of glucan synthesis.Pneumocandin Bo caused profound changes in hyphal growth; light micrographs showed abnormally swollen germ tubes, highly branched hyphal tips, and many cells with distended balloon shapes. Aspergillus electron micrographs confirmed that lipopeptides produce changes in cell walls; drug-treated germlings showed very stubby growth with thick walls and a conspicuous dark outer layer which was much thicker in the subapical regions. The rest of the hyphal tip ultrastructure was unaffected by the drug, indicating considerable specificity for the primary target. The drug-induced growth alteration produced very compact clumps in broth dilution wells, making it possible to score the morphological effect macroscopically. The morphological changes could be assayed quantitatively by using conventional broth microdilution susceptibility assay conditions. We defined the endpoint as the lowest concentration required to produce the morphological effect and called it the minimum effective concentration to distinguish it from the no-growth endpoints used in MIC determinations.The minimum effective concentration assay was related to inhibition of glucan synthase activity in vitro and may provide a starting point for development of susceptibility testing methods for lipopeptides.Echinocandins, pneumocandins, and papulacandins are antifungal agents which inhibit the synthesis of 1,3-p-D-glucan (22,39,43,44). ,B-Glucans are vital cell wall polymers in clinically important pathogenic fungi, including Candida and Aspergillus spp. (8). The proportion of this polysaccharide in the walls of different organisms varies, but at least 10% of the dry weight of the walls of these organisms is in the form of 1,3-p-D-glucan (4). This polysaccharide is synthesized by a membrane-associated activity which has been partially characterized in yeasts such as Candida albicans and Saccharomyces cerevisiae (31,40,46). The enzyme uses UDP-glucose as a substrate and catalyzes the polymerization of a linear polymer of 1,3-p-D-glucan reported to be 60 U (40) or as much as 700 U (35) long. The Km for UDP-glucose is approximately 2 mM, and the reaction is stimulated by nucleoside triphosphates, particularly GTP (31, 42). For both C. albicans and S. cerevisiae, the in vitro product, which is synthesized without a requirement for ...

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.Staphylococcus aureus is a gram-positive bacterium that is notable for the frequency and severity of infections that it causes in hospitalized patients. These infections range from localized skin infections to bacteremia and septic shock. In the past 20 years there has been a dramatic increase in the incidence of nosocomial staphylococcal infections; this increase parallels the increased use of intravascular devices and invasive procedures. S. aureus has been identified as one of the three most frequent nosocomial pathogens and is responsible for approximately 25% of the 2 million nosocomial infections reported in the United States each year (38, 39). A second trend has been the increase in the incidence of methicillin-resistant S. aureus, largely due to selective antibiotic pressure. Resistant strains were initially identified in tertiary care hospitals but have been increasingly reported among infections in the community (25, 30). Resistance to methicillin is often accompanied by resistance to other antibiotics; a CDC survey showed that the proportion of methicillin-resistant isolates which were susceptible only to vancomycin rose from 22.8% to 56. 2% from 1987 to 1997 (18). More recently, S. aureus strains with intermediate susceptibility or resistance to vancomycin have been reported (11,24,36). Infections caused by multidrug-resistant S. aureus limit therapeutic options, and they may be associated with higher mortality and higher costs than infections caused by susceptible staphylococci. There is clearly a need for new treatment and prevention strategies.In an immunological survey of S. aureus su...

The sphingolipid metabolites ceramide and sphingosine-1-phosphate are second messengers with opposing roles in mammalian cell growth arrest and survival; their relative cellular level has been proposed to be a rheostat that determines the fate of cells. This report demonstrates that this rheostat is an evolutionarily conserved stress-regulatory mechanism that inf luences growth and survival of yeast. Although the role of sphingosine-1-phosphate in yeast was not previously examined, accumulation of ceramide has been shown to induce G 1 arrest and cell death. We now have identified a gene in Saccharomyces cerevisiae, LBP1, that regulates the levels of phosphorylated sphingoid bases and ceramide. LBP1 was cloned from a yeast mutant that accumulated phosphorylated long-chain sphingoid bases and diverted sphingoid base intermediates from sphingolipid pathways to glycerophospholipid biosynthesis. LBP1 and its homolog, LBP2, encode very hydrophobic proteins that contain a novel-conserved sequence motif for lipid phosphatases, and both have long-chain sphingoid base phosphate phosphatase activity. In vitro characterization of Lbp1p shows that this phosphatase is Mg 2؉ -independent with high specificity for phosphorylated long-chain bases, phytosphingosine and sphingosine. The deletion of LBP1 results in the accumulation of phosphorylated long-chain sphingoid bases and reduced ceramide levels. Moreover, deletion of LBP1 and LBP2 results in dramatically enhanced survival upon severe heat shock. Thus, these phosphatases play a previously unappreciated role in regulating ceramide and phosphorylated sphingoid base levels in yeast, and they modulate stress responses through sphingolipid metabolites in a manner that is reminiscent of their effects on mammalian cells.Branching pathways of sphingolipid metabolism may mediate growth arrest, stress, or proliferative responses depending on the cell type and the nature of the stimulus. Ceramide is emerging as an important regulatory component of stress responses and programmed cell death, known as apoptosis (1-5). In contrast, another sphingolipid metabolite, sphingosine-1-phosphate (SPP), has been implicated as a second messenger in cellular proliferation (6) and antagonizes ceramide-mediated apoptosis (7). Thus, it has been suggested that the relative intracellular levels of ceramide and SPP are a critical factor for cell survival. Although the ceramide͞SPP rheostat may be an inherent characteristic of mammalian cells, external stimuli can reset this ratio (7-9). A variety of stress stimuli, including Fas ligand, TNF-␣, IL-1, growth factor withdrawal, anticancer drugs, oxidative stress, heat shock, and ionizing radiation, increase ceramide levels (1, 2, 10, 11), whereas platelet-derived growth factor and other growth factors stimulate rapid, transient elevations in SPP levels (6). The mechanisms that regulate the levels of these sphingolipid second messengers are under intense investigation with most of the attention focused on degradative pathways: sphingomyelinase, which...