Comparative genomic analysis of clinical Candida glabrata isolates identifies multiple polymorphic loci that can improve existing multilocus sequence typing strategy.

Candida glabrata is the second leading cause of candidemia in many countries and is one of the most concerning yeast species of nosocomial importance due to its increasing rate of antifungal drug resistance and emerging multidrug-resistant isolates. Application of multilocus sequence typing (MLST) to clinical C. glabrata isolates revealed an association of certain sequence types (STs) with drug resistance and mortality. The current C. glabrata MLST scheme is based on single nucleotide polymorphisms (SNPs) at six loci and is therefore relatively laborious and costly. Furthermore, only a few high-quality C. glabrata reference genomes are available, limiting rapid analysis of clinical isolates by whole genome sequencing. In this study we provide long-read based assemblies for seven additional clinical strains belonging to three different STs and use this information to simplify the C. glabrata MLST scheme. Specifically, a comparison of these genomes identified highly polymorphic loci (HPL) defined by frequent insertions and deletions (indels), two of which proved to be highly resolutive for ST. When challenged with 53 additional isolates, a combination of TRP1 (a component of the current MLST scheme) with either of the two HPL fully recapitulated ST identification. Therefore, our comparative genomic analysis identified a new typing approach combining SNPs and indels and based on only two loci, thus significantly simplifying ST identification in C. glabrata. Because typing tools are instrumental in addressing numerous clinical and biological questions, our new MLST scheme can be used for high throughput typing of C. glabrata in clinical and research settings.

Aspergillus fumigatus and aspergillosis: From basics to clinics.

The airborne fungus Aspergillus fumigatus poses a serious health threat to humans by causing numerous invasive infections and a notable mortality in humans, especially in immunocompromised patients. Mould-active azoles are the frontline therapeutics employed to treat aspergillosis. The global emergence of azole-resistant A. fumigatus isolates in clinic and environment, however, notoriously limits the therapeutic options of mould-active antifungals and potentially can be attributed to a mortality rate reaching up to 100 %. Although specific mutations in CYP 51A are the main cause of azole resistance, there is a new wave of azole-resistant isolates with wild-type CYP 51A genotype challenging the efficacy of the current diagnostic tools. Therefore, applications of whole-genome sequencing are increasingly gaining popularity to overcome such challenges. Prominent echinocandin tolerance, as well as liver and kidney toxicity posed by amphotericin B, necessitate a continuous quest for novel antifungal drugs to combat emerging azole-resistant A. fumigatus isolates. Animal models and the tools used for genetic engineering require further refinement to facilitate a better understanding about the resistance mechanisms, virulence, and immune reactions orchestrated against A. fumigatus. This review paper comprehensively discusses the current clinical challenges caused by A. fumigatus and provides insights on how to address them.

Multicenter study of epidemiological cutoff values and detection of resistance in Candida spp. to anidulafungin, caspofungin, and micafungin using the Sensititre YeastOne colorimetric method.

Neither breakpoints (BPs) nor epidemiological cutoff values (ECVs) have been established for Candida spp. with anidulafungin, caspofungin, and micafungin when using the Sensititre YeastOne (SYO) broth dilution colorimetric method. In addition, reference caspofungin MICs have so far proven to be unreliable. Candida species wild-type (WT) MIC distributions (for microorganisms in a species/drug combination with no detectable phenotypic resistance) were established for 6,007 Candida albicans, 186 C. dubliniensis, 3,188 C. glabrata complex, 119 C. guilliermondii, 493 C. krusei, 205 C. lusitaniae, 3,136 C. parapsilosis complex, and 1,016 C. tropicalis isolates. SYO MIC data gathered from 38 laboratories in Australia, Canada, Europe, Mexico, New Zealand, South Africa, and the United States were pooled to statistically define SYO ECVs. ECVs for anidulafungin, caspofungin, and micafungin encompassing ≥97.5% of the statistically modeled population were, respectively, 0.12, 0.25, and 0.06 µg/ml for C. albicans, 0.12, 0.25, and 0.03 µg/ml for C. glabrata complex, 4, 2, and 4 µg/ml for C. parapsilosis complex, 0.5, 0.25, and 0.06 µg/ml for C. tropicalis, 0.25, 1, and 0.25 µg/ml for C. krusei, 0.25, 1, and 0.12 µg/ml for C. lusitaniae, 4, 2, and 2 µg/ml for C. guilliermondii, and 0.25, 0.25, and 0.12 µg/ml for C. dubliniensis. Species-specific SYO ECVs for anidulafungin, caspofungin, and micafungin correctly classified 72 (88.9%), 74 (91.4%), 76 (93.8%), respectively, of 81 Candida isolates with identified fks mutations. SYO ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin, micafungin, and especially caspofungin, since testing the susceptibilities of Candida spp. to caspofungin by reference methodologies is not recommended.

Different risk factors for candidemia occur for Candida species belonging to the C. parapsilosis complex.

Previous studies have demonstrated reduced virulence in the species that comprise the Candida parapsilosis complex. We investigated a cohort of 93 patients with candidemia caused by this complex. Most infections were caused by C. parapsilosis (80.6%), followed by C. orthopsilosis (18.3%) and C. metapsilosis (1.1%). Renal failure (P < 0.001) and chronic liver diseases (P = 0.019) were more frequently encountered with infections caused by the C. orthopsilosis group, suggesting an association with patients who had a greater state of immune suppression in comparison with infections caused by C. parapsilosis sensu stricto.

Epidemiology and molecular characterization of bacteremia due to carbapenem-resistant Klebsiella pneumoniae in transplant recipients.

We conducted a retrospective study of 17 transplant recipients with carbapenem-resistant Klebsiella pneumoniae bacteremia, and described epidemiology, clinical characteristics and strain genotypes. Eighty-eight percent (15/17) of patients were liver or intestinal transplant recipients. Outcomes were death due to septic shock (18%), cure (24%) and persistent (>7 days) or recurrent bacteremia (29% each). Thirty- and 90-day mortality was 18% and 47%, respectively. Patients who were cured received at least one active antimicrobial agent and underwent source control interventions. Forty-one percent (7/17) of patients had intra-abdominal infections; all except one developed persistent/recurrent bacteremia despite drainage. Two patients tolerated persistent bacteremia for >300 days. All patients except one were infected with sequence type 258 (ST258), K. pneumoniae carbapenemase (KPC)-2-producing strains harboring a mutant ompK35 porin gene; the exception was infected with an ST37, KPC-3-producing strain. Seventy-one percent (12/17) of patients were infected with ST258 ompK36 mutant strains. In two patients, persistent bacteremia was caused by two strains with different ompK36 genotypes. Three ompK36 mutations were associated with significantly higher carbapenem minimum inhibitory concentrations than wild-type ompK36. Pulse-field gel electrophoresis identified a single ST258 lineage; serial strains from individual patients were indistinguishable. In conclusion, KPC-K. pneumoniae bacteremia exhibited highly diverse clinical courses following transplantation, and was caused by clonal ST258 strains with different ompK36 genotypes.

Isavuconazole activity against Aspergillus lentulus, Neosartorya udagawae, and Cryptococcus gattii, emerging fungal pathogens with reduced azole susceptibility.

Isavuconazole is an extended-spectrum triazole with in vitro activity against a wide variety of fungal pathogens. Clinical isolates of molds Aspergillus lentulus and Neosartorya udagawae and yeast Cryptococcus gattii VGII (implicated in the outbreak in the Pacific Northwest, North America) exhibit reduced susceptibilities to several azoles but higher susceptibilities to isavuconazole.

Clinical breakpoints for the echinocandins and Candida revisited: integration of molecular, clinical, and microbiological data to arrive at species-specific interpretive criteria.

The CLSI established clinical breakpoints (CBPs) for caspofungin (CSF), micafungin (MCF) and anidulafungin (ANF) versus Candida. The same CBP (susceptible (S): MIC ≤ 2 mcg/ml; non-S: MIC > 2 mcg/ml) was applied to all echinocandins and species. More data now allow reassessment of these CBPs. We examined cases of echinocandin failure where both MICs and fks mutations were assessed; wild type (WT) MICs and epidemiological cutoff values (ECVs) for a large Candida collection; molecular analysis of fks hotspots for Candida with known MICs; and pharmacokinetic and pharmacodynamic (PK/PD) data. We applied these findings to propose new species-specific CBPs for echinocandins and Candida. Of 18 candidiasis cases refractory to echinocandins and with fks mutations, 28% (CSF), 58% (ANF) and 66% (MCF) had MICs in the S category using CBP of ≤ 2 mcg/ml, while 0-8% would be S using CBP of ≤ 0.25 mcg/ml. WT MIC distributions revealed ECV ranges of 0.03-0.25 mcg/ml for all major species except C. parapsilosis (1-4 mcg/ml) and C. guilliermondii (4-16 mcg/ml). Among Candida tested for fks mutations, only 15.7-45.1% of 51 mutants were detected using the CBP for NS of >2 mcg/ml. In contrast, a cutoff of >0.25 mcg/ml for C. albicans, C. tropicalis, C. krusei, and C. dubliniensis detected 85.6% (MCF) to 95.2% (CSF) of 21 mutant strains. Likewise, a cutoff of >0.12 mcg/ml for ANF and CSF and of >0.06 mcg/ml for MCF detected 93% (ANF) to 97% (CSF, MCF) of 30 mutant strains of C. glabrata. These data, combined with PK/PD considerations, support CBPs of ≤ 0.25 mcg/ml (S), 0.5 mcg/ml (I), ≥ 1 (R) for CSF/MCF/ANF and C. albicans, C. tropicalis and C. krusei and ≤ 2 mcg/ml (S), 4 mcg/ml (I), and ≥ 8 mcg/ml (R) for these agents and C. parapsilosis. The CBPs for ANF and CSF and C. glabrata are ≤ 0.12 mcg/ml (S), 0.25 mcg/ml (I), and ≥ 0.5 mcg/ml (R), whereas those for MCF are ≤ 0.06 mcg/ml (S), 0.12 mcg/ml (I), and ≥ 0.25 mcg/ml (R). New, species-specific CBPs for Candida and the echinocandins are more sensitive to detect emerging resistance associated with fks mutations, and better able to predict risk for clinical failure.

Pharmacodynamics of echinocandins against Candida glabrata: requirement for dosage escalation to achieve maximal antifungal activity in neutropenic hosts.

Candida glabrata is a leading cause of disseminated candidiasis. The echinocandins are increasingly used as first-line agents for the treatment of patients with this syndrome, although the optimal regimen for the treatment of invasive Candida glabrata infections in neutropenic patients is not known. We studied the pharmacokinetics (PK) and pharmacodynamics (PD) of micafungin, anidulafungin, and caspofungin in a neutropenic murine model of disseminated Candida glabrata infection to gain further insight into optimal therapeutic options for patients with this syndrome. A mathematical model was fitted to the data and used to bridge the experimental results to humans. The intravenous inoculation of Candida glabrata in mice was followed by logarithmic growth throughout the experimental period (101 h). A dose-dependent decline in fungal burden was observed following the administration of 0.1 to 20 mg/kg of body weight every 24 h for all three agents. The exposure-response relationships for each drug partitioned into distinct fungistatic and fungicidal components of activity. Surprisingly, the average human drug exposures following currently licensed regimens were predicted to result in a fungistatic antifungal effect. Higher human dosages of all three echinocandins are required to induce fungicidal effects in neutropenic hosts.

Disseminated Candidiasis caused by Candida albicans with amino acid substitutions in Fks1 at position Ser645 cannot be successfully treated with micafungin.

The clinical utility of the echinocandins is potentially compromised by the emergence of drug resistance. We investigated whether Candida albicans with amino acid substitutions at position Ser645 in Fks1 can be treated with either a conventional or an elevated dosage of micafungin. We studied Candida albicans (wild-type SC5314; MIC, 0.06 mg/liter) and four fks1 mutants (one FKS1/fks1 heterozygote mutant [MIC, 0.5 mg/liter] and three fks1/fks1 homozygous mutants [MICs for all, 2 mg/liter]) with a variety of amino acid substitutions at Ser645. The pharmacokinetic and pharmacodynamic relationships were characterized in a persistently neutropenic murine model of disseminated candidiasis. A mathematical model was fitted to all pharmacokinetic and pharmacodynamic data. This mathematical model was then used to "humanize" the murine pharmacokinetics, and the predicted antifungal effect was determined. The estimated maximal rate of growth and ultimate fungal densities in the kidney for each of the strains were similar. The administration of micafungin at 1 mg/kg of body weight to the wild type resulted in moderate antifungal activity, whereas the administration of 5 and 20 mg/kg resulted in rapid fungicidal activity. In contrast, the FKS1/fks heterozygote was killed only with 20 mg/kg, and the homozygous fks1 mutants failed to respond to any dosage. The bridging study revealed that human dosages of 100 and 400 mg/day were active only against the wild type, with no activity against either the heterozygote or the homozygote mutants. Ser645 Fks1 Candida albicans mutants cannot be treated with either conventional or elevated dosages of micafungin and should be deemed resistant.

Use of a high-resolution melt assay to characterize codon 54 of the cyp51A gene of Aspergillus fumigatus on a Rotor-Gene 6000 instrument.

A high-resolution melt (HRM) assay using a Rotor-Gene 6000 instrument was developed to characterize the codon for glycine 54 in the cyp51A genes from 13 reference isolates and 12 clinical isolates of Aspergillus fumigatus. Mutations in this codon confer reduced susceptibility to itraconazole and posaconazole. The assay is simple to perform, and a result of "wild type" or "mutant" is available after approximately 1 h following DNA extraction using commercially available reagents and conventional primers.

Pleiotropic plasma membrane ATPase mutations of Saccharomyces cerevisiae.

We isolated a large number of mutations in the structural gene for the plasma membrane ATPase (PMA1) of Saccharomyces cerevisiae. These mutations were selected by their resistance to the aminoglycoside antibiotic hygromycin B. Biochemical analysis of purified membrane preparations showed that the plasma membrane ATPase activity of the mutants was reduced as much as 75%. Intragenic complementation of pma1 mutants suggested that the yeast plasma membrane ATPase was a multimeric enzyme. The pma1 mutants were apparently defective in maintaining internal pH; more than half of the mutants were unable to grow either at a low pH or in the presence of a weak acid. Most pma1 mutants were also osmotic pressure sensitive. At a very low temperature (5 degrees C) many pma1 mutants were unable to grow and were arrested as unbudded cells. The three most severely affected mutants were also unable to grow in the presence of NH4+. The most extreme mutant exhibited a severe defect in progression through the cell cycle; on synthetic medium, the cells progressively accumulated nucleus-containing small buds that generally failed to complete bud enlargement and cytokinesis. Most of the pleiotropic phenotypes of pma1 mutants could be suppressed by the addition of 50 mM KCl but not NaCl to the medium.

Effects of photosensitization by hematoporphyrin derivative on mitochondrial adenosine triphosphatase-mediated proton transport and membrane integrity of R3230AC mammary adenocarcinoma.

Photodynamic therapy, which consists of treatment with hematoporphyrin derivative (HPD) followed by photoradiation with visible light, is a promising approach to treatment of various cancers. To gain further understanding of the mechanisms whereby this therapy produces cytotoxicity, we undertook a study of the mitochondrial proton-translocating adenosine triphosphatase, an enzyme performing the critical role of coupling electrochemical proton gradients to the formation of adenosine triphosphate. Exposure of submitochondrial particles to HPD and photoradiation in vitro caused a marked inhibition of proton transport; inhibition displayed both drug-dose and light-dose relationships. Inhibition of proton transport was correlated with inhibition of adenosine triphosphate hydrolysis, both demonstrating an inhibition rate of 2% min-1. Investigation of effects of HPD plus light on membrane integrity, measured by [3H]sucrose leakage from submitochondrial particles and by K+ leakage from asolectin phospholipid vesicles, indicated that discrete membrane alterations were not the likely cause of initial loss of pH gradient formation. Likewise, photosensitization by HPD to inhibit adenosine triphosphate hydrolysis and proton transport was not coincident with cross-linking of the major subunits of the enzyme. We conclude that mitochondrial function is severely impaired by photodynamic therapy and offers a potentially important target site leading to cytotoxicity.

Modification of the N-terminal polyserine cluster alters stability of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae.

The N-terminus of the H(+)-ATPase from Saccharomyces cerevisiae contains a serine-rich cluster of 11 serine residues in the first 17 amino acids, including a stretch of eight consecutive serine residues. This cluster is conserved in the weakly expressed PMA2 gene from the same organism, but it is not present in PMA genes from other organisms suggesting that it is not likely to represent a conserved functional motif. To better understand whether this region plays a regulatory role, a series of mutant enzymes were generated in which the serine tract was systematically converted to alanine or deleted. Conversion of the first six serine residues to alanine or deletion of the entire serine tract had little effect on cell growth phenotypes. However, when eight or more serines were converted, the mutant cells displayed prominent hygromycin B-resistant and low pH-sensitive phenotypes indicative of reduced H(+)-ATPase function. The mutant enzymes were found to display relatively normal kinetic properties for ATP hydrolysis, but showed significantly decreased abundance in the plasma membrane under stress conditions when eight or more serine residues were converted to alanine. The reduced abundance of the enzyme appeared to be due to degradative turnover, as mutant enzymes with multiple alanine substitutions showed an accelerated rate of turnover relative to wild-type. The polyserine tract in the H(+)-ATPase does not appear to be important for catalysis, but may contribute to overall protein stability.

Mutations of G158 and their second-site revertants in the plasma membrane H(+)-ATPase gene (pma1) in Saccharomyces cerevisiae.

A G158D mutation residing near the cytoplasmic end of transmembrane segment 2 of the H(+)-ATPase from Saccharomyces cerevisiae appears to alter electrogenic proton transport by the proton pump (Perlin et al. (1988) J. Biol. Chem. 263, 18118-18122.) The mutation confers upon whole cells a pronounced growth sensitivity to low pH and a resistance to the antibiotic hygromycin B. The isolated enzyme retains high activity (70% of wild type) but is inefficient at pumping protons in a reconstituted vesicle system, suggesting that this enzyme may be partially uncoupled (Perlin et al. (1989) J. Biol. Chem. 264, 21857-21864). In this study, the acid-sensitive growth phenotype of the pma1-D158 mutant was utilized to isolate second site suppressor mutations in an attempt to probe structural interactions involving amino acid 158. Site-directed mutagenesis of the G158 locus was also performed to explore its local environment. Nineteen independent revertants of pma1-G158D were selected as low pH-resistant colonies. Four were full phenotypic revertants showing both low pH resistance and hygromycin B sensitivity. Of three full revertants analyzed further, one restored the original glycine residue at position 158 while the other two carried compensatory mutations V336A or F830S, in transmembrane segments 4 and 7, respectively. Partial revertants, which could grow on low pH medium but still retained hygromycin B resistance, were identified in transmembrane segments 1 (V127A) and 2 (C148T, G156C), as well as in the cytoplasmic N-terminal domain (E110K) and in the cytoplasmic loop between transmembrane segments 2 and 3 (D170N, L275S). Relative to the G158D mutant, all revertants showed enhanced net proton transport in whole-cell medium acidification assays and/or improved ATP hydrolysis activity. Small polar amino acids (Asp and Ser) could be substituted for glycine at the 158 position to produce active, albeit somewhat defective, enzymes; larger hydrophobic residues (Leu and Val) produced more severe phenotypes. These results suggest that G158 is likely to reside in a tightly packed polar environment which interacts, either directly or indirectly, with transmembrane segments 1, 4 and 7. The revertant data are consistent with transmembrane segments 1 and 2 forming a conformationally sensitive helical hairpin structure.

Assessing hydrophobic regions of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae.

The hydrophobic, photoactivatable probe TID [3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine] was used to label the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The H(+)-ATPase accounted for 43% of the total label associated with plasma membrane protein and incorporated 0.3 mol of [125I]TID per mol of 100 kDa polypeptide. The H(+)-ATPase was purified by octyl glucoside extraction and glycerol gradient centrifugation, and was cleaved by either cyanogen bromide digestion or limited tryptic proteolysis to isolate labeled fragments. Cyanogen bromide digestion resulted in numerous labeled fragments of mass less than 21 kDa. Seven fragments suitable for microsequence analysis were obtained by electrotransfer to poly(vinylidene difluoride) membranes. Five different regions of amino-acid sequence were identified, including fragments predicted to encompass both membrane-spanning and cytoplasmic protein structure domains. Most of the labeling of the cytoplasmic domain was concentrated in a region comprising amino acids 347 to 529. This catalytic region contains the site of phosphorylation and was previously suggested to be hydrophobic in character (Goffeau, A. and De Meis, L. (1990) J. Biol. 265, 15503-15505). Complementary labeling information was obtained from an analysis of limited tryptic fragments enriched for hydrophobic character. Six principal labeled fragments, of 29.6, 20.6, 16, 13.1, 11.4 and 9.7 kDa, were obtained. These fragments were found to comprise most of the putative transmembrane region and a portion of the cytoplasmic region that overlapped with the highly labeled active site-containing cyanogen bromide fragment. Overall, the extensive labeling of protein structure domains known to lie outside the bilayer suggests that [125I]TID labeling patterns cannot be unambiguously interpreted for the purpose of discerning membrane-embedded protein structure domains. It is proposed that caution should be applied in the interpretation of [125I]TID labeling patterns of the yeast plasma membrane H(+)-ATPase and that new and diverse approaches should be developed to provide a more definitive topology model.

Exploring an antifungal target in the plasma membrane H(+)-ATPase of fungi.

The plasma membrane H(+)-ATPase is a promising new antifungal target that is readily probed with the sulfhydryl-reactive reagent omeprazole. Inhibition of the H(+)-ATPase by omeprazole is closely linked to cell killing, and it has been suggested that enzyme inhibition may result from a covalent interaction within the first two transmembrane segments (M1 and M2) (Monk et al. (1995) Biochim. Biophys. Acta 1239, 81-90). In this study, the molecular nature of this interaction was examined by screening a series of 26 well-characterized pma1 mutations residing in the first two transmembrane segments of the H(+)-ATPase from Saccharomyces cerevisiae. Only two pma1 mutants, A135G and G158D,G156C, were found to significantly decrease the sensitivity of cells for omeprazole. In contrast, enhanced sensitivity was observed at a number of positions, with D140C(A) and M128C producing the most significant increases in sensitivity. The introduction of cysteine at various locations within this region only marginally affected omeprazole sensitivity, suggesting that this region was not a direct site of covalent modification. Rather, its conformation influences omeprazole binding at some other locus. In order to determine the sidedness of the omeprazole interaction, a novel in vitro assay system was exploited that utilized liposomes co-reconstituted with the H(+)-ATPase and the light-driven proton pump bacteriorhodopsin. Omeprazole was found to completely inhibit proton transport by the H(+)-ATPase at 50 microM in this system. An asymmetrically-distributed chemical trap system involving glutathione was used to demonstrate that this inhibition appears localized to the extracellular portion of the enzyme. This work indicates that omeprazole can inhibit the H(+)-ATPase from its extracellular face, and this inhibition is influenced by changes in the M1, M2 region of the protein.

Functional complementation between transmembrane loops of Saccharomyces cerevisiae and Candida albicans plasma membrane H(+)-ATPases.

Saccharomyces cerevisiae PMA1 sequences encoding a putative antifungal target site comprising transmembrane loops 1 + 2 and/or 3 + 4 were replaced with the homologous sequences from Candida albicans PMA1 by using PCR-mediated domain transfer. The chimeric pma1 mutants and an isogenic wild type S. cerevisiae strain had similar growth rates, growth yields, glucose-dependent proton pumping rates, acid-activated omeprazole sensitivities, salt tolerances and antifungal sensitivities. The yields and kinetic properties of H(+)-ATPases in plasma membranes of mutant and wild type strains were comparable. Single heterologous transmembrane loops caused deleterious phenotypes at low pH and elevated temperature. Inclusion of both heterologous transmembrane loops fully suppressed the temperature sensitivity caused by heterologous transmembrane loop 1 + 2, partially suppressed the pH sensitivity and gave Candida-like in vitro sensitivity to vanadate, suggesting that the loops operate as a domain. The fully functional chimeric H(+)-ATPase containing C. albicans transmembrane loops 1 + 2 and 3 + 4 demonstrates this domain's complementarity to the equivalent region of the S. cerevisiae enzyme and validates the wild type S. cerevisiae H(+)-ATPase as an antifungal screening target.

Inhibition of Escherichia coli H+-ATPase by venturicidin, oligomycin and ossamycin.

The antibiotics venturicidin, oligomycin and ossamycin were investigated as potential inhibitors of the Escherichia coli H+-ATPase. It was found that venturicidin strongly inhibited ATP-driven proton transport and ATP hydrolysis, while oligomycin weakly inhibited these functions. Inhibition of the H+-ATPase by venturicidin and oligomycin was correlated with inhibition of F0-mediate proton transport. Both inhibitors were found to interfere with the covalent reaction between dicyclohexyl[14C]carbodiimide and the F0 subunit c (uncE protein). Ossamycin had no direct inhibitory effect on E. coli F0 or F1; rather, it was found to uncouple ATP hydrolysis from proton transport.

The yeast plasma membrane proton pumping ATPase is a viable antifungal target. I. Effects of the cysteine-modifying reagent omeprazole.

The yeast plasma membrane proton pumping ATPase (H(+)-ATPase) was investigated as a potential molecular target for antifungal drug therapy by examining the inhibitory effects of the sulfhydryl-reactive reagent omeprazole on cell growth, glucose-induced medium acidification and H(+)-ATPase activity. Omeprazole inhibits the growth of Saccharomyces cerevisiae and the human pathogenic yeast Candida albicans in a pH dependent manner. Omeprazole action is closely correlated with inhibition of the H(+)-ATPase and is fungicidal. Glucose-dependent medium acidification is correspondingly blocked by omeprazole and appears to require the H(+)-ATPase to proceed through its reaction cycle. A strong correlation is observed between inhibition of medium acidification and H(+)-ATPase activity in plasma membranes isolated from treated cells. The inhibitory properties of omeprazole are blocked by pre-treatment of activated drug with beta-mercaptoethanol, which is consistent with the expected formation of a sulfhydryl-reactive sulfenamide derivative. Mutagenesis of the three putative membrane sector cysteine residues (C148S, C312S, C867A) in the S. cerevisiae H(+)-ATPase suggests that covalent modification of the conserved C148 residue may be important for inhibition of ATPase activity and cell growth. Other mutations (M128C and G158D/G156C) mapping near C148 support the importance of this region by modulating omeprazole inhibition of the H(+)-ATPase. These findings suggest that the plasma membrane H(+)-ATPase may serve as an important molecular target for antifungal intervention.

Probing the cytoplasmic LOOP1 domain of the yeast plasma membrane H(+)-ATPase by targeted factor Xa proteolysis.

The cytoplasmic domain linking transmembrane segments 2 and 3 (LOOP1) of the yeast H(+)-ATPase was probed by the introduction of unique factor Xa recognition sites. Three sites, I170EGR, I254EGR and I275EGR, representing different structural regions of the LOOP1 domain, were engineered by site-specific mutagenesis of the PMA1 gene. In each case, multiple amino acid substitutions were required to form the factor Xa sites, which enabled an analysis of clustered mutations. Both I170EGR and I275EGR-containing mutants grew at normal rates, but showed prominent growth resistance to hygromycin B and sensitivity to low external pH. The engineered I254EGR site within the predicted beta-strand region produced a recessive lethal phenotype, indicating that mutations G254I and F257R were not tolerated. Mutant I170EGR- and I275EGR-containing enzymes showed relatively normal Km and Vmax values, but they displayed a strong insensitivity to inhibition by vanadate. An I170EGR/I275EGR double mutant was more significantly perturbed showing a reduced Vmax and pronounced vanadate insensitivity. The I170EGR site within the putative alpha-helical stalk region was cleaved to a maximum of 10% by factor Xa under non-denaturing conditions resulting in a characteristic 81 kDa fragment, whereas the I275EGR site, near the end of the beta-strand region, showed about 30-35% cleavage with the appearance of a 70 kDa fragment. A I170EGR/I275EGR double mutant enzyme showed about 55-60% cleavage. The cleavage profile for the mutant enzymes was enhanced under denaturing conditions, but was unaffected by MgATP or MgATP plus vanadate. Cleavage at the I275EGR position had no adverse effects on ATP hydrolysis or proton transport by the H(+)-ATPase making it unlikely that this localized region of LOOP1 influences coupling. Overall, these results suggest that the local region encompassing I275EGR is accessible to factor Xa, while the region around I170EGR appears buried. Although there is no evidence for gross molecular motion at either site, the effects of multiple amino acid substitutions in these regions suggest that the LOOP1 domain is conformationally active, and that perturbations in this domain affect the distribution of conformational intermediates during steady-state catalysis.