D-glucan Synthase Research Articles

Antibiofilm Activity of Scutellaria baicalensis through the Inhibition of Synthesis of the Cell Wall (1, 3)-β-D-Glucan Polymer

Candida biofilms are self-organized microbial communities growing on the surfaces of host tissues and medical devices. These biofilms have been displaying increasing resistance against conventional antifungal agents. The roots of Scutellaria baicalensis have been widely used for medicinal purpose throughout East Asia. The aim of the present study was to evaluate the effect of S. baicalensis aqueous extract upon the preformed biofilms of 10 clinical C. albicans isolates, and assess the mechanism of the antibiofilm activity. Its effect on preformed biofilm was judged using an XTT reduction assay and the metabolic activity of all tested strains were reduced (%) at MIC values. The S. baicalenis extract inhibited (1, 3)--D-glucan synthase activity. The effect of S. baicalensis on the morphology of C. albicans was related to the changes in growth caused by inhibiting glucan synthesis; most cells were round and swollen, and cell walls were densely stained or ruptured. The anticandidal activity was fungicidal, and the extract also arrested C. albicans cells at . The data suggest that S. baicalensis has multiple fatal effects on target fungi, which ultimately result in cell wall disruption and killing by inhibiting (1, 3)--D-glucan synthesis. Therefore, S. baicalensis holds great promise for use in treating and eliminating biofilm-associated Candida infections.

Characterization of a Distinct Host Response Profile to Pneumocystismurina Asci during Clearance of Pneumocystis Pneumonia

Pneumocystis spp. are yeast-like fungi that cause pneumocystis pneumonia (PcP) in immunocompromised individuals and exacerbate chronic lung diseases in immunocompetent individuals. The Pneumocystis life cycle includes trophic forms and asci (cyst forms). The cell walls of Pneumocystis asci contain β-1,3-D-glucan, and treatment of PcP with β-1,3-D-glucan synthase inhibitors, such as anidulafungin, results in depletion of asci, but not trophic forms. The pulmonary host response during immune reconstitution (IR)-mediated clearance of PcP in anidulafungin-treated and untreated mice was characterized to identify ascus-specific responses. During IR, similar numbers of trophic forms were present in the anidulafungin-treated and untreated mice; however, asci were only present in the untreated mice. IR resulted in a significant reduction of trophic forms from the lungs in both groups and asci in the untreated group. The presence of asci in untreated mice correlated with increased β-glucan content in the lungs. The untreated mice mounted immune responses associated with a deleterious host inflammatory response, including increased CD8(+) T cell influx and expression of macrophage inflammatory response markers. A more robust cellular response was also observed in the untreated mice, with increased numbers of macrophages and neutrophils that were associated with greater lung damage. Markers of a Th17 response were also elevated in the untreated mice. These results suggest that the host mounts unique responses to asci and trophic forms. That these 2 life cycle stages provoked distinct host response profiles has significant implications for clearance and interpretation of the host immune responses to PcP.

Efficient Bioconversion of Echinocandin B to Its Nucleus by Overexpression of Deacylase Genes in Different Host Strains

Anidulafungin, which noncompetitively inhibits β-(1,3)-D-glucan synthase in fungal cell wall biosynthesis, is the newest antifungal drug to be developed. Echinocandin B deacylase from Actinoplanes utahensis NRRL 12052 catalyzes the cleavage of the linoleoyl group of echinocandin B, a key step in the process of manufacturing anidulafungin. Unfortunately, the natural yield of echinocandin B nucleus is low. In our study, the echinocandin B deacylase gene was systematically overexpressed by genetic engineering in its original producer, A. utahensis, and in the heterologous hosts Streptomyces lividans TK24 and Streptomyces albus. The introduction of additional copies of the gene, under the control of PermE* or its native promoter, into hosts showed significant increases in its transcription level and in the efficiency of the bioconversion of echinocandin B to its nucleus. The conditions for the cultivation and bioconversion of A. utahensis have been optimized further to improve production. As a result, while the wild-type strain initially produced 0.36 g/liter, a concentration of 4.21 g/liter was obtained after the generation of a strain with additional copies of the gene and further optimization of the reaction conditions. These results are useful for enhancing echinocandin B nucleus production in A. utahensis. Our study could enable the engineering of commercially useful echinocandin B nucleus-overproducing stains.

Synthesis and biological evaluation of antifungal derivatives of enfumafungin as orally bioavailable inhibitors of β-1,3-glucan synthase

Orally bioavailable inhibitors of β-(1,3)-d-glucan synthase have been pursued as new, broad-spectrum fungicidal therapies suitable for treatment in immunocompromised patients. Toward this end, a collaborative medicinal chemistry program was established based on semisynthetic derivatization of the triterpenoid glycoside natural product enfumafungin in order to optimize in vivo antifungal activity and oral absorption properties. In the course of these studies, it was hypothesized that the pharmacokinetic properties of the semisynthetic enfumafungin analog 3 could be improved by tethering the alkyl groups proximal to the basic nitrogen of the C3-aminoether side chain into an azacyclic system, so as to preclude oxidative N-demethylation. The results of this research effort are disclosed herein.

Synthesis and antifungal properties of papulacandin derivatives

Derivatives of an antifungal agent that targets the β-(1,3)-D-glucan synthase, papulacandin D, were synthesized and tested for activity. The papulacandin D structure contains a challenging benzannulated spiroketal unit, which is introduced in a palladium-catalyzed cross-coupling reaction of a glycal silanolate and an aryl iodide followed by an oxidative spiroketalization. Four different variants were made, differing in the nature of the acyl side chain with respect to the length, and in the number and stereochemistry of the double bonds. Moderate biological activity was observed for the derivatives with a side chain based on palmitic acid and linoleic acid.

β(1,3)-glucan synthase complex fromAlternaria infectoria, a rare dematiaceous human pathogen

The fungal cell wall polymer β-(1,3)-D-glucan is synthesized by the enzyme β-(1,3)- D-glucan synthase that is a complex composed of at least two proteins, Rho1p and Fks1p. Here, we report the nucleotide sequence of a single FKS gene and of the regulatory unit, RHO1 from the dematiaceous pathogenic fungus Alternaria infectoria. The predicted AiFks and AiRho share, respectively, 93% and 100% identity with that of Drechslera tritici-repentis. We also report that the sensitivity to caspofungin of eight different A. infectoria clinical strains is similar, with a MIC > 32 µg/ml and a MEC of 1 µg/ml, except for one strain which had a MEC of 1.4 µg/ml. This same strain exhibited one substitution at the hot spot 2, S1405A, compatible with less susceptible phenotypes, with the other seven strains having no mutations in either hot spot 1 or 2. The relative quantification of the expression of AiFKS and of AiRHO demonstrated a decrease in response to an exposure to caspofungin at 0.5 µg/ml.

A Genomewide Screen in Schizosaccharomyces pombe for Genes Affecting the Sensitivity of Antifungal Drugs That Target Ergosterol Biosynthesis

We performed a genomewide screen for altered sensitivity to antifungal drugs, including clotrimazole and terbinafine, that target ergosterol biosynthesis using a Schizosaccharomyces pombe gene deletion library consisting of 3,004 nonessential haploid deletion mutants. We identified 109 mutants that were hypersensitive and 11 mutants that were resistant to these antifungals. Proteins whose absence rendered cells sensitive to these antifungals were classified into various functional categories, including ergosterol biosynthesis, membrane trafficking, histone acetylation and deacetylation, ubiquitination, signal transduction, ribosome biosynthesis and assembly, regulation of transcription and translation, cell wall organization and biogenesis, mitochondrion function, amino acid metabolism, nucleic acid metabolism, lipid metabolism, meiosis, and other functions. Also, proteins whose absence rendered cells resistant to these antifungals were classified into functional categories including mitochondrion function, ubiquitination, membrane trafficking, cell polarity, chromatin remodeling, and some unknown functions. Furthermore, the 109 sensitive mutants were tested for sensitivity to micafungin, another antifungal drug that inhibits (1,3)-β-D-glucan synthase, and 57 hypersensitive mutants were identified, suggesting that these mutants were defective in cell wall integrity. Altogether, our findings in fission yeast have shed light on molecular pathways associated with the cellular response to ergosterol biosynthesis inhibitors and may provide useful information for developing strategies aimed at sensitizing cells to these drugs.

Regulation of (1,3;1,4)-β- d-glucan synthesis in developing endosperm of barley lys mutants

The mechanism behind altered content of (1,3;1,4)-β- d-glucan was investigated in developing endosperm of barley lys3 and lys5 mutants. Both types of mutants are primarily affected in starch biosynthesis, and hence effects on (1,3;1,4)-β- d-glucan are likely to be pleiotropic. The mutant alleles lys5f and lys5g exerted pronounced effects on the cell wall with increased level of (1,3;1,4)-β- d-glucan content. The low-starch high-(1,3;1,4)-β- d-glucan phenotype was most pronounced in lys5f. Among the Cellulose Synthase-Like ( CSL) gene members belonging to the families CSLF and CSLH, which all encode (1,3;1,4)-β- d-glucan synthase proteins, CSLF6 was by far the highest expressed in the wild type, whereas both lys5f and lys5g exhibited a decreased level of CSLF6 transcript. Thus, the lys5 mutants have increased (1,3;1,4)-β- d-glucan level in spite of lower transcript levels. This suggests the presence of a sensing and signaling system in the cell wall, which in the case of the lys5 mutants caused a decreased transcript level in response to the increased (1,3;1,4)-β- d-glucan levels. In the lys3a mutant we found a 1000-fold repression of the CSLF6 transcript throughout the whole endosperm development. Thus CSLF6 is under the control of the Lys3 transcriptional regulatory mechanism that operates during barley grain development.

Synthesis and evaluation of novel macrocyclic antifungal peptides

Echinocandins are a novel class of macrocyclic antifungal peptides that act by inhibiting the β-(1,3)-d-glucan synthase complex, which is not present in mammalian cells. Due to the large number of hydroxyl groups present in these complex macrocyclic lipopeptides, most structure–activity relationship studies have relied upon semisynthetic derivatives. In order to probe the influence of the cyclic peptide backbone on the antifungal activity we developed a successful strategy for the synthesis of novel echinocandins analogues by on-resin ring closing metathesis or disulfide formation. The specific minimum inhibitory activity of each mimic was determined against Candida albicans. Our results indicate that ring size is an important factor for antifungal activity.

Echinocandins in Children

Echinocandins in Children

Synthesis, anti-fungal and 1,3-β- d-glucan synthase inhibitory activities of caffeic and quinic acid derivatives

New derivatives of caffeic acid and quinic acid were synthesized and their anti-fungal and inhibitory activities on fungal 1,3-β-glucan synthase were determined in comparison with those of the corresponding chlorogenic acid derivatives. All the chlorogenic, quinic and caffeic acid derivatives that were coupled with an H 2N-orn-4-(octyloxy) aniline group ( 1, 1b and 1c) displayed potent activities in both anti-fungal and inhibition of 1,3-glucan synthase assays. Compounds 1 and 1c inhibited the fungal membrane enzyme with the potency comparable to that of a known 1,3-β- d-glucan synthase inhibitor, aculeacin A. The results revealed that the anti-fungal activity of the chlorogenic acid derivative with a free amino group was at least partly due to inhibition of the fungal 1,3-β-glucan synthase. These results suggest that further investigation on caffeic acid derivatives may lead to the discovery of novel anti-fungal agents with drug-like properties.

1,3-β- d-Glucan synthase of Paracoccidioides brasiliensis: recombinant protein, expression and cytolocalization in the yeast and mycelium phases

Paracoccidioides brasiliensis is a thermo-dimorphic human pathogenic fungus that in the mycelium phase lives at 23 °C in environment and in the yeast phase at 37 °C in the host tissues. In P. brasiliensis, the main polymers that compound the cell wall are chitin, 1,3-β- d-glucan and 1,3-α-glucan. They make a primary barrier responsible for the structural integrity and form of the cell wall. In P. brasiliensis, just one homologue of 1,3-β- d-glucan synthase gene ( PbFKS1) was found. Here, the active recombinant protein ( PbFks1pc) containing the catalytic region was obtained in Escherichia coli. In addition, a paradoxical dissociation was detected between the expression of the PbFKS1 transcript and the level of the corresponding protein PbFks1p, which was higher in the yeast phase, versus the amount of 1,3-β- d-glucan polymer, which was higher in the mycelium phase. Western blot analysis using protein extracts of cellular fractions showed that PbFks1p is present in the membrane-enriched fraction of mycelium and yeast cells and in the cell wall-enriched fractions of yeast cells. Confocal-immunocytolocalization of PbFks1p identified the protein in the apical growing region of the mycelium and distributed on the surface of the yeast cell. Two possible mechanisms could explain the above-mentioned discrepancy between the data: (a) overexpression of Rho1 GTPase as a regulator of 1,3-β- d-glucan synthase; (b) possible post-translational regulation of PbFks1p in P. brasiliensis isolates.

Novel FKS Mutations Associated with Echinocandin Resistance in Candida Species

We studied three clinical isolates of Candida spp. (one C. tropicalis isolate and two C. glabrata isolates) from patients with invasive candidiasis. The first isolate emerged during echinocandin treatment, while the others emerged after the same treatment. These strains harbored an amino acid substitution in Fksp never linked before with reduced echinocandin susceptibility in C. tropicalis or in C. glabrata. The molecular mechanism of reduced susceptibility was confirmed using a 1,3-beta-D-glucan synthase inhibition assay.

Echinocandins: The Newest Class of Antifungals

To review the mechanism of action, antifungal spectrum of activity, pharmacodynamics, pharmacokinetics, clinical efficacy, and safety of the echinocandins. A MEDLINE search (1982-May 2009) was conducted for articles published in the English language using the key words caspofungin, micafungin, anidulafungin, and echinocandins. Medicinal chemistry, in vitro, and animal studies, as well as human trials were reviewed for information on the pharmacodynamics, pharmacokinetics, efficacy, and safety of each echinocandin. Clinical trials were reviewed and included to compare and contrast the available echinocandins. Three echinocandin antifungal agents are currently approved for use in the US: caspofungin, micafungin, and anidulafungin. The echinocandins have a unique mechanism of action, inhibiting beta-(1,3)-D-glucan synthase, an enzyme that is necessary for the synthesis of an essential component of the cell wall of several fungi. The echinocandins display fungistatic activity against Aspergillus spp. and fungicidal activity against most Candida spp., including strains that are fluconazole-resistant. The echinocandins have been shown to be efficacious for the treatment of esophageal candidiasis, candidemia, and invasive candidiasis. In addition, caspofungin has demonstrated efficacy as empiric treatment of febrile neutropenia and salvage therapy for the treatment of invasive aspergillosis, and it is the only echinocandin approved for use in pediatric patients. Micafungin is the only echinocandin approved for use as prophylaxis against Candida infections in patients undergoing hematopoietic stem cell transplantation. Overall, resistance to echinocandins is still rare, and all agents are well tolerated, with similar adverse effect profiles and few drug-drug interactions. Echinocandins, the newest addition to the arsenal of antifungals, offer potential advantages over other classes of agents. Clinicians should assess their distinguishing characteristics, including route of metabolism, drug interaction profile, and approved indications for use, when determining which agent to include on a formulary.

Effect of Candida glabrata FKS1 and FKS2 Mutations on Echinocandin Sensitivity and Kinetics of 1,3-β- d -Glucan Synthase: Implication for the Existing Susceptibility Breakpoint

Thirteen Candida glabrata strains harboring a range of mutations in hot spot regions of FKS1 and FKS2 were studied. The mutations were linked to an echinocandin reduced susceptibility phenotype. Sequence alignments showed that 11 out of the 13 mutants harbored a mutation in FKS1 or FKS2 not previously implicated in echinocandin reduced susceptibility in C. glabrata. A detailed kinetic characterization demonstrated that amino acid substitutions in Fks1p and Fks2p reduced drug sensitivity in mutant 1,3-beta-D-glucan synthase by 2 to 3 log orders relative to that in wild-type enzyme. These mutations were also found to reduce the catalytic efficiency of the enzyme (Vmax) and to influence the relative expression of FKS genes. In view of the association of FKS mutations and reduced susceptibility of 1,3-beta-D-glucan synthase, an evaluation of the new CLSI echinocandin susceptibility breakpoint was conducted. Only 3 of 13 resistant fks mutants (23%) were considered anidulafungin or micafungin nonsusceptible (MIC > 2 microg/ml) by this criterion. In contrast, most fks mutants (92%) exceeded a MIC of >2 microg/ml with caspofungin. However, when MIC determinations were performed in the presence of 50% serum, all C. glabrata fks mutants showed MICs of > or = 2 microg/ml for the three echinocandin drugs. As has been observed with Candida albicans, the kinetic inhibition parameter 50% inhibitory concentration may be a better predictor of FKS-mediated resistance. Finally, the close association between FKS1/FKS2 hot spot mutations provides a basis for understanding echinocandin resistance in C. glabrata.

Analysis of the (1,3)-β- d-glucan synthase gene family of barley

Callose consists mostly of (1,3)-β- d-glucan and is synthesised in many plant tissues during growth and development, where it is believed to play a fundamental role in cell plate formation during cell division. Callose deposition also represents an important response to pathogen attack, wounding and to various abiotic stresses. Here, the transcription patterns of members of the callose synthase gene family from barley ( Hordeum vulgare) were defined. Thus, fragments of six barley (1,3)-β- d-glucan synthase-like ( GSL) cDNAs were obtained by PCR amplification using primers designed to barley expressed sequence tag (EST) sequences. The HvGSL genes, designated HvGSL2 to HvGSL7, were mapped to individual loci that were distributed across the barley genome on chromosomes 3H, 4H, 6H and 7H. The HvGSL1 gene has been isolated and characterised previously. Transcript levels for all the genes were analysed by quantitative real-time PCR in a range of barley tissues and organs, at various developmental stages. High levels of transcript for many of the HvGSL genes were detected in endosperm during the early stages of grain development, when cellularisation of the endosperm was occurring and it is likely that many of the genes participate in this process. Transcripts of HvGSL1 and HvGSL5 mRNAs were significantly more abundant than other GSL mRNAs in the roots of young seedlings, while HvGSL7 mRNA was detected at relatively high levels along the length of two week old shoots.

Correlating Echinocandin MIC and Kinetic Inhibition of fks1 Mutant Glucan Synthases for Candida albicans : Implications for Interpretive Breakpoints

A detailed kinetic characterization of echinocandin inhibition was performed for mutant 1,3-beta-d-glucan synthase enzymes from clinical isolates of Candida albicans with nine different FKS1 mutations resulting in high MICs. Among 14 mutant Fks1p enzymes studied, the kinetic parameters 50% inhibitory concentration and K(i) increased 50-fold to several thousandfold relative to those for the wild type. Enzymes with mutations at Ser645 (S645P, S645Y, and S645F) within hot spot 1 showed the most prominent decrease in sensitivity, while those with mutations at the N- and C-terminal ends of hot spot 1 generally retained greater sensitivity to all three drugs. Kinetic inhibitions by caspofungin, micafungin, and anidulafungin were comparable among the fks1 mutant enzymes, although absolute values did vary with specific mutations. Amino acid substitutions in Fks1p did not alter K(m) values, although some mutations decreased the V(max). Given the association of FKS1 mutations with clinical resistance, an evaluation of the kinetic parameters for the inhibition of mutant 1,3-beta-D-glucan synthase as a function of the MIC enabled an independent evaluation of the recently adopted susceptibility breakpoint for echinocandin drugs. Overall, a breakpoint MIC of >or=2 microg/ml for caspofungin captured nearly 100% of fks1 C. albicans strains when a kinetic inhibition rise threshold of <or=50-fold for the K(i) was used as a measure of susceptibility. A similar MIC breakpoint for micafungin and anidulafungin was less inclusive, and a projected MIC of >or=0.5 microg/ml was required for >95% coverage of clinical isolates. However, when MIC determinations were performed in the presence of 50% serum, all fks1 mutants showed MIC values of >or=2 microg/ml for the three echinocandin drugs. The 1,3-beta-D-glucan synthase kinetic inhibition data support the proposed susceptibility breakpoint for caspofungin in C. albicans, but a lower susceptibility breakpoint (<or=0.5 microg/ml) may be more appropriate for anidulafungin and micafungin. Overall, the data indicate that MIC testing with caspofungin may serve as a surrogate marker for resistance among the class of echinocandin drugs.

Mutations in the fks1 Gene in Candida albicans , C. tropicalis , and C. krusei Correlate with Elevated Caspofungin MICs Uncovered in AM3 Medium Using the Method of the European Committee on Antibiotic Susceptibility Testing

Mutations in two specific regions of the Fks1 subunit of 1,3-beta-D-glucan synthase are known to confer decreased caspofungin susceptibility on Candida spp. Clinical isolates of Candida spp. (404 Candida albicans, 62 C. tropicalis, and 21 C. krusei isolates) sent to the French National Reference Center were prospectively screened for susceptibility to caspofungin in vitro by the broth microdilution reference method of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST). Twenty-eight isolates (25 C. albicans, 2 C. tropicalis, and 1 C. krusei isolate) for which the caspofungin MIC was above the MIC that inhibited 90% of the isolates of the corresponding species (MIC(90)) were subjected to molecular analysis in order to identify mutations in the fks1 gene. Substitutions in the deduced protein sequence of Fks1 were found for 8 isolates, and 20 isolates had the wild-type sequence. Among the six C. albicans isolates harboring mutations, six patterns were observed involving amino acid changes at positions 641, 645, 649, and 1358. For C. tropicalis, one isolate showed an L644W mutation, and for one C. krusei isolate, two mutations, L658W and L701M, were found. Two media, RPMI medium and AM3, were tested for their abilities to distinguish between isolates with wild-type Fks1 and those with mutant Fks1. In RPMI medium, caspofungin MICs ranged from 0.25 to 2 microg/ml for wild-type isolates and from 1 to 8 micro for mutant isolates. A sharper difference was observed in AM3: all wild-type isolates were inhibited by 0.25 micro of caspofungin, while caspofungin MICs for all mutant isolates were >or=0.5 microg/ml. These data demonstrate that clinical isolates of C. albicans, C. tropicalis, and C. krusei with decreased susceptibility to caspofungin in vitro have diverse mutations in the fks1 gene and that AM3 is potentially a better medium than RPMI for distinguishing between mutant and wild-type isolates using the AFST-EUCAST method.

Efficacy of Caspofungin against Aspergillus flavus , Aspergillus terreus , and Aspergillus nidulans

The echinocandin caspofungin is a potent inhibitor of the activity of 1,3-beta-D-glucan synthase from Aspergillus flavus, Aspergillus terreus, and Aspergillus nidulans. In murine models of disseminated infection, caspofungin prolonged survival and reduced the kidney fungal burden. Caspofungin was at least as effective as amphotericin B against these filamentous fungi in vivo.

Echinocandins in the management of invasive fungal infections, part 2

The chemistry, pharmacology, spectrum of activity, resistance, pharmacokinetics, pharmacodynamics, clinical efficacy, adverse effects, drug interactions, dosage and administration, cost, and place in therapy of echinocandins are reviewed. Three echinocandins are currently available: caspofungin, micafungin, and anidulafungin. The principal mechanism of action of the echinocandins is the noncompetitive inhibition of beta-(1,3)-D-glucan synthase, an essential component of the cell wall of many fungi that is not present in mammalian cells. Echinocandins exhibit fungicidal activity against Candida species, including triazole-resistant isolates, and fungistatic activity against Aspergillus species. While fungistatic against mold, echinocandins may hold promise for the treatment of these pathogens when given in combination with amphotericin B or broad-spectrum triazoles, such as voriconazole. To date, resistance to echinocandins has been reported in only two patients. Echinocandins exhibit concentration-dependent activity against Candida species. In clinical trials, caspofungin has demonstrated efficacy in treating candidemia, esophageal candidiasis, and febrile neutropenia. Micafungin has demonstrated efficacy as antifungal prophylaxis in hematopoietic stem cell transplant recipients and in the treatment of esophageal candidiasis. Anidulafungin received approved labeling from the Food and Drug Administration in February 2006. Clinical efficacy data will be forthcoming. Echinocandins are fungicidal against yeast and fungistatic against mold. Their limited toxicity profile and minimal drug-drug interactions make them an attractive new option for the treatment of invasive fungal infections. Their cost may limit their use as initial therapy for patients with fungemia in medical centers or intensive care units with a high rate of triazoleresistant Candida infections.