ABSTRACT
With over 300 million severe cases and 1.5 million deaths annually, invasive fungal diseases (IFDs) are a major medical burden and source of global morbidity and mortality. The World Health Organization (WHO) recently released the first-ever fungal priority pathogens list including 19 fungal pathogens, considering the perceived public health importance. Most of the pathogenic fungi are opportunistic and cause diseases in patients under immunocompromised conditions such as HIV infection, cancer, chemotherapy, transplantation, and immune suppressive drug therapy. Worryingly, the morbidity and mortality caused by IFDs are continuously on the rise due to the limited available antifungal therapies, the emergence of drug resistance, and the increase of population that is vulnerable to IFDs. Moreover, the COVID-19 pandemic worsened IFDs as a globe health threat as it predisposes the patients to secondary life-threatening fungi. In this mini-review, we provide a perspective on the advances and strategies for combating IFDs with antifungal therapies.
Subject(s)
COVID-19 , HIV Infections , Invasive Fungal Infections , Humans , Antifungal Agents/therapeutic use , HIV Infections/drug therapy , Pandemics , COVID-19/epidemiology , Invasive Fungal Infections/drug therapyABSTRACT
The complete biosynthetic pathways of the potent antifungals AS2077715 (1) and funiculosin (2) are reconstituted and characterized. A five-enzyme cascade, including a multifunctional flavin-dependent monooxygenease and a repurposed O-methyltransferase, is involved to perform the dearomatization, stereoselective ring contraction, and redox transformations to morph a hydroxyphenyl-containing precursor into the unusual all-cis cyclopentanetetraol moiety.
Subject(s)
Antifungal Agents , Oxidation-ReductionABSTRACT
Aspergillus subgenus Nidulantes includes species with emericella-like ascomata and asexual species. Subgenus Nidulantes is the second largest subgenus of Aspergillus and consists of nine sections. In this study, agricultural soils were sampled from 12 provinces and autonomous regions in China. Based on primary BLAST analyses, seven of 445 Aspergillus isolates showed low similarity with existing species. A polyphasic investigation, including phylogenetic analysis of partial ITS, ß-tubulin, calmodulin, and RNA polymerase II second largest subunit genes, provided evidence that these isolates were distributed among four new species (Aspergillus guangdongensis, A. guangxiensis, A. sichuanensis and A. tibetensis) in sections Aenei, Ochraceorosei, and Sparsi of subgenus Nidulantes. Illustrated morphological descriptions are provided for each new taxon.
ABSTRACT
The genus Sphaerostilbella comprises fungi that colonize basidiomata of wood-inhabiting fungi, including important forest pathogens. Studies of fermentation cultures of an isolate (TFC201724) collected on the foothills of Himalayas, and closely related to S. broomeana isolates from Europe, led to the identification of a new cyclic octapeptide along with two closely related analogues (1-3) and four dioxopiperazines (4-7). The structure of the lead compound, broomeanamide A (1), was assigned mainly by analysis of 2D NMR and HRESIMS data. The structure consisted of one unit each of N-MeVal, Ala, N-MePhe, Pro, Val, and Ile and two N-MeLeu units. The amino acid sequence was determined on the basis of 2D NMR and HRESIMSMS data. NMR and HRMS data revealed that the other two new peptides have the same amino acid composition except that the Ile unit was replaced with Val in one instance (2) and the N-MeVal unit was replaced with Val in the other (3). The absolute configuration of 1 was assigned by analysis of the acid hydrolysate by application of Marfey's method using both C18 and C3 bonded-phase columns. Broomeanamide A (1) showed antifungal activity against Cryptococcus neoformans and Candida albicans, with MIC values of 8.0 and 64 µg/mL, respectively.
Subject(s)
Antifungal Agents/pharmacology , Hypocreales/chemistry , Peptides, Cyclic/pharmacology , Amino Acid Sequence , Antifungal Agents/isolation & purification , Candida albicans/drug effects , Cryptococcus neoformans/drug effects , India , Molecular Structure , Peptides, Cyclic/isolation & purificationABSTRACT
Lanosterol 14α-demethylase (CYP51) is an important target in the development of antifungal drugs. The fungal-derived restricticin 1 and related molecules are the only examples of natural products that inhibit CYP51. Here, using colocalizations of genes encoding self-resistant CYP51 as the query, we identified and validated the biosynthetic gene cluster (BGC) of 1. Additional genome mining of related BGCs with CYP51 led to production of the related lanomycin 2. The pathways for both 1 and 2 were identified from fungi not known to produce these compounds, highlighting the promise of the self-resistance enzyme (SRE) guided approach to bioactive natural product discovery.
Subject(s)
14-alpha Demethylase Inhibitors/metabolism , Biological Products/metabolism , Cytochrome P450 Family 51/genetics , Antifungal Agents/chemistry , Antifungal Agents/metabolism , Biological Products/chemistry , Cytochrome P450 Family 51/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fungi/genetics , Multigene Family , Pyrans/chemistry , Pyrans/metabolismABSTRACT
Cryptococcus neoformans is a serious human pathogen with limited options for treatment. We have interrogated extracts from fungal fermentations to find Cryptococcus-inhibiting natural products using assays for growth inhibition and differential thermosensitivity. Extracts from fermentations of four fungal strains from wild and domestic animal dung from Arkansas and West Virginia, USA were identified as Preussia typharum. The extracts exhibited two antifungal regions. Purification of one region yielded new 24-carbon macrolides incorporating both a phosphoethanolamine unit and a bridging tetrahydrofuran ring. The structures of these metabolites were established mainly by analysis of high-resolution mass spectrometry and 2D NMR data. Relative configurations were assigned using NOESY data, and the structure assignments were supported by NMR comparison with similar compounds. These new metabolites are designated preussolides A and B. The second active region was caused by the cytotoxin, leptosin C. Genome sequencing of the four strains revealed biosynthetic gene clusters consistent with those known to encode phosphoethanolamine-bearing polyketide macrolides and the biosynthesis of dimeric epipolythiodioxopiperazines. All three compounds showed moderate to potent and selective antifungal activity toward the pathogenic yeast C. neoformans.
Subject(s)
Cryptococcus neoformans , Macrolides , Animals , Antifungal Agents/pharmacology , Ascomycota , Ethanolamines , Humans , Indole Alkaloids , Macrolides/pharmacologyABSTRACT
Sphaerostilbella toxica is a mycoparasitic fungus that can be found parasitizing wood-decay basidiomycetes in the southern USA. Organic solvent extracts of fermented strains of S. toxica exhibited potent antimicrobial activity, including potent growth inhibition of human pathogenic yeasts Candida albicans and Cryptococcus neoformans, the respiratory pathogenic fungus Aspergillus fumigatus, and the Gram-positive bacterium Staphylococcus aureus. Bioassay-guided separations led to the purification and structure elucidation of new peptaibiotics designated as sphaerostilbellins A and B. Their structures were established mainly by analysis of NMR and HRMS data, verification of amino acid composition by Marfey's method, and by comparison with published data of known compounds. They incorporate intriguing structural features, including an N-terminal 2-methyl-3-oxo-tetradecanoyl (MOTDA) residue and a C-terminal putrescine residue. The minimal inhibitory concentrations for sphaerostilbellins A and B were measured as 2 µM each for C. neoformans, 1 µM each for A. fumigatus, and 4 and 2 µM, respectively, for C. albicans. Murine macrophage cells were unaffected at these concentrations.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Infective Agents/chemistry , Antifungal Agents/pharmacology , Basidiomycota/chemistry , Animals , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/pharmacology , Antifungal Agents/chemistry , Aspergillus fumigatus/drug effects , Aspergillus fumigatus/pathogenicity , Candida albicans/drug effects , Candida albicans/pathogenicity , Cryptococcus neoformans/drug effects , Cryptococcus neoformans/pathogenicity , Humans , Macrophages/drug effects , Mice , Microbial Sensitivity Tests , Staphylococcus aureus/pathogenicityABSTRACT
Campafungin A is a polyketide that was recognized in the Candida albicans fitness test due to its antiproliferative and antihyphal activity. Its mode of action was hypothesized to involve inhibition of a cAMP-dependent PKA pathway. The originally proposed structure appeared to require a polyketide assembled in a somewhat unusual fashion. However, structural characterization data were never formally published. This background stimulated a reinvestigation in which campafungin A and three closely related minor constituents were purified from fermentations of a strain of the ascomycete fungus Plenodomus enteroleucus. Labeling studies, along with extensive NMR analysis, enabled assignment of a revised structure consistent with conventional polyketide synthetic machinery. The structure elucidation of campafungin A and new analogues encountered in this study, designated here as campafungins B, C, and D, is presented, along with a proposed biosynthetic route. The antimicrobial spectrum was expanded to methicillin-resistant Staphylococcus aureus, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Aspergillus fumigatus, and Schizosaccharomyces pombe, with MICs ranging as low as 4-8 µg mL-1 in C. neoformans. Mode-of-action studies employing libraries of C. neoformans mutants indicated that multiple pathways were affected, but mutants in PKA/cAMP pathways were unaffected, indicating that the mode of action was distinct from that observed in C. albicans.
Subject(s)
Candida albicans/drug effects , Cryptococcus neoformans/drug effects , Hyphae/drug effects , Hyphae/growth & development , Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Ascomycota/chemistry , Ascomycota/metabolism , Bacteria/drug effects , Cyclic AMP-Dependent Protein Kinases/drug effects , Fermentation , Fungi/drug effects , Microbial Sensitivity Tests , Molecular Structure , Polyketides/pharmacology , Signal Transduction/drug effectsABSTRACT
Cryptococcus neoformans is an important human pathogen with limited options for treatments. We have interrogated extracts from fungal fermentations to find Cryptococcus-inhibiting natural products using assays for growth inhibition, differential thermosensitivity, and synergy with existing antifungal drugs. Extracts from fermentations of strains of Discosia rubi from eastern Texas showed anticryptococcal bioactivity with preferential activity in agar zone of inhibition assays against C. neoformans at 37°C versus 25°C. Assay-guided fractionation led to the purification and identification of chaetoglobosin P as the active component of these extracts. Genome sequencing of these strains revealed a biosynthetic gene cluster consistent with chaetoglobosin biosynthesis and ß-methylation of the tryptophan residue. Proximity of genes of the actin-binding protein twinfilin-1 to the chaetoglobosin P and K gene clusters suggested a possible self-resistance mechanism involving twinfilin-1 which is consistent with the predicted mechanism of action involving interference with the polymerization of the capping process of filamentous actin. A C. neoformans mutant lacking twinfilin-1 was hypersensitive to chaetoglobosin P. Chaetoglobosins also potentiated the effects of amphotericin B and caspofungin on C. neoformans.
ABSTRACT
The genus Stromatoneurospora was erected in 1973 by Jong and Davis to accommodate the pyrophilic pyrenomycete Sphaeria phoenix and has traditionally been placed in the family Xylariaceae based on morphological features. However, no living culture of this genus has so far been available in the public domain. Molecular data were restricted to an internal transcribed spacer (ITS) sequence that only confirmed the familial position, and was generated from a strain that is not deposited in a public culture collection. We have recently collected fresh material and were able to culture this fungus from Thailand. The secondary metabolites of this strains were analysed after fermentation in multiple media. The the prominent components of these fermentation were purified, using preparative chromatography. Aside from two new eremophilane sesquiterpenoids named phoenixilanes A-B (1-2), four other components that are known from species of the xylariaceous genera Xylaria and Poronia were identified by spectral methods (nuclear magnetic resonance spectroscopy and high resolution mass spectrometry). Notably, (-)-(R)-6-hydroxy-3-methyl-4-dihydroisocoumarin-5-carboxylic acid (6) has not been reported as a natural product before. Moreover, DNA sequences of Stromatoneurospora phoenix clustered with members of the genera Poronia and Podosordaria in a multi-locus molecular phylogeny. These results confirmed that the genus belongs to the same evolutionary lineage as the coprophilic Xylariaceae. The results also suggest that this lineage has evolved independently from the plant-inhabiting saprotrophs and endophytes that are closely related to the genus Xylaria. These findings are discussed in relation to some theories about the endophytic vs. the pyrophilic/coprophilic fungal life style.
ABSTRACT
The antifungal echinocandin lipopeptide, acrophiarin, was circumscribed in a patent in 1979. We confirmed that the producing strain NRRL 8095 is Penicillium arenicola and other strains of P. arenicola produced acrophiarin and acrophiarin analogues. Genome sequencing of NRRL 8095 identified the acrophiarin gene cluster. Penicillium arenicola and echinocandin-producing Aspergillus species belong to the family Aspergillaceae of the Eurotiomycetes, but several features of acrophiarin and its gene cluster suggest a closer relationship with echinocandins from Leotiomycete fungi. These features include hydroxy-glutamine in the peptide core instead of a serine or threonine residue, the inclusion of a non-heme iron, α-ketoglutarate-dependent oxygenase for hydroxylation of the C3 of the glutamine, and a thioesterase. In addition, P. arenicola bears similarity to Leotiomycete echinocandin-producing species because it exhibits self-resistance to exogenous echinocandins. Phylogenetic analysis of the genes of the echinocandin biosynthetic family indicated that most of the predicted proteins of acrophiarin gene cluster exhibited higher similarity to the predicted proteins of the pneumocandin gene cluster of the Leotiomycete Glarea lozoyensis than to those of the echinocandin B gene cluster from A. pachycristatus. The fellutamide gene cluster and related gene clusters are recognized as relatives of the echinocandins. Inclusion of the acrophiarin gene cluster into a comprehensive phylogenetic analysis of echinocandin gene clusters indicated the divergent evolutionary lineages of echinocandin gene clusters are descendants from a common ancestral progenitor. The minimal 10-gene cluster may have undergone multiple gene acquisitions or losses and possibly horizontal gene transfer after the ancestral separation of the two lineages.
Subject(s)
Anti-Infective Agents/metabolism , Ascomycota , Aspergillus , Echinocandins , Lipopeptides , Penicillium , Ascomycota/genetics , Aspergillus/genetics , Echinocandins/biosynthesis , Echinocandins/genetics , Lipopeptides/biosynthesis , Lipopeptides/genetics , Multigene Family , Penicillium/geneticsABSTRACT
Aspergillus pachycristatus is an industrially important fungus for the production of the antifungal echinocandin B and is closely related to model organism A. nidulans. Its secondary metabolism is largely unknown except for the production of echinocandin B and sterigmatocystin. We constructed mutants for three genes that regulate secondary metabolism in A. pachycristatus NRRL 11440, and evaluated the secondary metabolites produced by wild type and mutants strains. The secondary metabolism was explored by metabolic networking of UPLC-HRMS/MS data. The genes and metabolites of A. pachycristatus were compared to those of A. nidulans FGSC A4 as a reference to identify compounds and link them to their encoding genes. Major differences in chromatographic profiles were observable among the mutants. At least 28 molecules were identified in crude extracts that corresponded to nine characterized gene clusters. Moreover, metabolic networking revealed the presence of a yet unexplored array of secondary metabolites, including several undescribed fellutamides derivatives. Comparative reference to its sister species, A. nidulans, was an efficient way to dereplicate known compounds, whereas metabolic networking provided information that allowed prioritization of unknown compounds for further metabolic exploration. The mutation of global regulator genes proved to be a useful tool for expanding the expression of metabolic diversity in A. pachycristatus.
Subject(s)
Aspergillus/genetics , Aspergillus/metabolism , Data Mining , Genome, Fungal , Secondary Metabolism/genetics , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Biosynthetic Pathways/genetics , Chromatography, High Pressure Liquid , Metabolic Networks and Pathways/genetics , Multigene Family , Oligopeptides/pharmacologyABSTRACT
During investigation of the secondary metabolism of four strains of Penicillium arenicola, two new depsides, arenicolins A (1) and B (2), were isolated and characterized. Their structures were established mainly by analysis of NMR and HRMS data and by comparison with known compounds. These depsides incorporate intriguing structural features, including dual alkyl side chains and a C-glycosyl unit, with 1 also containing an acylated 2-hydroxymethyl-4,5,6-trihydroxycyclohexenone moiety. Although the arenicolins were produced by all strains tested, arenicolin A (1) was obtained using only one of five medium compositions employed, while arenicolin B (2) was produced in all media tested. Neither compound showed antibacterial or antifungal activity, but 1 exhibited cytotoxicity toward mammalian cell lines, including colorectal carcinoma (HCT-116), neuroblastoma (IMR-32), and ductal carcinoma (BT-474), with IC50 values of 7.3, 6.0, and 9.7 µM, respectively.
Subject(s)
Antineoplastic Agents/pharmacology , Depsides/pharmacology , Penicillium/chemistry , Antineoplastic Agents/isolation & purification , Biological Products/chemistry , Cell Line, Tumor , Depsides/isolation & purification , Glycosylation , Humans , Molecular StructureABSTRACT
The impact of the global secondary metabolite regulators LaeA and VeA on echinocandin B production and morphological development was evaluated in the industrial production strain Aspergillus pachycristatus NRRL 11440. Other representative secondary metabolites were examined as well to determine if the velvet complex functions as in A. nidulans and other species of fungi. Genetic methods used for gene manipulations in A. nidulans were applied to A. pachycristatus. Separate deletions of genes Apc.laeA and Apc.veA resulted in similar yet differing phenotypes in strain NRRL 11440. Disruption of Apc.laeA and Apc.veA significantly reduced, but did not eliminate, the production of echinocandin B. Similar to what has been observed in A. nidulans, the production of sterigmatocystin was nearly eliminated in both mutants. Quantitative reverse transcription PCR analyses confirmed that selected genes of both the echinocandin B and sterigmatocystin gene clusters were down-regulated in both mutant types. The two mutants differed with respect to growth of aerial hyphae, pigmentation, development of conidiophores, conidial germination rate, and ascospore maturation. Further functional annotation of key regulatory genes in A. pachycristatus and related Aspergillus species will improve our understanding of regulation of echinocandin production and co-produced metabolites.
Subject(s)
Aspergillus/metabolism , Echinocandins/metabolism , Fungal Proteins/metabolism , Secondary Metabolism , Aspergillus/genetics , Gene Expression Regulation, Fungal , Genome, Fungal , Multigene Family , Spores, FungalABSTRACT
The Pleuroascaceae (Leotiomycetes) is introduced for Phialophora hyalina (section Catenulatae) and its closest relatives based on analyses of DNA sequences of five gene regions and the comparison of cultural and micromorphological characters. The family is resolved as a strongly supported clade that encompasses Pleuroascus and the new anamorph genera Entimomentora and Venustampulla. The latter includes V. parva, a species placed formerly in Scopulariopsis, and V. echinocandica, which is established for the echinocandin-producing isolate BP-5553. Entimomentora includes E. hyalina, a species based on the ex-type strain of Ph. hyalina. Additional isolates identified as Ph. hyalina are distantly related to the Pleuroacaceae and include Psychrophila antarctica (Arachnopezizaceae) and Cryonesomyces dreyfussii, the sole member of the new genus Cryonesomyces (incertae sedis). Isolates identified or deposited as Ph. alba are also not closely related; they include a species for which we propose the name Neobulgaria koningiana (Gelatinodiscaceae) and a second psychrophilic species that we describe as Psychrophila lagodekhiensis. Of the 13 isolates assessed for in vitro antifungal activity, only V. echinocandica inhibited the growth of Candida albicans.
Subject(s)
Microbiological Techniques , Microscopy , Phialophora/classification , Phialophora/genetics , Phylogeny , Antifungal Agents/analysis , Antifungal Agents/pharmacology , Chromatography, High Pressure Liquid , Cluster Analysis , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Genes, rRNA , Mass Spectrometry , Microbial Sensitivity Tests , Phialophora/growth & development , RNA Polymerase II/genetics , RNA, Fungal/genetics , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 28S/genetics , Sequence Analysis, DNAABSTRACT
Three new species, closely related to Sphaerostilbella broomeana, are described from the USA and India. These species form septate conidia from simple conidiophores with individual branches terminating in a single phialide and chlamydospores. Teleomorphs, known for S. broomeana and S. appalachiensis, are characterised by hairy perithecia and fusiform, apiculate, and conspicuously warted ascospores. This combination of characters distinguishes the S. broomeana-group from other members of Sphaerostilbella that all form gliocladium-type anamorphs and mostly grow on basidiomata of Stereum spp. Like in other species of the genus, the majority of hosts of the species described in this paper belong to wood-inhabiting taxa of Russulales. Sphaerostilbella broomeana had been recorded from a few regions in Europe and exclusively on Heterobasidion annosum. Herein, it is reported also from H. parviporum in many other localities and on H. insulare s.l. at the foothills of the Himalayas. Its sister species, found in the same region in northern India on another member of Russulales (Dichostereum effuscatum), is described as S. himalayensis. The two species described from North America colonize polypores from various taxa. Whereas S. appalachiensis occurs in eastern USA, with H. irregulare among its hosts, S. toxica is so far known only from two locations in eastern Texas, growing on Gloeophyllum striatum (Polyporales). Despite their great similarity in morphology and ITS rDNA, TEF1 sequences clearly distinguish these two North-American species. Moreover, the two strains of S. toxica appeared metabolically distinct as their organic extracts strongly inhibited the growth of human pathogenic microbes grown in vitro. Phylogenetic analysis of rDNA sequences supports monophyly of the genus Sphaerostilbella and the included S. broomeana-group, established here.
ABSTRACT
Two new species and a new combination of Hypoxylon from Texas were identified and described based on morphological, multigene phylogenetic (ITS [nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2], 28S [5' 1200 bp of nuc 28S rDNA], RPB2 [partial second largest subunit of the DNA-directed RNA polymerase II], TUB2 [partial ß-tubulin]), and chemotaxonomic data. Hypoxylon olivaceopigmentum is characterized by its pulvinate to glomerate stromata, olivaceous KOH-extractable pigments, equilateral ascospores, and indehiscent perispore. Hypoxylon texense can be distinguished from morphologically similar species by its rust to dark brick KOH-extractable pigments and the high-performance liquid chromatography (HPLC) profile of its stromatal secondary metabolites. Hypoxylon hinnuleum is proposed as the sexual morph of Nodulisporium hinnuleum, featuring dark vinaceous glomerate stromata with dark brick KOH-extractable pigments composed of cohaerin-type azaphilones and smooth equilateral ascospores with indehiscent perispore. Based on these diagnostic characters, H. hinnuleum forms a complex with H. croceum and H. minicroceum. More than 50 ITS sequences with high identity originating from North American and East Asian environmental isolates formed a well-supported clade with the type of N. hinnuleum, demonstrating the widespread distribution of the species complex. In addition, updated descriptions and comprehensive illustrations with detailed information on the diagnostic features of H. fendleri and H. perforatum are provided. The multilocus phylogenetic reconstruction of Hypoxylon supported the status of the new species and broadened the knowledge about intergeneric relationships.
Subject(s)
Environmental Microbiology , Phylogeny , Spores, Fungal/cytology , Xylariales/classification , Xylariales/isolation & purification , Cluster Analysis , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Microscopy , Pigments, Biological/analysis , RNA Polymerase II/genetics , RNA, Ribosomal, 28S/genetics , Sequence Analysis, DNA , Texas , Tubulin/genetics , Xylariales/genetics , Xylariales/physiologyABSTRACT
In the course of our studies of coprophilous fungi as sources of antifungal agents, a strain of an undescribed species in the genus Niesslia (TTI-0426) was isolated from horse dung collected in Texas. An extract from fermentation cultures of this strain afforded two new antifungal wortmannin derivatives, wortmannins C and D (1 and 2), as well as four additional new related compounds, wortmannines B1-B4 (3-6), containing an unusual ring system. The structures of these metabolites were established mainly by analysis of HRESIMS and 2D NMR data. Relative configurations were assigned using NOESY data, and the structure assignments were supported by NMR comparison with similar compounds. Wortmannins C and D showed activity against Cryptococcus neoformans and Candida albicans in disk assays, but low MIC potency observed for 1 was suggested to be due in part to efflux processes on the basis of assay results for a Schizosaccharomyces pombe efflux mutant in comparison to wild-type.
Subject(s)
Hypocreales/chemistry , Wortmannin/isolation & purification , Candida albicans/drug effects , Cryptococcus neoformans/drug effects , Fermentation , Microbial Sensitivity Tests , Molecular Structure , Schizosaccharomyces/drug effects , Spectrum Analysis/methods , Wortmannin/chemistry , Wortmannin/pharmacologyABSTRACT
Enfumafungin is a glycosylated fernene-type triterpenoid produced by the fungus Hormonema carpetanum. Its potent antifungal activity, mediated by its interaction with ß-1,3-glucan synthase and the fungal cell wall, has led to its development into the semi-synthetic clinical candidate, ibrexafungerp (=SCY-078). We report on the preliminary identification of the enfumafungin biosynthetic gene cluster (BGC) based on genome sequencing, phylogenetic reconstruction, gene disruption, and cDNA sequencing studies. Enfumafungin synthase (efuA) consists of a terpene cyclase domain (TC) fused to a glycosyltransferase (GT) domain and thus represents a novel multifunctional enzyme. Moreover, the TC domain bears a phylogenetic relationship to bacterial squalene-hopene cyclases (SHC) and includes a typical DXDD motif within the active centre suggesting that efuA evolved from SHCs. Phylogenetic reconstruction of the GT domain indicated that this portion of the fusion gene originated from fungal sterol GTs. Eleven genes flanking efuA are putatively involved in the biosynthesis, regulation, transport and self-resistance of enfumafungin and include an acetyltransferase, three P450 monooxygenases, a dehydrogenase, a desaturase and a reductase. A hypothetical scheme for enfumafungin assembly is proposed in which the E-ring is oxidatively cleaved to yield the four-ring system of enfumafungin. EfuA represents the first member of a widespread lineage of fungal SHCs.