Pentaketide melanin biosynthesis in Aspergillus fumigatus requires chain-length shortening of a heptaketide precursor

Chain lengths and cyclization patterns of microbial polyketides are generally determined by polyketide synthases alone. Fungal polyketide melanins are often derived from a pentaketide 1,8-dihydroxynaphthalene, and pentaketide synthases are used for synthesis of the upstream pentaketide precursor, 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN). However, Aspergillus fumigatus, a human fungal pathogen, uses a heptaketide synthase (Alb1p) to synthesize its conidial pigment through a pentaketide pathway similar to that which produces 1,8-dihydroxynaphthalene-melanin. In this study we demonstrate that a novel protein, Ayg1p, is involved in the formation of 1,3,6,8-THN by chain-length shortening of a heptaketide precursor in A. fumigatus. Deletion of the ayg1 gene prevented the accumulation of 1,3,6,8-THN suggesting the involvement of ayg1 in 1,3,6,8-THN production. Genetic analyses of double-gene deletants suggested that Ayg1p catalyzes a novel biosynthetic step downstream of Alb1p and upstream of Arp2p (1,3,6,8-THN reductase). Further genetic and biochemical analyses of the reconstituted strains carrying alb1, ayg1, or alb1 + ayg1 indicated that Ayg1p is essential for synthesis of 1,3,6,8-THN in addition to Alb1p. Cell-free enzyme assays, using the crude Ayg1p protein extract, revealed that Ayg1p enzymatically shortened the heptaketide product of Alb1p to 1,3,6,8-THN. Thus, the protein Ayg1p facilitates the participation of a heptaketide synthase in a pentaketide pathway via a novel polyketide-shortening mechanism in A. fumigatus.     

Aspergillus fumigatus arp1 modulates conidial pigmentation and complement deposition

Aspergillus fumigatus is an important pathogen causing invasive pulmonary aspergillosis in immunocompromised patients. The fungus propagates by conidia, which are the infectious structures inhaled by the human host. Opsonophagocytosis is thought to contribute to clearance of the inhaled conidia, a process that is facilitated by complement deposition on conidial surfaces. We now show that conidial colour mutants exhibit significant increases in C3 binding capacity compared with wild type. A reddish-pink mutation that led to enhanced C3 binding was complemented by a cosmid clone. A 3.3 kb DNA fragment from the subsequently rescued cosmid was sufficient to restore the bluish-green conidial pigment. The bluish-green transformant exhibited a level of C3 binding similar to that of the parental strain. A gene, designated arp1 , was responsible for the complementation. Comparison of the genomic and cDNA sequences of arp1 revealed that it has two introns and encodes a putative protein of 168 amino acids. Arp1 is very similar to scytalone dehydratase, an enzyme involved in 1,8-dihydroxynaphthalene-melanin synthesis in Colletotrichum lagenarium and Magnaporthe grisea . Northern hybridization analysis revealed that arp1 is developmentally regulated, being expressed during conidiation. Disruption of arp1 resulted in reddish-pink conidia and increased C3 binding. Our studies suggest that arp1 modulates the bluish-green pigmentation of conidia as well as complement deposition.     

The molecular and genetic basis of conidial pigmentation in Aspergillus niger

A characteristic hallmark of Aspergillus niger is the formation of black conidiospores. We have identified four loci involved in spore pigmentation of A. niger by using a combined genomic and classical complementation approach. First, we characterized a newly isolated color mutant, colA, which lacked pigmentation resulting in white or colorless conidia. Pigmentation of the colA mutant was restored by a gene (An12g03950) which encodes a putative 4'phosphopantetheinyl transferase protein (PptA). 4'Phosphopantetheinyl transferase activity is required for the activation of Polyketide Synthases (PKSs) and/or Non-Ribosomal Peptide Synthases (NRPSs). The loci whose mutation resulted in fawn, olive, and brown color phenotypes were identified by complementation. The fawn phenotype was complemented by a PKS protein (FwnA, An09g05730), the ovlA mutant by An14g05350 (OlvA) and the brnA mutant by An14g05370 (BrnA), the respective homologs of alb1/pksP, ayg1 and abr1 in A. fumigatus. Targeted disruption of the pptA, fwnA, olvA and brnA genes confirmed the complementation results. Disruption of the pptA gene abolished synthesis of all polyketides and non-ribosomal peptides, while the naphtho-γ-pyrone subclass of polyketides were specifically dependent on fwnA, and funalenone on fwnA, olvA and brnA. Thus, secondary metabolite profiling of the color mutants revealed a close relationship between polyketide synthesis and conidial pigmentation in A. niger.     

What makes Aspergillus fumigatus a successful pathogen? Genes and molecules involved in invasive aspergillosis

Aspergillus fumigatus is an opportunistic pathogen that causes 90% of invasive aspergillosis (IA) due to Aspergillus genus, with a 50-95% mortality rate. It has been postulated that certain virulence factors are characteristic of A. fumigatus, but the "non-classical" virulence factors seem to be highly variable. Overall, published studies have demonstrated that the virulence of this fungus is multifactorial, associated with its structure, its capacity for growth and adaptation to stress conditions, its mechanisms for evading the immune system and its ability to cause damage to the host. In this review we intend to give a general overview of the genes and molecules involved in the development of IA. The thermotolerance section focuses on five genes related with the capacity of the fungus to grow at temperatures above 30°C (thtA, cgrA, afpmt1, kre2/afmnt1, and hsp1/asp f 12). The following sections discuss molecules and genes related to interaction with the host and with the immune responses. These sections include β-glucan, α-glucan, chitin, galactomannan, galactomannoproteins (afmp1/asp f 17 and afmp2), hydrophobins (rodA/hyp1 and rodB), DHN-melanin, their respective synthases (fks1, rho1-4, ags1-3, chsA-G, och1-4, mnn9, van1, anp1, glfA, pksP/alb1, arp1, arp2, abr1, abr2, and ayg1), and modifying enzymes (gel1-7, bgt1, eng1, ecm33, afpigA, afpmt1-2, afpmt4, kre2/afmnt1, afmnt2-3, afcwh41 and pmi); several enzymes related to oxidative stress protection such as catalases (catA, cat1/catB, cat2/katG, catC, and catE), superoxide dismutases (sod1, sod2, sod3/asp f 6, and sod4), fatty acid oxygenases (ppoA-C), glutathione tranferases (gstA-E), and others (afyap1, skn7, and pes1); and efflux transporters (mdr1-4, atrF, abcA-E, and msfA-E). In addition, this review considers toxins and related genes, such as a diffusible toxic substance from conidia, gliotoxin (gliP and gliZ), mitogillin (res/mitF/asp f 1), hemolysin (aspHS), festuclavine and fumigaclavine A-C, fumitremorgin A-C, verruculogen, fumagillin, helvolic acid, aflatoxin B1 and G1, and laeA. Two sections cover genes and molecules related with nutrient uptake, signaling and metabolic regulations involved in virulence, including enzymes, such as serine proteases (alp/asp f 13, alp2, and asp f 18), metalloproteases (mep/asp f 5, mepB, and mep20), aspartic proteases (pep/asp f 10, pep2, and ctsD), dipeptidylpeptidases (dppIV and dppV), and phospholipases (plb1-3 and phospholipase C); siderophores and iron acquisition (sidA-G, sreA, ftrA, fetC, mirB-C, and amcA); zinc acquisition (zrfA-H, zafA, and pacC); amino acid biosynthesis, nitrogen uptake, and cross-pathways control (areA, rhbA, mcsA, lysF, cpcA/gcn4p, and cpcC/gcn2p); general biosynthetic pathway (pyrG, hcsA, and pabaA), trehalose biosynthesis (tpsA and tpsB), and other regulation pathways such as those of the MAP kinases (sakA/hogA, mpkA-C, ste7, pbs2, mkk2, steC/ste11, bck1, ssk2, and sho1), G-proteins (gpaA, sfaD, and cpgA), cAMP-PKA signaling (acyA, gpaB, pkaC1, and pkaR), His kinases (fos1 and tcsB), Ca(2+) signaling (calA/cnaA, crzA, gprC and gprD), and Ras family (rasA, rasB, and rhbA), and others (ace2, medA, and srbA). Finally, we also comment on the effect of A. fumigatus allergens (Asp f 1-Asp f 34) on IA. The data gathered generate a complex puzzle, the pieces representing virulence factors or the different activities of the fungus, and these need to be arranged to obtain a comprehensive vision of the virulence of A. fumigatus. The most recent gene expression studies using DNA-microarrays may be help us to understand this complex virulence, and to detect targets to develop rapid diagnostic methods and new antifungal agents.     

Isolation and disruption of the melanin pathway polyketide synthase gene of the softwood deep stain fungus Ceratocystis resinifera

Ceratocystis resinifera hyphae produce a black melanin pigment causing a deep stain in softwood logs. We exploited the homology of polyketide synthases to clone PKS1, a gene responsible for dihydroxynaphthalene-melanin biosynthesis in C. resinifera. Sequence analysis indicated that PKS1 has two introns near its 5(') end and encodes a 2188-amino acid polypeptide with five functional domains: beta-ketoacyl synthase, acyl transferase, two acyl carrier proteins and a thioesterase/Claisen cyclase. A gene disruption construct designed to replace a portion of PKS1 with a hygromycin resistance cassette was transformed into C. resinifera through Agrobacterium tumefaciens-mediated transformation. PKS1 null mutants had an albino phenotype, and pigmentation was restored by the addition of scytalone, a melanin pathway intermediate. The disruption of PKS1 and restoration of pigmentation with scytalone confirmed the presence of a dihydroxynaphthalene-melanin pathway in C. resinifera. The transformation method described in this paper is the first reported for a Ceratocystis species.     

Melanin biosynthesis and the metabolism of flaviolin and 2-hydroxyjuglone inWangiella dermatitidis

Melanin biosynthesis in the human pathogenWangiella dermatitidis was inhibited by tricyclazole, causing pentaketide melanin metabolites to accumulate in the cultures. One of these metabolites, scytalone, was racemic and thus different than the (+)-enantiomer fromVerticillium dahliae. An albino mutant ofW. dermatitidis metabolized scytalone to a pigment ultrastructurally identical to wild-type melanin. Cell-free homogenates of the wild type carried out typical reductive and dehydrative reactions with known melanin intermediates and the reductive reactions were inhibited by tricyclazole. Other reductive and dehydrative reactions that utilize flaviolin and 2-hydroxyjuglone were studied anaerobically with homogenates from both the wild type and the albino mutant. The homogenates converted flaviolin to 5-hydroxyscytalone and products identical to those obtained from 2-hydroxyjuglone. The albino, in culture, carried out the same reactions with 2-hydroxyjuglone but metabolized flaviolin to a number of unknown colored products apparently through oxidative reactions. Similarities between the melanin pathway and the flaviolin and 2-hydroxyjuglone branch pathways are discussed and tricyclazole is shown to inhibit reductive reactions with naphthols in the three pathways.Abbreviations DHN dihydroxynaphtalene - HJ hydroxyjuglone - THT trihydroxytetralone - THN trihydroxynaphthalene or tetrahydroxynaphthalene - DTT dithiothreitol - HS hydroxyscytalone - PHN pentahydroxynaphthalene     

肉桂醛、柠檬醛对烟曲霉色素及关键基因alb1mRNA表达的影响

研究了肉桂醛、柠檬醛对烟曲霉色素及其关键基因alb1 mRNA表达的影响。结果表明随着肉桂醛、柠檬醛浓度的增加,菌苔逐渐变稀、薄且烟绿色色素逐渐变淡,甚至白化;肉桂醛、柠檬醛对alb1基因mRNA表达有明显抑制作用,随着药物浓度的降低这种抑制作用亦相应呈现不同程度的减弱,肉桂醛、柠檬醛可能通过抑制alb1 mRNA的表达导致烟曲霉缺失烟绿色色素。     

Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus

Aspergillus fumigatus is an important pathogen of the immunocompromised host. Previously, it was shown that the polyketide synthase encoded by the pksP (alb1) gene represents a virulence determinant. pksP is part of a gene cluster involved in dihydroxynaphthalene (DHN)-like melanin biosynthesis. Because a putative laccase-encoding gene (abr2) is also part of the cluster and a laccase was found to represent a virulence factor in Cryptococcus neoformans, here, the Abr2 laccase was characterised. Deletion of the abr2 gene changed the gray-green conidial pigment to a brown color and the ornamentation of conidia was reduced compared with wild-type conidia. In contrast to the white pksP mutant, the susceptibility of the Δabr2 mutant against reactive oxygen species (ROS) was not increased, suggesting that the intermediate of DHN-like melanin produced up to the step catalysed by Abr2 already possesses ROS scavenging activity. In an intranasal mouse infection model, the Δabr2 mutant strain showed no reduction in virulence compared with the wild type. In the Δabr2 mutant, overall laccase activity was reduced only during sporulation, but not during vegetative growth. An abr2p-lacZ gene fusion was expressed during sporulation, but not during vegetative growth confirming the pattern of laccase activity due to Abr2.     

Characterization of melanin pigment produced by <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

Although most of the Ascomycetes present DHN-melanin, some reports suggest that A. nidulans does not produce this type of melanin. In this study, we analyzed the pigment extracted from highly melanized strains (MEL1 and MEL2) of Aspergillus nidulans to determine the type of melanin present in this fungus. Our results showed that the pigment produced by MEL1 and MEL2 mutants possesses physical and chemical properties and UV- and IR-spectra very similar to synthetic DOPA-melanin. The characterization of this pigment in terms of its degradation products indicated the presence of indolic units, which were also found in synthetic DOPA-melanin. The analyses of the elemental composition showed that the pigment extracted from these mutants has a high percentage of nitrogen and, therefore, it cannot be DHN-melanin, which presents only trace of nitrogen. This observation was confirmed in the test with tricyclazole because this inhibitor of DHN-melanin biosynthesis did not suppress pigment production in the MEL1 and MEL2 strains. On the other hand, in a medium containing tropolone, an inhibitor of DOPA-melanin biosynthesis, the dark pigmentation of the colonies was not observed indicating that this compound inhibited melanin production in these strains. Taken together, the results obtained in this study indicate that melanin produced by these mutants is DOPA type, representing the first report on characterization of this type of melanin in A. nidulans.     

1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis inhibitors increase erythritol production in Torula corallina,and DHN-melanin inhibits erythrose reductase

The yeast Torula corallina is a strong erythritol producer that is used in the industrial production of erythritol. However, melanin accumulation during culture represents a serious problem for the purification of erythritol from the fermentation broth. Melanin biosynthesis inhibitors such as 3,4-dihydroxyphenylalanine and 1,8-dihydroxynaphthalene (DHN)-melanin inhibitors were added to the T. corallina cultures. Only the DHN-melanin inhibitors showed an effect on melanin production, which suggests that the melanin formed during the culturing of T. corallina is derived from DHN. This finding was confirmed by the detection of a shunt product of the pentaketide pathway, flaviolin, and elemental analysis. Among the DHN-melanin inhibitors, tricyclazole was the most effective. Supplementation with tricyclazole enhanced the production of erythritol while significantly inhibiting the production of DHN-melanin and DHN-melanin biosynthetic enzymes, such as trihydroxynaphthalene reductase. The erythrose reductase from T. corallina was purified to homogeneity by ion-exchange and affinity chromatography. Purified erythrose reductase was significantly inhibited in vitro in a noncompetitive manner by elevated levels of DHN-melanin. In contrast, the level of erythrose reductase activity was unaffected by increasing concentrations of tricyclazole. These results suggest that supplemental tricyclazole reduces the production of DHN-melanin, which may lead to a reduction in the inhibition of erythrose reductase and a higher yield of erythritol. This is the first report to demonstrate that melanin biosynthesis inhibitors increase the production of a sugar alcohol in T. corallina.     

1,8-Dihydroxynaphthalene (DHN)-Melanin Biosynthesis Inhibitors Increase Erythritol Production in Torula corallina, and DHN-Melanin Inhibits Erythrose Reductase

The yeast Torula corallina is a strong erythritol producer that is used in the industrial production of erythritol. However, melanin accumulation during culture represents a serious problem for the purification of erythritol from the fermentation broth. Melanin biosynthesis inhibitors such as 3,4-dihydroxyphenylalanine and 1,8-dihydroxynaphthalene (DHN)-melanin inhibitors were added to the T. corallina cultures. Only the DHN-melanin inhibitors showed an effect on melanin production, which suggests that the melanin formed during the culturing of T. corallina is derived from DHN. This finding was confirmed by the detection of a shunt product of the pentaketide pathway, flaviolin, and elemental analysis. Among the DHN-melanin inhibitors, tricyclazole was the most effective. Supplementation with tricyclazole enhanced the production of erythritol while significantly inhibiting the production of DHN-melanin and DHN-melanin biosynthetic enzymes, such as trihydroxynaphthalene reductase. The erythrose reductase from T. corallina was purified to homogeneity by ion-exchange and affinity chromatography. Purified erythrose reductase was significantly inhibited in vitro in a noncompetitive manner by elevated levels of DHN-melanin. In contrast, the level of erythrose reductase activity was unaffected by increasing concentrations of tricyclazole. These results suggest that supplemental tricyclazole reduces the production of DHN-melanin, which may lead to a reduction in the inhibition of erythrose reductase and a higher yield of erythritol. This is the first report to demonstrate that melanin biosynthesis inhibitors increase the production of a sugar alcohol in T. corallina.     

Apd1(+), a Gene Required for Red Pigment Formation in Ade6 Mutants of Schizosaccharomyces Pombe, Encodes an Enzyme Required for Glutathione Biosynthesis: A Role for Glutathione and a Glutathione-Conjugate Pump

Mutants in the adenine biosynthetic pathway of yeasts (ade1 and ade2 of Saccharomyces cerevisiae, ade6 and ade7 of Schizosaccharomyces pombe) accumulate an intense red pigment in their vacuoles when grown under adenine-limiting conditions. The precise events that determine the formation of the pigment are however, still unknown. We have begun a genetic investigation into the nature and cause of pigmentation of ade6 mutants of S. pombe and have discovered that one of these pigmentation defective mutants, apd1 (adenine pigmentation defective), is a strict glutathione auxotroph. The gene apd1(+) was found to encode the first enzyme in glutathione biosynthesis, γ-glutamylcysteine synthetase, gcs1(+). This gene when expressed in the mutant could confer both glutathione prototrophy and the characteristic red pigmentation, and disruption of the gene led to a loss in both phenotypes. Supplementation of glutathione in the medium, however, could only restore growth but not the pigmentation because the cells were unable to achieve sufficient intracellular levels of glutathione. Disruption of the second enzyme in glutathione biosynthesis, glutathione synthetase, gsh2(+), also led to glutathione auxotrophy, but only a partial defect in pigment formation. A reevaluation of the major amino acids previously reported to be present in the pigment indicated that the pigment is probably a glutathione conjugate. The ability of vanadate to inhibit pigment formation indicated that the conjugate was transported into the vacuole through a glutathione-conjugate pump. This was further confirmed using strains of S. cerevisiae bearing disruptions in the recently identified glutathione-conjugate pump, YCF1, where a significant reduction in pigment formation was observed. The pump of S. pombe is distinct from the previously identified vacuolar pump, hmt1p, for transporting cadystin peptides into vacuoles of S. pombe.     

Functional identification of the DHN melanin synthesis gene cluster and its role in UV-C tolerance in citrus postharvest pathogenic fungus Penicillium digitatum

The filamentous fungus Penicillium digitatum brings out great losses in citrus fruits by causing citrus green mold disease during the postharvest period. Previously, we obtained a T-DNA insertion mutant N2130 of P. digitatum, which produced albino conidia. To understand the role of green-grey conidial pigment in P. digitatum, we identified the insertion site and deeply explored the 1,8-dihydroxynaphtsalene (DHN)-melanin synthesis gene cluster in this phytopathogen. In this study, we deleted five genes in P. digitatum, PdPksP, PdAbr1, PdArp1, PdArp2, and PdAyg1, and the experiments were further performed on phenotype analyses, including pigmentation, UV-C tolerance, virulence, growth rate, conidiation, stress (osmotic-, oxidative-, cell wall disturbing-, and high temperature-) tolerance, fungicide resistance, and conidial hydrophobicity. The results showed that the five deletion mutants (ΔPdPksP, ΔPdAbr1, ΔPdArp1, ΔPdArp2 and ΔPdAyg1) produced albino, brownish, brown, reddish-brown, and Yellowish green conidia, respectively. In addition, the survival colony forming units (CFUs) of the deletion mutants, under the treatment of UV-C radiation (261.4 mJ/cm²), were 0.3- to 0.6-fold of those surviving in wild-type strain N1. Moreover, after 522.8 mJ/cm²-UV-C-irradiation on conidia, the deletion mutants showed a larger decrease in pathogenicity on Valencia Orange fruits compared with strain N1. However, there were no significant differences among other phenotypes tested in this study. Collectively, our research reported the DHN-melanin synthesis pathway in P. digitatum for the first time, and revealed that DHN-melanin is important for P. digitatum to tolerate UV-C irradiation.     

玉米大斑病菌小柱孢酮脱水酶基因(scd)的克隆与功能分析

根据已知植物病原真菌黑色素生物合成相关基因scd(scytalone dehydratase)氨基酸序列保守区域设计简并引物,分别以玉米大斑病菌基因组DNA和cDNA为模板,通过PCR技术获得scd基因的同源片段,利用SMART-RACE技术和3′RACE技术获得了scd的cDNA全长序列。并根据scd基因cDNA全长序列设计基因特异性引物扩增玉米大斑病菌基因组DNA获得了该基因DNA全长。通过DNA序列和cDNA序列对比分析发现scd基因编码一个180个氨基酸的开放阅读框架,DNA序列含有两个分别为50bp和78bp的内含子。生物信息学分析表明其氨基酸序列与水稻胡麻叶斑病菌的scd基因的相似性很高。DHN黑色素生物合成途径特异性抑制剂—Carpropamid处理玉米大斑病菌,在12~24h之内可以抑制病菌分生孢子的萌发和附着胞的产生,但随着处理时间的延长抑制剂的抑制作用变弱,并且经过抑制剂处理的病菌不能侵入寄主组织或不能在寄主组织内扩展。初步明确了scd与玉米大斑病菌黑色合成途径及致病性的关系。     

Novel polyketide synthase from Nectria haematococca

We identified a polyketide synthase (PKS) gene, pksN, from a strain of Nectria haematococca by complementing a mutant unable to synthesize a red perithecial pigment. pksN encodes a 2,106-amino-acid polypeptide with conserved motifs characteristic of type I PKS enzymatic domains: beta-ketoacyl synthase, acyltransferase, duplicated acyl carrier proteins, and thioesterase. The pksN product groups with the Aspergillus nidulans WA-type PKSs involved in conidial pigmentation and melanin, bikaverin, and aflatoxin biosynthetic pathways. Inactivation of pksN did not cause any visible change in fungal growth, asexual sporulation, or ascospore formation, suggesting that it is involved in a specific developmental function. We propose that pksN encodes a novel PKS required for the perithecial red pigment biosynthesis.     

The Aspergillus fumigatus cellobiohydrolase B (cbhB) promoter is tightly regulated and can be exploited for controlled protein expression and RNAi

The utility of the Aspergillus fumigatus cellobiohydrolase cbhB promoter for controlled gene expression has been investigated. cbhB message was present at high levels in the presence of carboxymethylcellulose and undetected in the presence of glucose. A reporter construct using the cbhB promoter showed similar behaviour and gave lower message levels than the Aspergillus nidulans alcA promoter under repressing conditions. An RNAi construct driven by the cbhB promoter was used to down-regulate the alb1 gene; transformants showed low alb1 message levels and a loss-of-function phenotype with carboxymethylcellulose, while both wild-type message levels and phenotype were seen with glucose. The cbhB promoter is therefore tightly controlled and can be exploited for the study of A. fumigatus.