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1.
IUBMB Life ; 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39011777

RESUMO

Cryomyces antarcticus, a melanized cryptoendolithic fungus endemic to Antarctica, can tolerate environmental conditions as severe as those in space. Particularly, its ability to withstand ionizing radiation has been attributed to the presence of thick and highly melanized cell walls, which-according to a previous investigation-may contain both 1,8-dihydroxynaphthalene (DHN) and L-3,4 dihydroxyphenylalanine (L-DOPA) melanin. The genes putatively involved in the synthesis of DHN melanin were identified in the genome of C. antarcticus. Most important is capks1 encoding a non-reducing polyketide synthase (PKS) and being the ortholog of the functionally characterized kppks1 from the rock-inhabiting fungus Knufia petricola. The co-expression of CaPKS1 or KpPKS1 with a 4'-phosphopantetheinyl transferase in Saccharomyces cerevisiae resulted in the formation of a yellowish pigment, suggesting that CaPKS1 is the enzyme providing the precursor for DHN melanin. To dissect the composition and function of the melanin layer in the outer cell wall of C. antarcticus, non-melanized mutants were generated by CRISPR/Cas9-mediated genome editing. Notwithstanding its slow growth (up to months), three independent non-melanized Δcapks1 mutants were obtained. The mutants exhibited growth similar to the wild type and a light pinkish pigmentation, which is presumably due to carotenoids. Interestingly, visible light had an adverse effect on growth of both melanized wild-type and non-melanized Δcapks1 strains. Further evidence that light can pass the melanized cell walls derives from a mutant expressing a H2B-GFP fusion protein, which can be detected by fluorescence microscopy. In conclusion, the study reports on the first genetic manipulation of C. antarcticus, resulting in non-melanized mutants and demonstrating that the melanin is rather of the DHN type. These mutants will allow to elucidate the relevance of melanization for surviving extreme conditions found in the natural habitat as well as in space.

2.
World J Microbiol Biotechnol ; 40(10): 323, 2024 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-39292329

RESUMO

The important role of dihydroxynaphthalene-(DHN) melanin in enhancing fungal stress resistance and its importance in fungal development and pathogenicity are well-established. This melanin also aids biocontrol fungi in surviving in the environment and effectively infecting insects. However, the biosynthetic origin of melanin in the biocontrol agents, Metarhizium spp., has remained elusive due to the complexity resulting from the divergence of two DHN-like biosynthetic pathways. Through the heterologous expression of biosynthetic enzymes from these two pathways in baker's yeast Saccharomyces cerevisiae, we have confirmed the presence of DHN biosynthesis in M. roberstii, and discovered a novel naphthopyrone intermediate, 8, that can produce a different type of pigment. These two pigment biosynthetic pathways differ in terms of polyketide intermediate structures and subsequent modification steps. Stress resistance studies using recombinant yeast cells have demonstrated that both DHN and its intermediates confer resistance against UV light prior to polymerization; a similar result was observed for its naphthopyrone counterpart. This study contributes to the understanding of the intricate and diverse biosynthetic mechanisms of fungal melanin and has the potential to enhance the application efficiency of biocontrol fungi such as Metarhizium spp. in agriculture.


Assuntos
Vias Biossintéticas , Melaninas , Metarhizium , Saccharomyces cerevisiae , Metarhizium/metabolismo , Metarhizium/genética , Melaninas/metabolismo , Melaninas/biossíntese , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Naftóis/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Raios Ultravioleta
3.
Int J Mol Sci ; 24(8)2023 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-37108573

RESUMO

Anthracnose of poplar caused by Colletotrichum gloeosporioides is a leaf disease that seriously affects poplar growth. The pathogen invades the host in the form of adherent cells, which generate turgor pressure through the metabolism of intracellular substances prior to penetrating the epidermis of poplar leaves. In this study, the expansion-related pressure of the mature appressorium of the wild-type C. gloeosporioides was approximately 13.02 ± 1.54 MPa at 12 h, whereas it was 7.34 ± 1.23 MPa and 9.34 ± 2.22 MPa in the melanin synthesis-related gene knockout mutants ΔCgCmr1 and ΔCgPks1, respectively. The CgCmr1 and CgPks1 genes were highly expressed at 12 h in the wild-type control, implying that the DHN melanin biosynthesis pathway may play an important role in the mature appressorium stage. The transcriptome sequencing analysis indicated that the upregulated melanin biosynthesis genes in C. gloeosporioides, such as CgScd1, CgAyg1, CgThr1, CgThr2, and CgLac1, are involved in specific KEGG pathways (i.e., fatty acid biosynthesis, fatty acid metabolism, and biotin metabolism). Therefore, we speculate that the melanin synthesis-related genes and fatty acid metabolism pathway genes contribute to the regulation of the turgor pressure in the mature C. gloeosporioides appressorium, ultimately leading to the formation of infection pegs that enter plant tissues. These observations may reflect the co-evolution of C. gloeosporioides and its host.


Assuntos
Colletotrichum , Transcriptoma , Melaninas/metabolismo , Perfilação da Expressão Gênica , Ácidos Graxos/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia
4.
Int J Mol Sci ; 24(21)2023 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-37958874

RESUMO

Colletotrichum higginsianum is a major pathogen causing anthracnose in Chinese flowering cabbage (Brassica parachinensis), posing a significant threat to the Chinese flowering cabbage industry. The conidia of C. higginsianum germinate and form melanized infection structures called appressoria, which enable penetration of the host plant's epidermal cells. However, the molecular mechanism underlying melanin biosynthesis in C. higginsianum remains poorly understood. In this study, we identified two enzymes related to DHN-melanin biosynthesis in C. higginsianum: ChPks and ChThr1. Our results demonstrate that the expression levels of genes ChPKS and ChTHR1 were significantly up-regulated during hyphal and appressorial melanization processes. Furthermore, knockout of the gene ChPKS resulted in a blocked DHN-melanin biosynthetic pathway in hyphae and appressoria, leading to increased sensitivity of the ChpksΔ mutant to cell-wall-interfering agents as well as decreased turgor pressure and pathogenicity. It should be noted that although the Chthr1Δ mutant still exhibited melanin accumulation in colonies and appressoria, its sensitivity to cell-wall-interfering agents and turgor pressure decreased compared to wild-type strains; however, complete loss of pathogenicity was not observed. In conclusion, our results indicate that DHN-melanin plays an essential role in both pathogenicity and cell wall integrity in C. higginsianum. Specifically, ChPks is crucial for DHN-melanin biosynthesis while deficiency of ChThr1 does not completely blocked melanin production.


Assuntos
Colletotrichum , Melaninas , Virulência , Melaninas/metabolismo , Parede Celular/metabolismo
5.
Mol Plant Microbe Interact ; 35(3): 244-256, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34813706

RESUMO

Most plant fungal pathogens that cause worldwide crop losses are understudied, due to various technical challenges. With the increasing availability of sequenced whole genomes of these non-model fungi, effective genetic analysis methods are highly desirable. Here, we describe a newly developed pipeline, which combines forward genetic screening with high-throughput next-generation sequencing to enable quick gene discovery. We applied this pipeline in the notorious soilborne phytopathogen Sclerotinia sclerotiorum and identified 32 mutants with various developmental and growth deficiencies. Detailed molecular studies of three melanization-deficient mutants provide a proof of concept for the effectiveness of our method. A master transcription factor was found to regulate melanization of sclerotia through the DHN (1,8-dihydroxynaphthalene) melanin biosynthesis pathway. In addition, these mutants revealed that sclerotial melanization is important for sclerotia survival under abiotic stresses, sclerotial surface structure, and sexual reproduction. Foreseeably, this pipeline can be applied to facilitate efficient in-depth studies of other non-model fungal species in the future.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.


Assuntos
Ascomicetos , Basidiomycota , Ascomicetos/fisiologia , Basidiomycota/genética , Regulação da Expressão Gênica , Testes Genéticos
6.
Genomics ; 113(4): 1962-1975, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33901575

RESUMO

Many genes responsible for melanin biosynthesis in fungi were physically linked together. The PKS gene clusters in most of the melanin-producing fungi were regulated by the Cmr1. It was found that a close rearrangement of the PKS gene clusters had evolved in most of the melanin-producing fungi and various functions of melanin in them were beneficial to their adaptation to the changing environments. The melanin-producing fungi had undergone at least five large-scale differentiations, making their PKS gene clusters be quickly evolved and the fungi be adapted to different harsh environments. The recent gene losses and expansion were remarkably frequent in the PKS gene clusters, leading to their rapid evolution and adaptation of their hosts to different environments. The PKS gene and the CMR1 gene in them were subject to a strong co-evolution, but the horizontal gene transfer events might have occurred in the genome-duplicated species, Aspergillus and Penicillium.


Assuntos
Melaninas , Família Multigênica , Evolução Molecular , Fungos/genética , Transferência Genética Horizontal , Melaninas/genética
7.
J Basic Microbiol ; 61(3): 203-211, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33576034

RESUMO

Melanins are a diverse group of dark pigments with similar properties. In fungi, the most studied is the dihydroxynaphtalene (DHN)-melanin, present in several species including all the chromoblastomycosis agents, a chronic, disabling, and recalcitrant subcutaneous mycosis. It is synthesized in a pathway known as the pentaketide pathway, which has the agrochemical tricyclazole as an inhibitor, widely used in in vitro studies because it does not prevent the growth of fungi. There are different methodologies for qualitative and quantitative analyses of DHN-melanin, which made it possible to discover its important structural and antioxidant functions, with melanin acting as a protective factor against the host's immune system. Also, it can interact with some of the main antifungals of medical interest, reducing its activity and the susceptibility of fungi to these agents. This review aims to discuss the aspects of DHN-melanin, focusing on chromoblastomycosis, bringing the main findings of the published scientific studies, and highlighting the need for further research to understand this important fungal pathogenicity and a virulence factor.


Assuntos
Antifúngicos/farmacologia , Antioxidantes/farmacologia , Ascomicetos/efeitos dos fármacos , Cromoblastomicose/tratamento farmacológico , Melaninas/farmacologia , Naftóis/farmacologia , Ascomicetos/patogenicidade , Humanos , Melaninas/análise , Naftóis/análise
8.
Angew Chem Int Ed Engl ; 60(32): 17464-17471, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-33913253

RESUMO

Melanosomes in nature have diverse morphologies, including spheres, rods, and platelets. By contrast, shapes of synthetic melanins have been almost entirely limited to spherical nanoparticles with few exceptions produced by complex templated synthetic methods. Here, we report a non-templated method to access synthetic melanins with a variety of architectures including spheres, sheets, and platelets. Three 1,8-dihydroxynaphthalene dimers (4-4', 2-4' and 2-2') were used as self-assembling synthons. These dimers pack to form well-defined structures of varying morphologies depending on the isomer. Specifically, distinctive ellipsoidal platelets can be obtained using 4-4' dimers. Solid-state polymerization of the preorganized dimers generates polymeric synthetic melanins while maintaining the initial particle morphologies. This work provides a new route to anisotropic synthetic melanins, where the building blocks are preorganized into specific shapes, followed by solid-state polymerization.


Assuntos
Corantes/química , Naftóis/química , Polímeros/química , Anisotropia , Corantes/síntese química , Naftóis/síntese química , Polimerização , Polímeros/síntese química
9.
Mol Plant Microbe Interact ; 33(11): 1315-1329, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32815478

RESUMO

The role of NADPH oxidases (NOXs) in pathogenesis and development in the Curvularia leaf spot agent Curvularia lunata remains poorly understood. In this study, we identified C. lunata ClNOX2, which localized to the plasma membrane and was responsible for reactive oxygen species (ROS) generation. Scavenging the ROS production inhibited the conidial germination and appressorial formation. The ClNOX2 and ClBRN1 deletion mutants were defective in 1,8-dihydroxynaphthalene (DHN) melanin accumulation, appressorial formation, and cellulase synthesis and exhibited lower virulence. However, disruption of the ClNOX2 and ClBRN1 genes facilitated hyphal growth, enhanced stress adaptation to cell-wall-disrupting agents, and promoted developmental processes such as conidiation, conidial germination, and pseudothecium and ascus formation. Interestingly, loss of ClM1, the cell wall integrity (CWI) mitogen-activated protein kinase gene in C. lunata, led to morphology and pathogenicity phenotypes similar to ClNOX2 and ClBRN1 deletion mutants such as abnormal conidia, fewer appressoria, less melanin, increased hyphal growth, and enhanced tolerance to Congo red (CR). These results indicated that the ClNOX2 gene plays an important role in C. lunata development and virulence via regulating intracellular DHN melanin biosynthesis. Quantitative reverse-transcription PCR revealed that the ClNOX2-related ROS signaling pathway and ClM1-mediated CWI signaling pathway are cross-linked in regulating DHN melanin biosynthesis. Our findings provide new insights into how ClNOX2 participates in pathogenesis and development in hemibiotrophic plant fungal pathogens.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.


Assuntos
Ascomicetos/enzimologia , Melaninas/biossíntese , NADPH Oxidases , Doenças das Plantas/microbiologia , Ascomicetos/patogenicidade , Proteínas Fúngicas/genética , NADPH Oxidases/genética , Espécies Reativas de Oxigênio/metabolismo , Esporos Fúngicos , Virulência
10.
Microbiol Spectr ; : e0064524, 2024 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-39329485

RESUMO

Alternaria infectoria causes opportunistic human infections and is a source of allergens leading to respiratory allergies. In this work, we prepared cell wall nanoparticles (CWNPs) as a novel approach to study macrophage immunomodulation by fungal hyphal cell walls. A. infectoria was grown in the presence of caspofungin, an inhibitor of ß(1,3)-glucan synthesis; nikkomycin Z, an inhibitor of chitin synthases; and pyroquilon, an inhibitor of dihydroxynaphthalene (DHN)-melanin synthesis. Distinct CWNPs were obtained from these cultures, referred to as casCWNPs, nkCWNPs, and pyrCWNPs, respectively. CWNPs are round-shaped particles with a diameter of 70-200 nm diameter particles that when added to macrophages are taken up by membrane ruffling. CWNPs with no DHN-melanin and more glucan (pyrCWNPs) caused early macrophage activation and lowest viability, with the cells exhibiting ultrastructural modifications such as higher vacuolization and formation of autophagy-like structures. CasCWNPs promoted the highest tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß) increase, also resulting in the release of partially degraded chitin, an aspect never observed in macrophage-like cells and fungi. After 6 h of interaction with CWNPs, only half were viable, except with control CWNPs. Overall, this work indicates that compounds that modify the fungal cell wall led to CWNPs with new properties that may have implications for the effects of drugs during antifungal therapy. CWNPs provide a new tool to study the interaction of hyphal fungal cell wall components with phagocytic cells and enable to show how the modification of cell wall components in A. infectoria can modulate the response by macrophages.IMPORTANCEAlternaria species are ubiquitous environmental fungi to which the human host can continuously be exposed, through the inhalation of fungal spores but also of fragments of hyphae, from desegregated mycelia. These fungi are involved in hypersensitization and severe respiratory allergies, such as asthma, and can cause opportunistic infections in immunodepressed human host leading to severe disease. The first fungal structures to interact with the host cells are the cell wall components, and their modulation leads to differential immune responses. Here, we show that fungal cells grown with cell wall inhibitors led to cell wall nanoparticles with new properties in their interaction with macrophages. With this strategy, we overcame the limitation of in vitro assays interacting with filamentous fungi and showed that the absence of DNH-melanin leads to higher virulence, while caspofungin leads to cells walls that trigger higher hydrolysis of chitin and higher production of cytokines.

11.
Microbiol Spectr ; : e0507822, 2023 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-37707447

RESUMO

Sporothrix brasiliensis is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis by contaminated cats. Within 25 years, the disease has spread not only throughout Brazil but now to neighboring countries in Latin America. Thermo-dimorphism, melanin, glycans, adhesins, and secreted vesicles have been associated with the ability of Sporothrix species to cause disease in the mammalian host. Although certain virulence factors have been proposed as potential determinants for sporotrichosis, the scarcity of molecular tools for performing reverse genetics in Sporothrix has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous gene expression in S. brasiliensis, S. schenckii, and S. chilensis. Combined with CRISPR/Cas9 gene editing, this transformation protocol enabled the deletion of the putative DHN-melanin synthase gene pks1, which is a proposed virulence factor of Sporothrix species. To improve in locus integration of deletion constructs, we deleted the KU80 homolog that is critical for non-homologous end-joining DNA repair. The use of Δku80 strains from S. brasiliensis enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion in combination with CRISPR/Cas9. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in Sporothrix species. IMPORTANCE Sporotrichosis caused by Sporothrix brasiliensis is a disease that requires long periods of treatment and is rapidly spreading across Latin America. The virulence of this fungus and the surge of atypical and more severe presentations of the disease raise the need for an understanding of the molecular mechanisms underlying sporotrichosis, as well as the development of better diagnostics and antifungal therapies. By developing molecular tools for accurate genetic manipulation in Sporothrix, this study addresses the paucity of reliable and reproducible tools for stable genetic engineering of Sporothrix species, which has represented a major obstacle for studying the virulence determinants and their roles in the establishment of sporotrichosis.

12.
J Fungi (Basel) ; 9(2)2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36836250

RESUMO

Scedosporium apiospermum is a saprophytic filamentous fungus involved in human infections, of which the virulence factors that contribute to pathogenesis are still poorly characterized. In particular, little is known about the specific role of dihydroxynaphtalene (DHN)-melanin, located on the external layer of the conidia cell wall. We previously identified a transcription factor, PIG1, which may be involved in DHN-melanin biosynthesis. To elucidate the role of PIG1 and DHN-melanin in S. apiospermum, a CRISPR-Cas9-mediated PIG1 deletion was carried out from two parental strains to evaluate its impact on melanin biosynthesis, conidia cell-wall assembly, and resistance to stress, including the ability to survive macrophage engulfment. ΔPIG1 mutants did not produce melanin and showed a disorganized and thinner cell wall, resulting in a lower survival rate when exposed to oxidizing conditions, or high temperature. The absence of melanin increased the exposure of antigenic patterns on the conidia surface. PIG1 regulates the melanization of S. apiospermum conidia, and is involved in the survival to environmental injuries and to the host immune response, that might participate in virulence. Moreover, a transcriptomic analysis was performed to explain the observed aberrant septate conidia morphology and found differentially expressed genes, underlining the pleiotropic function of PIG1.

13.
Braz J Microbiol ; 53(4): 2093-2100, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36152271

RESUMO

The present study reports a new occurrence of Rhinocladiella similis isolated as an endophytic fungus in the Caatinga dry tropical forest in Brazil and describes its antifungal susceptibility. The isolate R. similis URM 7800 was obtained from leaves of the medicinal plant Myracrodruon urundeuva. Its morphological characterization was performed on potato dextrose agar medium and molecular analysis using the ITS rDNA sequence. The antifungal susceptibility profile was defined using the Clinical and Laboratory Standards Institute (CLSI) protocol M38-A2. The colony of isolate URM 7800 showed slow growth, with an olivaceous-gray color and powdery mycelium; in microculture, it showed the typical features of R. similis. In the antifungal susceptibility test, isolate URM 7800 showed high minimal inhibitory concentration (MIC) values for amphotericin B (>16 µg/mL), voriconazole (16 µg/mL), terbinafine (>0.5 µg/mL), and caspofungin (>8 µg/mL), among other antifungal drugs. Pathogenic melanized fungi are frequently isolated in environments where humans may be exposed, and these data show that it is essential to know if these isolates possess antifungal resistance.


Assuntos
Antifúngicos , Ascomicetos , Humanos , Antifúngicos/farmacologia , Brasil , Ascomicetos/genética , Florestas
14.
Fungal Biol ; 126(9): 566-575, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36008049

RESUMO

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.


Assuntos
Citrus , Penicillium , Citrus/microbiologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Melaninas/metabolismo , Família Multigênica , Esporos Fúngicos
15.
mSphere ; 7(1): e0087421, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-34986316

RESUMO

Previously, 1,8-dihydroxynaphthalene (DHN)-melanin was described to protect Aspergillus fumigatus against hydrogen peroxide (H2O2), thereby protecting this opportunistic human pathogen from reactive oxygen species generated by the immune system. This was based on the finding that the ATCC 46645 mutant with mutations in the pksP gene of the DHN-melanin synthesis pathway showed increased sensitivity to reactive oxygen species compared to the wild type. Here, it is shown that deletion of the pksP gene in A. fumigatus strain CEA10 did not affect sensitivity for H2O2 and superoxide in a plate stress assay. In addition, direct exposure of the dormant white conidia of the pksP deletion strains to H2O2 did not result in increased sensitivity. Moreover, complementation of the ATCC 46645 pksP mutant strain with the wild-type pksP gene did result in pigmented conidia but did not rescue the H2O2-sensitive phenotype observed in the plate stress assay. Genome sequencing of the ATCC 46645 pksP mutant strain and its complemented strain revealed a mutation in the cat1 gene, likely due to the UV mutagenesis procedure used previously, which could explain the increased sensitivity toward H2O2. In summary, DHN-melanin is not involved in protection against H2O2 or superoxide and, thus, has no role in survival of conidia when attacked by these reactive oxygen species. IMPORTANCE Opportunistic pathogens like Aspergillus fumigatus have strategies to protect themselves against reactive oxygen species like hydrogen peroxides and superoxides that are produced by immune cells. DHN-melanin is the green pigment on conidia of Aspergillus fumigatus and more than 2 decades ago was reported to protect conidia against hydrogen peroxide. Here, we correct this misinterpretation by showing that DHN-melanin actually is not involved in protection of conidia against hydrogen peroxide. We show that UV mutagenesis that was previously used to select a pksP mutant generated many more genome-wide mutations. We discovered that a mutation in the mycelial catalase gene cat1 could explain the observed phenotype of increased hydrogen peroxide sensitivity. Our work shows that UV mutagenesis is not the preferred methodology to be used for generating mutants. It requires genome sequencing with single-nucleotide polymorphism analysis as well as additional validations to discard unwanted and confirm correct phenotypes.


Assuntos
Aspergillus fumigatus , Superóxidos , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Melaninas/genética , Melaninas/metabolismo , Naftóis , Espécies Reativas de Oxigênio/metabolismo , Esporos Fúngicos/genética , Superóxidos/metabolismo
16.
Front Fungal Biol ; 3: 897954, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37746219

RESUMO

Aspergillus fumigatus is a potentially deadly opportunistic human pathogen. A. fumigatus has evolved a variety of mechanisms to evade detection by the immune system. For example, the conidium surface is covered in a layer of 1,8-dihydroxynaphthalene (DHN) melanin which masks the antigen macrophages use for recognition. DHN melanin also protects conidia from ultraviolet radiation and gives A. fumigatus conidia their characteristic green-grayish color. Here, we conducted genomic analysis of two closely related white-spore natural variants of A. fumigatus in comparison to two closely related green-spore isolates to identify a genetic basis of the white-spore phenotype. Illumina whole-genome resequencing data of the four isolates was used to identify variants that were shared in the white-spore isolates and different from both the green-spore isolates and the Af293 reference genome (which is also a green-spore isolate). We identified 4,279 single nucleotide variants and 1,785 insertion/deletions fitting this pattern. Among these, we identified 64 variants predicted to be high impact, loss-of-function mutations. One of these variants is a single nucleotide deletion that results in a frameshift in pksP (Afu2g17600), the core biosynthetic gene in the DHN melanin encoding gene cluster. The frameshift mutation in the white-spore isolates leads to a truncated protein in which a phosphopantetheine attachment site (PP-binding domain) is interrupted and an additional PP-binding domain and a thioesterase domain are omitted. Growth rate analysis of white-spore and green-spore isolates at 37°C and 48°C revealed that white-spore isolates are thermosensitive. Growth rate of A. fumigatus Af293 and a pksP null mutant in the Af293 background suggests pksP is not directly involved in the thermosensitivity phenotype. Further, our study identified a mutation in a gene (Afu4g04740) associated with thermal sensitivity in yeasts which could also be responsible for the thermosensitivity of the white-spore mutants. Overall, we used comparative genomics to identify the mutation and protein alterations responsible for the white-spore phenotype of environmental isolates of A. fumigatus.

17.
Pest Manag Sci ; 78(11): 4557-4568, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35833811

RESUMO

BACKGROUND: The natural 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis inhibitors (MBIs) are one of the promising approaches to the integrated management of plant diseases but have received scarce attention until now. Herein, to explore the natural DHN MBIs used in the control of northern corn leaf blight (NCLB), a library of 53 essential oil compounds was used to screen the MBIs against Exserohilum turcicum, the causal pathogen of NCLB, using tricyclazole as a reference compound. RESULTS: The results of morphological change in the colony, thermogravimetric analysis, ultraviolet-visible spectroscopy, and transmission electron microscopy confirmed that guaiacol could effectively inhibit the melanin production at 50 µg/mL under in vitro conditions. The in vitro bioassay results indicated that this inhibition effect was concentration-dependent and the minimum inhibition concentration of guaiacol was 50 µg/mL. The in vivo experimental results demonstrated that guaiacol significantly inhibited appressorium formation and penetration on corn leaf sheaths at the concentration of 500 µg/mL. The pot experiment results revealed that there were no differences between guaiacol (500 µg/mL) and tricyclazole (100 µg/mL) in control efficacy. The enzymatic assay suggested that guaiacol might exert the activity through inhibiting DHN polymerization to form melanins, which was distinct from tricyclazole. CONCLUSIONS: Taken together, this study screened out guaiacol as a natural MBI from 53 essential oil compounds and verified its effectiveness in the control of NCLB at 500 µg/mL. Above all, this research opened an avenue for exploring natural DHN MBIs in the integrated management of plant diseases. © 2022 Society of Chemical Industry.


Assuntos
Melaninas , Óleos Voláteis , Ascomicetos , Guaiacol , Melaninas/química , Óleos Voláteis/farmacologia , Tiazóis
18.
Front Fungal Biol ; 3: 862429, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37746170

RESUMO

Black microcolonial fungi (Ascomycetes from Arthonio-, Dothideo-, and Eurotiomycetes) are stress-tolerant and persistent dwellers of natural and anthropogenic extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures and accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) melanin in the multilayered cell walls. To understand how black fungi live, survive, colonize mineral substrates, and interact with phototrophs genetic methods are needed to test these functions and interactions. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a model for developing methods for genetic manipulation. Here, we report on the expansion of the genetic toolkit by more efficient multiplex CRISPR/Cas9 using a plasmid-based system for expression of Cas9 and multiple sgRNAs and the implementation of the three resistance selection markers genR (geneticin/nptII), baR (glufosinate/bar), and suR (chlorimuron ethyl/sur). The targeted integration of expression constructs by replacement of essential genes for pigment synthesis allows for an additional color screening of the transformants. The black-pink screening due to the elimination of pks1 (melanin) was applied for promoter studies using GFP fluorescence as reporter. The black-white screening due to the concurrent elimination of pks1 and phs1 (carotenoids) allows to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. The co-localization and interaction of the two K. petricola White Collar orthologs were demonstrated. Two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for a broad implementation in other fungi. This variety of genetic tools is opening a completely new perspective for mechanistic and very detailed study of expression, functioning and regulation of the genes/proteins encoded by the genomes of black fungi.

19.
Front Fungal Biol ; 3: 908343, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37746208

RESUMO

Aspergillus fumigatus is the main cause of life-threatening invasive aspergillosis. Despite the availability of various antifungals, therapy remains challenging and requires further studies. Accordingly, the clinical A. fumigatus isolate NIH4215 deriving from a fatal case of human pulmonary aspergillosis has frequently been used in drug efficacy studies. Unexpectedly, our initial attempts to generate a bioluminescent reporter of strain NIH4215 for in vivo drug efficacy studies failed, as NIH4215 was unable to grow on defined minimal medium. Subsequent analyses discovered a previously undescribed thiamine auxotrophy of strain NIH4215 and transformation with thiamine biosynthesis genes from A. fumigatus strain Af293 identified the nmt1 gene as cause of the thiamine auxotrophy. Sequencing of the defective nmt1 gene revealed the loss of a cysteine codon within an essential iron-binding motif. Subsequently, the wild-type nmt1 gene was successfully used to generate a bioluminescent reporter strain in NIH4215 by simultaneously deleting the akuB locus. The resulting bioluminescent ΔakuB strains showed a high frequency of homologous integration as confirmed by generation of pyrG and niaD deletion mutants. When tested in a Galleria mellonella infection model, neither thiamine auxotrophy nor the deletion of the akuB locus had a significant effect on virulence. However, besides thiamine auxotrophy, sectors with altered morphology and albino mutants frequently arose on colony edges of strain NIH4215 and its derivatives, and stable albino mutants were successfully isolated. A proposed increased mutation rate of NIH4215 was confirmed by screening for spontaneous occurrence of fluoorotic acid resistant mutants. Independent mutations in the pyrG and pyrE gene were identified in the fluoroorotic acid resistant NIH4215 isolates and the frequency of mutation was by at least one order of magnitude higher than that observed for the clinical A. fumigatus isolate CBS144.89. In summary, despite its virulence in animal models, strain NIH4215 is a thiamine auxotroph and prone to accumulate mutations. Our results suggest that thiamine biosynthesis is dispensable for host infection and mutation-prone strains such as NIH4215 could potentially facilitate the evolution of azole resistant strains as increasingly observed in the environment.

20.
mBio ; 12(2)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758088

RESUMO

In filamentous fungi, 1,8-dihydroxynaphthalene (DHN) melanin is a major component of the extracellular matrix, endowing fungi with environmental tolerance and some pathogenic species with pathogenicity. However, the subcellular location of the melanin biosynthesis pathway components remains obscure. Using the gray mold pathogen Botrytis cinerea, the DHN melanin intermediate scytalone was characterized via phenotypic and chemical analysis of mutants, and the key enzymes participating in melanin synthesis were fused with fluorescent proteins to observe their subcellular localizations. The Δbcscd1 mutant accumulated scytalone in the culture filtrate rather than in mycelium. Excessive scytalone appears to be self-inhibitory to the fungus, leading to repressed sclerotial germination and sporulation in the Δbcscd1 mutant. The BcBRN1/2 enzymes responsible for synthesizing scytalone were localized in endosomes and found to be trafficked to the cell surface, accompanied by the accumulation of BcSCD1 proteins in the cell wall. In contrast, the early-stage melanin synthesis enzymes BcPKS12/13 and BcYGH1 were localized in peroxisomes. Taken together, the results of this study revealed the subcellular distribution of melanin biosynthetic enzymes in B. cinerea, indicating that the encapsulation and externalization of the melanin synthetic enzymes need to be delicately orchestrated to ensure enzymatic efficiency and protect itself from the adverse effect of the toxic intermediate metabolite.IMPORTANCE The devastating gray mold pathogen Botrytis cinerea propagates via melanized conidia and sclerotia. This study reveals that the sclerotial germination of B. cinerea is differentially affected by different enzymes in the melanin synthesis pathway. Using gene knockout mutants and chemical analysis, we found that excessive accumulation of the melanin intermediate scytalone is inhibitory to B. cinerea. Subcellular localization analysis of the melanin synthesis enzymes of B. cinerea suggested two-stage partitioning of the melanogenesis pathway: the intracellular stage involves the steps until the intermediate scytalone was translocated to the cell surface, whereas the extracellular stage comprises all the steps occurring in the wall from scytalone to final melanin formation. These strategies make the fungus avert self-poisoning during melanin production. This study opens avenues for better understanding the mechanisms of secondary metabolite production in filamentous fungi.


Assuntos
Vias Biossintéticas/genética , Botrytis/genética , Melaninas/biossíntese , Melaninas/genética , Naftóis/metabolismo , Vias Biossintéticas/fisiologia , Botrytis/enzimologia , Botrytis/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Mutação , Naftóis/análise , Metabolismo Secundário
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