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Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Depsides have garnered substantial interest due to the diverse biological activities exhibited by members of this class. Among these are the antibacterial aquastatins, glycosylated heteromeric depsides formed through the condensation of orsellinic acid with corticiolic acid. In this work, we isolated aquastatins and the recently described geministatins, along with several novel aquastatin-related depsides with different alkyl side chains from the fungus Austroacremonium gemini MST-FP2131. The structures were determined through comprehensive spectroscopic analysis and chemical degradation. Genome mining and heterologous expression in Aspergillus nidulans and Saccharomyces cerevisiae revealed that aquastatin biosynthesis requires only two genes: a non-reducing polyketide synthase (SAT-KS-AT-PT-ACP-TE) and a glycosyltransferase. We demonstrated that the single polyketide synthase can synthesise an acetyl-primed orsellinic acid and alkylresorcylate with various chain lengths (C14, C16, or C18) by incorporating different long-chain acyl-CoAs as starter units, and then join these as heteromeric depsides. Using chemical degradation, we generated a series of analogues and showed that several aglycone depsides exhibit antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), as well as antifungal and cytotoxic activities. Interestingly, heterologous expression of the aquastatin gene cluster in A. nidulans produced higher levels of geministatins with Δ 15,16 and Δ 18,19 double bonds, which have superior bioactivities compared to the aquastatins but are only present as minor compounds in the native fungus A. gemini ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (This journal is © The Royal Society of Chemistry.)

Two new depside antibiotics, geministatins A (1) and B (2), were isolated from the fungus Austroacremonium gemini MST-FP2131 (Sordariomycetes, Ascomycota), which was recovered from rotting wood in the wet tropics of northern Australia. The structures of the geministatins were elucidated by detailed spectroscopic analysis, chemical degradation and comparison with literature values. Chemical degradation of 1 and 2 yielded three new analogues, geministatins C-E (3-5), as well as a previously reported compound dehydromerulinic acid A (6). Compounds 1, 2 and 6 exhibited antibacterial activity against the Gram-positive bacteria Bacillus subtilis (MIC 0.2-1.6 µg mL -1 ) and Staphylococcus aureus (MIC 0.78-6.3 µg mL -1 ), including methicillin-resistant S. aureus (MRSA), while 4 exhibited antifungal activity against the yeast Saccharomyces cerevisiae (MIC 13 µg mL -1 )., (© 2024. The Author(s).)

Episomal AMA1-based plasmids are increasingly used for expressing biosynthetic pathways and CRISPR/Cas systems in filamentous fungi cell factories due to their high transformation efficiency and multicopy nature. However, the gene expression from AMA1 plasmids has been observed to be highly heterogeneous in growing mycelia. To overcome this limitation, here we developed next-generation AMA1-based plasmids that ensure homogeneous and strong expression. We achieved this by evaluating various degradation tags fused to the auxotrophic marker gene on the AMA1 plasmid, which introduces a more stringent selection pressure throughout multicellular fungal growth. With these improved plasmids, we observed in Aspergillus nidulans a 5-fold increase in the expression of a fluorescent reporter, a doubling in the efficiency of a CRISPRa system for genome mining, and a up to a 10-fold increase in the production of heterologous natural product metabolites. This strategy has the potential to be applied to diverse filamentous fungi., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

The fungus Parastagonospora nodorum causes septoria nodorum blotch on wheat. The role of the fungal Velvet-family transcription factor VeA in P. nodorum development and virulence was investigated here. Deletion of the P. nodorum VeA ortholog, PnVeA, resulted in growth abnormalities including pigmentation, abolished asexual sporulation and highly reduced virulence on wheat. Comparative RNA-Seq and RT-PCR analyses revealed that the deletion of PnVeA also decoupled the expression of major necrotrophic effector genes. In addition, the deletion of PnVeA resulted in an up-regulation of four predicted secondary metabolite (SM) gene clusters. Using liquid-chromatography mass-spectrometry, it was observed that one of the SM gene clusters led to an accumulation of the mycotoxin alternariol. PnVeA is essential for asexual sporulation, full virulence, secondary metabolism and necrotrophic effector regulation., (© 2024. The Author(s).)

Cocultivation of the fungi Penicillium brasilianum MST-FP1927 and Aspergillus nomius MST-FP2004 resulted in the reciprocal induction of two new compounds, miktospiromide A ( 1 ) from A . nomius and kitrinomycin A ( 2 ) from P . brasilianum . A third new compound, kitrinomycin B ( 3 ), was also identified from an axenic culture of P . brasilianum , along with the previously reported compounds austalide K ( 4 ), 17 S -dihydroaustalide K ( 5 ), verruculogen ( 6 ), and fumitremorgin B ( 7 ). The structures of 1 - 3 were elucidated by detailed spectroscopic analysis and DFT calculations, while 4 - 7 were identified by comparison to authentic standards. The genome of A . nomius MST-FP2004 was sequenced, and a putative biosynthetic gene cluster for 1 was identified. Compound 2 showed activity against murine melanoma NS-1 cells (LD 99 7.8 μM) and the bovine parasite Tritrichomonas foetus (LD 99 4.8 μM).

The concept of "one target, one drug, one disease" is not always true, as compounds with previously described therapeutic applications can be useful to treat other maladies. For example, acridine derivatives have several potential therapeutic applications. In this way, identifying new potential targets for available drugs is crucial for the rational management of diseases. Computational methodologies are interesting tools in this field, as they use rational and direct methods. Thus, this study focused on identifying other rational targets for acridine derivatives by employing inverse virtual screening (IVS). This analysis revealed that chitinase enzymes can be potential targets for these compounds. Subsequently, we coupled molecular docking consensus analysis to screen the best chitinase inhibitor among acridine derivatives. We observed that 3 compounds displayed potential enhanced activity as fungal chitinase inhibitors, showing that compound 5 is the most active molecule, with an IC 50 of 0.6 ng/µL. In addition, this compound demonstrated a good interaction with the active site of chitinases from Aspergillus fumigatus and Trichoderma harzianum. Additionally, molecular dynamics and free energy demonstrated complex stability for compound 5. Therefore, this study recommends IVS as a powerful tool for drug development. The potential applications are highlighted as this is the first report of spiro-acridine derivatives acting as chitinase inhibitors that can be potentially used as antifungal and antibacterial candidates., (© 2023. The Author(s).)

Here, we report the occurrence and complete genome sequence of a novel victorivirus infecting Metarhizium anisopliae, named "Metarhizium anisopliae victorivirus 1" (MaVV1). The genome is 5353 bp in length and contains two open reading frames (ORFs), encoding a coat protein and an RNA-dependent RNA polymerase (RdRp), that overlap at the octanucleotide sequence AUGAGUAA. These ORFs showed sequence similarity to the corresponding ORFs of Ustilaginoidea virens RNA virus L (68.23%) and Ustilaginoidea virens RNA virus 13 (58.11%), respectively, both of which belong to the family Totiviridae. Phylogenetic analysis based on RdRp sequences revealed that MaVV1 clustered with members of the genus Victorivirus. This is the first genome sequence reported for a virus belonging to the genus Victorivirus infecting the entomopathogenic fungus M. anisopliae., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The genus Paracoccidioides comprises the species complex causing paracoccidioidomycoses (PCM). These fungi are a serious public health problem due to the long-term drug therapy, follow-up treatment, and frequent sequelae generated by the infection, such as pulmonary fibrosis. In this sense, the objective of this work was to generate bioluminescent reporter strains of Paracoccidioides spp. harboring a thermostable, red-shifted luciferase gene under the control of different constitutive promoters. The strains were generated by the integration of a species-specific codon-optimized luciferase gene upon actin or enolase promoter's control. The insertion of the constructs in Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells were performed through Agrobacterium tumefaciens-mediated transformation. The results demonstrated the presence of several transformants harboring the luciferase gene. These transformants were further confirmed by the expression of luciferase and by the presence of the hygromycin resistance gene. Moreover, the luciferase activity could be detected in in vitro bioluminescence assays and in vivo models of infection. In general, this work presents the methodology for the construction of bioluminescent strains of Paracoccidioides spp., highlighting potential promoters and proposing an in vivo model, in which those strains could be used for the systemic study of PCM., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Species from the Metarhizium genus are the causal agents of the green muscardine disease of insects. These fungi have been successfully employed for the biological control of pests over decades. Besides the biocontrol applications, recent efforts for genome sequencing of species in this genus have revealed a great diversity of biosynthetic gene clusters potentially associated with secondary metabolite synthesis. Amongst such molecules are the pseurotins, compounds with several activities, as chitin synthase inhibitors, and immunoglobulin E suppressors. Here, we report, for the first time, the isolation of pseurotin A from the culture broth of M. anisopliae, as well as the characterization of the effects of this compound over the model-arthropod Galleria mellonella. Pseurotin A displayed dose-dependent reversible paralysis effects when injected into the larvae hemocoel. However, the posterior challenge of the treated insects with M. anisopliae conidia did not lead to increased mortality, suggesting that pseurotin A treatment did not increase larvae susceptibility to the green muscardine disease. Although apparent insecticidal effects were not observed for pseurotin A, the paralysis effect observed can be important in M. anisopliae infection development., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Filamentous fungi are the organisms of choice for most industrial biotechnology. Some species can produce a variety of secondary metabolites and enzymes of commercial interest, and the production of valuable molecules has been enhanced through different molecular tools. Methods for genetic manipulation and transformation have been essential for the optimization of these organisms. The genus Simplicillium has attracted increased attention given several potential biotechnological applications. The Simplicillium genus harbors several entomopathogenic species and some isolates have been explored for bioremediation of heavy metal contaminants. Furthermore, the myriad of secondary metabolites isolated from Simplicillium spp. render these organisms as ideal targets for deep exploration and further biotechnological mining possibilities. However, the lack of molecular tools hampered the exploration of this genus. Thus, an Agrobacterium tumefaciens-mediated transformation method was established for Simplicillium subtropicum, employing the far-red fluorescent protein TURBOFP635/Katushka, as a visual marker, and the selection marker SUR gene, that confers resistance to chlorimuron ethyl. Notably, one round of transformation using the established method yielded almost 400 chlorimuron resistant isolates. Furthermore, these transformants displayed mitotic stability for, at least, five generations. We anticipate that this method can be useful for deep molecular exploration and improvement of strains in the Simplicillium genus.

Metarhizium anisopliae is an important entomopathogenic species and model for arthropod-fungus interaction studies. This fungus harbors a diverse arsenal of unexplored secondary metabolite biosynthetic gene clusters, which are suggested to perform diverse roles during host interaction and soil subsistence as a saprophytic species. Here we explored an unusual carnitine acyltransferase domain-containing highly reducing polyketide synthase found in the genome of M. anisopliae. Employing heterologous expression in Aspergillus nidulans, two new polyketides were obtained, named BAA and BAB, as well as one known polyketide [(2Z,4E,6E)-octa-2,4,6-trienedioic acid]. Intra-hemocoel injection of the most abundant compound (BAA) in the model-arthropod Galleria mellonella larvae did not induce mortality or noticeable alterations, suggesting that this compound may not harbor insecticidal activity. Also, the potential role of such molecules in polymicrobial interactions was evaluated. Determination of minimum inhibitory concentration assays using distinct fungal species revealed that BAA and BAB did not alter Cryptococcus neoformans growth, while BAA exhibited weak antifungal activity against Saccharomyces cerevisiae. Unexpectedly, these compounds increased Candida albicans growth compared to control conditions. Furthermore, BAA can mitigate the fungicidal effects of fluconazole over C. albicans. Although the exact role of these compounds on the M. anisopliae life cycle is elusive, the described results add up to the complexity of secondary metabolites produced by Metarhizium spp. Moreover, up to our knowledge, these are the first polyketides isolated from filamentous fungi that can boost the growth of another fungal species., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The first line of the Arthropods defense against infections is the hard-structured exoskeleton, a physical barrier, usually rich in insoluble chitin. For entomopathogenic fungi that actively penetrate the host body, an arsenal of hydrolytic enzymes (as chitinases and N-acetylglucosaminidases), that break down chitin, is essential. Notably, twenty-one putative chitinase genes have been identified in the genome of Metarhizium anisopliae, a generalist entomopathogenic fungus. As a multigenic family, with enzymes that, presumably, perform redundant functions, the main goal is to understand the singularity of each one of such genes and to discover their precise role in the fungal life cycle. Specially chitinases that can act as virulence determinants are of interest since these enzymes can lead to more efficient biocontrol agents. Here we explored a horizontally acquired chitinase from M. anisopliae, named chiMaD1. The deletion of this gene did not lead to phenotypic alterations or diminished supernatant's chitinolytic activity. Surprisingly, chiMaD1 deletion enhanced M. anisopliae virulence to the cattle tick (Rhipicephalus microplus) larvae and engorged females, while did not alter the virulence to the mealworm larvae (Tenebrio molitor). These results add up to recent reports of deleted genes that enhanced entomopathogenic virulence, showing the complexity of host-pathogen interactions., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Cryptococcus gattii is one of the causes of cryptococcosis, a life-threatening disease generally characterized by pneumonia and/or meningitis. Zinc is an essential element for life, being required for the activity of many proteins with catalytic and structural roles. Here, we characterize ZRG1 (zinc-related gene 1), which codes a product involved in zinc metabolism. Transcriptional profiling revealed that zinc availability regulated the expression of ZRG1, and its null mutants demonstrated impaired growth in zinc- and nitrogen-limiting conditions. Moreover, zrg1 strains displayed alterations in the expression of the zinc homeostasis-related genes ZAP1 and ZIP1. Notably, cryptococcal cells lacking Zrg1 displayed upregulation of autophagy-like phenotypes. Despite no differences were detected in the classical virulence-associated traits; cryptococcal cells lacking ZRG1 displayed decreased capacity for survival inside macrophages and attenuated virulence in an invertebrate model. Together, these results indicate that ZRG1 plays an important role in proper zinc metabolism, and is necessary for cryptococcal fitness and virulence., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cryptococcosis is a fungal infection caused mainly by the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii . The infection initiates with the inhalation of propagules that are then deposited in the lungs. If not properly treated, cryptococci cells can disseminate and reach the central nervous system. The current recommended treatment for cryptococcosis employs a three-stage regimen, with the administration of amphotericin B, flucytosine and fluconazole. Although effective, these drugs are often unavailable worldwide, can lead to resistance development, and may display toxic effects on the patients. Thus, new drugs for cryptococcosis treatment are needed. Recently, an iridoid named plumieridine was found in Allamanda polyantha seed extract; it exhibited antifungal activity against C. neoformans with a MIC of 250 μg/mL. To address the mode of action of plumieridine, several in silico and in vitro experiments were performed. Through a ligand-based a virtual screening approach, chitinases were identified as potential targets. Confirmatory in vitro assays showed that C. neoformans cell-free supernatant incubated with plumieridine displayed reduced chitinase activity, while chitinolytic activity was not inhibited in the insoluble cell fraction. Additionally, confocal microscopy revealed changes in the distribution of chitooligomers in the cryptococcal cell wall, from a polarized to a diffuse cell pattern state. Remarkably, further assays have shown that plumieridine can also inhibit the chitinolytic activity from the supernatant and cell-free extracts of bacteria, insect and mouse-derived macrophage cells (J774.A1). Together, our results suggest that plumieridine can be a broad-spectrum chitinase inhibitor., (Copyright © 2020 Silva e Souza, Barcellos, Sbaraini, Reuwsaat, Schneider, da Silva, Garcia, von Poser, Barbosa, Lima and Vainstein.)

Iron is an essential nutrient for all organisms. For pathogenic fungi, iron is essential for the success of infection. Thus, these organisms have developed high affinity iron uptake mechanisms to deal with metal deprivation imposed by the host. Siderophore production is one of the mechanisms that fungal pathogens employ for iron acquisition. Paracoccidioides spp. present orthologous genes encoding the enzymes necessary for the biosynthesis of hydroxamates, and plasma membrane proteins related to the transport of these molecules. All these genes are induced in iron deprivation. In addition, it has been observed that Paracoccidioides spp. are able to use siderophores to scavenge iron. Here we observed that addition of the xenosiderophore ferrioxamine B FOB) to P. brasiliensis culture medium results in repression (at RNA and protein levels) of the SidA, the first enzyme of the siderophore biosynthesis pathway. Furthermore, SidA activity was reduced in the presence of FOB, suggesting that P. brasiliensis blocks siderophores biosynthesis and can explore siderophores in the environment to scavenge iron. In order to support the importance of siderophores on Paracoccidioides sp. life and infection cycle, silenced mutants for the sidA gene were obtained by antisense RNA technology. The obtained AsSidA strains displayed decreased siderophore biosynthesis in iron deprivation conditions and reduced virulence to an invertebrate model., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Background: The Metarhizium genus harbors important entomopathogenic fungi. These species have been widely explored as biological control agents, and strategies to improve the fungal virulence are under investigation. Thus, the interaction between Metarhizium species and susceptible hosts have been explored employing different methods in order to characterize putative virulence determinants. However, the impact of epigenetic modulation on the infection cycle of Metarhizium is still an open topic. Among the different epigenetic modifications, DNA methylation of cytosine bases is an important mechanism to control gene expression in several organisms. To better understand if DNA methylation can govern Metarhizium-host interactions, the genome-wide DNA methylation profile of Metarhizium anisopliae was explored in two conditions: tick mimicked infection and a saprophytic-like control., Results: Using a genome wide DNA methylation profile based on bisulfite sequencing (BS-Seq), approximately 0.60% of the total cytosines were methylated in saprophytic-like condition, which was lower than the DNA methylation level (0.89%) in tick mimicked infection condition. A total of 670 mRNA genes were found to be putatively methylated, with 390 mRNA genes uniquely methylated in the tick mimicked infection condition. GO terms linked to response to stimuli, cell wall morphogenesis, cytoskeleton morphogenesis and secondary metabolism biosynthesis were over-represented in the tick mimicked infection condition, suggesting that energy metabolism is directed towards the regulation of genes associated with infection. However, recognized virulence determinants known to be expressed at distinct infection steps, such as the destruxin backbone gene and the collagen-like protein gene Mcl1, were found methylated, suggesting that a dynamic pattern of methylation could be found during the infectious process. These results were further endorsed employing RT-qPCR from cultures treated or not with the DNA methyltransferase inhibitor 5-Azacytidine., Conclusions: The set of genes here analyzed focused on secondary metabolites associated genes, known to be involved in several processes, including virulence. The BS-Seq pipeline and RT-qPCR analysis employing 5-Azacytidine led to identification of methylated virulence genes in M. anisopliae. The results provided evidences that DNA methylation in M. anisopliae comprises another layer of gene expression regulation, suggesting a main role of DNA methylation regulating putative virulence determinants during M. anisopliae infection cycle.

Cell walls are involved in manifold aspects of fungi maintenance. For several fungi, chitin synthesis, degradation and recycling are essential processes required for cell wall biogenesis; notably, the activity of β-N-acetylglucosaminidases (NAGases) must be present for chitin utilization. For entomopathogenic fungi, such as Metarhizium anisopliae, chitin degradation is also used to breach the host cuticle during infection. In view of the putative role of NAGases as virulence factors, this study explored the transcriptional profile and evolution of putative GH20 NAGases (MaNAG1 and MaNAG2) and GH3 NAGases (MaNAG3 and MaNAG4) identified in M. anisopliae. While MaNAG2 orthologs are conserved in several ascomycetes, MaNAG1 clusters only with Aspergilllus sp. and entomopathogenic fungal species. By contrast, MaNAG3 and MaNAG4 were phylogenetically related with bacterial GH3 NAGases. The transcriptional profiles of M. anisopliae NAGase genes were evaluated in seven culture conditions showing no common regulatory patterns, suggesting that these enzymes may have specific roles during the Metarhizium life cycle. Moreover, the expression of MaNAG3 and MaNAG4 regulated by chitinous substrates is the first evidence of the involvement of putative GH3 NAGases in physiological cell processes in entomopathogens, indicating their potential influence on cell differentiation during the M. anisopliae life cycle.

Colletotrichum musae is an important cosmopolitan pathogenic fungus that causes anthracnose in banana fruit. The entire genome of C. musae isolate GM20 (CMM 4420), originally isolated from infected banana fruit from Alagoas State, Brazil, was sequenced and annotated. The pathogen genomic DNA was sequenced on HiSeq Illumina platform. The C. musae GM20 genome has 50,635,197 bp with G + C content of 53.74% and in its present assembly has 2763 scaffolds, harboring 13,451 putative genes with an average length of 1626 bp. Gene prediction and annotation was performed by Funannotate pipeline, using a pattern for gene identification based on BUSCO.

The emergence of new microbial pathogens can result in destructive outbreaks, since their hosts have limited resistance and pathogens may be excessively aggressive. Described as the major ecological incident of the twentieth century, Dutch elm disease, caused by ascomycete fungi from the Ophiostoma genus, has caused a significant decline in elm tree populations ( Ulmus sp.) in North America and Europe. Genome sequencing of the two main causative agents of Dutch elm disease ( Ophiostoma ulmi and Ophiostoma novo-ulmi ), along with closely related species with different lifestyles, allows for unique comparisons to be made to identify how pathogens and virulence determinants have emerged. Among several established virulence determinants, secondary metabolites (SMs) have been suggested to play significant roles during phytopathogen infection. Interestingly, the secondary metabolism of Dutch elm pathogens remains almost unexplored, and little is known about how SM biosynthetic genes are organized in these species. To better understand the metabolic potential of O. ulmi and O. novo-ulmi , we performed a deep survey and description of SM biosynthetic gene clusters (BGCs) in these species and assessed their conservation among eight species from the Ophiostomataceae family. Among 19 identified BGCs, a fujikurin-like gene cluster (OpPKS8) was unique to Dutch elm pathogens. Phylogenetic analysis revealed that orthologs for this gene cluster are widespread among phytopathogens and plant-associated fungi, suggesting that OpPKS8 may have been horizontally acquired by the Ophiostoma genus. Moreover, the detailed identification of several BGCs paves the way for future in-depth research and supports the potential impact of secondary metabolism on Ophiostoma genus' lifestyle.

Background: The described species from the Metarhizium genus are cosmopolitan fungi that infect arthropod hosts. Interestingly, while some species infect a wide range of hosts (host-generalists), other species infect only a few arthropods (host-specialists). This singular evolutionary trait permits unique comparisons to determine how pathogens and virulence determinants emerge. Among the several virulence determinants that have been described, secondary metabolites (SMs) are suggested to play essential roles during fungal infection. Despite progress in the study of pathogen-host relationships, the majority of genes related to SM production in Metarhizium spp. are uncharacterized, and little is known about their genomic organization, expression and regulation. To better understand how infection conditions may affect SM production in Metarhizium anisopliae, we have performed a deep survey and description of SM biosynthetic gene clusters (BGCs) in M. anisopliae, analyzed RNA-seq data from fungi grown on cattle-tick cuticles, evaluated the differential expression of BGCs, and assessed conservation among the Metarhizium genus. Furthermore, our analysis extended to the construction of a phylogeny for the following three BGCs: a tropolone/citrinin-related compound (MaPKS1), a pseurotin-related compound (MaNRPS-PKS2), and a putative helvolic acid (MaTERP1)., Results: Among 73 BGCs identified in M. anisopliae, 20 % were up-regulated during initial tick cuticle infection and presumably possess virulence-related roles. These up-regulated BGCs include known clusters, such as destruxin, NG39x and ferricrocin, together with putative helvolic acid and, pseurotin and tropolone/citrinin-related compound clusters as well as uncharacterized clusters. Furthermore, several previously characterized and putative BGCs were silent or down-regulated in initial infection conditions, indicating minor participation over the course of infection. Interestingly, several up-regulated BGCs were not conserved in host-specialist species from the Metarhizium genus, indicating differences in the metabolic strategies employed by generalist and specialist species to overcome and kill their host. These differences in metabolic potential may have been partially shaped by horizontal gene transfer (HGT) events, as our phylogenetic analysis provided evidence that the putative helvolic acid cluster in Metarhizium spp. originated from an HGT event., Conclusions: Several unknown BGCs are described, and aspects of their organization, regulation and origin are discussed, providing further support for the impact of SM on the Metarhizium genus lifestyle and infection process.

Background: Metarhizium anisopliae is an entomopathogenic fungus used in the biological control of some agricultural insect pests, and efforts are underway to use this fungus in the control of insect-borne human diseases. A large repertoire of proteins must be secreted by M. anisopliae to cope with the various available nutrients as this fungus switches through different lifestyles, i.e., from a saprophytic, to an infectious, to a plant endophytic stage. To further evaluate the predicted secretome of M. anisopliae, we employed genomic and transcriptomic analyses, coupled with phylogenomic analysis, focusing on the identification and characterization of secreted proteins., Results: We determined the M. anisopliae E6 genome sequence and compared this sequence to other entomopathogenic fungi genomes. A robust pipeline was generated to evaluate the predicted secretomes of M. anisopliae and 15 other filamentous fungi, leading to the identification of a core of secreted proteins. Transcriptomic analysis using the tick Rhipicephalus microplus cuticle as an infection model during two periods of infection (48 and 144 h) allowed the identification of several differentially expressed genes. This analysis concluded that a large proportion of the predicted secretome coding genes contained altered transcript levels in the conditions analyzed in this study. In addition, some specific secreted proteins from Metarhizium have an evolutionary history similar to orthologs found in Beauveria/Cordyceps. This similarity suggests that a set of secreted proteins has evolved to participate in entomopathogenicity., Conclusions: The data presented represents an important step to the characterization of the role of secreted proteins in the virulence and pathogenicity of M. anisopliae.

Fungal chitin metabolism involves diverse processes such as metabolically active cell wall maintenance, basic nutrition, and different aspects of virulence. Chitinases are enzymes belonging to the glycoside hydrolase family 18 (GH18) and 19 (GH19) and are responsible for the hydrolysis of β-1,4-linkages in chitin. This linear homopolymer of N-acetyl-β-D-glucosamine is an essential constituent of fungal cell walls and arthropod exoskeletons. Several chitinases have been directly implicated in structural, morphogenetic, autolytic and nutritional activities of fungal cells. In the entomopathogen Metarhizium anisopliae, chitinases are also involved in virulence. Filamentous fungi genomes exhibit a higher number of chitinase-coding genes than bacteria or yeasts. The survey performed in the M. anisopliae genome has successfully identified 24 genes belonging to glycoside hydrolase family 18, including three previously experimentally determined chitinase-coding genes named chit1, chi2 and chi3. These putative chitinases were classified based on domain organization and phylogenetic analysis into the previously described A, B and C chitinase subgroups, and into a new subgroup D. Moreover, three GH18 proteins could be classified as putative endo-N-acetyl-β-D-glucosaminidases, enzymes that are associated with deglycosylation and were therefore assigned to a new subgroup E. The transcriptional profile of the GH18 genes was evaluated by qPCR with RNA extracted from eight culture conditions, representing different stages of development or different nutritional states. The transcripts from the GH18 genes were detected in at least one of the different M. anisopliae developmental stages, thus validating the proposed genes. Moreover, not all members from the same chitinase subgroup presented equal patterns of transcript expression under the eight distinct conditions studied. The determination of M. anisopliae chitinases and ENGases and a more detailed study concerning the enzymes' roles in morphological or nutritional functions will allow comprehensive insights into the chitinolytic potential of this highly infective entomopathogenic fungus.