Agrupamentos gênicos envolvidos na biosíntese de metabólitos secundários no fungo entomopatogênico Metarhizium anisopliae : identificação genômica e padrões de expressão em cutículas do carrapato Rhipicephalus microplus

O gênero Metarhizium abriga fungos cosmopolitas que infectam hospedeiros artrópodes. Curiosamente, enquanto algumas espécies do gênero infectam um amplo espectro de hospedeiros (hospedeiro-generalistas), outras espécies infectam apenas alguns artrópodes (hospedeiro-especialistas). Esse traço evolutivo singular permite comparações únicas, a fim de determinar como patógenos e fatores de virulência surgem. Dentre os diversos fatores de virulência descritos, se destacam os metabólitos secundários que hipoteticamente desempenhem papéis essenciais na infecção fúngica. No entanto, a maioria dos genes para a produção de metabólitos secundários em Metarhizium spp. não foram ainda caracterizados, e pouco se sabe sobre a organização gênomica, expressão e regulação destes genes. A fim de melhor compreender estes aspectos, nós realizamos uma análise e descrição detalhada de agrupamentos gênicos (clusters) envolvidos na biossíntese de metabólitos secundários em M. anisopliae, analisamos dados de um estudo transcritômico onde o fungo foi cultivado em cutículas de carrapato avaliando a expressão diferencial destes clusters, bem como avaliamos a conservação destes genes entre espécies do gênero Metarhizium. Ademais, nossa análise se estendeu avaliando aspectos evolutivos e filogenéticos para três clusters: MaPKS1 cujo produto é putativamente assemelhado a tropolonas e citrininas, MaNRPS-PKS2 cujo produto é putativamente assemelhado a pseurotina e MaTERP1 cujo produto putativo é o ácido helvólico. Dentre os 73 clusters identificados no genoma de M. anisopliae, 20 % estavam positivamente regulados na condição experimental de infecção inicial, com presumível papel na virulência do fungo. Dentre os clusters positivamente regulados estão genes já caracterizados envolvidos na biossíntese de destruxinas, NG39x e ferricrocina, em conjunto com genes putativamente envolvidos na biossíntese do ácido helvólico, produtos assemelhados a pseurotina e tropolonas e citrininas, além de genes envolvidos na biossíntese de compostos desconhecidos. Curiosamente, diversos clusters positivamente regulados na condição de infecção inicial não estão presentes em espécies hospedeiro-especialistas do gênero Metarhizium, indicando que existem diferenças nas estratégias metabólicas empregadas por espécies hospedeiro-generalistas e hospedeiro-especialistas no ciclo infeccioso. Estas diferenças no potencial metabólico podem ter sido parcialmente moldadas por eventos de transferência horizontal, conforme sugere nossa análise filogenética sobre a origem do o cluster putativamente envolvido na biossíntese do ácido helvólico em Metarhizium spp. Em conclusão, diversos clusters desconhecidos são descritos e aspectos da sua organização, regulação e origem são discutidos, fornecendo evidências sobre o impacto dos metabólitos secundários no ciclo de vida e infecção de espécies do gênero Metarhizium.The Metarhizium genus harbors 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. However, 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 these aspects, 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 within 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). 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. 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. In conclusion several unknown BGCs are described, and aspects of their organization, regulation and origin are discussed, providing evidence for the impact of SMs on the Metarhizium genus lifestyle and infection process

Secondary metabolite gene clusters in the entomopathogen fungus Metarhizium anisopliae : genome identification and patterns of expression in a cuticle infection model

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

Genome-wide DNA methylation analysis of Metarhizium anisopliae during tick mimicked infection condition

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

A plumieridine-rich fraction from Allamanda polyantha inhibits chitinolytic activity and exhibits antifungal properties against Cryptococcus neoformans

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

Análise funcional de quatro genes putativos da família GH18 de glicosil hidrolases no fungo entomopatogênico Metarhizium anisopliae

Metarhizium anisopliae é um fungo filamentoso entomopatogênico, da ordem Hypocreales, que infecta um grande número de espécies de artrópodes. Este fungo caracteriza-se por ser um dos primeiros organismos a ser aplicado em larga escala para o controle de insetos-praga na Agricultura. A penetração através da carapaça do hospedeiro constitui sua via preferencial de infecção, sendo esta a primeira barreira a ser superada. Com composições variáveis, na maioria dos artrópodes a carapaça é, geralmente, rica em quitina. A quitina é um polissacarídeo composto por monômeros de N-acetilglicosamina (GlcNAc) que também está presente na parede celular de fungos filamentosos. Para transpassar essa barreira, o fungo combina diferentes estratégias, como pressão mecânica e a dissolução da cutícula pela secreção de diversas enzimas hidrolíticas. Dentre as enzimas secretadas estão as quitinases, responsáveis por catalisar a hidrólise de ligações glicosídicas β-1,4 entre monômeros GlcNAc. Em fungos, as quitinases não apresentam apenas importância nutricional, participando ativamente de processos morfogênicos e autolíticos. Quitinases e endo-β-D-N-acetilglicosaminidases (ENGases) são membros da família GH18 de glicosil hidrolases em fungos filamentosos As ENGases catalisam uma reação de hidrólise semelhante à das quitinases, porém utilizando N-oligossacarídeos como substrato, em detrimento a quitina, em uma reação de deglicosilação. Uma análise genômica realizada em nosso laboratório, na linhagem E6 de M. anisopliae, identificou vinte e quatro genes putativos contendo o domínio GH18, os quais foram classificados em cinco subgrupos, baseado em similaridade de sequências, relações filogenéticas, presença de domínios e características funcionais. Com nove genes agrupados no subgrupo A, sete genes no B, quatro genes no C, um gene no subgrupo D e três genes em um subgrupo contendo ENGases. A importância do gene do subgrupo D (chimaD1) e dos genes do subgrupo das ENGases (MaEng18A, MaEng18B e MaEng18C), permanece ainda desconhecida em M. anisopliae. O presente estudo visou avaliar, pela construção de mutantes funcionais, a importância destes genes no ciclo de vida do fungo Análises filogenéticas foram conduzidas, levantando hipóteses sobre a evolução do gene ChimaD1, e das ENGases de fungos. Diferindo de outros fungos filamentos analisados, que possuem apenas uma ENGase putativa secretada e outra cópia com possível atividade citosólica, M. anisopliae possui dois possíveis parálogos com peptídeo sinal predito. A presença destes dois genes pode estar correlacionada ao estilo de vida de do fungo. Um mutante nulo para o gene chimaD1 foi construído, e construções de mutantes nulos para os demais genes estão em andamento. Futuras análises fenotípicas destes mutantes devem auxiliar na compreensão de diversos aspectos do ciclo de vida e da importância da família GH18 em fungos.Metarhizium anisopliae is an entomopathogenic fungus of Hypocreales order that infects a huge range of arthropod species worldwide, being one of the first organisms widely applied to control crop pests. The fungal penetration through host cuticle constitutes the preferential route to establish M. anisopliae infection, and it is the first barrier to be surpassed to obtain a successful infection. Exhibiting a variable composition, the cuticle is a chitin-rich structure in many arthropods. The chitin, an N-acetylglucosamine (GlcNAc) polysaccharide is also found in filamentous fungi cell wall. In order to surpass the host cuticle barrier, the fungus combines different strategies, such as mechanical pressure and secretion of several hydrolytic enzymes. Among these secreted enzymes there are chitinases. These enzymes are responsible for catalyze the hydrolysis of β-1,4 glycosidic bonds between GlcNAc monomers. In fungi, chitinases presents not only nutritional importance, but also exhibit morphogenic and autolytic functions, acting at different processes of fungal development and life cycle maintenance. In filamentous fungi glycoside hydrolase family 18 (GH18) contains enzymes with chitinase and endo-β-D-N-acetylglucosaminidases (ENGases) activity. The ENGases are similar to chitinases, catalyzing a similar hydrolysis reaction but using N-oligosaccharides as substrate instead of chitin, in a deglycosylation A genomic analysis performed in our laboratory, in M. anisopliae E6 strain, identified twenty-four putative genes containing the GH18 domain, which were classified five subgroups. Nine genes were assigned to subgroup A, seven genes to B, four to C, one gene to D and three genes to a subgroup containing only ENGases. This classification was based on sequence similarity, domains organization, phylogenetic relationship and functional features. The importance of subgroup D gene (chimaD1) and the genes from ENGase subgroup (MaEng18A, MaEng18B and MaEng18C) remains unknown in M. anisopliae. The present study aims to evaluate, through the construction of knockout mutants, the function of these genes in the fungus life cycle. Differing from other filamentous fungi analyzed, which have two ENGases, one putatively secreted and other with possible cytosolic activity, M. anisopliae has two putative secreted ENGases. This difference could be related with fungus life style. Phylogenetic analyses were conducted, raising hypotheses about chimaD1 and fungal ENGases evolution. A ChimaD1 knockout strain has been created, as well as knockout strains for the others genes are in progress. Future phenotypic analysis of these knockout strains could help explaining some aspects from fungi life cycle and the importance of GH18 family

Molecular evolution and transcriptional profile of GH3 and GH20 β-N-acetylglucosaminidases in the entomopathogenic fungus Metarhizium anisopliae

Abstract 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