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Social insects establish complex interactions with microorganisms, some of which play defensive roles in colony protection. The important role of pollinators such as the stingless bee Melipona scutellaris in nature encouraged us to pursue efforts to study its associated microbiota. Here we describe the discovery of two novel cyclic hexadepsipeptides, meliponamycin A (1) and meliponamycin B (2), from Streptomyces sp. ICBG1318 isolated from M. scutellaris nurse bees. Their structures were established by interpretation of NMR and MS data, and the absolute configuration of the constituent amino acids was determined by the advanced Marfey's method. Compounds 1 and 2 showed strong activity against the entomopathogen Paenibacillus larvae and human pathogens Staphylococcus aureus and Leishmania infantum.

Amphibians are known to possess a wide variety of compounds stored in their skin glands. While significant progress has been made in understanding the chemical diversity and biological relevance of alkaloids, amines, steroids, and peptides, most aspects of the odorous secretions are completely unknown. In this study, we examined sexual variations in the volatile profile from the skin of the tree frog Boana prasina and combined culture and culture-independent methods to investigate if microorganisms might be a source of these compounds. We found that sesquiterpenes, thioethers, and methoxypyrazines are major contributors to the observed sex differences. We also observed that each sex has a distinct profile of methoxypyrazines, and that the chemical origin of these compounds can be traced to a Pseudomonas sp. strain isolated from the frog´s skin. This symbiotic bacterium was present in almost all individuals examined from different sites and was maintained in captive conditions, supporting its significance as the source of methoxypyrazines in these frogs. Our results highlight the potential relevance of bacteria as a source of chemical signals in amphibians and contribute to increasing our understanding of the role that symbiotic associations have in animals. Fil: Brunetti, Andrés Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina. Universidade de Sao Paulo; Brasil Fil: Lucio Lyra, Mariana. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Melo, Weilan G. P.. Universidade de Sao Paulo; Brasil Fil: Andrade, Laura Elena. Universidade de Sao Paulo; Brasil Fil: Palacios Rodríguez, Pablo. Universidad de los Andes; Colombia Fil: Prado, Bárbara M.. Universidade de Sao Paulo; Brasil Fil: Baptista Haddad, Célio Fernando. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Tallarico Pupo, Monica. Universidade de Sao Paulo; Brasil Fil: Peporine Lopes, Norberto. Universidade de Sao Paulo; Brasil

Made available in DSpace on 2021-06-25T10:56:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 The leaf-cutter ant Acromyrmex balzani is responsible for causing important losses in reforestation areas, crops, and pastures, and is frequently found in the Brazilian savanna (Cerrado). So far, there is no information regarding the yeast communities that occur in their nests. Here, we evaluated the diversity, composition, and structure of yeast communities in both fungus gardens (FG) and external refuse dump (RD) of this ant species (Palmas, Tocantins, northern Brazil). A total of 720 yeasts were isolated, comprising 52 species distributed in 29 genera. The RDs have significantly richer and more diverse yeast communities than the fungus gardens, regardless of the season and the level of preservation in the area. The isolates produced a wide range of carbon polymer-degrading enzymes and were able to assimilate carbon-sources present in plant materials. We observed a different proportion of enzyme-producers and carbon-assimilation found in external refuse dump and fungus gardens from preserved and disturbed areas, suggesting that this interaction may vary depending on the environmental conditions. A. balzani nests in the savanna biome are a hotspot of yeast species with ecological, clinical, and biotechnological implications. Centre for the Study of Social Insects São Paulo State University (UNESP) Laboratory of Environmental Microbiology and Biotechnology Federal University of Tocantins (UFT) Department of Pharmaceutical Science School of Pharmaceutical Sciences of Ribeirão Preto University of São Paulo Department of Biology Faculty of Philosophy Sciences and Letters of Ribeirão Preto University of São Paulo Centre for the Study of Social Insects São Paulo State University (UNESP)

In the myrmecophytic mutualistic relationship between Azteca ants and Cecropia plants both species receive protection and exchange nutrients. The presence of microorganisms in this symbiotic system has been reported, and the symbiotic role of some fungi involved in the myrmecophytic interactions has been described. In this work we focus on bacteria within this mutualism, conducting isolations and screening for antimicrobial activities, genome sequencing, and biochemical characterization. We show that Pantoea, Rhizobium, Methylobacterium, Streptomyces and Pseudomonas are the most common cultivable genera of bacteria. Interestingly, Pseudomonas spp. isolates showed potent activity against 83% of the pathogens tested in our antimicrobial activity assays, including a phytopathogenic fungus isolated from Cecropia samples. Given the predicted nitrogen limitations associated with the fungal patches within this myrmecophyte, we performed nitrogen fixation analyses on the bacterial isolates within the Proteobacteria and show the potential for nitrogen fixation in Pseudomonas strains. The genome of one Pseudomonas strain was sequenced and analyzed. The gene cluster involved in the biosynthesis of cyclic lipodepsipeptides (CLPs) was identified, and we found mutations that may be related to the loss of function in the dual epimerization/condensation domains. The compound was isolated, and its structure was determined, corresponding to the antifungal viscosinamide. Our findings of diazotrophy and production of viscosinamide in multiple Pseudomonas isolates suggests that this bacterial genus may play an important role in the Cecropia-Azteca symbiosis.

Three antifungal macrolides cyphomycin (1), caniferolide C (2) and GT-35 (3) were isolated from Streptomyces sp. ISID311, a bacterial symbiont associated with Cyphomyrmex fungus-growing ants. The planar structures of these compounds were established by 1 and 2D NMR data and MS analysis. The relative configurations of 1–3 were established using Kishi's universal NMR database method, NOE/ROE analysis and coupling constants analysis assisted by comparisons with NMR data of related compounds. Detailed bioinformatic analysis of cyphomycin biosynthetic gene cluster confirmed the stereochemical assignments. Compounds 1–3 displayed high antagonism against different strains of Escovopsis sp., pathogen fungi specialized to the fungus-growing ant system. Compounds 1–3 also exhibited potent antiprotozoal activity against intracellular amastigotes of the human parasite Leishmania donovani with IC50 values of 2.32, 0.091 and 0.073 µM, respectively, with high selectivity indexes.

Fungus-growing ants engage in a multilateral symbiosis: they cultivate a fungal garden as their primary food source and host symbiotic actinobacteria (Pseudonocardia spp.) that provide chemical defenses. The bacterial symbionts produce small specialized metabolites that protect the fungal garden from specific fungal pathogens (Escovopsis spp.), and in return, they are fed by the ant hosts. Multiple studies on the molecules underlying this symbiotic system have led to the discovery of a large number of structurally diverse antifungal molecules, but somewhat surprisingly no shared structural theme emerged from these studies. A large systematic study of Brazilian nests led to the discovery of the widespread production of a potent but overlooked antifungal agent, which we named attinimicin, by nearly two-thirds of all Pseudonocardia strains from multiple sites in Brazil. Here we report the structure of attinimicin, its putative biosynthetic gene cluster, and the evolutionary relationship between attinimicin and two related peptides, oxachelin A and cahuitamycin A. All three nonribosomal peptides are structural isomers with different primary peptide sequences. Attinimicin shows iron-dependent antifungal activity against specific environmental fungal parasites but no activity against the fungal cultivar. Attinimicin showed potent in vivo activity in a mouse Candida albicans infection model comparable to clinically used azole-containing antifungals. In situ detection of attinimicin in both ant nests and on worker ants supports an ecological role for attinimicin in protecting the fungal cultivar from pathogens. The geographic spread of the attinimicin biosynthetic gene cluster in Brazilian Pseudonocardia spp. marks attinimicin as the first specialized metabolite from ant-associated bacteria with broad geographic distribution., Attinimicin, the product of an evolutionarily conserved combinatorial three-module nonribosomal peptide biosynthetic pathway, provides iron-dependent chemical defenses to fungus-growing ants over a broad geographical swath.

In the myrmecophytic mutualistic relationship between Azteca ants and Cecropia plants both species receive protection and exchange nutrients. The presence of microorganisms in this symbiotic system has been reported, and the symbiotic role of some fungi involved in the myrmecophytic interactions has been described. In this work we focus on bacteria within this mutualism, conducting isolations and screening for antimicrobial activities, genome sequencing, and biochemical characterization. We show that Pantoea, Rhizobium, Methylobacterium, Streptomyces and Pseudomonas are the most common cultivable genera of bacteria. Interestingly, Pseudomonas spp. isolates showed potent activity against 83% of the pathogens tested in our antimicrobial activity assays, including a phytopathogenic fungus isolated from Cecropia samples. Given the predicted nitrogen limitations associated with the fungal patches within this myrmecophyte, we performed nitrogen fixation analyses on the bacterial isolates within the Proteobacteria and show the potential for nitrogen fixation in Pseudomonas strains. The genome of one Pseudomonas strain was sequenced and analyzed. The gene cluster involved in the biosynthesis of cyclic lipodepsipeptides (CLPs) was identified, and we found mutations that may be related to the loss of function in the dual epimerization/condensation domains. The compound was isolated, and its structure was determined, corresponding to the antifungal viscosinamide. Our findings of diazotrophy and production of viscosinamide in multiple Pseudomonas isolates suggests that this bacterial genus may play an important role in the Cecropia-Azteca symbiosis.

Social insects are frequently observed in symbiotic association with bacteria that produce antimicrobial natural products as a defense mechanism. There is a lack of studies on the microbiota associated with stingless bees and their antimicrobial compounds. To the best of our knowledge, this study is the first to report the isolation of Paenibacillus polymyxa ALLI-03-01 from the larval food of the stingless bee Melipona scutellaris. The bacterial strain was cultured under different conditions and produced (L)-(-)-3-phenyllactic acid and fusaricidins, which were active against entomopathogenic fungi and Paenibacillus larvae. Our results indicate that such natural products could be related to colony protection, suggesting a defense symbiosis between P. polymyxa ALLI-03-01 and Melipona scutellaris.

Bacterial strains isolated from attine ants showed activity against the insect specialized fungal pathogen Escovopsis and also against the human protozoan parasite Leishmania donovani. The bioassay guided fractionation of extracts from cultures of Streptomyces sp. ICBG292, isolated from the exoskeleton of Cyphomyrmex workers, led to the isolation of Mer-A2026B (1), piericidin-A1 (2) and nigericin (3). Nigericin (3) presented high activity against intracellular amastigotes of L. donovani (IC50 0.129 ± 0.008 μM). Streptomyces puniceus ICBG378, isolated from workers of Acromyrmex rugosus rugosus, produced dinactin (4) with potent anti-L. donovani activity against intracellular amastigotes (IC50 0.018 ± 0.003 μM). Compounds 3 and 4 showed good selectivity indexes, 88.91 and 656.11 respectively, and were more active than positive control, miltefosine. Compounds 1–4 were also active against some Escovopsis strains. Compounds 1 and 2 were also produced by Streptomyces sp. ICBG233, isolated from workers of Atta sexdens, and detected in ants’ extracts by mass spectrometry, suggesting they are produced in the natural environment as defensive compounds involved in the symbiotic interaction., Author summary Visceral leishmaniasis, caused by Leishmania infantum and L. donovani, is characterized by high rate mortality worldwide. Current treatments for this disease suffer from toxicity, variable efficacy, requirements for parenteral administration and length of treatment regimens. New chemical entities and development of new drugs are important to overcome the impact of this protozoan disease. Actinobacterial strains, such as Streptomyces, have been a source of most naturally derived antibiotics, as well as anticancer, anthelmintic, and antifungal drugs. These microorganisms also produce small molecules important in symbiotic interactions with insects, such as fungus-growing ants, fungus-growing termites, beetles and wasps against pathogens. Several novel compounds have been reported from these microorganisms with promising biological activities. In this work we show an interesting ecologic approach for drug discovery that also shows promise for the identification of antileishmanial natural products from fungus-growing ant ecosystem. Two compounds isolated from Streptomyces strains showed potent activity against L. donovani, higher than the positive control (miltefosine) with high selectivity indexes.

Social insects can establish nutritional, communication and defensive symbiotic associations with microorganisms, among which actinobacteria play important roles in these symbioses. Stingless bees are characterized by keeping their colonies free of diseases and parasites. These bees have evolved different protective strategies, but most are still unknown. Here we explore the potential of stingless bee Melipona scutellaris to associate with actinobacteria to suppress pathogenic microbes. Seven actinobacterial strains were isolated from foraging and nurse bees and identified through 16S rRNA sequencing. Three of these strains exhibited pronounced growth inhibition against the specialized pathogen of honey bees Paenibacillus larvae, the causative agent of American Foulbrood. Bioassay-guided HPLC fractionation, followed by NMR and mass spectrometry-based structure elucidation, revealed the presence of lobophorin (1–4) and anthracycline (5–15) families isolated from strains Streptomyces sp. ICBG1323 and Micromonospora sp. ICBG1321, all responsible for this activity. Our results suggest that M. scutellaris bees engage in defensive symbiosis with actinobacteria that produce antibacterial small molecules, helping to keep their colonies healthy.

The antibiotic streptazolin (1), its E-isomer (2), along with the stereoisomers strepchazolin A (3) and strepchazolin B (4) and the inorganic compound cyclooctasulfur (5) were produced in solid culture by Streptomyces chartreusis ICBG377, which was isolated from the fungal garden of the leaf-cutter ant Acromyrmex subterraneus brunneus. This is the first time compound 2 is reported as a natural product. Compound 5, which showed antagonist activity against the specialized pathogenic fungus Escovopsis sp., was also produced by Streptomyces chartreusis ICBG323, isolated from the exoskeleton of winged male of Mycocepurus goeldii. The absolute configurations of 3 and 4 were confirmed by the combination of vibrational circular dichroism (VCD) spectroscopy and density functional theory (DFT) calculations. These results clearly demostrate the power of VCD to tell apart epimeric natural products. Compounds 1, 3 and 4 were produced by geographically distant but phylogenetically close strains, S. chartreusis ICBG 377 isolated in Brazil, and S. chartreusis NA02069, a marine sediment strain isolated in China.

Endophytic actinobacteria from the Brazilian medicinal plant Lychnophora ericoides were isolated for the first time, and the biological potential of their secondary metabolites was evaluated. A phylogenic analysis of isolated actinobacteria was accomplished with 16S rRNA gene sequencing, and the predominance of the genus Streptomyces was observed. All strains were cultured on solid rice medium, and ethanol extracts were evaluated with antimicrobial and cytotoxic assays against cancer cell lines. As a result, 92% of the extracts showed a high or moderate activity against at least one pathogenic microbial strain or cancer cell line. Based on the biological and chemical analyses of crude extracts, three endophytic strains were selected for further investigation of their chemical profiles. Sixteen compounds were isolated, and 3-hydroxy-4-methoxybenzamide (9) and 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone (15) are reported as natural products for the first time in this study. The biological activity of the pure compounds was also assessed. Compound 15 displayed potent cytotoxic activity against all four tested cancer cell lines. Nocardamine (2) was only moderately active against two cancer cell lines but showed strong activity against Trypanosoma cruzi. Our results show that endophytic actinobacteria from L. ericoides are a promising source of bioactive compounds.

Antimicrobial resistance is a global health crisis and few novel antimicrobials have been discovered in recent decades. Natural products, particularly from Streptomyces, are the source of most antimicrobials, yet discovery campaigns focusing on Streptomyces from the soil largely rediscover known compounds. Investigation of understudied and symbiotic sources has seen some success, yet no studies have systematically explored microbiomes for antimicrobials. Here we assess the distinct evolutionary lineages of Streptomyces from insect microbiomes as a source of new antimicrobials through large-scale isolations, bioactivity assays, genomics, metabolomics, and in vivo infection models. Insect-associated Streptomyces inhibit antimicrobial-resistant pathogens more than soil Streptomyces. Genomics and metabolomics reveal their diverse biosynthetic capabilities. Further, we describe cyphomycin, a new molecule active against multidrug resistant fungal pathogens. The evolutionary trajectories of Streptomyces from the insect microbiome influence their biosynthetic potential and ability to inhibit resistant pathogens, supporting the promise of this source in augmenting future antimicrobial discovery., Host microbiomes are feasible sources for drug discovery. Here, using large-scale isolations, bioactivity assays and omics, the authors uncover the antimicrobial potential of insect-associated Streptomyces and identify a compound, cyphomycin, active against multidrug-resistant fungal pathogens.

O presente trabalho teve como objetivo testar um protocolo de avaliação rápida (PAR) para avaliação da microbacia do Córrego Sussuapara, um córrego urbano em Palmas, Tocantins. A integridade de ecossistemas aquáticos em áreas urbanas é ameaçada pelas atividades humanas em sua bacia. Assim, PAR podem ser ferramentas auxiliares ao monitoramento microbiológico e físico-químico das águas. O protocolo elaborado teve como atributos os parâmetros substrato de fundo, complexidade do habitat, qualidade dos remansos, velocidade da corrente, proteção e estabilidade dos barrancos, cobertura vegetal das margens, extensão da mata ciliar e ação antrópica, para os quais foram atribuídos uma entre quatro qualificações conforme a percepção das pessoas envolvidas. Dois grupos de aplicadores foram selecionados, estudantes de graduação do curso de Engenharia Ambiental da Universidade Federal do Tocantins e professores do ensino fundamental do município, com diferentes formações em ensino superior. O resultado foi comparado com o monitoramento físico-quimico e microbiológico. O protocolo aplicado mostrou-se de fácil entendimento e rápida aplicação por ambos os grupos, mostrando resultados similares da avaliação da integridade ambiental de um trecho do Córrego Sussuapara, situado em área urbana de Palmas. A avaliação feita pelos dois grupos refletiu os impactos resultantes das falhas da drenagem urbana de Palmas sobre o córrego, refletido na baixa qualidade do substrato de fundo, na qualidade da proteção e estabilidade dos barrancos e no grau de atividade antrópica, atributos com classificação razoável e ruim pelos atores envolvidos.

The known antibiotic and cytotoxic compounds griseorhodin A (1) and griseorhodin C (2) were produced in solid culture by Streptomyces puniceus AB10, which was isolated from the leaf-cutter ant Acromyrmex rugosus rugosus. Their absolute configurations were unambiguously established as 6S,6aR,7S,8S and 6R,6aR,7S,8R, respectively, using vibrational circular dichroism (VCD) and density functional theory (DFT) calculations.

A novel yeast species was recovered from the fungus garden of the leaf-cutting ant Acromyrmex balzani (Hymenoptera: Formicidae). The growth of the novel yeast species is limited by its ability to metabolize only a few carbon and nitrogenous compounds. A remarkable characteristic of this strain is the vigorous growth in 1 % acetic acid. Sequence analysis of the D1/D2 domains of the LSU rRNA gene showed that the novel species belongs to the Starmerella clade and is phenotypically and genetically divergent from currently recognized species in this clade. Described here as Starmerella aceti f.a., sp. nov., it differs by 37 nucleotide substitutions in the D1/D2 region from Starmerella jinningensis CBS 11864T, the most closely related species. The type strain of Starmerella aceti sp. nov. is TO 125T ( = CBMAI 1594T = CBS 13086T).

The known antibiotic and cytotoxic compounds griseorhodin A ( 1 ) and griseorhodin C ( 2 ) were produced in solid culture by Streptomyces puniceus AB10, which was isolated from the leaf-cutter ant Acromyrmex rugosus rugosus . Their absolute configurations were unambiguously established as 6 S ,6a S ,7 S ,8 S and 6 R ,6a S ,7 S ,8 R , respectively, using vibrational circular dichroism (VCD) and density functional theory (DFT) calculations.

Two novel yeast species, Wickerhamomyces queroliae sp. nov. and Candida jalapaonensis sp. nov., were isolated, respectively, from larvae of Anastrepha mucronata (Diptera: Tephritidae) collected from ripe fruit of Peritassa campestris ('Bacupari', Hippocrateaceae) and from flowers of Centropogon cornutus (Campanulaceae) in the Cerrado ecosystem of the state of Tocantins, Brazil. Analysis of the D1/D2 large-subunit rRNA gene sequences placed W. queroliae in the Wickerhamomyces clade near Wickerhamomyces ciferri and Candida silvicultrix. Candida jalapaonensis belongs to the Wickerhamiella clade and is related to Candida drosophilae. The type strain of Wickerhamomyces queroliae is UFMG-05-T200.1(T) (=CBS 10936(T)=NRRL Y-48478(T)) and the type strain of Candida jalapaonensis is UFMG-03-T210(T) (=CBS 10935(T)=NRRL Y-48477(T)).