The Entomopathogenic Fungus Beauveria bassiana Shows Its Toxic Side within Insects: Expression of Genes Encoding Secondary Metabolites during Pathogenesis

Entomopathogenic fungi are extensively used for the control of insect pests worldwide. Among them, Beauveria bassiana (Ascomycota: Hypocreales) produce a plethora of toxic secondary metabolites that either facilitate fungal invasion or act as immunosuppressive compounds. These toxins have different chemical natures, such as nonribosomal peptides and polyketides. Even though their precise role is poorly understood, they are usually linked to virulence. These fungal secondary metabolites are produced by the expression of gene clusters encoding the various proteins needed for their biosynthesis. Each cluster includes synthetases for nonribosomal peptides (NRPS), polyketides (PKS), or hybrid NRPS–PKS genes. The aim of this review is to summarize the information available from transcriptomics and quantitative PCR studies related to the expression of B. bassiana NRPS and PKS genes inside different insects as the infection progresses; as for the host immune response, to help understand the mechanisms that these toxins trigger as virulence factors, antimicrobials, or immunosuppressives within the context of a fungus–insect interaction.Fil: Pedrini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin

Natural occurrence of the entomopathogenic genus Pandora on spittlebug pests of crops and pastures in Argentina

The natural occurrence of entomophathogenic fungi infecting spittlebugs (Hemiptera: Cercopidae) considered serious pests to pasture grasses and crops in Argentina was investigated during summer˗autumn (December to May) from 2013 to 2016. Adults and nymphs of spittlebugs were collected from Sorghum halepense and Setaria parviflora var. parviflora in San Miguel de Tucumán, Tucumán province. The entomopathogenic fungal species were characterized on the basis of morphological keys and molecular techniques. Microscopic characters were described from material mounted in lactophenol/aceto-orcein (1% w/v), and the amplification of the fungal SSU rDNA was carried out using the universal primers nu-SSU-0021-5′ and nu-SSU-1780-3′. Summarized information about occurrence of fungal infections on spittlebugs populations is provided. This study reports for the first time the occurrence of the genus Pandora infecting adults of the economically important spittlebugs Deois (Deois) mourei, D. (D.) knoblauchii, Isozulia christenseni christenseni and Notozulia entreriana from Argentina expanding the host range and geographical distribution of entomophthoralean fungi.Facultad de Ciencias Naturales y Muse

CYP52X1, representing new cytochrome P450 subfamily, displays fatty acid hydroxylase activity and contributes to virulence and growth on insect cuticular substrates in entomopathogenic fungus Beauveria bassiana

Infection of insects by the entomopathogenic fungus Beauveria bassiana proceeds via attachment and penetration of the host cuticle. The outermost epicuticular layer or waxy layer of the insect represents a structure rich in lipids including abundant amounts of hydrocarbons and fatty acids. A member of a novel cytochrome P450 subfamily, CYP52X1, implicated in fatty acid assimilation by B. bassiana was characterized. B. bassiana targeted gene knockouts lacking Bbcyp52x1 displayed reduced virulence when topically applied to Galleria mellonella, but no reduction in virulence was noted when the insect cuticle was bypassed using an intrahemoceol injection assay. No significant growth defects were noted in the mutant as compared with the wild-type parent on any lipids substrates tested including alkanes and fatty acids. Insect epicuticle germination assays, however, showed reduced germination of ΔBbcyp52x1 conidia on grasshopper wings as compared with the wild-type parent. Complementation of the gene-knock with the full-length gene restored virulence and insect epicuticle germination to wild-type levels. Heterologous expression of CYP52X1 in yeast was used to characterize the substrate specificity of the enzyme. CYP52X1 displayed the highest activity against midrange fatty acids (C12:0 and C14:0) and epoxy stearic acid, 4–8-fold lower activity against C16:0, C18:1, and C18:2, and little to no activity against C9:0 and C18:0. Analyses of the products of the C12:0 and C18:1 reactions confirmed NADPH-dependent regioselective addition of a terminal hydroxyl to the substrates (ω-hydroxylase). These data implicate CYP52X1 as contributing to the penetration of the host cuticle via facilitating the assimilation of insect epicuticle lipids.Fil: Zhang, Shizhu. Nanjing Normal University; China. University of Florida; Estados UnidosFil: Widemann, Emilie. Université de Strasbourg; FranciaFil: Bernard, Grausem. Université de Strasbourg; FranciaFil: Lesot, Agnes. Université de Strasbourg; FranciaFil: Pinot, Franck. Université de Strasbourg; FranciaFil: Pedrini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Keyhani, Nemat O.. University of Florida; Estados Unido

Tracing the coevolution between <i>Triatoma infestans</i> and its fungal pathogen <i>Beauveria bassiana</i>

The chemical control of Triatoma infestans, the major Chagas disease vector in southern South America, has been threatened in the last years by the emergence of pyrethroid-resistant bug populations. As an alternative approach, the efficacy of the entomopathogenic fungus Beauveria bassiana to control T. infestans populations (regardless their pyrethroid susceptibility) has been demonstrated. Growing research efforts on the interaction between T. infestans and B. bassiana by molecular, ecological, biochemical and behavioral traits has allowed framing such interaction as an evolutionary arms race. This review will focus on the relationships established in this particular host-pathogen system, compiling available data on the relevance of fungal pathogenesis, insect behavior, population dynamics and human intervention to favor fungal dissemination in bug populations. The current snapshot shows the fungus ahead in the evolutionary arms race and predicts a promissory landscape for the biological control of Chagas disease vectors.Instituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias Exacta

Tracing the coevolution between Triatoma infestans and its fungal pathogen Beauveria bassiana

The chemical control of Triatoma infestans, the major Chagas disease vector in southern South America, has been threatened in the last years by the emergence of pyrethroid-resistant bug populations. As an alternative approach, the efficacy of the entomopathogenic fungus Beauveria bassiana to control T. infestans populations (regardless their pyrethroid susceptibility) has been demonstrated. Growing research efforts on the interaction between T. infestans and B. bassiana by molecular, ecological, biochemical and behavioral traits has allowed framing such interaction as an evolutionary arms race. This review will focus on the relationships established in this particular host-pathogen system, compiling available data on the relevance of fungal pathogenesis, insect behavior, population dynamics and human intervention to favor fungal dissemination in bug populations. The current snapshot shows the fungus ahead in the evolutionary arms race and predicts a promissory landscape for the biological control of Chagas disease vectors.Fil: Mannino, Maria Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Juarez, Marta Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Pedrini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin

Cytochrome P450 Genes of the CYP4 Clan and Pyrethroid Resistance in Chagas Disease Vectors

Triatomine insects are vectors of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. Although residual pyrethroid spraying has been a successful vector control strategy for many years, a growing number of pyrethroid-resistance foci is being documented, mainly in Triatoma infestans, that led to failures in vector elimination. Insecticide resistance is a multifactorial phenomenon that often implies a combination of three different mechanisms: increased insecticide detoxification, reduced affinity of the site of action, and reduced insecticide penetration through the cuticle. All three mechanisms were reported in pyrethroid-resistant T. infestans. Cytochrome P450s are enzymes involved in the metabolism of xenobiotics and endogenous chemicals. They are encoded by CYP genes and classified into different families and clans. In triatomines, the CYP4 clan is divided in two families, CYP3093 and CYP4, and both exhibit genome-wide, triatomine-specific gene expansions. Some members from each family have been reported to be involved in two of the mechanisms mentioned above, i.e., they participate in insecticide detoxification in different organs and tissues, and in the synthesis of cuticular hydrocarbons, which ultimately can contribute to a reduced insecticide penetration. The aim of this manuscript is to review the current state of knowledge of P450 genes belonging to the CYP4 clan in triatomines and to highlight their potential role in insecticide resistance.Fil: Dulbecco, Andrea Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Pedrini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Calderón Fernández, Gustavo Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin

Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana

The insect epicuticle or waxy layer comprises a heterogeneous mixture of lipids that include abundant levels of long-chain alkanes, alkenes, wax esters and fatty acids. This structure represents the first barrier against microbial attack and for broad-host-range insect pathogens, such as Beauveria bassiana, it is the initial interface mediating the host-pathogen interaction, since these organisms do not require any specialized mode of entry and infect target hosts via the cuticle. B. bassiana is able to grow on straight chain alkanes up to n-C33 as a sole source of carbon and energy. The cDNA and genomic sequences, including putative regulatory elements, for eight cytochrome P450 enzymes, postulated to be involved in alkane and insect epicuticle degradation, were isolated and characterized. Expression studies using a range of alkanes as well as an insect-derived epicuticular extract from the blood-sucking bug Triatomas infestans revealed a differential expression pattern for the P450 genes examined, and suggest that B. bassiana contains a series of hydrocarbon-assimilating enzymes with overlapping specificity in order to target the surface lipids of insect hosts. Phylogenetic analysis of the translated ORFs of the sequences revealed that the enzyme which displayed the highest levels of induction on both alkanes and the insect epicuticular extract represents the founding member of a new cytochrome P450 family, with three of the other sequences assigned as the first members of new P450 subfamilies. The remaining four proteins clustered with known P450 families whose members include alkane monooxygenases.Facultad de Ciencias Exacta

Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana

The insect epicuticle or waxy layer comprises a heterogeneous mixture of lipids that include abundant levels of long-chain alkanes, alkenes, wax esters and fatty acids. This structure represents the first barrier against microbial attack and for broad-host-range insect pathogens, such as Beauveria bassiana, it is the initial interface mediating the host-pathogen interaction, since these organisms do not require any specialized mode of entry and infect target hosts via the cuticle. B. bassiana is able to grow on straight chain alkanes up to n-C33 as a sole source of carbon and energy. The cDNA and genomic sequences, including putative regulatory elements, for eight cytochrome P450 enzymes, postulated to be involved in alkane and insect epicuticle degradation, were isolated and characterized. Expression studies using a range of alkanes as well as an insect-derived epicuticular extract from the blood-sucking bug Triatomas infestans revealed a differential expression pattern for the P450 genes examined, and suggest that B. bassiana contains a series of hydrocarbon-assimilating enzymes with overlapping specificity in order to target the surface lipids of insect hosts. Phylogenetic analysis of the translated ORFs of the sequences revealed that the enzyme which displayed the highest levels of induction on both alkanes and the insect epicuticular extract represents the founding member of a new cytochrome P450 family, with three of the other sequences assigned as the first members of new P450 subfamilies. The remaining four proteins clustered with known P450 families whose members include alkane monooxygenases.Facultad de Ciencias Exacta

Alkane-grown Beauveria bassiana produce mycelial pellets displaying peroxisome proliferation, oxidative stress, and cell surface alterations

The entomopathogenic fungus Beauveria bassiana is able to grow on insect cuticle18 hydrocarbons as the sole carbon source, inducing several enzymes involved in alkane19 assimilation and concomitantly increasing virulence against insect hosts. In this study, we20 describe some physiological and molecular processes implicated in growth, nutritional21 stress response, and cellular alterations found in alkane-grown fungi. The fungal cytology22 was investigated using light and transmission electron microscopy (TEM) while the surface23 topography was examined using atomic force microscopy (AFM). Fungal hydrophobicity24 was also measured on the cell surface. Additionally, the expression pattern of several genes25 associated with oxidative stress, peroxisome biogenesis, and hydrophobicity were analysed26 by qPCR. We found a novel type of growth in alkane-cultured B. bassiana similar to27 mycelial pellets described in other alkane-free fungi, which were able to germinate and28 produce viable conidia in media without a carbon source and to be pathogenic against29 larvae of the beetles Tenebrio molitor and Tribolium castaneum. Optical microscopy and30 TEM showed that pellets were formed by hyphae cumulates with high peroxidase activity,31 exhibiting peroxisome proliferation and an apparent surface thickening. Alkane-grown32 conidia appeared to be more hydrophobic and cell surfaces displayed different topography33 than glucose-grown cells, as it was observed by AFM. We also found a significant34 induction in several genes encoding for peroxins, catalases, superoxide dismutases, and35 hydrophobins. These results show that both morphological and metabolic changes are36 triggered in mycelial pellets derived from alkane-grown B. bassiana.Fil: Huarte Bonnet, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Santos Da Paixao, Flavia Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Ponce, Juan C. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Santana, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Prieto, Eduardo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Pedrini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; Argentin

The limpet transcription factors of Triatoma infestans regulate the response to fungal infection and modulate the expression pattern of defensin genes

As part of the innate humoral response to microbial attack, insects activate the expression of antimicrobial peptides (AMP). Understanding the regulatory mechanisms of this response in the Chagas disease vector Triatoma infestans is important since biological control strategies against pyrethroid-resistant insect populations were recently addressed by using the entomopathogenic fungus Beauveria bassiana. By bioinformatics, gene expression, and silencing techniques in T. infestans nymphs, we achieved sequence and functional characterization of two variants of the limpet transcription factor (Tilimpet) and studied their role as regulators of the AMP expression, particularly defensins, in fungus-infected insects. We found that Tilimpet variants may act differentially since they have divergent sequences and different relative expression ratios, suggesting that Tilimpet-2 could be the main regulator of the higher expressed defensins and Tilimpet-1 might play a complementary or more general role. Also, the six defensins (Tidef-1 to Tidef-6) exhibited different expression levels in fungus-infected nymphs, consistent with their phylogenetic clustering. This study aims to contribute to a better understanding of T. infestans immune response in which limpet is involved, after challenge by B. bassiana infection.Fil: Mannino, Maria Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Santos Da Paixao, Flavia Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Pedrini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin