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1.
PLoS Pathog ; 16(9): e1008738, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946515

RESUMO

Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidian species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 µm⋅s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.


Assuntos
Microscopia/métodos , Microsporídios/patogenicidade , Organelas/imunologia , Organelas/ultraestrutura , Esporos Fúngicos/imunologia , Esporos Fúngicos/ultraestrutura , Proteínas Fúngicas/metabolismo , Microsporídios/imunologia , Microsporídios/ultraestrutura , Esporos Fúngicos/crescimento & desenvolvimento
2.
Pestic Biochem Physiol ; 170: 104698, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980053

RESUMO

Essential oils and their main compounds, monoterpenoids, are considered as alternative control systems for phytopathogenic fungi, particularly those related to late diseases of fruits and vegetables, like anthracnose caused by Colletotrichum species. In this context, we studied the effect of twenty monoterpenoids on Colletotrichum fructicola and C. acutatum to elucidate their effectiveness and mechanisms of action. Thus, we analyzed mycelial growth and conidial inhibitory concentration, as well as the effect of selected monoterpenoids on membrane integrity and cell vitality, reactive oxygen species (ROS) accumulation, and mitochondrial membrane potential by flow cytometry. The results showed that oxygenated monoterpenoids (alcohols and aldehydes) exhibited higher antifungal activity than their corresponding hydrocarbons, esters, and cyclic counterparts. Indicating that OH- and O- radicals react with cellular components affecting fungal homeostasis. In this sense, selected monoterpenoids (citral, citronellol, geraniol, carvacrol, and thymol) inhibited conidial germination of C. acutatum in a dose-dependent manner. The inhibition of conidial germination is associated with a loss of membrane integrity, a decrease of cell metabolism, and a dose-dependent accumulation of ROS, which was non-directly associated with modifications on mitochondrial membrane potential. Membrane dysfunction and ROS accumulation may be responsible for the necrotic behavior induced by high monoterpenoids concentrations, and possible apoptotic response in sub dosages of these compounds.


Assuntos
Colletotrichum , Antifúngicos/farmacologia , Frutas , Monoterpenos/farmacologia , Esporos Fúngicos
3.
Nat Commun ; 11(1): 3897, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753587

RESUMO

Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development.


Assuntos
Quitina/análogos & derivados , Quitina/metabolismo , Fungos/crescimento & desenvolvimento , Fungos/metabolismo , Transdução de Sinais/fisiologia , Ascomicetos/crescimento & desenvolvimento , Basidiomycota/crescimento & desenvolvimento , Ecologia , Ácidos Graxos/metabolismo , Micorrizas/fisiologia , Rhizobium/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Simbiose/fisiologia
4.
PLoS One ; 15(8): e0233665, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804955

RESUMO

Oligomycins are macrolide antibiotics, produced by Streptomyces spp. that show antagonistic effects against several microorganisms such as bacteria, fungi, nematodes and the oomycete Plasmopara viticola. Conidiogenesis, germination of conidia and formation of appressoria are determining factors pertaining to pathogenicity and successful diseases cycles of filamentous fungal phytopathogens. The goal of this research was to evaluate the in vitro suppressive effects of two oligomycins, oligomycin B and F along with a commercial fungicide Nativo® 75WG on hyphal growth, conidiogenesis, conidial germination, and appressorial formation of the wheat blast fungus, Magnaporthe oryzae Triticum (MoT) pathotype. We also determined the efficacy of these two oligomycins and the fungicide product in vivo in suppressing wheat blast with a detached leaf assay. Both oligomycins suppressed the growth of MoT mycelium in a dose dependent manner. Between the two natural products, oligomycin F provided higher inhibition of MoT hyphal growth compared to oligomycin B with a minimum inhibitory concentration of 0.005 and 0.05 µg/disk, respectively. The application of the compounds completely halted conidial formation of the MoT mycelium in agar medium. Further bioassays showed that these compounds significantly inhibited MoT conidia germination and induced lysis. The compounds also caused abnormal germ tube formation and suppressed appressorial formation of germinated spores. Interestingly, the application of these macrolides significantly inhibited wheat blast on detached leaves of wheat. This is the first report on the inhibition of mycelial growth, conidiogenesis, germination of conidia, deleterious morphological changes in germinated conidia, and suppression of blast disease of wheat by oligomycins from Streptomyces spp. Further study is needed to unravel the precise mode of action of these natural compounds and consider them as biopesticides for controlling wheat blast.


Assuntos
Magnaporthe/efeitos dos fármacos , Magnaporthe/patogenicidade , Oligomicinas/farmacologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Triticum/microbiologia , Agentes de Controle Biológico/farmacologia , Grão Comestível/microbiologia , Microbiologia de Alimentos , Fungicidas Industriais/farmacologia , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Magnaporthe/crescimento & desenvolvimento , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento
5.
Int J Syst Evol Microbiol ; 70(8): 4798-4807, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32783804

RESUMO

Leptographium panxianense and L. puerense are proposed as new taxa based on sequence data and morphological characters. The phylogenetic analyses based on ITS2-partial LSU rDNA region, ß-tubulin and elongation factor 1-α genes showed that L. panxianense and L. puerense formed well-supported clades and were closely related to L. yunnanense, L. wushanense and L. conjunctum, and then nested within the L. lundbergii complex. The two species differ in their conidial size and shape. The conidia of L. panxianense are larger than those of L. puerense while the conidial shape of L. puerense is more ovovoid. The optimal growth temperature of both L. panxianense and L. puerense is at 20 °C, which is different from those of L. yunnanense, L. wushanense and L. conjunctum. Comparison of sequence data and morphological characters confirmed the placement of the two undescribed taxa in the genus of Leptographium.


Assuntos
Besouros/microbiologia , Ophiostomatales/classificação , Filogenia , Pinus , Animais , China , DNA Fúngico/genética , DNA Ribossômico/genética , Técnicas de Tipagem Micológica , Ophiostomatales/isolamento & purificação , Fator 1 de Elongação de Peptídeos/genética , Análise de Sequência de DNA , Esporos Fúngicos , Tubulina (Proteína)/genética
6.
Gene ; 762: 145042, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32777529

RESUMO

OBJECTIVES: Inhibitory effect of allicin with broad-spectrum antimicrobial activity on A. fumigatus and the regulation mechanism of inflammation and autophagy in vitro and in vivo. METHODS: The corresponding concentration of allicin was prepared according to the needs of the experiment. In vitro, 2 ml 5 × 104 of fungal spores suspension was added to the 6-well plate per hole, and different final concentrations of allicin (1 µl/ml, 2.5 µl/ml, 5 µl/ml, 10 µl/ml, 20 µl/ml, 30 µl/ml) were added. The fungal spores were stained by fluorescent dye SYTO 9 (green) every day, and the spore germination inhibition was detected by flow cytometry in different PH. RAW264.7 cells were cultured and stimulated by A. fumigatus spores for 3 h, then allicin solution was added. Then some cells were stained with ROS probe (green) and hochest33342 (blue). The effect of allicin on ROS was observed by fluorescence microscope. The other part of cells extracted protein from cell lysate and detected the effect of allicin on inflammatory factors and autophagy by Western-blotting. The green and red spots of RAW264.7 cells stably transfected with GFP-RFP-LC3 were observed by fluorescence microscopy. In vivo, A. fumigatus spore was injected intratracheally into mice, then allicin was injected intravenously at a concentration of 5 mg/kg/day for 7 consecutive days. The survival status, pulmonary fungal load and weight of mice was recorded continuously for 30 days and detected the changes of lung by pathological examination and immunohistochemistry. RESULTS: In vitro, allicin significantly inhibited the spore germination of A. fumigatus within 24 h in a dose-dependent manner and it had a stable inhibition on the spore germination of A. fumigatus in acidic environment. Cell experiments showed that allicin inhibited intracellular spore germination by inhibiting ROS production, inflammation and autophagy. In the animal experiment, the survival rate and body weight of allicin injection group were higher than that of non injection group, while the spore load of lung was lower than that of non injection group (P < 0.05). CONCLUSIONS: These results support that allicin reduces inflammation and autophagy resistance to A. fumigatus infection, It also provides a possible treatment for Aspergillus infectious diseases, i.e. early anti-inflammation, antibiotics or drugs that inhibit excessive autophagy.


Assuntos
Anti-Inflamatórios/uso terapêutico , Aspergilose/tratamento farmacológico , Autofagia , Depuradores de Radicais Livres/uso terapêutico , Ácidos Sulfínicos/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Aspergillus fumigatus/patogenicidade , Aspergillus fumigatus/fisiologia , Feminino , Depuradores de Radicais Livres/farmacologia , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Esporos Fúngicos/efeitos dos fármacos , Ácidos Sulfínicos/farmacologia
7.
Plant Dis ; 104(10): 2585-2597, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32813612

RESUMO

Quinoa black stem is a new disease that affects the stems of quinoa plants and is more likely to develop under cool conditions (15 to 25°C, RH = 55 ± 2%). The typical symptoms include the formation of black necrotic lesions on the stem, which can completely wrap around the stem, causing lodging and blanking (development of 'empty' and sterile grain on the panicle). Furthermore, the pycnidia form small round protrusions on the surface of the lesions. Phylogenetic analysis revealed that representative isolates LMHS-3 and LMHS-5 were closely related to Ascochyta caulina (teleomorph: Neocamarosporium calvescens). Comprehensive morphological and molecular characterizations confirmed A. caulina as the pathogen that caused quinoa black stem. A. caulina mainly infected quinoa stems and could produce many pycnidia, but it rarely infected quinoa leaves. Pathogenicity testing showed that the most suitable temperature for the onset of quinoa black stem was from 15 to 25°C. When the temperature was increased above 30°C, the conidial germination of A. caulina became malformed, and when the temperature was decreased below 5°C, mycelium growth of A. caulina became extremely slow; thus, both extreme high and low temperatures affected the pathogenicity of A. caulina. Mancozeb and azoxystrobin fungicides were revealed to have had the strongest inhibitory effects on the conidial germination of A. caulina, and in some cases caused malformations in conidial germination. Tebuconazole and difenoconazole had the strongest inhibitory effects on A. caulina mycelial growth and less on the effects on the conidial germination. The results of the present study provide a basis for the recognition and management of quinoa black stem.


Assuntos
Chenopodium quinoa , Fungicidas Industriais/farmacologia , China , Filogenia , Doenças das Plantas , Esporos Fúngicos/efeitos dos fármacos , Virulência
8.
Environ Monit Assess ; 192(8): 537, 2020 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-32696086

RESUMO

A thunderstorm is a risk factor for severe respiratory allergy or asthma attacks in patients suffering from pollen/spore allergy. This study aimed to investigate the changes in the spectrum and quantity of pollen and fungal spores in the air of Bratislava during summer storms as well as the impact of selected environmental parameters on these changes. Pollen/spore samples were collected using a Burkard volumetric aerospore trap during summer 2016. To identify those types of pollen/spores that may harm human health during the storm episodes, we analysed how the concentration of individual bioparticles in the air changed during pre-storm/storm/post-storm periods. The effect of environmental variables on the concentration of selected pollen/spore types was evaluated through Spearman's correlation analysis. The results of our study suggest that thunderstorm-related respiratory allergy symptoms in the study area may be caused by (1) spores of Myxomycetes, the airborne concentration of which increases due to an increase in wind speed during the pre-storm period; (2) ruptured pollen and Diatripaceae spores, the concentration of which increases due to increase in precipitation and relative air humidity, respectively, during the storm period; and (3) spores of Fusarium and Leptosphaeria, the concentration of which increases due to increase in precipitation and air temperature, respectively, during the post-storm period.


Assuntos
Microbiologia do Ar , Poluentes Atmosféricos/análise , Alérgenos/análise , Monitoramento Ambiental , Europa (Continente) , Humanos , Estações do Ano , Esporos Fúngicos
9.
PLoS One ; 15(7): e0235855, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658904

RESUMO

This paper represents the first article in a series on Yunnanese microfungi. We herein provide insights into Magnolia species associated with microfungi. All presented data are reported from the Kunming Botanical Gardens. Final conclusions were derived from the morphological examination of specimens coupled with phylogenetic sequence data to better integrate taxa into appropriate taxonomic ranks and infer their relationships. Shearia formosa, the type species of Shearia, lacks type material, and its phylogenetic position accordingly remains unresolved. A fresh collection of Shearia formosa, obtained from Magnolia denudata and M. soulangeana in China, therefore, designated a neotype for stabilizing the application of the species and/or genus name. Phylogenetic analyses of a combined DNA data matrix containing SSU, LSU, RPB2 and TEF loci of representative Pleosporales revealed that the genera Crassiperidium, Longiostiolum and Shearia are a well-defined monophylum. It is recognized as the family Longiostiolaceae and strongly supported by Bayesian and Maximum Likelihood methods. Its members are characterized by immersed to semi-immersed, globose to subglobose ascomata with a central, periphysate ostiole, a peridium composed of rectangular to polygonal cells, cylindrical to clavate asci, broadly fusiform, hyaline to pale brown ascospores, a coelomycetous asexual morph with pycnidial conidiomata, enteroblastic, annellidic, ampulliform, doliiform or cylindrical conidiogenous cells and cylindrical to fusiform, transverse and sometimes laterally distoseptate conidia without a sheath or with a basal lateral sheath. Nigrograna magnoliae sp. nov. is introduced from Magnolia denudata with both asexual and sexual morphs. We observed the asexual morph of Brunneofusispora sinensis from the culture and therefore amended the generic and species descriptions of Brunneofusispora.


Assuntos
Fungos/classificação , Magnolia/microbiologia , Filogenia , Código de Barras de DNA Taxonômico , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/citologia , Fungos/genética , Fungos/patogenicidade , Esporos Fúngicos/citologia
10.
Water Res ; 185: 116218, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32726715

RESUMO

Fungal contamination of drinking water sources is increasingly threatening the environment and human health. In this study, the inactivation of three genera of dominant fungi in drinking water sources using ozone was first reported. The inactivation of the fungal spores by ozone could be divided into two distinct stages: first a rapid reduction in survival, and then the inactivation at a slower rate. The secondary stage inactivation fitted the Chick-Watson model well, and there was no significant difference in the second-order inactivation rate constants of the three fungal spores (0.199-0.209 L mg-1 min-1). The inactivation rate constants of fungal spores by molecular ozone were much lower than those of viruses, which were equivalent to that of Cryptosporidium. The increase in pH and temperature showed a positive effect on the inactivation rate. Damage to cell membranes, leakage of intracellular compounds, and changes of reactive oxygen species and esterase activity in the spores were detected after inactivation. The results indicated that ozone inactivated fungal spores by firstly destroying cell walls and membranes and then causing the release of intracellular compounds. The fungicidal efficiency of ozone was superior to those of chlorine and chlorine dioxide. In addition, the inactivation efficiency of ozone on fungal spores in real water matrices was reduced to 50.7-91.2% of the efficiency in phosphate buffer. In conclusion, ozone showed high efficiency in the inactivation of fungal spores and could be used as an alternative disinfectant for fungal contamination in drinking water sources.


Assuntos
Criptosporidiose , Cryptosporidium , Ozônio , Purificação da Água , Animais , Desinfecção , Cinética , Esporos Fúngicos , Água
11.
Int J Food Microbiol ; 330: 108783, 2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-32659523

RESUMO

The contamination of Aspergillus carbonarius causes decreases and great decay of agricultural products, and threatens the human and animal health by producing mycotoxins, especially ochratoxin A. Bacillus subtilis has been proved to efficiently inhibit the growth of A. carbonarius. Revealing the major active compound and the mechanisms for the antifungal of B. subtilis are essential to enhance its antifungal activity and control the quality of antifungal products made of it. In this study, we determined that iturin A is the major compound that inhibits Aspergillus carbonarius, a widespread fungal pathogen of grape and other fruits. Iturin A significantly inhibited growth and ochratoxin A production of A. carbonarius with minimal inhibitory concentrations (MICs) of 10 µg/mL and 0.312 µg/mL, respectively. Morphological observations revealed that iturin A caused swelling of the fungal cells and thinning of the cell wall and membrane at 1/2 MIC, whereas it inhibited fungal spore germination and caused mitochondrial swelling at higher concentrations. A differential transcriptomic analysis indicated that the mechanisms used by iturin A to inhibit A. carbonarius were to downregulate the expression of genes related to cell membrane, transport, osmotic pressure, oxidation-reduction processes, and energy metabolism. Among the down-regulated genes, those related to the transport capacity were most significantly influenced, including the increase of energy-related transport pathways and decrease of other pathways. Notably, the genes related to taurine and hypotaurine metabolism were also decreased, indicating iturin A potentially cause the occurrence of osmotic imbalance in A. carbonarius, which may be the intrinsic cause for the swelling of fungal cells and mitochondria. Overall, iturin A produced by B. subtilis played important roles to inhibit A. carbonarius via changing the fungal cell structure and causing perturbations to energy, transport and osmotic pressure metabolisms in fungi. The results indicated a new direction for researches on the mechanisms for lipopeptides and provided useful information to develop more efficient antifungal agents, which are important to agriculture and biomedicine.


Assuntos
Antifúngicos/farmacologia , Aspergillus/efeitos dos fármacos , Aspergillus/metabolismo , Bacillus subtilis/metabolismo , Peptídeos Cíclicos/farmacologia , Antifúngicos/metabolismo , Aspergillus/genética , Aspergillus/crescimento & desenvolvimento , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Micotoxinas/metabolismo , Pressão Osmótica/efeitos dos fármacos , Peptídeos Cíclicos/metabolismo , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Transcriptoma
12.
Gene ; 759: 145002, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32726608

RESUMO

Recent evidence has shown that microRNAs are transferred from one species to another through cross-species transmission and exhibit biological activities in the receptor. However, the cross-kingdom regulation of pathogen virulence by plant-derived miRNAs is rarely reported. This study investigated the regulatory role of novel tomato miRNA miR1001 in the growth and development of Botrytis cinerea. Results showed that miR1001 inhibited the virulence of B. cinerea-infected plants, and the inhibitory effect of miR1001/miR1001* was stronger than that of miR1001. Moreover, miR1001 exerted a significant inhibitory effect on the conidiospore germination of B. cinerea. Degradome-seq experiment showed that miR1001 can directly target the Bcin03g02170.1 and Bcin10g01400.1 genes, which respectively encode the ATP-dependent metallopeptidase and cysteine-type endopeptidase, in B. cinerea. The interactions of both targets with miR1001 were further confirmed by using transient co-expression in tobacco. Real-time RT-PCR analysis showed that the expression levels of the two target genes were significantly downregulated in B. cinerea with miR1001 treatment. Our findings provide new evidence into the coevolution of pathogens and host plants, as well as new directions for the use of plant-derived miRNAs to control pathogens.


Assuntos
Botrytis/patogenicidade , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno , Lycopersicon esculentum/genética , MicroRNAs/metabolismo , RNA de Plantas/metabolismo , Botrytis/fisiologia , Proteínas Fúngicas/metabolismo , Lycopersicon esculentum/microbiologia , MicroRNAs/genética , RNA de Plantas/genética , Esporos Fúngicos/fisiologia
13.
J Vis Exp ; (159)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32478725

RESUMO

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A polyethylene glycol (PEG)-mediated protoplast transformation method is generally used for the introduction of heterologous genes into A. oryzae. The conventional method typically requires three weeks for the screening of favorable transformants. Here, a new technique, the direct liquid-culture (DLC) screening method, is introduced which reduces the screening time to six days in a 200 mL flask format or to 10 days in a 24 well microplate format. The DLC screening method ensures the acquisition of positive transformants and evaluation of the secretory production of heterologous proteins in a single step, unlike the conventional screening method where two separate steps are required for the same. The protocol for PEG-mediated protoplast transformation of A. oryzae is described, which consists of five steps: preparation of fresh spore suspension, preculture, preparation of protoplasts, introduction of DNA, and DLC screening. For successful results in DLC screening, it is critical to use a nutrient-rich medium with optimized osmotic pressure. The protocol should further popularize the use of A. oryzae as a host of choice in the industrial production of proteins.


Assuntos
Aspergillus oryzae/genética , Bioquímica/métodos , Proteínas Fúngicas/biossíntese , Mutação/genética , Aspergillus oryzae/metabolismo , DNA/metabolismo , Polietilenoglicóis/química , Protoplastos/metabolismo , Esporos Fúngicos/metabolismo
14.
J Vis Exp ; (159)2020 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-32478760

RESUMO

Invasive aspergillosis (IA) is one of the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause >50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.


Assuntos
Aspergilose/imunologia , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Larva/microbiologia , Peixe-Zebra/microbiologia , Animais , Imunossupressores , Esporos Fúngicos/imunologia
15.
Int J Food Microbiol ; 328: 108687, 2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32474227

RESUMO

Penicilium griseofulvum, the causal agent of apple blue mold, is able to produce in vitro and on apple a broad spectrum of secondary metabolites (SM), including patulin, roquefortine C and griseofulvin. Among them, griseofulvin is known for its antifungal and antiproliferative activity, and has received interest in many sectors, from medicine to agriculture. The biosynthesis of SM is finely regulated by filamentous fungi and can involve global regulators and pathway specific regulators, which are usually encoded by genes present in the same gene cluster as the backbone gene and tailoring enzymes. In the griseofulvin gene cluster, two putative transcription factors were previously identified, encoded by genes gsfR1 and gsfR2, and their role has been investigated in the present work. Analysis of P. griseofulvum knockout mutants lacking either gene suggest that gsfR2 forms part of a different pathway and gsfR1 exhibits many spectra of action, acting as regulator of griseofulvin and patulin biosynthesis and influencing conidia production and virulence on apple. The analysis of gsfR1 promoter revealed that the regulation of griseofulvin biosynthesis is also controlled by global regulators in response to many environmental stimuli, such as carbon and nitrogen. The influence of carbon and nitrogen on griseofulvin production was further investigated and verified, revealing a complex network of response and confirming the central role of gsfR1 in many processes in P. griseofulvum.


Assuntos
Griseofulvina/biossíntese , Patulina/biossíntese , Penicillium/metabolismo , Penicillium/patogenicidade , Esporos Fúngicos/crescimento & desenvolvimento , Carbono/metabolismo , Microbiologia de Alimentos , Griseofulvina/metabolismo , Malus/microbiologia , Família Multigênica , Nitrogênio/metabolismo , Patulina/metabolismo , Esporos Fúngicos/metabolismo , Fatores de Transcrição/genética , Virulência
16.
PLoS Negl Trop Dis ; 14(6): e0008386, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32542003

RESUMO

Chromoblastomycosis (CBM) is a chronic worldwide subcutaneous mycosis, caused by several dimorphic, pigmented dematiaceous fungi. It is difficult to treat patients with the disease, mainly because of its recalcitrant nature. The correct activation of host immune response is critical to avoid fungal persistence in the tissue and disease chronification. CD4+ T cells are crucial for the development of protective immunity to F. pedrosoi infection. Here, we investigated T helper cell response dynamics during experimental CBM. Following footpad injection with F. pedrosoi hyphae and conidia, T cells were skewed towards a Th17 and Th1 phenotype. The Th17 population was the main Th cell subset found in the infected area during the early stages of experimental murine CBM, followed by Th1 predominance in the later stages, coinciding with the remission phase of the disease in this experimental model. Depletion of CD25+ cells, which leads to a reduction of Treg cells in the draining lymph node, resulted in decline in fungal burden after 14 days of infection. However, fungal cells were not cleared in the later stages of the disease, prolonging CBM clinical features in those animals. IL-17A and IFN-γ neutralization hindered fungal cell elimination in the course of the disease. Similarly, in dectin-2 KO animals, Th17 contraction in the course of experimental CBM was accompanied by fungal burden decrease in the first 14 days of infection, although it did not affect disease resolution. In this study, we gained insight into T helper subsets' dynamics following footpad injections of F. pedrosoi propagules and uncovered their contribution to disease resolution. The Th17 population proved to be important in eliminating fungal cells in the early stages of infection. The Th1 population, in turn, closely assisted by Treg cells, proved to be relevant not only in the elimination of fungal cells at the beginning of infection but also essential for their complete elimination in later stages of the disease in a mouse experimental model of CBM.


Assuntos
Ascomicetos/imunologia , Cromoblastomicose/imunologia , Lectinas Tipo C/metabolismo , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Cromoblastomicose/microbiologia , Cromoblastomicose/patologia , Modelos Animais de Doenças , Humanos , Hifas , Interferon gama/metabolismo , Interleucina-17/metabolismo , Lectinas Tipo C/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esporos Fúngicos
17.
Mycorrhiza ; 30(5): 611-621, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32556837

RESUMO

The use of genetically modified (GM) plants has increased in recent decades, but there are uncertainties about their effects on soil microbial communities. Aiming to quantify root colonization and characterize arbuscular mycorrhizal fungi (AMF) communities associated with roots and rhizosphere soil of different maize genotypes, a field trial was carried out in Southern Brazil with three maize genotypes as follows: a GM hybrid (DKB 240 VTPRO), its non-modified isoline (DKB 240), and a landrace (Pixurum). Soil samples were collected to evaluate the occurrence of AMF during the growth of corn genotypes at sowing and V3 (vegetative), R1 (flowering), and R3 (grain formation) stages of the crop. The occurrence of AMF was determined by the morphological identification of spores, and by analyzing AMF community composition in soil and roots of maize, using PCR-DGGE. The GM genotype of maize promoted lower mycorrhizal colonization in the vegetative stage and had lower sporulation at grain development than the conventional hybrid and the landrace maize. Twenty AMF morphotypes were identified and 13 were associated with all maize genotypes. The genera Acaulospora, Glomus, and Dentiscutata had the largest numbers of species. There were no differences in AMF community composition due to maize genotypes or genetic modification, but crop phenological stages affected AMF communities associated with maize roots.


Assuntos
Micobioma , Micorrizas , Brasil , Raízes de Plantas , Microbiologia do Solo , Esporos Fúngicos , Zea mays
18.
Int J Syst Evol Microbiol ; 70(7): 4217-4223, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32589574

RESUMO

Five yeast strains were isolated from the gut of the groundbeetle Pterostichus gebleri and rotting wood, which were collected from two different localities in China. These strains were identified as representing two novel species of the genus Blastobotrys through comparison of sequences in the D1/D2 domains of the LSU rRNA gene and other taxonomic characteristics. Blastobotrys baotianmanensis sp. nov. produces two to three spherical ascospores per ascus, and is most closely related to the type strains of B. elegans, B. capitulata, B. arbuscula, and an undescribed species represented by strain BG02-7-20-006A-3-1. Blastobotrys baotianmanensis sp. nov. differed from these strains by 3.6-8.4 % divergence (21-46 substitutions and 0-4 gaps) in the D1/D2 sequences. Blastobotrys xishuangbannaensis f.a., sp. nov. is closely related to B. nivea, B. elegans and B. aristata but the formation of ascospores was not observed on various sporulation media, and it differed from its relatives by 6.2-8.5 % divergence (34-43 substitutions and 2-6 gaps) in the D1/D2 sequences. The holotype of Blastobotrys baotianmanensis sp. nov. is NYNU 1581 and the holotype of Blastobotrys xishuangbannaensis f.a., sp. nov. is NYNU 181030.


Assuntos
Besouros/microbiologia , Filogenia , Saccharomycetales/classificação , Madeira/microbiologia , Animais , China , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Técnicas de Tipagem Micológica , Saccharomycetales/isolamento & purificação , Análise de Sequência de DNA , Esporos Fúngicos
19.
Int J Food Microbiol ; 326: 108649, 2020 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-32402917

RESUMO

In this study we assessed the efficacy of chitosan (CHI) (2%) emulsion added with Ruta graveolens L. essential oil (REO) at different concentrations (0.5%, 1.0% and 1.5%) to control C. gloesporioides grows both "in situ" and "in vitro" in papaya Maradol (Carica papaya L.). In vitro studies showed a decrease on fungal growth (mycelia diameter) with the increase of REO concentration, while 0.5% of REO induce a reduction of 56.42%, REO at 1.0% and 1.5% induced a reduction of 97%. Microscopic analysis showed irreversible deleterious morphological and ultrastructural alterations as well as changes in conidia morphology, and conidia germination inhibition up to 90%. Among the most abundant REO constituents, 2-Nonanol showed strong antifungal activity followed by 2-Undecanone, Benzyl acetate, 2-Nonanone, 2-Tridecanone and 2-Dodecanone. Studies "in situ" on papaya fruit during 12 days at 20 °C, showed a reduction of the C. gloesporioides lesion expansion by 50% using CHI-REO 0.5% emulsions and by 100% with treatments of CHI-REO 1.0 and 1.5%, in addition the emulsions were efficacious to reduce the fruit surface microbiota. On the other hand, physicochemical analysis of the papaya fruits demonstrated that CHI-REO emulsions treatment delayed papaya ripening without affecting the organoleptic characteristics. All these results demonstrated for the first time the application of coatings CHI-REO as a postharvest treatment for the control of anthracnose on papaya fruit.


Assuntos
Antifúngicos/farmacologia , Carica/microbiologia , Quitosana/farmacologia , Colletotrichum/efeitos dos fármacos , Óleos Voláteis/farmacologia , Ruta/metabolismo , Emulsões , Frutas/microbiologia , Micélio/crescimento & desenvolvimento , Esporos Fúngicos/crescimento & desenvolvimento
20.
Laryngorhinootologie ; 99(5): 292-298, 2020 05.
Artigo em Alemão | MEDLINE | ID: mdl-32384555

RESUMO

Alternaria alternata spores are important allergens causing "hayfever". Outdoor fungal exposure is primarily associated with increased asthma symptoms and increased risk of asthma exacerbations. In our outpatient-clinic, we investigated patients who presented with symptoms of allergic conjunctivitis and respiratory symptoms in the summertime. We found a sensitisation to Alternaria alternata in about 6 % of the investigated patients. Especially in children and teenagers, the sensitisation is clinically important. The spores of Alternaria alternata are responsible for the development and persistence of nonspecific airway hyperresponsiveness and in a group of patients also for a difficult-to-treat asthma in the summer. We should always bear in mind that prolonged symptoms (after end of June) in grass pollen allergic patients might be caused by additional sensitisation to Alternaria alternata spores or mugwort pollen. In our patients, we were able to show a sensitisation to Alternaria alternata in grass pollen allergic patients rather frequently. In the summertime, high counts of Alternaria spores and pollen often appear simultaneously. Our findings show a considerable effect of Alternaria sensitisation on the asthma morbidity and exacerbations. In this article we refer to the necessity of keeping a patient diary of allergic symptoms and the importance of information from regional pollen traps about the counts of pollen and Alternaria spores. Only then we will be able to differentiate, which allergen is responsible for the severe "hay fever", especially in patients with severe asthma episodes. Before the start of a specific immunotherapy with Alternaria alternata it is recommended to perform a mucosal provocation test, to prove the clinical significance of the sensitisation. It has been shown that also in Germany, there are high counts of Alternaria spores in the summer. The results of the pollen and alternaria count in the year 2009, measured in the regional pollen trap (Treuenbrietzen), are shown in Abb. 1, 2, 3, 4, 5. We have also found broken Alternaria spores. They are associated with increased asthma exacerbations and appear often during thunderstorms and harvesting. An allergen-specific immunotherapy with Alternaria alternata is a safe treatment and it is effective in improving respiratory function. The safety and efficacy is comparable to the one found in the allergen-specific immunotherapy with pollen.


Assuntos
Asma , Rinite Alérgica Sazonal , Adolescente , Alérgenos , Asma/terapia , Criança , Alemanha , Humanos , Esporos Fúngicos/imunologia
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