RESUMO
Fungal plant pathogens are responsible for serious losses in many economically important crop species worldwide. Due to the use of fungicides and the fungi genome plasticity, multi-drug resistant strains are emerging as a new generation of pathogens, causing an expansive range of superficial and systemic plant infections, or new opportunistic fungal pathogens for humans. The group of antagonistic fungi Trichoderma spp. has been widely used to enhance plant growth and for the control of different pathogens affecting crops. Although Neurospora crassa is not a mycoparasitic fungus, its secretion of secondary metabolites with antimicrobial activity has been described. In this work, the effect of crude extract of the monoculture of Trichoderma asperellum T8a or the co-culture with N. crassa as an inhibitory treatment against the fungal pathogens Botrytis cinerea and Fusarium solani was evaluated. The findings demonstrate that the secondary metabolites contained in the T. asperellum crude extract have a clear fungistatic activity against B. cinerea and F. solani. Interestingly, this fungistatic activity highly increases when T. asperellum is co-cultivated with the non-pathogenic fungus N. crassa. Moreover, the co-culture crude extract also showed antifungal activity on post-harvest fruits, and no toxic effects on Murine fibroblast L929 (CCL-1) and murine macrophages RAW 264.7 (TIB-71) were observed. All these results together are solid evidence of the potential of the co-culture crude extract of T. asperellum and N. crassa, as an antifungal agent against phytopathogenic fungi, or post-harvest fruits during the transportation or commercialization time.
Assuntos
Botrytis , Técnicas de Cocultura , Frutas , Fusarium , Trichoderma , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Frutas/microbiologia , Frutas/química , Botrytis/efeitos dos fármacos , Botrytis/crescimento & desenvolvimento , Trichoderma/metabolismo , Trichoderma/genética , Animais , Camundongos , Antifúngicos/farmacologia , Antifúngicos/metabolismo , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/metabolismo , Células RAW 264.7 , Misturas Complexas/farmacologia , Misturas Complexas/químicaRESUMO
In Neurospora crassa, caffeine and other methylxanthines are known to inhibit phosphodiesterase (PDE) activity, leading to augmented cAMP levels. In this organism, it has also been shown that the addition of these drugs significantly lengthens the circadian period, as seen by conidiation rhythms. Utilizing in vivo bioluminescence reporters, pharmacological inhibitors, and cAMP analogs, we revisited the effect of methylxanthines and the role of cAMP signaling in the Neurospora clockworks. We observed that caffeine, like all tested methylxanthines, led to significant period lengthening, visualized with both core-clock transcriptional and translational reporters. Remarkably, this phenotype is still observed when phosphodiesterase (PDE) activity is genetically or chemically (via 3-isobutyl-1-methylxanthine) abrogated. Likewise, methylxanthines still exert a period effect in several cAMP signaling pathway mutants, including adenylate cyclase (cr-1) and protein kinase A (PKA) (Δpkac-1) mutants, suggesting that these drugs lead to circadian phenotypes through mechanisms different from the canonical PDE-cAMP-PKA signaling axis. Thus, this study highlights the strong impact of methylxanthines on circadian period in Neurospora, albeit the exact mechanisms somehow remain elusive. IMPORTANCE Evidence from diverse organisms show that caffeine causes changes in the circadian clock, causing period lengthening. The fungus Neurospora crassa is no exception; here, several methylxanthines such as caffeine, theophylline, and aminophylline cause period lengthening in a concentration-dependent manner. Although methylxanthines are expected to inhibit phosphodiesterase activity, we were able to show by genetic and pharmacological means that these drugs exert their effects through a different mechanism. Moreover, our results indicate that increases in cAMP levels and changes in PKA activity do not impact the circadian period and therefore are not part of underlying effects of methylxanthine. These results set the stage for future analyses dissecting the molecular mechanisms by which these drugs dramatically modify the circadian period.
Assuntos
Cafeína , Neurospora crassa , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/fisiologia , Ritmo Circadiano/efeitos dos fármacos , AMP Cíclico/metabolismo , Cafeína/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/antagonistas & inibidores , 1-Metil-3-Isobutilxantina , Proteínas Quinases/metabolismo , Transdução de SinaisRESUMO
In the negative feedback loop composing the Neurospora circadian clock, the core element, FREQUENCY (FRQ), binds with FRQ-interacting RNA helicase (FRH) and casein kinase 1 to form the FRQ-FRH complex (FFC) which represses its own expression by interacting with and promoting phosphorylation of its transcriptional activators White Collar-1 (WC-1) and WC-2 (together forming the White Collar complex, WCC). Physical interaction between FFC and WCC is a prerequisite for the repressive phosphorylations, and although the motif on WCC needed for this interaction is known, the reciprocal recognition motif(s) on FRQ remains poorly defined. To address this, we assessed FFC-WCC in a series of frq segmental-deletion mutants, confirming that multiple dispersed regions on FRQ are necessary for its interaction with WCC. Biochemical analysis shows that interaction between FFC and WCC but not within FFC or WCC can be disrupted by high salt, suggesting that electrostatic forces drive the association of the two complexes. As a basic sequence on WC-1 was previously identified as a key motif for WCC-FFC assembly, our mutagenetic analysis targeted negatively charged residues of FRQ, leading to identification of three Asp/Glu clusters in FRQ that are indispensable for FFC-WCC formation. Surprisingly, in several frq Asp/Glu-to-Ala mutants that vastly diminish FFC-WCC interaction, the core clock still oscillates robustly with an essentially wildtype period, indicating that the interaction between the positive and negative elements in the feedback loop is required for the operation of the circadian clock but is not a determinant of the period length.
Assuntos
Relógios Circadianos , Proteínas Fúngicas , Neurospora crassa , Relógios Circadianos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/genética , Neurospora crassa/metabolismo , Fatores de Transcrição/metabolismo , Domínios Proteicos , Deleção de Genes , Cloreto de Sódio/farmacologia , Mutação , Expressão GênicaRESUMO
The emergence of antifungal resistance, especially to the most widely used azole class of ergosterol biosynthesis inhibitors, makes fungal infections difficult to treat in clinics and agriculture. When exposed to azoles, fungi can make adaptive responses to alleviate azole toxicity and produce azole tolerance. However, except for azole efflux pumps and ergosterol biosynthesis genes, the role of most azole responsive genes in azole resistance is unknown. In this study, STK-17, whose transcription is upregulated by azoles, was characterized as a novel kinase that is required for azole resistance. Deletion or dysfunction of STK-17 led to azole hypersensitivity in Neurospora crassa and to other ergosterol biosynthesis inhibitors such as amorolfine, terbinafine, and amphotericin B, but not fatty acid and ceramide biosynthesis inhibitors. STK-17 was also required for oxidative stress resistance, but this was not connected to azole resistance. RNA-seq results showed that stk-17 deletion affected the basal expression and the response to ketoconazole of some membrane protein genes, indicating functional association of STK-17 with the membrane. Notably, deletion of stk-17 affected the normal response to azoles of erg genes, including the azole target-encoding gene erg11, and erg2, erg6, and erg24, and led to abnormal accumulation of sterols in the presence of azoles. HPLC-MS/MS analysis revealed increased intracellular azole accumulation in the stk-17 mutant, possibly due to enhanced azole influx and reduced azole efflux that was independent of the major efflux pump CDR4. Importantly, STK-17 was widely distributed and functionally conserved among fungi, thus providing a potential antifungal target. IMPORTANCE Antifungal resistance is increasing worldwide, especially to the most widely used azole class of ergosterol biosynthesis inhibitors, making control of fungal infections more challenging. A lot of effort has been expended in elucidating the mechanism of azole resistance and revealing potential antifungal targets. In this study, by analyzing azole-responsive genes in Neurospora crassa, we discovered STK-17, a novel kinase, that is required for azole resistance in several types of fungi. It has a role in regulating membrane homeostasis, responses to azole by ergosterol biosynthesis genes and azole accumulation, thus, deepening our understanding on the mechanism of azole stress response. Additionally, STK-17 is conserved among fungi and plays important roles in fungal development and stress resistance. Kinase inhibitors are broadly used for treating diseases, and our study pinpoints a potential drug target for antifungal development.
Assuntos
Antifúngicos/metabolismo , Azóis/metabolismo , Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/enzimologia , Proteínas Quinases/metabolismo , Antifúngicos/farmacologia , Azóis/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Farmacorresistência Fúngica , Ergosterol/biossíntese , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Homeostase , Testes de Sensibilidade Microbiana , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/genética , Neurospora crassa/metabolismo , Proteínas Quinases/genéticaRESUMO
DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.
Assuntos
Bioensaio/métodos , Dano ao DNA , Reparo do DNA , Alquilação , Técnicas de Cultura de Células , Linhagem Celular , Células Cultivadas , Humanos , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/genética , Fatores de Tempo , Fluxo de TrabalhoRESUMO
Eisosomes are stable protein complexes at the plasma membrane, with punctate distributional patterns. Their formation and how their locations are determined remain unclear. The current study discovered that the formation and distribution of eisosomes are influenced by the cytoskeleton. Disassembly of either the F-actin or the microtubules leads to eisosome localization at hyphal tips of germinated macroconidia in Neurospora crassa, and treatment with a high concentration of the microtubule-inhibitor benomyl results in the production of filamentous eisosome patterns. The defect in the cytoskeleton caused by the disassembly of microtubules or F-actin leads to an increased formation of eisosomes.
Assuntos
Proteínas Fúngicas/metabolismo , Neurospora crassa/metabolismo , Actinas/metabolismo , Benomilo/farmacologia , Citoesqueleto/metabolismo , Fungicidas Industriais/farmacologia , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Microdomínios da Membrana/metabolismo , Microtúbulos/metabolismo , Complexos Multiproteicos/metabolismo , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/ultraestrutura , FenótipoRESUMO
Fungal diseases are responsible for the deaths of over 1.5 million people worldwide annually. Antifungal peptides represent a useful source of antifungals with novel mechanisms-of-action, and potentially provide new methods of overcoming resistance. Here we investigate the mode-of-action of the small, rationally designed synthetic antifungal peptide PAF26 using the model fungus Neurospora crassa. Here we show that the cell killing activity of PAF26 is dependent on extracellular Ca2+ and the presence of fully functioning fungal Ca2+ homeostatic/signaling machinery. In a screen of mutants with deletions in Ca2+ -signaling machinery, we identified three mutants more tolerant to PAF26. The Ca2+ ATPase NCA-2 was found to be involved in the initial interaction of PAF26 with the cell envelope. The vacuolar Ca2+ channel YVC-1 was shown to be essential for its accumulation and concentration within the vacuolar system. The Ca2+ channel CCH-1 was found to be required to prevent the translocation of PAF26 across the plasma membrane. In the wild type, Ca2+ removal from the medium resulted in the peptide remaining trapped in small vesicles as in the Δyvc-1 mutant. It is, therefore, apparent that cell killing by PAF26 is complex and unusually dependent on extracellular Ca2+ and components of the Ca2+ -regulatory machinery.
Assuntos
Cálcio/metabolismo , Oligopeptídeos/metabolismo , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Cálcio/fisiologia , Canais de Cálcio/metabolismo , Parede Celular/metabolismo , Homeostase , Testes de Sensibilidade Microbiana , Neurospora crassa/efeitos dos fármacos , Oligopeptídeos/fisiologia , Vacúolos/metabolismoRESUMO
Work on the filamentous fungus Neurospora crassa has contributed to or pioneered many aspects of research on circadian clock mechanism, a process that is functionally conserved across eukaryotes. Biochemical assays of the fungal circadian clock typically involve growth in liquid medium where Neurospora forms a spherical ball of submerged mycelium. Here, we revive a method for dispersed growth of Neurospora in batch culture using polyacrylic acid as an additive to the medium. We demonstrate that dispersed growth cultures utilize more carbon than mycelial balls, but nonetheless retain a functional circadian clock. This culturing method is suited for use in circadian experiments where uniform exposure to nutrients and/or increased biomass is required.
Assuntos
Ritmo Circadiano , Glucose , Neurospora crassa , Ritmo Circadiano/efeitos dos fármacos , Técnicas de Cultura , Glucose/farmacologia , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/crescimento & desenvolvimentoRESUMO
Neurospora crassa is an excellent model fungus for studies on molecular genetics, biochemistry, physiology, and molecular cell biology. Along with the rapid progress of Neurospora research, new tools facilitating more efficient and accurate genetic analysis are in high demand. Here, we tested whether the dominant selective makers widely used in yeasts are applicable in N. crassa. Among them, we found that the strains of N. crassa are sensitive to the aminoglycoside antibiotics, G418 and nourseothricin. 1000 µg/mL of G418 or 50 µg/mL of nourseothricin is sufficient to inhibit Neurospora growth completely. When the neomycin phosphotransferase gene (neo) used in mammalian cells is expressed, N. crassa shows potent resistance to G418. This establishes G418-resistant marker as a dominant selectable marker to use in N. crassa. Similarly, when the nourseothricin acetyltransferase gene (nat) from Streptomyces noursei is induced by qa-2 promoter in the presence of quinic acid (QA), N. crassa shows potent resistance to nourseothricin. When nat is constitutively expressed by full-length or truncated versions of the promoter from the N. crassa cfp gene (NCU02193), or by the trpC promoter of Aspergillus nidulans, the growth of N. crassa in the presence of nourseothricin is proportional to the expression levels of Nat. Finally, these two markers are used to knock-out wc-2 or al-1 gene from the N. crassa genome. The successful development of these two markers in this study expands the toolbox for N. crassa and very likely for other filamentous fungi as well.
Assuntos
Farmacorresistência Fúngica/genética , Marcadores Genéticos , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/genética , Acetiltransferases/genética , Antibacterianos/farmacologia , Elementos de DNA Transponíveis , Farmacorresistência Fúngica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Genes Dominantes , Gentamicinas/farmacologia , Canamicina Quinase/genética , Microrganismos Geneticamente Modificados , Regiões Promotoras Genéticas , Ácido Quínico/farmacologia , Estreptotricinas/farmacologiaRESUMO
The development of a tetO/TetR system in the fungus Neurospora crassa is described. The system includes (i) a synthetic gene encoding a TetR variant fused to GFP, and (ii) a standard tetO array integrated homologously, as a proof of principle, near the his-3 gene. The localization of TetR-GFP at the tetO array (observed by fluorescence microscopy) can be disrupted by the application of tetracycline. The full-length array is stable during vegetative growth, but it triggers strong repeat-induced point mutation (RIP) by the RID-dependent as well as the DIM-2-dependent pathways during the sexual phase. Thus, both RIP pathways must be inactivated to allow the faithful inheritance of the unmodified construct. In summary, this study introduces a new molecular tool into Neurospora research, and suggests that the standard tetO array can self-engage in recombination-independent homologous pairing.
Assuntos
Neurospora crassa/genética , Regiões Operadoras Genéticas , Proteínas Repressoras/genética , Tetraciclina/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/efeitos dos fármacos , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Genes Sintéticos , Genoma Fúngico , Recombinação Homóloga , Neurospora crassa/efeitos dos fármacos , Fotomicrografia , Mutação Puntual , Proteínas Recombinantes de Fusão/genética , Sequências Repetitivas de Ácido Nucleico , Proteínas Repressoras/efeitos dos fármacos , SaporinasRESUMO
Plasma membrane damage commonly occurs during cellular growth and development. To counteract these potentially lethal injuries, membrane repair mechanisms have evolved, which promote the integrity of the lipid bilayer. Although the membrane of fungi is the target of important clinical drugs and agricultural fungicides, the molecular mechanisms which mediate membrane repair in these organisms remain elusive. Here we identify the penta-EF-hand protein PEF1 of the genetic model fungus Neurospora crassa as part of a cellular response mechanism against different types of membrane injury. Deletion of the pef1 gene in the wild type and different lysis-prone gene knockout mutants revealed a function of the protein in maintaining cell integrity during cell-cell fusion and in the presence of pore-forming drugs, such as the plant defense compound tomatine. By fluorescence and live-cell imaging we show that green fluorescent protein (GFP)-tagged PEF1 accumulates at the sites of membrane injury in a Ca2+-dependent manner. Site-directed mutagenesis identified Ca2+-binding domains essential for the spatial dynamics and function of the protein. In addition, the subcellular localization of PEF1 revealed that the syncytial fungal colony undergoes compartmentation in response to antifungal treatment. We propose that plasma membrane repair in fungi constitutes an additional line of defense against membrane-disturbing drugs, thereby expanding the current model of fungal drug resistance mechanisms.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Fusão de Membrana , Antifúngicos/farmacologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Membrana Celular/efeitos dos fármacos , Motivos EF Hand , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Ionóforos/farmacologia , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/genética , Neurospora crassa/metabolismo , Tomatina/farmacologiaRESUMO
Aminopyrifen, 4-phenoxybenzyl 2-amino-6-methylnicotinate, strongly inhibited the mycelial growth of a wild-type Neurospora crassa strain on Vogel's minimal medium containing 1.2% sucrose, with a 0.001â¯mg/L concentration required for 50% growth inhibition. Similar to micafungin, an inhibitor of beta-1, 3-glucan synthetase, aminopyrifen further inhibited the growth of N. crassa deletion mutants of MAP kinase cascade genes, such as mak-1 and mak-2, than the wild-type strain, suggesting that aminopyrifen perturbs cell wall-related processes. Furthermore, we found that three chitin synthase gene mutants (chs-1, chs-5, and chs-7) were highly sensitive to both chemicals; however, aminopyrifen, but not micafungin, induced a swollen germ tube from the conidia of chs-5 and chs-7 mutants on Vogel's medium containing 1.2% sucrose. To elucidate the target protein of aminopyrifen, we isolated mutants resistant to aminopyrifen after UV treatment of conidia of the wild-type strain or the chs-5 strain. The resistance mutations were localized to the gwt-1 gene that encodes an acyltransferase, GWT-1, which participates in the biosynthesis of the glycosylphosphatidylinositol (GPI) precursor, and were found to result in S180F and V178A alterations in the protein. These results strongly suggest that aminopyrifen works as an inhibitor targeting GWT-1, a protein involved in GPI-anchor biosynthesis.
Assuntos
Proteínas Fúngicas/metabolismo , Fungicidas Industriais/farmacologia , Glicosilfosfatidilinositóis/metabolismo , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/metabolismo , Quitina Sintase/genética , Quitina Sintase/metabolismo , Proteínas Fúngicas/genética , Mutagênese Sítio-DirigidaRESUMO
We studied the molecular mechanism of neuronal calcium sensor-1 (NCS-1) signaling pathway for tolerance to Ca2+ stress in Neurospora crassa. Increasing concentration of Ca2+ increased the expression of ncs-1; however, the calcineurin inhibitor FK506 severely reduced ncs-1 mRNA transcript levels. Chromatin immunoprecipitation (ChIP) studies revealed that the transcription factor calcineurin responsive zinc finger-1 (CRZ-1) binds to the ncs-1 promoter, and CRZ-1 binding upregulated ncs-1 expression under high Ca2+ concentrations. These results suggested the regulation of NCS-1 function through calcineurin- CRZ-1 signaling pathway. Furthermore, the electrophoretic mobility shift assay (EMSA) revealed that the CRZ-1 binds specifically to an 8 bp sequence 5'-CCTTCACA-3' in the ncs-1 promoter 216 bp upstream of the ATG start codon. We also showed that NCS-1 binds to the Ca2+ permeable channel MID-1 for tolerance to Ca2+ stress. Therefore, CRZ-1 binds to a unique sequence in the ncs-1 promoter, causing upregulation of NCS-1 that binds to MID-1 for tolerance to Ca2+ stress.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Neurospora crassa/genética , Regiões Promotoras Genéticas , Dedos de Zinco , Calcineurina , Inibidores de Calcineurina/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Sinalização do Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicoproteínas de Membrana/genética , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/metabolismo , Estresse Fisiológico , Tacrolimo/farmacologia , Regulação para CimaRESUMO
Azoles are the most widely used antifungals for controlling fungal infections in clinic and agriculture. Fungi can adapt to azole stress by rapidly activating the transcription of a number of genes, and some of these genes can elevate resistance to azoles. We had reported the transcription factor CCG-8 as a new regulator in the adaptation to antifungal azole stress in Neurospora crassa and Fusarium verticillioides. In this study, we further investigate the mechanisms by which CCG-8 promotes fungal adaptation to azole stress using N. crassa as a model. While deletion of ccg-8 made N. crassa hypersensitive to azoles, ccg-8 overexpression strain was more resistant to azoles than wild type, which further confirmed the positive role of ccg-8 in the adaptation to antifungal azoles. Liquid chromatography-mass spectrometry analysis showed that deletion of ccg-8 resulted in decrease of ergosterol biosynthesis, and high accumulation of toxic sterol 14α-methyl-3,6-diol and ketoconazole (KTC) in the cells, whereas intracellular accumulation of ketoconazole was decreased in the ccg-8 overexpression strain as compared to wild type. For analyzing the effect of CCG-8 on azole export, we tested the contribution of predicted multidrug transporters to azole resistance and found that CDR4 is the major contributor for azole efflux in N. crassa. Interestingly, overexpression of cdr4 or erg11 in the ccg-8 deletion mutant restored its hypersensitive phenotype and overexpression of cdr4 can reduce the level of intracellular KTC. However, the double mutant of ccg-8 and cdr4 was more sensitive than each single mutant, suggesting that drug efflux pump CDR4 plays less contribution for intracellular azole accumulation in the ccg-8 deletion mutant, and CCG-8 may regulate drug uptake. Together, our results revealed that CCG-8 plays a pivotal role in azole adaptive responses of N. crassa by regulating the drug accumulation in the cells.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Azóis/farmacologia , Farmacorresistência Fúngica , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Neurospora crassa/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Antifúngicos/farmacologia , Proteínas Fúngicas/genética , Testes de Sensibilidade Microbiana , Fatores de Transcrição/genéticaRESUMO
Calcium ions (Ca2+) play an important role in the toxicity of the cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum: high extracellular Ca2+ levels reduce the toxicity of PAF in the sensitive model fungus Neurospora crassa in a concentration dependent way. However, little is known about the mechanistic details of the Ca2+ ion impact and the Ca2+ binding capabilities of PAF outside the fungal cell, which might be the reason for the activity loss. Using nuclear magnetic resonance (NMR), isothermal titration calorimetry and molecular dynamics (MD) simulations we demonstrated that PAF weakly, but specifically binds Ca2+ ions. MD simulations of PAF predicted one major Ca2+ binding site at the C-terminus involving Asp53 and Asp55, while Asp19 was considered as putative Ca2+ binding site. The exchange of Asp19 to serine had little impact on the Ca2+ binding, however caused the loss of antifungal activity, as was shown in our recent study. Now we replaced the C-terminal aspartates and expressed the serine variant PAFD53S/D55S. The specific Ca2+ binding affinity of PAFD53S/D55S decreased significantly if compared to PAF, whereas the antifungal activity was retained. To understand more details of Ca2+ interactions, we investigated the NMR and MD structure/dynamics of the free and Ca2+-bound PAF and PAFD53S/D55S. Though we found some differences between these protein variants and the Ca2+ complexes, these effects cannot explain the observed Ca2+ influence. In conclusion, PAF binds Ca2+ ions selectively at the C-terminus; however, this Ca2+ binding does not seem to play a direct role in the previously documented modulation of the antifungal activity of PAF.
Assuntos
Cálcio/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Penicillium chrysogenum/crescimento & desenvolvimento , Sítios de Ligação , Calorimetria , Proteínas Fúngicas/genética , Proteínas Fúngicas/toxicidade , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Neurospora crassa/efeitos dos fármacos , Penicillium chrysogenum/metabolismo , Ligação ProteicaRESUMO
Implant-related bacterial infections remain a serious problem that is not solved yet. Herein we combined several antibacterial agents to achieve synergistic effects and broader protection of widely used metallic implants. Titanium samples with microcontainers for drug, produced by selective laser sintering, were coated with Ag-doped biocompatible and bioactive TiCaPCON film and loaded with an antibiotic (gentamicin or a mixture of gentamicin and amphotericin B). Bactericide release tests demonstrated that the release rate of one bactericide agent (Ag+ ions or gentamicin) depended on the presence of the other antibacterial component. The antibacterial activity of the biocide-doped samples was evaluated against clinically isolated Escherichia coli O78 (E. coli), Staphylococcus aureus (S. aureus) bacteria, and Neurospora crassa wt-987 (N. crassa) spores. It was found that samples loaded with low gentamicin concentration (0.2 and 0.02â¯mg/cm2), i.e. 10 and 100 times less than the standard gentamicin concentration (2â¯mg/cm2), demonstrated a superb antibacterial activity against E. coli bacteria. We showed that a crosslinking reaction between gentamicin and TiCaPCON film proceeded either through the formation of amide bonds or via the electrostatic interaction between amine groups of gentamicin and COOH groups of TiCaPCON and led to the formation of relatively stable drug/film conjugates that prevented a rapid dissolution of gentamicin and ensured its long-term (for 72â¯h) antibacterial protection. Leaching of silver ions provided an effective antibacterial protection after the depletion of the drug reservoirs. The obtained results clearly show a synergistic antibacterial action of Ag+ ions and gentamicin against S. aureus bacteria. In addition, in the presence of Ag+ ions, the antifungal activity of samples loaded with a mixture of gentamicin and amphotericin B against N. crassa fungus was observed to increase. Thus, it is demonstrated that silver can be successfully coupled with different types of antibiotics to provide innovative hybrid metal-ceramic bioconstructions that are able to deliver precise doses of bactericide agents within a certain period of time and are equally effective against Gram-negative E. coli bacteria, Gram-positive S. aureus, and N. crassa fungus.
Assuntos
Antibacterianos/química , Antifúngicos/química , Gentamicinas/química , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Neurospora crassa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Titânio/químicaRESUMO
Defensins are cysteine-rich cationic antimicrobial peptides contributing to the innate immunity in plants. A unique gene encoding a highly cationic bi-domain defensin MtDef5 has been identified in a model legume Medicago truncatula. MtDef5 consists of two defensin domains of 50 amino acids each linked by a 7-amino acid peptide. It exhibits broad-spectrum antifungal activity against filamentous fungi at submicromolar concentrations. It rapidly permeabilizes the plasma membrane of the ascomycete fungi Fusarium graminearum and Neurospora crassa and induces accumulation of reactive oxygen species. It is internalized by these fungi, but uses spatially distinct modes of entry into these fungi. It co-localizes with cellular membranes, travels to nucleus and becomes dispersed in other subcellular locations. It binds to several membrane-resident phospholipids with preference for phosphatidylinositol monophosphates and forms oligomers. Mutations of the cationic amino acids present in the two γ-core motifs of this defensin that eliminate oligomerization also knockout its ability to induce membrane permeabilization and fungal growth arrest. MtDef5 is the first bi-domain plant defensin that exhibits potent broad-spectrum antifungal activity, recruits multiple membrane phospholipids and forms oligomers in their presence. These findings raise the possibility that MtDef5 might be useful as a novel antifungal agent in transgenic crops.
Assuntos
Antifúngicos/química , Defensinas/química , Fosfolipídeos/química , Antifúngicos/farmacologia , Fusarium/efeitos dos fármacos , Fusarium/metabolismo , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
To elucidate genetic mechanisms affecting the lifespan of the filamentous fungus Neurospora crassa, we attempted to identify a gene of which a defect causes a short-lifespan. By screening a Neurospora knockout library, provided by the Fungal Genetics Stock Center at Kansas State University, several KO strains with a short-lifespan were isolated. FGSC#11693 is one of these, which shows similar phenotypes to known Neurospora short-lifespan mutants as follows: 1) hyphal growth ceases after about 2weeks of cultivation, despite that of the wild-type continuing for over 2years, 2) viability of conidia is lower than that of the wild-type, and 3) high sensitivity to mutagens such as methyl methanesulfonate, ultraviolet radiation, and hydroxyl urea is exhibited. The NCU number of the knocked-out gene in the KO strain is NCU02695, and recovery from the short-lifespan and mutagen sensitivity was achieved by the introduction of this gene from the wild-type. The putative amino acid sequence of the knocked-out gene contains two high mobility group box domains and a mitochondrial localization signal is found at the N-terminal of this sequence. Upon analyzing the subcellular localization of the gene product fused with GFP, GFP signals were detected in mitochondria. From these observations, the gene and KO strain were named mitochondrial high mobility group box protein 1 (MHG1) and mhg1KO strain, respectively. The amount of mtDNA relative to the nuclear amount was lower in the mhg1KO strain than in the wild-type. mtDNA aberration was also observed in the mhg1KO strain. These results suggest that the MHG1 protein plays an important role in the maintenance of mitochondrial DNA, and mitochondrial abnormality caused by mtDNA aberration is responsible for the short-lifespan of the mhg1KO strain.
Assuntos
DNA Fúngico/metabolismo , DNA Mitocondrial/metabolismo , Proteínas Fúngicas/genética , Proteínas HMGB/genética , Neurospora crassa/genética , Sequência de Aminoácidos , Proteínas Fúngicas/metabolismo , Técnicas de Silenciamento de Genes , Genes Fúngicos , Proteínas HMGB/metabolismo , Longevidade/genética , Mutagênicos/farmacologia , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/isolamento & purificação , Neurospora crassa/metabolismo , Fenótipo , Domínios ProteicosRESUMO
Neuronal calcium sensor-1 (NCS-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (Ca2+) binding EF-hand domains. We performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the N-terminal myristoylation, a conserved glutamic acid residue responsible for Ca2+ binding in the third EF-hand (EF3), and an unusual non-conserved amino acid arginine at position 175 in the Neurospora crassa NCS-1. The N. crassa strains possessing the ncs-1 mutant allele of these three amino acid residues showed impairment in functions ranging from growth, Ca2+ stress tolerance, and ultraviolet survival. In addition, heterologous expression of the NCS-1 from Rattus norvegicus in N. crassa confirmed its interspecies functional conservation. Moreover, functions of glutamic acid at position 120, the first Ca2+ binding residue among all the EF-hands of the R. norvegicus NCS-1 was found conserved. Thus, we identified three critical amino acid residues of N. crassa NCS-1, and demonstrated its functional conservation across species using the orthologue from R. norvegicus.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Sensoras de Cálcio Neuronal/química , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Neurospora crassa/metabolismo , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Proteínas Fúngicas/genética , Mutação , Proteínas Sensoras de Cálcio Neuronal/genética , Neuropeptídeos/genética , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/efeitos da radiação , Ratos , Raios UltravioletaRESUMO
Catalase-3 (CAT-3) constitutes the main catalase activity in growing hyphae of Neurospora crassa, and its activity increases during exponential growth or is induced under different stress conditions. Although extensive progress has been made to identify catalase regulators, the regulation mechanism of CAT-3 at the chromatin level still remains unclear. Here, we aim at investigating the molecular regulation mechanisms of cat-3 at the chromatin level. We found that CAT-3 protein levels increased in mutants defective in proper global heterochromatin formation. Bioinformatics analysis identified a 5-kb AT-rich sequence adjacent to the cat-3 promoter as a heterochromatin region because of its enrichment of H3K9me3 and HP1. Expression of CAT-3 was induced by H2O2 treatment in wild-type and such change occurred along with the accumulation of histone H3 acetylation at 5-kb heterochromatin boundaries and cat-3 locus, but without alteration of its H3K9me3 repressive modification. Moreover, disruption of 5-kb heterochromatin region results in elevated cat-3 expression, and higher levels of cat-3 expression were promoted by the combination with global heterochromatin defective mutants. Interestingly, the molecular weight and activity bands of CAT-3 protein are different in heterochromatin defective mutants compared with those in wild-type, suggesting that its N-terminal processing and modification may be altered. Our study indicates that the local chromatin structure creates a heterochromatin repressive environment to repress nearby gene expression.