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1.
Fungal homologues of human Rac1 as emerging players in signal transduction and morphogenesis.
Hühn, J, Musielak, M, Schmitz, HP, Heinisch, JJ
International microbiology : the official journal of the Spanish Society for Microbiology. 2020;(1):43-53
Abstract
A wealth of data is accumulating on the physiological functions of human Rac1, a member of the Rho GTPase family of molecular switches and substrate of botulinum toxin, which was first identified as a regulator of cell motility through its effect on the actin cytoskeleton. Later on, it was found to be involved in different diseases like cancers, cardiac function, neuronal disorders, and apoptotic cell death. Despite the presence of Rac1 homologues in most fungi investigated so far, including Rho5 in the genetically tractable model yeast Saccharomyces cerevisiae, knowledge on their physiological functions is still scarce, let alone the details of the molecular mechanisms of their actions and interactions. Nevertheless, all functions proposed for human Rac1 seem to be conserved in one or the other fungus. This includes the regulation of MAPK cascades, polarized growth, and actin dynamics. Moreover, both the production and response to reactive oxygen species, as well as the reaction to nutrient availability, can be affected. We here summarize the studies performed on fungal Rac1 homologues, with a special focus on S. cerevisiae Rho5, which may be of use in drug development in medicine and agriculture.
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2.
Increased glycosidase activities improved the production of wine varietal odorants in mixed fermentation of P. fermentans and high antagonistic S. cerevisiae.
Li, N, Wang, QQ, Xu, YH, Li, AH, Tao, YS
Food chemistry. 2020;:127426
Abstract
A selected Pichia fermentans strain was simultaneously and sequentially inoculated in synthetic and real juice with S. cerevisiae strains of different antagonistic activities in a ratio 1:1 to observe the correlation between varietal odorants and glycosidase activities. Fermentations using pure S. cerevisiae strains were used for comparison. Yeast biomass and glycosidase activities were monitored, varietal odorants were detected using HS-SPME-GC/MS during fermentation. The final wine aroma attributes were analyzed by trained panelists. Results showed that co-inoculation with high antagonistic S. cerevisiae resulted in higher glycosidase activities than others. Pearson correlation analysis indicated that yeast biomass was positively related to glycosidase activities during fermentation. The increase in glycosidase activities was the main reason for the higher production of terpenes and C13-norisoprenoids, and for the lower C6 compound content, which lead to superior fruity and floral aromas in the final wine samples of the high antagonistic S. cerevisiae group.
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3.
Zinc Finger Proteins in the Human Fungal Pathogen Cryptococcus neoformans.
Li, YH, Liu, TB
International journal of molecular sciences. 2020;(4)
Abstract
Zinc is one of the essential trace elements in eukaryotes and it is a critical structural component of a large number of proteins. Zinc finger proteins (ZNFs) are zinc-finger domain-containing proteins stabilized by bound zinc ions and they form the most abundant proteins, serving extraordinarily diverse biological functions. In recent years, many ZNFs have been identified and characterized in the human fungal pathogen Cryptococcus neoformans, a fungal pathogen causing fatal meningitis mainly in immunocompromised individuals. It has been shown that ZNFs play important roles in the morphological development, differentiation, and virulence of C. neoformans. In this review, we, first, briefly introduce the ZNFs and their classification. Then, we explain the identification and classification of the ZNFs in C. neoformans. Next, we focus on the biological role of the ZNFs functionally characterized so far in the sexual reproduction, virulence factor production, ion homeostasis, pathogenesis, and stress resistance in C. neoformans. We also discuss the perspectives on future function studies of ZNFs in C. neoformans.
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4.
Cutinases as stereoselective catalysts: Specific activity and enantioselectivity of cutinases and lipases for menthol and its analogs.
Su, A, Kiokekli, S, Naviwala, M, Shirke, AN, Pavlidis, IV, Gross, RA
Enzyme and microbial technology. 2020;:109467
Abstract
The specific activity and enantioselectivity of immobilized cutinases from Aspergillus oryzae (AoC) and Humicola insolens (HiC) were compared with those of lipases from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML) and Lipase B from Candida antarctica (CALB) for menthol and its analogs that include isopulegol, trans-2-tert-butylcyclohexanol (2TBC), and dihydrocarveol (DHC). Common features of these alcohols are two bulky substituents: a cyclohexyl ring and an alkyl substituent. Dissimilarities are that the alkyl group reside at different positions or have dissimilar structures. The aim was to develop an understanding at a molecular level of similarities and differences in the catalytic behavior of the selected cutinases and lipases as a function of substrate structural elements. The experimental results reflect the (-)-enantioselectivity for AoC, HiC, TLL, and RML, while CALB is only active on DHC with (+)-enantioselectivity. In most cases, AoC has the highest activity while HiC is significantly more active than other enzymes on 2TBC. The E values of AoC, HiC, TLL, and RML for menthol are 27.8, 16.5, 155, and 125, respectively. HiC has a higher activity (>10-fold) on (-)-2TBC than AoC while they exhibit similar activities on menthol. Docking results reveal that the bulky group adjacent to the hydroxyl group determines the enantioselectivity of AoC, HiC, TLL, and RML. Amino acid residues that dominate the enantioselectivity of these enzymes are AoC's Phe195 aromatic ring; HiC's hydrophobic Leu 174 and Ile 169 groups; TLL's ring structures of Trp89, His258 and Tyr21; and Trp88 for RML. Results of this study highlight that cutinases can provide important advantages relative to lipases for enantioselective transformation, most notably with bulky and sterically hindered substrates.
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5.
Carbon sources and XlnR-dependent transcriptional landscape of CAZymes in the industrial fungus Talaromyces versatilis: when exception seems to be the rule.
Llanos, A, Déjean, S, Neugnot-Roux, V, François, JM, Parrou, JL
Microbial cell factories. 2019;(1):14
Abstract
BACKGROUND Research on filamentous fungi emphasized the remarkable redundancy in genes encoding hydrolytic enzymes, the similarities but also the large differences in their expression, especially through the role of the XlnR/XYR1 transcriptional activator. The purpose of this study was to evaluate the specificities of the industrial fungus Talaromyces versatilis, getting clues into the role of XlnR and the importance of glucose repression at the transcriptional level, to provide further levers for cocktail production. RESULTS By studying a set of 62 redundant genes representative of several categories of enzymes, our results underlined the huge plasticity of transcriptional responses when changing nutritional status. As a general trend, the more heterogeneous the substrate, the more efficient to trigger activation. Genetic modifications of xlnR led to significant reorganisation of transcriptional patterns. Just a minimal set of genes actually fitted in a simplistic model of regulation by a transcriptional activator, and this under specific substrates. On the contrary, the diversity of xlnR+ versus ΔxlnR responses illustrated the existence of complex and unpredicted patterns of co-regulated genes that were highly dependent on the culture condition, even between genes that encode members of a functional category of enzymes. They notably revealed a dual, substrate-dependant repressor-activator role of XlnR, with counter-intuitive transcripts regulations that targeted specific genes. About glucose, it appeared as a formal repressive sugar as we observed a massive repression of most genes upon glucose addition to the mycelium grown on wheat straw. However, we also noticed a positive role of this sugar on the basal expression of a few genes, (notably those encoding cellulases), showing again the strong dependence of these regulatory mechanisms upon promoter and nutritional contexts. CONCLUSIONS The diversity of transcriptional patterns appeared to be the rule, while common and stable behaviour, both within gene families and with fungal literature, the exception. The setup of a new biotechnological process to reach optimized, if not customized expression patterns of enzymes, hence appeared tricky just relying on published data that can lead, in the best scenario, to approximate trends. We instead encourage preliminary experimental assays, carried out in the context of interest to reassess gene responses, as a mandatory step before thinking in (genetic) strategies for the improvement of enzyme production in fungi.
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6.
Structural and immunological characterization of a new nucleotidyltransferase-like antigen from Paracoccidioides brasiliensis.
Coitinho, JB, Costa, MAF, Melo, EM, Morais, EA, de Andrade, LGA, da Rocha, AM, de Magalhães, MTQ, Favaro, DC, Bleicher, L, Pedroso, ERP, et al
Molecular immunology. 2019;:151-162
Abstract
Pb27 antigen is an interesting alternative to immunological diagnosis of Paracoccidioidomycosis (PCM) and has demonstrated to be protective in experimental PCM. Its tertiary structure and possible function remained unknown till now. To study Pb27 at the atomic level, the recombinant protein was expressed in Escherichia coli BL21(DE3), purified, and its three-dimensional structure was solved by X-ray crystallography. Based on this structure, we performed a residue correlation analysis and in silico ligand search assays to address a possible biological function to Pb27. We identified Pb27 as a member of the extensive nucleotidyltransferase superfamily. The protein has an αβαβαβ topology with two domains (N- and C-terminal domains) and adopts a monomeric form as its biological unit in solution. Structural comparisons with similar members of the superfamily clearly indicate Pb27 C-terminal domain is singular and may play an important role in its biological function. Bioinformatics analysis suggested that Pb27 might bind to ATP and CTP. This suggestion is corroborated by the fact that a magnesium cation is coordinated by two aspartic acid residues present at the active site (between N- and C-terminal domains), as evidenced by X-ray diffraction data. Besides, NMR assays (1H-15N HSQC spectra) confirmed the binding of CTP to Pb27, demonstrating for the first time an interaction between a nucleotide and this protein. Moreover, we evaluated the reactivity of sera from patients with Paracoccidioides brasiliensis infection against the recombinant form of Pb27 and showed that it was recognized by sera from infected and treated patients. Predicted B and T cell epitopes were synthesized and further evaluated against sera of PCM patients, providing information of the most reactive peptides in Pb27 primary structure which interact with specific Pb27 antibodies.
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7.
The elicitin β-cryptogein's activity in tomato is mediated by jasmonic acid and ethylene signalling pathways independently of elicitin-sterol interactions.
Starý, T, Satková, P, Piterková, J, Mieslerová, B, Luhová, L, Mikulík, J, Kašparovský, T, Petřivalský, M, Lochman, J
Planta. 2019;(3):739-749
Abstract
The level of resistance induced in different tomato genotypes after β-CRY treatment correlated with the upregulation of defence genes, but not sterol binding and involved ethylene and jasmonic acid signalling. Elicitins, a family of small proteins secreted by Phytophthora and Pythium spp., are the most well-known microbe-associated molecular patterns of oomycetes, a lineage of fungus-like organisms that include many economically significant crop pathogens. The responses of tomato plants to elicitin INF1 produced by Phytophthora infestans have been studied extensively. Here, we present studies on the responses of three tomato genotypes to β-cryptogein (β-CRY), a potent elicitin secreted by Phytophthora cryptogea that induces hypersensitive response (HR) cell death in tobacco plants and confers greater resistance to oomycete infection than acidic elicitins like INF1. We also studied β-CRY mutants impaired in sterol binding (Val84Phe) and interaction with the binding site on tobacco plasma membrane (Leu41Phe), because sterol binding was suggested to be important in INF1-induced resistance. Treatment with β-CRY or the Val84Phe mutant induced resistance to powdery mildew caused by the pathogen Pseudoidium neolycopersici, but not the HR cell death observed in tobacco and potato plants. The level of resistance induced in different tomato genotypes correlated with the upregulation of defence genes including defensins, β-1,3-glucanases, heveins, chitinases, osmotins, and PR1 proteins. Treatment with the Leu41Phe mutant did not induce this upregulation, suggesting similar elicitin recognition in tomato and tobacco. However, here β-CRY activated ethylene and jasmonic acid signalling, but not salicylic acid signalling, demonstrating that elicitins activate different downstream signalling processes in different plant species. This could potentially be exploited to enhance the resistance of Phytophthora-susceptible crops.
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8.
Light in the Fungal World: From Photoreception to Gene Transcription and Beyond.
Corrochano, LM
Annual review of genetics. 2019;:149-170
Abstract
Fungi see light of different colors by using photoreceptors such as the White Collar proteins and cryptochromes for blue light, opsins for green light, and phytochromes for red light. Light regulates fungal development, promotes the accumulation of protective pigments and proteins, and regulates tropic growth. The White Collar complex (WCC) is a photoreceptor and a transcription factor that is responsible for regulating transcription after exposure to blue light. In Neurospora crassa, light promotes the interaction of WCCs and their binding to the promoters to activate transcription. In Aspergillus nidulans, the WCC and the phytochrome interact to coordinate gene transcription and other responses, but the contribution of these photoreceptors to fungal photobiology varies across fungal species. Ultimately, the effect of light on fungal biology is the result of the coordinated transcriptional regulation and activation of signal transduction pathways.
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9.
The Fusarium graminearum cerato-platanins loosen cellulose substrates enhancing fungal cellulase activity as expansin-like proteins.
Quarantin, A, Castiglioni, C, Schäfer, W, Favaron, F, Sella, L
Plant physiology and biochemistry : PPB. 2019;:229-238
Abstract
Cerato-platanin proteins (CPPs) are small non-catalytic, cysteine-rich hydrophobic proteins produced by filamentous fungi. The genome of Fusarium graminearum, the causal agent of Fusarium head blight disease of wheat and other cereal grains, contains two genes putatively encoding for CPPs. To better characterize their features, the two FgCPPs were heterologously expressed in Pichia pastoris. The recombinant FgCPPs reduced the viscosity of a cellulose soluble derivate (carboxymethyl cellulose, CMC). The same effect was not observed on other polysaccharide substrates such as chitin, 1,3-β-glucan, xylan and pectin. Indeed, differently from other fungal CPPs and similarly to expansins, FgCPPs are trapped by cellulose and not by chitin, thus suggesting that these proteins interact with cellulose. A double knock-out mutant deleted of both FgCPPs encoding genes produces much more cellulase activity than the corresponding wild type strain when grown on CMC, likely compensating the absence of FgCPPs. This result prompted us to investigate a possible synergistic effect of these proteins with fungal cellulases. The incubation of FgCPPs in the presence of a fungal cellulase (EC 3.2.1.4) determines an increased enzymatic activity on CMC, filter paper and wheat cell walls. The observation that FgCPPs act with a non-hydrolytic mechanism indicates that these proteins favor fungal cellulase activity in an expansin-like manner. Though the disruption of the FgCPP genes had no demonstrable impact on fungal virulence, our experimental data suggest their probable involvement in virulence, thus we refer to them as accessory virulence genes. Our results suggest also that the FgCPPs could be exploited for future biotechnological application in second-generation biofuels production on lignocellulosic biomasses rich in cellulose. Finally, we demonstrate that FgCPPs act as elicitors of defense responses on Arabidopsis leaves, increasing resistance to Botrytis cinerea infections.
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10.
Fungal co-culture increases ligninolytic enzyme activities: statistical optimization using response surface methodology.
Jiménez-Barrera, D, Chan-Cupul, W, Fan, Z, Osuna-Castro, JA
Preparative biochemistry & biotechnology. 2018;(9):787-798
Abstract
The optimization of ligninolytic enzyme (LE) activities in a novel fungal co-culture between Pycnoporus sanguineus and Beauveria brongniartii were studied using a Plackett-Burman experimental design (PBED) and a central composite design (CCD). In addition, H2O2 role was analyzed. Laccase (EC. 1.10.3.2) and MnP (EC 1.11.1.14) activities of P. sanguineus increased 6.0- and 2.3-fold, respectively, in the co-culture with B. brongniartii. The H2O2 content was higher in the co-culture (0.33-7.12-fold) than in the P. sanguineus monoculture. The PBED revealed that yeast extract (YE), FeSO4, and inoculum amount were significant factors for laccase and MnP activities and H2O2 production in the co-culture, which increased by 8.2-, 5.2- and 1.03-fold, respectively. The YE and FeSO4 were studied using a CCD to optimize the studied response variables. Laccase activity was enhanced 1.5-fold by CCD, the optimal amount of YE was 0.366 g L-1. Quadratic term of FeSO4 modulated MnP activity and was associated with a 4.28-fold increase compared to the PBED. Both YE and its quadratic term significantly affected H2O2 production; however, the CCD did not enable an increase in H2O2 production. Pearson correlation indicated an increase in laccase (r2=0.4411, p = 0.0436) and MnP (r2=0.5186, p = 0.0198) activities following increases in H2O2 in the co-culture system.