RESUMEN
Aspergillus fumigatus is a filamentous fungus that can infect the lungs of patients with immunosuppression and/or underlying lung diseases. The mortality associated with chronic and invasive aspergillosis infections remain very high, despite availability of antifungal treatments. In the last decade, there has been a worrisome emergence and spread of resistance to the first-line antifungals, the azoles. The mortality caused by resistant isolates is even higher, and patient management is complicated as the therapeutic options are reduced. Nevertheless, treatment failure is also common in patients infected with azole-susceptible isolates, which can be due to several non-mutually exclusive reasons, such as poor drug absorption. In addition, the phenomena of tolerance or persistence, where susceptible pathogens can survive the action of an antimicrobial for extended periods, have been associated with treatment failure in bacterial infections, and their occurrence in fungal infections already proposed. Here, we demonstrate that some isolates of A. fumigatus display persistence to voriconazole. A subpopulation of the persister isolates can survive for extended periods and even grow at low rates in the presence of supra-MIC of voriconazole and seemingly other azoles. Persistence cannot be eradicated with adjuvant drugs or antifungal combinations and seemed to reduce the efficacy of treatment for certain individuals in a Galleria mellonella model of infection. Furthermore, persistence implies a distinct transcriptional profile, demonstrating that it is an active response. We propose that azole persistence might be a relevant and underestimated factor that could influence the outcome of infection in human aspergillosis. IMPORTANCE The phenomena of antibacterial tolerance and persistence, where pathogenic microbes can survive for extended periods in the presence of cidal drug concentrations, have received significant attention in the last decade. Several mechanisms of action have been elucidated, and their relevance for treatment failure in bacterial infections demonstrated. In contrast, our knowledge of antifungal tolerance and, in particular, persistence is still very limited. In this study, we have characterized the response of the prominent fungal pathogen Aspergillus fumigatus to the first-line therapy antifungal voriconazole. We comprehensively show that some isolates display persistence to this fungicidal antifungal and propose various potential mechanisms of action. In addition, using an alternative model of infection, we provide initial evidence to suggest that persistence may cause treatment failure in some individuals. Therefore, we propose that azole persistence is an important factor to consider and further investigate in A. fumigatus.
RESUMEN
Aspergillus fumigatus is the most important airborne fungal pathogen and allergen of humans causing high morbidity and mortality worldwide. The factors that govern pathogenicity of this organism are multi-factorial and are poorly understood. Molecular tools to dissect the mechanisms of pathogenicity in A. fumigatus have improved significantly over the last 20 years however many procedures have not been standardised for A. fumigatus. Here, we present a new genomic safe-haven locus at the site of an inactivated transposon, named SH-aft4, which can be used to insert DNA sequences in the genome of this fungus without impacting its phenotype. We show that we are able to effectively express a transgene construct from the SH-aft4 and that natural regulation of promoter function is conserved at this site. Furthermore, the SH-aft4 locus is highly conserved in the genome of a wide range of clinical and environmental isolates including the isolates commonly used by many laboratories CEA10, Af293 and ATCC46645, allowing a wide range of isolates to be manipulated. Our results show that the aft4 locus can serve as a site for integration of a wide range of genetic constructs to aid functional genomics studies of this important human fungal pathogen.
Asunto(s)
Aspergilosis , Aspergillus fumigatus , Aspergilosis/microbiología , Genoma Fúngico/genética , Genómica , Humanos , Virulencia/genéticaRESUMEN
Aspergillus fumigatus is a saprophytic fungal pathogen that is the cause of more than 300,000 life-threatening infections annually. Our understanding of pathogenesis and factors contributing to disease progression are limited. Development of rapid and versatile gene editing methodologies for A. fumigatus is essential. CRISPR-Cas9 mediated transformation has been widely used as a novel genome editing tool and has been used for a variety of editing techniques, such as protein tagging, gene deletions and site-directed mutagenesis in A. fumigatus. However, successful genome editing relies on time consuming, multi-step cloning procedures paired with the use of selection markers, which can result in a metabolic burden for the host and/or unintended transcriptional modifications at the site of integration. We have used an in vitro CRISPR-Cas9 assembly methodology to perform selection-free genome editing, including epitope tagging of proteins and site-directed mutagenesis. The repair template used during this transformation use 50 bp micro-homology arms and can be generated with a single PCR reaction or by purchasing synthesised single stranded oligonucleotides, decreasing the time required for complex construct synthesis.
Asunto(s)
Aspergillus fumigatus/genética , Epítopos/genética , Mutagénesis Sitio-Dirigida , Micosis/genética , Aspergillus fumigatus/patogenicidad , Sistemas CRISPR-Cas/genética , Proteínas Fúngicas/genética , Edición Génica/tendencias , Humanos , Micosis/microbiologíaRESUMEN
To sustain iron homeostasis, microorganisms have evolved fine-tuned mechanisms for uptake, storage and detoxification of the essential metal iron. In the human pathogen Aspergillus fumigatus, the fungal-specific bZIP-type transcription factor HapX coordinates adaption to both iron starvation and iron excess and is thereby crucial for virulence. Previous studies indicated that a HapX homodimer interacts with the CCAAT-binding complex (CBC) to cooperatively bind bipartite DNA motifs; however, the mode of HapX-DNA recognition had not been resolved. Here, combination of in vivo (genetics and ChIP-seq), in vitro (surface plasmon resonance) and phylogenetic analyses identified an astonishing plasticity of CBC:HapX:DNA interaction. DNA motifs recognized by the CBC:HapX protein complex comprise a bipartite DNA binding site 5'-CSAATN12RWT-3' and an additional 5'-TKAN-3' motif positioned 11-23 bp downstream of the CCAAT motif, i.e. occasionally overlapping the 3'-end of the bipartite binding site. Phylogenetic comparison taking advantage of 20 resolved Aspergillus species genomes revealed that DNA recognition by the CBC:HapX complex shows promoter-specific cross-species conservation rather than regulon-specific conservation. Moreover, we show that CBC:HapX interaction is absolutely required for all known functions of HapX. The plasticity of the CBC:HapX:DNA interaction permits fine tuning of CBC:HapX binding specificities that could support adaptation of pathogens to their host niches.
Asunto(s)
Aspergillus fumigatus/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factor de Unión a CCAAT/metabolismo , Proteínas Fúngicas/metabolismo , Hierro/metabolismo , Regiones Promotoras Genéticas , Secuencia Rica en At , Aspergillus fumigatus/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Sitios de Unión , ADN de Hongos/química , ADN de Hongos/metabolismo , Evolución Molecular , Proteínas Fúngicas/química , Mutación , Motivos de Nucleótidos , Unión Proteica , Dominios Proteicos , Regulón , Sideróforos/metabolismo , Resonancia por Plasmón de Superficie , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
The frequency of antifungal resistance, particularly to the azole class of ergosterol biosynthetic inhibitors, is a growing global health problem. Survival rates for those infected with resistant isolates are exceptionally low. Beyond modification of the drug target, our understanding of the molecular basis of azole resistance in the fungal pathogen Aspergillus fumigatus is limited. We reasoned that clinically relevant antifungal resistance could derive from transcriptional rewiring, promoting drug resistance without concomitant reductions in pathogenicity. Here we report a genome-wide annotation of transcriptional regulators in A. fumigatus and construction of a library of 484 transcription factor null mutants. We identify 12 regulators that have a demonstrable role in itraconazole susceptibility and show that loss of the negative cofactor 2 complex leads to resistance, not only to the azoles but also the salvage therapeutics amphotericin B and terbinafine without significantly affecting pathogenicity.
Asunto(s)
Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Farmacorresistencia Fúngica , Proteínas Fúngicas/metabolismo , Anfotericina B/farmacología , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Azoles/farmacología , Proteínas Fúngicas/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
We consider the situation where a temporal process is composed of contiguous segments with differing slopes and replicated noise-corrupted time series measurements are observed. The unknown mean of the data generating process is modelled as a piecewise linear function of time with an unknown number of change-points. We develop a Bayesian approach to infer the joint posterior distribution of the number and position of change-points as well as the unknown mean parameters. A-priori, the proposed model uses an overfitting number of mean parameters but, conditionally on a set of change-points, only a subset of them influences the likelihood. An exponentially decreasing prior distribution on the number of change-points gives rise to a posterior distribution concentrating on sparse representations of the underlying sequence. A Metropolis-Hastings Markov chain Monte Carlo (MCMC) sampler is constructed for approximating the posterior distribution. Our method is benchmarked using simulated data and is applied to uncover differences in the dynamics of fungal growth from imaging time course data collected from different strains. The source code is available on CRAN.
Asunto(s)
Bioestadística , Crecimiento , Modelos Estadísticos , Simulación por Computador , Hongos/crecimiento & desarrollo , Cadenas de Markov , Método de MontecarloRESUMEN
The antifungal drug 5-flucytosine (5FC), a derivative of the nucleobase cytosine, is licensed for the treatment of fungal diseases; however, it is rarely used as a monotherapeutic to treat Aspergillus infection. Despite being potent against other fungal pathogens, 5FC has limited activity against Aspergillus fumigatus when standard in vitro assays are used to determine susceptibility. However, in modified in vitro assays where the pH is set to pH 5, the activity of 5FC increases significantly. Here we provide evidence that fcyB, a gene that encodes a purine-cytosine permease orthologous to known 5FC importers, is downregulated at pH 7 and is the primary factor responsible for the low efficacy of 5FC at pH 7. We also uncover two transcriptional regulators that are responsible for the repression of fcyB and, consequently, mediators of 5FC resistance, the CCAAT binding complex (CBC) and the pH regulatory protein PacC. We propose that the activity of 5FC might be enhanced by the perturbation of factors that repress fcyB expression, such as PacC or other components of the pH-sensing machinery.
Asunto(s)
Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Flucitosina/farmacología , Proteínas Fúngicas/metabolismo , Factores de Transcripción/metabolismo , Aspergillus fumigatus/metabolismo , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Concentración de Iones de Hidrógeno , Pruebas de Sensibilidad Microbiana , Factores de Transcripción/genéticaRESUMEN
The GH10 xylanase XYNIII is expressed in the hyper-cellulase-producing mutant PC-3-7, but not in the standard strain QM9414 of Trichoderma reesei. The GH11 xylanase gene xyn1 is induced by cellulosic and xylanosic carbon sources while xyn3 is induced only by cellulosic carbon sources in the PC-3-7 strain. In this study, we constructed a modified xyn3 promoter in which we replaced the cis-acting region of the xyn3 promoter by the cis-acting region of the xyn1 promoter. The resulting xyn3 chimeric promoter exhibited improved inductivity against cellulosic carbon over the wild-type promoter and acquired inductivity against xylanosic carbon. Furthermore, PC-3-7 expressing the heterologous ß-glycosidase gene, Aspergillus aculeatus bgl1, under the control of the xyn3 chimeric promoter, showed enhanced saccharification ability through increased cellobiase activity. We also show that the xyn3 chimeric promoter is also functional in the QM9414 strain. Our results indicate that the xyn3 chimeric promoter is very efficient for enzyme expression.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Ingeniería Metabólica/métodos , Regiones Promotoras Genéticas , Trichoderma/enzimología , Trichoderma/genética , Xilosidasas/genética , Aspergillus/enzimología , Aspergillus/genética , Celulosa/metabolismo , Trichoderma/metabolismo , Xilanos/metabolismo , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1005775.].
RESUMEN
Azole drugs selectively target fungal sterol biosynthesis and are critical to our antifungal therapeutic arsenal. However, resistance to this class of drugs, particularly in the major human mould pathogen Aspergillus fumigatus, is emerging and reaching levels that have prompted some to suggest that there is a realistic probability that they will be lost for clinical use. The dominating class of pan-azole resistant isolates is characterized by the presence of a tandem repeat of at least 34 bases (TR34) within the promoter of cyp51A, the gene encoding the azole drug target sterol C14-demethylase. Here we demonstrate that the repeat sequence in TR34 is bound by both the sterol regulatory element binding protein (SREBP) SrbA, and the CCAAT binding complex (CBC). We show that the CBC acts complementary to SrbA as a negative regulator of ergosterol biosynthesis and show that lack of CBC activity results in increased sterol levels via transcriptional derepression of multiple ergosterol biosynthetic genes including those coding for HMG-CoA-synthase, HMG-CoA-reductase and sterol C14-demethylase. In agreement with these findings, inactivation of the CBC increased tolerance to different classes of drugs targeting ergosterol biosynthesis including the azoles, allylamines (terbinafine) and statins (simvastatin). We reveal that a clinically relevant mutation in HapE (P88L) significantly impairs the binding affinity of the CBC to its target site. We identify that the mechanism underpinning TR34 driven overexpression of cyp51A results from duplication of SrbA but not CBC binding sites and show that deletion of the 34 mer results in lack of cyp51A expression and increased azole susceptibility similar to a cyp51A null mutant. Finally we show that strains lacking a functional CBC are severely attenuated for pathogenicity in a pulmonary and systemic model of aspergillosis.
Asunto(s)
Aspergilosis/metabolismo , Aspergillus fumigatus/metabolismo , Factor de Unión a CCAAT/metabolismo , Farmacorresistencia Fúngica/fisiología , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Antifúngicos , Azoles , Inmunoprecipitación de Cromatina , Sistema Enzimático del Citocromo P-450/metabolismo , Modelos Animales de Enfermedad , Proteínas Fúngicas/metabolismo , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , Esteroles/biosíntesisRESUMEN
The filamentous fungus Trichoderma reesei is a potent cellulase producer and the best-studied cellulolytic fungus. A lot of investigations not only on glycoside hydrolases produced by T. reesei, but also on the machinery controlling gene expression of these enzyme have made this fungus a model organism for cellulolytic fungi. We have investigated the T. reesei strain including mutants developed in Japan in detail to understand the molecular mechanisms that control the cellulase gene expression, the biochemical and morphological aspects that could favor this phenotype, and have attempted to generate novel strains that may be appropriate for industrial use. Subsequently, we developed recombinant strains by combination of these insights and the heterologous-efficient saccharifing enzymes. Resulting enzyme preparations were highly effective for saccharification of various biomass. In this review, we present some of the salient findings from the recent biochemical, morphological, and molecular analyses of this remarkable cellulase hyper-producing fungus.
Asunto(s)
Celulasa/biosíntesis , Hongos/metabolismo , Trichoderma/metabolismo , Biomasa , Celulasa/química , Celulasa/genética , Celulosa/química , Celulosa/metabolismo , Hongos/química , Hongos/genética , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Japón , Mutación , Trichoderma/química , Trichoderma/genéticaRESUMEN
Although lactose is a preferred cellulase inducer in the industrial production of cellulase by Trichoderma reesei, the mechanism of induction is not fully understood. Because sugar transporters might be involved at an early step of induction by oligosaccharides, we sought permeases associated with cellulase induction by lactose. Two such MFS sugar transporters in the T. reesei hyper-cellulolytic PC-3-7 strain, an industrial cellulase producer developed in Japan, were identified in a screening for lactose permeases. Disruption of the genes encoding these two transporters resulted in decreased lactose uptake and delayed growth in lactose culture. Further, the deletion strains produced less cellulase when cultivated on lactose. No substantial differences were observed in cellulase production when PC-3-7 was cultivated in cellulose-based medium. The present work provides evidence that these transporters are critical for cellulase production in lactose culture.
Asunto(s)
Proteínas Bacterianas/metabolismo , Celulasa/biosíntesis , Lactosa/farmacología , Trichoderma/crecimiento & desarrollo , Trichoderma/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico , Celulosa/metabolismo , Medios de Cultivo/química , Técnicas de Cultivo , Inducción Enzimática/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación , Filogenia , Trichoderma/efectos de los fármacos , Trichoderma/enzimologíaRESUMEN
The ascomycete Trichoderma reesei is known as one of the most prolific producers of plant biomass-degrading enzymes. While several mutant strains have been developed by mutagenesis to improve enzyme productivity for a variety of industrial applications, little is known about the mechanical basis of these improvements. A genomic sequence comparison of mutant and wild-type strains was undertaken to provide new insights in this regard. We identified a number of single-nucleotide polymorphisms (SNPs) after sequencing the genome of a hyper-cellulolytic T. reesei strain, PC-3-7, with a next-generation sequencer. Of these, the SNP detected in cre1, the carbon catabolite repressor gene, was found to be responsible for increased cellulase production. Further comparative genomic analysis enabled the identification of an SNP that correlated well with high cellulase production in a T. reesei mutant. These results provide a better understanding of the genetic changes induced by classical mutagenesis and how they correlate with desirable phenotypes in filamentous fungi.
Asunto(s)
Celulosa/metabolismo , Mutación , Polimorfismo de Nucleótido Simple , Trichoderma/genética , Secuencia de Aminoácidos , Celulasa/biosíntesis , ADN de Hongos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Glucosa/metabolismo , Japón , Datos de Secuencia Molecular , Mutagénesis , Estructura Terciaria de Proteína , Trichoderma/enzimología , Trichoderma/metabolismoRESUMEN
Ethanol production by Flammulina velutipes from high substrate concentrations was evaluated. F. velutipes produces approximately 40-60 g l(-1) ethanol from 15% (w/v) D-glucose, D-fructose, D-mannose, sucrose, maltose, and cellobiose, with the highest conversion rate of 83% observed using cellobiose as a carbon source. We also attempted to assess direct ethanol fermentation from sugarcane bagasse cellulose (SCBC) by F. velutipes. The hydrolysis rate of 15% (w/v) SCBC with commercial cellulase was approximately 20%. In contrast, F. velutipes was able to produce a significant amount of ethanol from 15% SCBC with the production of ß-glucosidase, cellobohydrolase, and cellulase, although the addition of a small amount of commercial cellulase to the culture was required for the conversion. When 9 mg g(-1) biomass of commercial cellulase was added to cultures, 0.36 g of ethanol was produced from 1 g of cellulose, corresponding to an ethanol conversion rate of 69.6%. These results indicate that F. velutipes would be useful for consolidated bioprocessing of lignocellulosic biomass to bioethanol.
Asunto(s)
Celulasa/química , Celulosa/metabolismo , Etanol/metabolismo , Flammulina/metabolismo , Celulasa/metabolismo , Celulosa/química , Fermentación , Hidrólisis , Microbiología IndustrialRESUMEN
BglR (PI: 52368, beta-glucosidaseregulator) was identified as a new transcription factor that up-regulates expression of specific genes encoding ß-glucosidases. Based on a comparative genomic analysis to verify SNPs between Trichoderma reesei mutant PC-3-7 and its parent KDG-12, 19 were confirmed. One of the SNPs was found to cause a missense mutation close to the end of the DNA-binding region of BglR that turned out to be a Zn(II)(2)Cys(6)-type fungal-specific transcription factor. BglR was found to share little homologous to amyR of Aspergillus oryzae that is commonly considered a key regulator of starch degradation. A mutant lacking the bglr gene as well as the PC-3-7 mutant exhibited elevated cellulase production during growth on cellobiose. Reversion of the SNP missence mutation within bglr to the wild-type allele resulted in reduced cellulase production. Expression of specific ß-glucosidase genes in a bglr gene disruptant was repressed with the mutant exhibiting little ability to hydrolyze cellobiose during early log phase even when induced. Thus, one of the functions of BglR is to up-regulate specific ß-glucosidase genes (with the exception of bgl1, which is seemingly under the direct control of Xyr1). The glucose produced then triggers carbon catabolite repression in cellobiose culture.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Factores de Transcripción/metabolismo , Transcripción Genética , Trichoderma/enzimología , Trichoderma/genética , beta-Glucosidasa/biosíntesis , Secuencia de Aminoácidos , Aspergillus oryzae/enzimología , Aspergillus oryzae/genética , Celobiosa/metabolismo , Celulasa/metabolismo , Eliminación de Gen , Datos de Secuencia Molecular , Mutación Missense , Homología de Secuencia de Aminoácido , Supresión Genética , Factores de Transcripción/genética , Trichoderma/crecimiento & desarrollo , Trichoderma/metabolismoRESUMEN
One of the limiting factors for the application of Trichoderma reesei to degrade cellulosic biomass is its low beta-glucosidase activity, required to convert cellobiose to glucose. The egl3 and the xyn3 promoters were used to express beta-glucosidase 1 gene bgl1 through homologous recombination to improve the cellulose degradation ability of T. reesei. The recombinant strains expressing beta-glucosidase 1 (BGLI) under the control of either the egl3 or the xyn3 promoter had 4.0 and 7.5 fold higher beta-glucosidase activity than the native strain, which compares well to the finding that in wild-type T. reesei PC-3-7, the levels of egl3 and xyn3 mRNA expression were 6.0 and 12 fold higher respectively than that of bgl1. Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis of proteins secreted by the recombinant strains demonstrated that BGLI was overproduced. The increase in the transcription of bgl1 and the concomitant elevated level of BGLI in these recombinant strains were sufficient to degrade the cellobiose and cellotriose formed during the degradation of pretreated cedar powder.
Asunto(s)
Celulasa/genética , Espacio Extracelular/metabolismo , Regiones Promotoras Genéticas/genética , Ingeniería de Proteínas/métodos , Trichoderma/enzimología , Xilosidasas/genética , beta-Glucosidasa/biosíntesis , Animales , Bovinos , Cedrus/metabolismo , Celobiosa/metabolismo , Perfilación de la Expresión Génica , ARN Mensajero/metabolismo , Trichoderma/metabolismo , beta-Glucosidasa/genéticaRESUMEN
The transcriptional activator XYR1 is the central regulator that governs cellulolytic and xylanolytic gene expression in Trichoderma reesei. However, despite its biological importance, relatively little is known about its functional binding sequences. In the present study, we investigated the binding characteristics and specific target for XYR1 by using DNase I footprinting analysis and electrophoretic mobility shift assays. We demonstrate that XYR1 can interact not only with the 5'-GGCTAA-3' motif but also with several 5'-GGC(A/T)(3)-3' motifs. In silico analysis revealed that the 5'-GGC(A/T)(3)-3' motifs are widespread as single site in 5'-upstream region of all the XYR1-regulated genes. Furthermore, we defined the important nucleotides within the binding site that contribute to specific interaction with XYR1. Our results suggest that, together with the inverted repeat motifs, the single 5'-GGC(A/T)(4)-3' motifs play important roles as functional XYR1-binding sites in the regulation of cellulase and xylanase gene expression in T. reesei.
Asunto(s)
Sitios de Unión , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Redes y Vías Metabólicas/genética , Elementos Reguladores de la Transcripción , Transactivadores/metabolismo , Trichoderma/fisiología , Celulosa/metabolismo , Huella de ADN/métodos , Ensayo de Cambio de Movilidad Electroforética/métodos , Genes Fúngicos , Unión Proteica , Trichoderma/genética , Xilanos/metabolismoRESUMEN
Comprehensive analyses on promoters of four cellulase and one xylanase genes of Trichoderma reesei were performed expressing a single reporter uidA from Escherichia coli to construct highly functional cellulase-overproducing strains. GUS amount expressed under each promoter correlated entirely with each mRNA amount, suggesting that GUS production was controlled at the transcriptional level. The uidA transcript levels were much lower than the native gene mRNAs, but they were produced in proportion to the mRNA of native cellulase and xylanase genes driven by the same promoters except for the cbh2 promoter. Cellulose-degrading activity and protein amount was reduced in cbh1 and cbh2 disruptant mutants compared to the wild-type T. reesei PC-3-7 and other uidA transformants. The cbh1 disruptant strain was observed to produce more CBH II, EG I, EG III, and xylanases than native PC-3-7 and the other uidA transformants with the same amounts of protein in SDS-PAGE gels. This observation was further analyzed by measuring mRNA levels of cellulase and xylanase genes in the disruptants using quantitative real-time PCR. In the Pcbh1-gus, mRNA levels for cbh2 and egl1 genes were higher than those in native T. reesei PC-3-7 and all other disruptant strains. The cbh2 disruptant strain had the highest amount of cbh1 mRNA among the strains tested. Homologous integration of uidA at the egl1, egl3, and xyn3 loci was also found to cause a slight increased level of cbh1 mRNA, whereas mRNA levels for egl1, egl3, and xyn3 in all the disruptants were similar to those of T. reesei PC-3-7.
Asunto(s)
Celulasas/genética , Endo-1,4-beta Xilanasas/genética , Regiones Promotoras Genéticas , Trichoderma , Celulasas/metabolismo , Celulosa/metabolismo , ADN Recombinante/análisis , ADN Recombinante/biosíntesis , Electroforesis en Gel de Poliacrilamida , Endo-1,4-beta Xilanasas/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Genes Bacterianos , Genes Reporteros , Microbiología Industrial/métodos , ARN Mensajero/biosíntesis , ARN Mensajero/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Trichoderma/enzimología , Trichoderma/genéticaRESUMEN
The xylanase III gene (xyn3) from the filamentous fungus Trichoderma reesei PC-3-7 is only induced by cellulose, its derivatives and L-sorbose, but not by xylan. In this study, we defined three cis-acting elements within the xyn3 upstream region by using detailed deletion and mutation analysis. In addition to the Xyr1/ACEII-binding motif (5'-GGCTAA-3'), the analogous motifs 5'-GGCTAT-3' and 5'-GGCAAA-3' presented as an inverted repeat spaced by 16-bp internal sequences were identified as essential elements for xyn3 expression. Electrophoretic mobility shift assay using heterologously expressed Xyr1 demonstrates that all the identified cis-acting elements are able to interact with Xyr1. Furthermore, no xyn3 transcripts were formed in the xyr1-knockout strain upon induction by sophorose and L-sorbose. These results indicate that xyn3 expression is transcriptionally regulated by Xyr1, and suggest that the 5'-GGCTAT-3' and 5'-GGCAAA-3' motifs play roles in Xyr1-mediated cellulase and xylanase gene expression in T. reesei.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Elementos de Respuesta , Transcripción Genética , Trichoderma/enzimología , Trichoderma/genética , Xilosidasas/genética , Secuencia de Bases , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Reporteros , Eliminación de Secuencia , Trichoderma/metabolismo , Xilosidasas/metabolismoRESUMEN
In the filamentous fungus Trichoderma reesei, endoglucanase III (EGIII) is coordinately expressed with other cellulases during growth on cellulose, its derivatives, and L-sorbose. To elucidate EGIII induction mechanism, we cloned and sequenced the upstream region of egl3 encoding EGIII. Two GGCTAA motifs, a putative binding site for ACEII and xylanase regulator Xyr1, were found on the template strand of the egl3 upstream region. Deletion analysis of the egl3 upstream region using the beta-glucuronidase (GUS) reporter system revealed that removal of regions containing the GGCTAA motifs and the region between -1,045 and -1,002 bp containing GGCTAT motif severely affected GUS inducibility. Furthermore, mutation of the two GGCTAA motifs and the GGCTAT motif of this region led to a significant decrease in GUS activity. These data indicate that both GGCTAA and GGCTAT are key motifs for egl3 expression, and that egl3 induction may also be controlled by Xyr1. This hypothesis was supported by in vitro electrophoretic mobility shift assay, in which heterologously expressed Xyr1 specifically bound not only GGCTAA but also GGCTAT motif.