Proteome profiling of enriched membrane-associated proteins unraveled a novel sophorose and cello-oligosaccharide transporter in Trichoderma reesei

BackgroundTrichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized.ResultsTo address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose.ConclusionThis study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production

Phosphoproteomic analysis of the fungus Trichoderma reesei during degradation of sugar cane bagasse

Trichoderma reesei é o principal produtor de celulases e hemicelulases. Entretanto, a regulação da expressão dessas enzimas não é totalmente compreendida. Sabe-se que os eventos de fosforilação estão envolvidos na regulação de vários processos, incluindo metabolismo, transcrição, regulação da tradução, degradação de proteínas, homeostase, sinalização e secreção proteica, entre outros. Em T. reesei, o padrão de fosforilação da proteína muda de maneira dependente da fonte de carbono. Para compreender os eventos de fosforilação e como este processo afeta diferentes funções celulares em T. reesei em presença de bagaço de cana-de-açúcar, a principal biomassa para produção de bioetanol no Brasil, realizamos um experimento no qual identificamos proteínas fosforiladas por LC-MS / MS. Um total de 114 proteínas fosforiladas foram identificadas. Fosfosserina e fosfotreonina (ST) corresponderam a 89,6% dos fosfitoses e 10,4% relativos à fosfotirosina (Y). Entre as proteínas identificadas, 65% são fosforiladas isoladamente, 19% são duplamente fosforiladas, 12% são triplamente fosforiladas e 4% são fosforiladas, incluindo um peptídeo que apresentou 11 fosfopeptídeos. Após a aplicação dos critérios de filtragem NetworKIN (Score String ≥0,6 e Escore NetworKIN ≥1,0), 75 kinases foram preditas para fosforilar os locais identificados neste trabalho, compreendendo 19 famílias de serina / treonina quinase para todos os locais de fosforilação identificados. As famílias de serina / treonina quinase mais frequentes foram PKC1. Entre as proteínas fosforiladas, quatro hidrolases glicosiladas eram conhecidas por serem secretadas (uma glucuronidase, uma beta-xilosidase, uma exo-glucanase e uma celobiohidrolase - CBHI). O CBHI foi purificado a partir do secretoma de T. reesei e desfosforilado para detectar qualquer diferença de atividade enzimática causada pela fosforilação. A atividade da CBHI foi cerca de 60% menor após a desfosforilação. O padrão de fosforilação ampla que ocorre em T. reesei quando cultivado em bagaço de cana-de-açúcar fornece novas informações sobre várias proteínas não descritos anteriormente. Este trabalho mostra pela primeira vez que as glicosil-hidrolases secretadas são fosforiladas na condição testada. Nossos resultados sugerem que a atividade da CBHI é modulada pela fosforilação.Trichoderma reesei is the major producer of cellulases and hemicellulases. However, the regulation of these enzymes expression is not fully understood. Phosphorylation events are known to be involved in the regulation of various processes, including metabolism, transcription, translation regulation, protein degradation, homeostasis, signaling, and protein secretion, among others. In T. reesei, protein phosphorylation pattern changes in a carbon dependent way. In order to have a broad understanding of the phosphorylation events and how it affects different cellular functions, once T. reesei is grown in sugarcane bagasse, the main biomass for bioethanol production in Brazil, we conducted an experiment in which we identified phosphorylated proteins by LC-MS/MS. A total of 114 phosphorylated proteins were identified. Phosphoserine and phosphotreonine (ST) corresponded to 89.6% of the phosphosites and 10.4% relative to phosphotyrosine (Y). Among the proteins identified, 65% are singly phosphorylated, 19% are doubly phosphorylated, 12% are triply phosphorylated and 4% are higher phosphorylated, including one peptide that presented 11 phosphopeptides. After applying NetworKIN filtering criteria (String score ≥0.6 and NetworKIN score ≥1.0), 75 kinases were predicted to phosphorylate the sites identified in this work, comprising 19 serine/threonine kinase families for all identified phosphorylation sites. The most frequent serine/threonine kinase families were PKC1. Among the phosphorylated proteins, four glycosyl hydrolases supposed to be secreted were identified (a glucuronidase, a beta-xylosidase, an exo-glucanase and a cellobiohydrolase - CBHI). CBHI was purified from the secretome of T. reesei and dephosphorylated in order to detect any enzymatic activity difference caused by phosphorylation. CBHI activity was about 60% less after dephosphorylation. The broad phosphorylation pattern that happens in T. reesei when it is cultivated in sugarcane bagasse provides novel information about several proteins and phosphosites not previously described. This work shows for the first time that secreted glycosyl hydrolases are phosphorylated in the condition tested. Our results suggest CBHI activity is modulated by phosphorylation

Deletion of pH Regulator pac-3 Affects Cellulase and Xylanase Activity during Sugarcane Bagasse Degradation by Neurospora crassa.

Microorganisms play a vital role in bioethanol production whose usage as fuel energy is increasing worldwide. The filamentous fungus Neurospora crassa synthesize and secrete the major enzymes involved in plant cell wall deconstruction. The production of cellulases and hemicellulases is known to be affected by the environmental pH; however, the regulatory mechanisms of this process are still poorly understood. In this study, we investigated the role of the pH regulator PAC-3 in N. crassa during their growth on sugarcane bagasse at different pH conditions. Our data indicate that secretion of cellulolytic enzymes is reduced in the mutant Δpac-3 at alkaline pH, whereas xylanases are positively regulated by PAC-3 in acidic (pH 5.0), neutral (pH 7.0), and alkaline (pH 10.0) medium. Gene expression profiles, evaluated by real-time qPCR, revealed that genes encoding cellulases and hemicellulases are also subject to PAC-3 control. Moreover, deletion of pac-3 affects the expression of transcription factor-encoding genes. Together, the results suggest that the regulation of holocellulase genes by PAC-3 can occur as directly as in indirect manner. Our study helps improve the understanding of holocellulolytic performance in response to PAC-3 and should thereby contribute to the better use of N. crassa in the biotechnology industry

Toxoplasma gondii Chitinase Induces Macrophage Activation.

Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 °C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection

Schematic representation of putative PAC-3 binding sites (5′ -BGCCVAGV-3′).

<p>The analysis was performed using the region 1.0-kbp upstream of cellulolytic and xylanolytic genes. The position of the motifs is relative to the translation initiation codon (ATG). B = C or G or T; V = A or C or G, according to International Union of Pure and Applied Chemistry (IUPAC) norms.</p

PAC-3 influences holocellulase activity in <i>N</i>. <i>crassa</i>.

<p>Total cellulolytic (FPase) (A), endoglucanase (CMCase) (B), and xylanolytic activities (C) were assayed in the supernatant of the strains 74A, Δ<i>mus-52</i>, and Δ<i>pac-3</i> after their cultivation for eight days on sugarcane bagasse. Values show the mean of three replicates. The error bar indicates the standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.</p

The model proposed for the regulation of holocellulolytic-encoding genes in <i>N</i>. <i>crassa</i> by PAC-3.

<p>Changes in the pH environment lead to activation of PAC-3, which directly or indirectly regulates transcription of cellulases and xylanases as well as the transcription factors XLR-1, CLR-1, and CLR-2 genes. The transcription factor CLR-1 regulate transcription of both <i>xlr-1</i> and <i>clr-2</i> genes. These transcription factors can also regulate cellulases and xylanases. XLR-1 regulate transcription of <i>gh3-1</i>, <i>gh11-1</i>, and <i>gh43-5</i> genes (purple arrows); CLR-1 regulate transcription of <i>cbh-1</i> and <i>gh7-1</i> genes (green arrows), and CLR-2 regulate transcription of <i>cbh-1</i>, <i>gh7-1</i>, and <i>gh11-1</i> genes (red arrows). (?) Missing component in regulation of <i>cre-1</i> by PAC-3.</p

Effect of <i>pac-3</i> deletion on transcriptional factors gene expression.

<p>The strains 74A, Δ<i>mus-52</i>, and Δ<i>pac-3</i> grown on sugarcane bagasse at different pH (3.0, 5.0, 7.0, 8.0, and 10.0) were used for RT-qPCR experiments. (A) <i>xlr-1</i> = Xylan degradation regulator-1 NCU06971; (B) <i>cre-1</i> = Carbon catabolite regulator NCU08807; (C) <i>clr-1</i> = Cellulose degradation regulator-1 NCU07705 and (D) <i>clr-2</i> = Cellulose degradation regulator-2 NCU08042. Values show the mean of three replicates. The error bar indicates the standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.</p

Gene expression profiles of holocellulolytic enzymes in <i>N</i>. <i>crassa</i>.

<p>The strains 74A, Δ<i>mus-52</i>, and Δ<i>pac-3</i> grown on sugarcane bagasse at different pH (3.0, 5.0, 7.0, 8.0, and 10.0) were used for RT-qPCR experiments. (A) <i>cbh-1</i> = Cellobiohydrolase 1 (exoglucanase) NCU07340; (B) <i>gh7-1</i> = Endoglucanase 1 NCU05057; (C) <i>gh3-2</i> = β-glucosidase 1 NCU08054; (D) <i>gh11-1</i> = Endo-1,4-β-xylanase 1 NCU02855; (E) <i>gh10-4</i> = Endo-1,4-β-xylanase 2 NCU07130; (F) <i>gh43-5</i> = β-xylosidase NCU09652. Values show the mean of three replicates. The error bar indicates the standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.</p

Western blot analysis shows a reduced amount of cellulase (CBHI) at both neutral and alkaline pH.

<p>The analysis was performed using supernatants collected from the <i>N</i>. <i>crassa</i> 74A, Δ<i>mus-52</i>, and Δ<i>pac-3</i> strains after growth for eight days on sugarcane bagasse at pH 3.0, 5.0, 7.0, 8.0, and 10.0. Anti-cellulase antibody from <i>Trichoderma viride</i> were used to detect CBH-1.</p