Extracellular vesicles carry cellulases in the industrial fungus Trichoderma reesei

Abstract

Background: Trichoderma reesei is the most important industrial producer of lignocellulolytic enzymes. These enzymes play an important role in biomass degradation leading to novel applications of this fungus in the biotechnology industry, specifically biofuel production. The secretory pathway of fungi is responsible for transporting proteins addressed to different cellular locations involving some cellular endomembrane systems. Although protein secretion is an extremely efficient process in T. reesei, the mechanisms underlying protein secretion have remained largely uncharacterized in this organism.

Results: Here, we report for the first time the isolation and characterization of T. reesei extracellular vesicles (EVs). Using proteomic analysis under cellulose culture condition, we have confidently identified 188 vesicular proteins belonging to different functional categories. Also, we characterized EVs production using transmission electron microscopy in combination with light scattering analysis. Biochemical assays revealed that T. reesei extracellular vesicles have an enrichment of filter paper (FPase) and β-glucosidase activities in purified vesicles from 24, 72 and 96, and 72 and 96 h, respectively. Furthermore, our results showed that there is a slight enrichment of small RNAs inside the vesicles after 96 h and 120 h, and presence of hsp proteins inside the vesicles purified from T. reesei grown in the presence of cellulose.

Conclusions: This work points to important insights into a better understanding of the cellular mechanisms underlying the regulation of cellulolytic enzyme secretion in this fungus.

Keywords: Cellulases; Extracellular vesicles; Proteome; Secretion; Trichoderma reesei.

Fig. 1

Nanoparticle-tracking analysis (NTA) of extracellular vesicles (EVs) produced by T. reesei. a Histogram showing the EV particle-size distribution (EVs × 10⁸/mL vs size in nanometers) from the cellulose-supernatant culture at 24, 48, 72, 96 and 120 h post-induction in the presence of cellulose. b Histogram showing the calculated mean ± SD of size distribution by NTA analysis of purified T. reesei EVs. c Screenshots from video recorded using NanoSight NS300, showing the distribution of EVs from the cellulose-supernatant culture at 24, 48, 72, 96 and 120 h. These results are based on three replicates of three independent experiments

Fig. 2

TEM analyses of vesicles in Trichoderma reesei mycelium cells. a–d The occurrence of vesicles in association with the cytoplasmic membrane and cell wall is evident after growth for 48 h in the presence of cellulose. Black arrows indicate the cell wall (CW) and cell membrane (CM). Red arrows indicate the T. reesei vesicles. Bars, 500 nm (a–c) and 200 nm (d)

Fig. 3

Proteomic analysis of T. reesei EVs. a Expression pattern of the proteins identified in T. reesei EVs at 24, 48, 72, 96 and 120 h in the presence of cellulose. Venn diagram clustering was designed using Bioinformatics & Evolutionary Genomics tools (http://bioinformatics.psb.ugent.be/webtools/Venn/). b KOG term of 188 proteins identified in T. reesei EVs. c Molecular function and biological process of proteins identified at 24, 48, 72, 96 and 120 h in the presence of cellulose. The proteins are classified in terms of Gene Ontology (GO) being molecular function related to proteins features, such as binding or catalysis, and finally, biological process, classified the proteins according to the processes that it involved. These results are based on three replicates of three independent experiments

Fig. 4

Cellulolytic activities from culture supernatant and purified T. reesei EVs after induction in the presence of cellulose. a Filter paper activity (FPase) and b β-glucosidase activity from culture supernatant (blue bars) and purified T. reesei EVs (red bars) grown at 24, 48, 72, 96 and 120 h in the presence of cellulose. ****Significantly different (P < 0.001). These results are based on three replicates of three independent experiments and are expressed as mean ± standard deviation