Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• We established that protein glycosylation plays a critical role in protein secretion and hydrolytic activity in industrially important Trichoderma fungi.
• We established a close correlation between glycosylation status and the creation of pathogenic features in Candida albicans.
• We showed that DPM synthase from S. cerevisiae creates a complex with the regulatory subunit Yil102c-A, a homologue of the DPMII subunit from Trichoderma.
• We found that filamentous fungi that were isolated from natural resources could outgrow sewage waste, reducing its weight and humidity.
• We established that municipal waste that is overgrown with fungi loses its odor.

Opis badań

• Candida albicans: defects in the glycosylation-limited formation of pathogenic features

To study consequences of glycosylation defects in Candida, several genes were cloned and analyzed for homology based on heterologous expression in mutants of S. cerevisiae (Juchimiuk et al. 2014 [RER2 gene]; Juchimiuk et al. 2015 [DPM1, DPM2, DPM3 genes); Niewiadomska et al. 2017 (ALG13 gene); Janik et al. 2020 (CWH8 gene); DFG10 and ROT1 genes [unpublished]). Next, we created Candida mutants using the URA blaster method. The analyzed genes are mostly essential. To study their function in Candida, one copy of the gene was deleted, and the second was expressed under the regulatory promoter MET3 (repressed by methionine) or TRp (repressed by doxycycline). We created dpm1∆/TRpDPM1, dpm2∆/TRpDPM2, dpm3∆/TRpDPM3, and alg13∆/TRp-ALG13 (mutants with impairments in in protein glycosylation) and rer2Δ/pMET3RER2, dfg10Δ/dfg10Δ, and cwh8Δ/cwh8Δ (mutants with alterations of dolichol synthesis).

The transition of Candida from commensal to pathogenic includes the morphological transition between yeast and hyphal forms, the expression of adhesins and invasins on the cell surface, the formation of biofilm, and the secretion of hydrolytic enzymes. We found that this transition can be affected by changes in protein glycosylation and dolichol synthesis. Our mutants exhibited a decrease in glycosylation and the activity of model N– and O-glycosylated proteins. Adhesins and hydrolytic enzymes are heavily glycosylated, and we observed a direct effect of our mutations on their modification and activity. Furthermore, we found that our mutants exhibited significant alterations of cell wall carbohydrates. All mutations that generally resulted in a low concentration of mannose in the cell wall mostly compensated with higher chitin content, with the exception of the ALG13 mutant. Finally, all of the mutants exhibited impairments in the yeast-to-hyphae transition and biofilm formation.

• Biocontrol activity

Soilborn Trichoderma spp are ubiquitous colonizers of cellulosic materials and can thus often be found where decaying plant material is available and in the rhizosphere of plants where they can promote systemic resistance to pathogens. The biocontrol activity of Trichoderma depends on the efficient secretion of enzymes that hydrolyze the cell wall of fungal pathogens. These enzymes are heavily glycosylated. The effective synthesis of these enzymes requires efficient glycosylation; otherwise, unfolded proteins accumulate and cause endoplasmic reticulum stress. We enhanced glycosylation potential by overexpressing DPM synthase in T. atroviride (Zembek et al. 2011; Graczyk et al. 2020; patent no. P-401450, P-402603). Transformants exhibited two-times higher protein secretion, significantly higher hydrolytic enzyme activity, the higher protection of germinating seeds against pathogens, and the promotion of plant growth. We also showed importance of the mevalonate pathway in the biocontrol activity of Trichoderma. Overexpression of the ERG20 gene (which encodes farnesyl pyrophosphate synthase, an enzyme that produces substrates for the synthesis of dolichol and sterols) elevated the activity of glycosylation and enhanced plant protection against fungal pathogens.

Unexpectedly, we observed that overexpression of the ERG20 gene from S. cerevisiae in Trichoderma (Graczyk et al. 2020) enhanced the sterol branch of the mevalonate pathway, whereas overexpression of the erg20 gene from Trichoderma resulted in enhancement of the dolichol branch and a significant decrease in sterol branch activity (Piłsyk et al. 2014). Despite this, ergosterol levels remained unchanged in both transformants.

• Structure of dolichols regulates the activity of dolichol-dependent enzymes and alters physicochemical properties of model membranes

Glycosylation in fungi depends on dolichol phosphate, a lipid carrier of carbohydrates. Filamentous fungi produced original poly-saturated dolichols (Gryz et al. 2019). We showed that Trichoderma had dolichols with up to 10 additional saturated bonds that were located at their omega end. To determine the consequences of the presence of such dolichols in the cell, we compared the influence of typical mono-saturated dolichols vs. poly-saturated dolichols on the activity of dolichyl phosphate mannose (DPM) synthase and the properties of model lipid membranes. Our study revealed that poly-saturated dolichols were a poor substrate for DPM synthase, even for this enzyme from Trichoderma. Studies of model membranes showed that the two types of dolichols differed in their membrane-destabilizing effects, which also depended on membrane composition (Gryz et al. 2019). Our results suggest a regulatory role for the dolichol structure in dolichol-dependent enzymatic activity directly or via the membrane structure.

• Gene silencing by its overexpression (Górka-Nieć et al. 2011)

We found that the integration of additional copies of the pmt1 gene into the genome of T. reesei results in the silencing of gene expression, and the mechanism of this silencing was related to DNA methylation. Such a mechanism has not been observed previously in Trichoderma. In Acobolus immersus, methylation was induced premeiotically, and repeated genes that were obtained by integrative transformation lost their expression during the sexual phase. We observed this mechanism, although Trichoderma as an imperfect fungus does not reproduce sexually. The silencing of pmt1 expression resulted in the lower activity of mannosyltransferases, the lower glycosylation of secreted proteins, and changes in cell wall composition.

• Fungal cell wall

We found that structure and composition of the cell wall in fungi is influenced by protein glycosylation (Górka-Nieć et al. 2011; Janik et al. 2012; Kruszewska and Piłsyk 2013; Juchimiuk et al. 2014; Niewiadomska et al. 2017; in addition to our earlier studies). In our review article, we presented current knowledge of the ways in which targeted changes in the structure of the cell wall can lead to the emergence of super secretory strains that are important for industry (Kruszewska and Piłsyk 2013).

We also suggested that the cell wall is a natural target for antifungal drugs. Knowledge about the cell wall structure in pathogenic fungi may inspire future searches for new cell wall-related targets beyond the currently used inhibitors of chitin synthase (nikkomycins) and β-1,3-glucan synthase (echinocandins; Muszewska et al. 2018).

• Three species of Trichoderma: genomic and lifestyle differences

We compared genome integrity, chromatin, photobiology, glycobiology, lipids, and sulfur and nitrogen metabolism in T. reesei, T. atroviride, and T. virens. Highlights of our analyses included overall carbohydrate cleavage preference that was attributable to the different genomic contents and regulation of respective genes. We also compared mycoparasitic and defense activities that were largely lost in T. reesei during evolution (Schmoll et al. 2016).

• Fungi in fungi

The fungal microbiome of fruiting bodies of Tuber species has not been previously analyzed. We found fruiting bodies of T. aestivum that were collected in Poland host bacteria, yeast, and filamentous fungi and discussed possible causes of differences among specimens.

Metodologia

Standard methods of genetic engineering are used (cloning, sequencing, transformation, DNA and RNA analysis, electrophoresis, and hybridization, among others). Biochemical analysis is performed using radiolabeled substrates, chemical analysis, spectrophotometric methods, and chromatography (TLC, HPLC). Microbiological methods include cultivation, sensitivity to drugs, microscopy, and stereoscopy.

Unique equipment includes a Dionex ICS-3000 Ion Chromatography System with a Carbo Pac PA10 analytical column that is used for carbohydrate analysis.

Wybrane publikacje

• Schmoll M., Dattenböck Ch., Carreras-Villaseñor N., Mendoza-Mendoza A., Tisch D., Alemán M.I., Baker S.E., Brown Ch., Cervantes-Badillo M.G., Cetz-Chel J., Cristobal-Mondragon G.R., Delaye L., Esquivel-Naranjo E.U., Frischmann A., Gallardo-Negrete J. J., García-Esquivel M., Gomez-Rodriguez E.Y., Greenwood D.R., Hernández-Oñate M., Kruszewska J.S., Lawry R., Mora-Montes H.M., Muñoz-Centeno T., Nieto-Jacobo M.F., Lopez G.N., Olmedo-Monfil V., Osorio-Concepcion M., Piłsyk S., Pomraning K.R., Rodriguez-Iglesias A., Rosales-Saavedra M.T., Sánchez-Arreguín J.A., Seidl-Seiboth V., Stewart A., Uresti-Rivera E.E., Wang Chih-L., Wang T.F., Zeilinger S., Casas-Flores S., Herrera-Estrella A. (2016) The genome of three uneven siblings : foodprints of the lifestyles of three Trichoderma species. Microbiol. Mol. Biol Rev. 80, 205-327. doi: 10.1128/MMBR.00040-15.
• Niewiadomska, M., Janik, A., Perlińska-Lenart, U., Piłsyk, S., Palamarczyk, G., Kruszewska J.S. (2017) The role of Alg13 N-acetylglucosaminyl transferase in the expression of pathogenic features of Candida albicans. BBActa General Subjects 1861, 789-801. doi: 10.1016/j.bbagen.2017.01.019
• Graczyk S, Perlinska-Lenart U, Górka-Nieć W, Lichota R, Piłsyk S, Zembek P, Lenart J, Bernat P, Gryz E, Augustyniak J, Palamarczyk G, Kruszewska JS (2020) Increased activity of the sterol branch of the mevalonate pathway elevates glycosylation of secretory proteins and improves antifungal properties of Trichoderma atroviride. Fungal Gen. Biol. 137, 103334. doi: 10.1016/j.fgb.2020.103334
• Perlinska-Lenart U, Piłsyk S, Gryz E, Turło J, Hilszczańska D, Kruszewska JS (2020) Identification of bacteria and fungi inhabiting fruiting bodies of Burgundy truffle (Tuber aestivum Vittad.) Archives of Microbiology 202, 2727-2738 doi: 10.1007/s00203-020-02002-x.

Współpraca

• Prof. Jerzy Długoński, prof. Przemysław Bernat – University of Lodz
• Prof. Kazimierz Strzałka, dr Małgorzata Jemioła-Rzemińska – Jagiellonian University, Cracov
• Prof. Krzysztof Ginalski, dr hab. Kamil Steczkiewicz,– CENT Warsaw University
• Dr Hanna Gawińska-Urbanowicz- The Plant Breeding and Acclimatization Institute, Bonin
• Prof. Elżbieta Salińska, dr Jacek Lenart, dr Justyna Augustyniak - Mossakowski Medical Research Centre, PAS
• Prof. Jadwiga Turło – Warsaw Medical University
• Dr hab. Dorota Hilszczańska – Forest Research Institute, Sękocin
• Prof. Ewa Świeżewska, dr hab. Anna Muszewska Dr Adam Mieczkowski Prof. Andrzej Paszewski, dr Marzena Sieńko, dr Renata Natorff, dr hab. Marek Skoneczny - Institute of Biochemistry and Biophysics PAS
• Dr Agata Wawrzyniak –University of Rzeszow
• Mgr Dariusz Teresiński, dr Krzysztof Nowak dr Andrzej Skalmowski mgr Piotr Szymanek – Demeter Ltd
• Dr hab. Prof. PW Artur Badyda Warsaw Technical University
• Dr Gabriela Smoleńska-Sym National Institute of Public Health
• Prof. Robert Mach, dr Kurt Brunner, dr Barbara Reithner – Technical University of Vienna, Austria
• Dr Monika Schmoll – Technological Institute of Tulln, Austria