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Cell Wall Biosynthesis in the Pathogenic Oomycete Saprolegnia Parasitica

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Cell Wall Biosynthesis in the Pathogenic Oomycete Saprolegnia Parasitica Cell wall biosynthesis in the pathogenic oomycete Saprolegnia parasitica Elzbieta Rzeszutek Doctoral Thesis in Biotechnology School of Engineering Sciences in Chemistry, Biotechnology and Health Royal Institute of Technology Stockholm, Sweden 2019 © Elzbieta Rzeszutek, Stockholm, 2019 ISBN: 978-91-7873-370-5 TRITA-CBH-FOU-2019:61 KTH School of Engineering Sciences in Chemistry, Biotechnology and Health Royal Institute of Technology AlbaNova University Center 106 91 Stockholm Sweden Printed by Universitetsservice US-AB, Stockholm 2019 Abstract The oomycete Saprolegnia parasitica is a fungus-like microorganism responsible for the fish disease saprolegniosis, which leads to important economic losses in aquaculture. Currently, there is no efficient method to control the infection and therefore methods for disease management are urgently needed. One of the promising approaches to tackle the pathogen is the inhibition of cell wall biosynthesis, specifically the enzymes involved in carbohydrate biosynthesis. The cell wall of S. parasitica consists mainly of cellulose, β-1,3 and β-1,6-glucans, whereas chitin is present in minute amounts only. The available genome sequence allowed the identification of six putative chitin (Chs) and cellulose (CesA) synthase genes. The main objective of this work was to characterize CHSs and CesAs from S. parasitica and test the effect of cell wall related inhibitors on pathogen growth. The tested inhibitors included nikkomycin Z, a competitive inhibitor of CHS as well as inhibitors of cellulose biosynthesis, namely flupoxam, CGA325'615 and compound I (CI). All drugs strongly reduced the growth of S. parasitica and inhibited the in vitro formation of chitin or cellulose, demonstrated by the use of a radiometric assay. The chemicals also affected the expression of some of the corresponding Chs and CesA genes. One of the CHSs, namely SpCHS5, was successfully expressed in yeast and purified to homogeneity as a full length protein. The recombinant enzyme was biochemically characterized and demonstrated to form chitin crystallites in vitro. Moreover, our data indicate that SpCHS5 most likely occurs as a homodimer which can further assemble into larger multi- subunit complexes. Point mutations of conserved amino acids allowed us to identify the essential residues for activity and processivity of the enzyme. In addition to the cell wall related inhibitors, a biosurfactant naturally produced by Pseudomonas species, massetolide A, was tested, showing strong inhibition of S. parasitica growth. Altogether, our data provide key information on the fundamental mechanisms of chitin and cellulose biosynthesis in oomycetes and the biochemical properties of the enzymes involved. They also demonstrate that the enzymes involved in cell wall biosynthesis represent promising targets for anti-oomycete drugs, even when the corresponding polysaccharides, such as chitin, occur in small amounts in the cell wall. Keywords: Cellulose biosynthesis; chitin biosynthesis; cellulose synthase genes; chitin synthase genes; oomycetes; Saprolegnia parasitica i Sammanfattning Oomyceten Saprolegnia parasitica är en svampliknande mikroorganism som orsakar fisksjukdomen saprolegniosis, vilken leder till stora ekonomiska förluster inom vattenbruk. För närvarande finns det ingen effektiv metod för att kontrollera infektionen och därför är behovet av metoder för att begränsa sjukdom stort. Ett lovande tillvägagångssätt för att angripa patogenen är genom att hämma biosyntes av cellväggen, särskilt de enzymer som är involverade i kolhydratbiosyntes. Cellväggen hos S. parasitica består huvudsakligen av cellulosa, ß-1,3 och ß-1,6- glukaner, samt små mängder kitin. Den tillgängliga genomsekvensen möjliggjorde identifiering av sex förmodade kitin (Chs) och cellulosa (CesA) -syntasgener. Huvudsyftet med denna avhandling var att karakterisera CHS och CesA från S. parasitica och testa effekten av cellväggsrelaterade inhibitorer på patogenens tillväxt. De testade inhibitorerna inkluderade nikcomycin Z, en kompetitiv inhibitor av CHS, samt flera inhibitorer av cellulosabiosyntes, nämligen flupoxam, CGA325'615 och compund I (Cl). Alla inhibitorer reducerade kraftigt tillväxten av S. parasitica och genom användning av en radiometrisk analysmetod visades att in vitro-bildningen av kitin och cellulosa inhiberades. Kemikalierna påverkade också uttrycket av några av Chs- och CesA-generna. En av CHS, nämligen SpCHS5, uttrycktes framgångsrikt i jäst och renades till homogenitet som ett fullängdsprotein. Det rekombinanta enzymet karakteriserades biokemiskt och visade sig bilda kitinkristalliter in vitro. Dessutom indikerar våra data att SpCHS5 troligen är en homodimer som kan bilda större komplex bestående av flera subenheter. Punktmutationer av konserverade aminosyror tillät oss att identifiera de aminosyror som är väsentliga för enzymets aktivitet och processivitet. Förutom de cellväggsrelaterade inhibitorerna testades även biosurfaktanten massetolid A som produceras naturligt av Pseudomonas- arter. Denna visade sig ha en starkt inhiberande verkan på S. parasitica- tillväxt. Sammantaget bidrar våra data med viktig information rörande de grundläggande mekanismerna för biosyntes av kitin och cellulosa i oomyceter och de biokemiska egenskaperna av de involverade enzymerna. Resultaten visar också att enzymerna som är involverade i biosyntes av cellväggen är lovande mål för bekämpningsmedel mot oomyceter även då motsvarande polysackarider, såsom kitin, förekommer i små mängder i cellväggen. Nyckelord: Cellulosabiosyntes; kitinbiosyntes; cellulosasyntasgener; kitin- syntasgener; Oomyceter; Saprolegnia parasitica ii List of publications Publication I Jiang, R. H., de Bruijn, I., Haas, B. J., Belmonte, R., Löbach, L., Christie, J., van den Ackerveken, G., Bottin, A., Bulone, V., Díaz-Moreno, S. M., Dumas, B., Fan, L., Gaulin, E., Govers, F., Grenville-Briggs, L. J., Horner, N. R., Levin, J. Z., Mammella, M., Meijer, H. J. G., Morris, P., Nusbaum, C., Oome, S., Phillips, A. J., van Rooyen, D., Rzeszutek, E., Saraiva, M., Secombes, C. J., Seidl, M. F., Snel, B., Stassen, J. H. M., Sykes,S., Tripathy, S., van den Berg, H., Vega-Arreguin, J. C., Wawra, S., Young, S. K., Zeng, Q.,Dieguez-Uribeondo, J., Russ, C., Tyler, B. M., and Pieter van West (2013). Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica. PLoS Genetics, 9(6), e1003272. Publication II Rzeszutek, E., Díaz-Moreno, S. M., and Bulone, V. (2019). Identification and characterization of the chitin synthase genes from the fish pathogen Saprolegnia parasitica. Accepted, Frontiers in Microbiology. Publication III Rzeszutek*, E., Díaz-Moreno*, S. M., Ampomah, O. Y., Inman, A., Sirvastava, V., Zhou, Q., and Bulone, V. (2019). Novel insights into chitin biosynthesis through heterologous expression and biochemical characterization of chitin synthase 5 from the pathogenic oomycete Saprolegnia parasitica. Manuscript. Publication IV Rzeszutek, E., Díaz-Moreno, S. M., Klinter, S., and Bulone, V. (2019). Analysis of the cellulose synthase genes in the oomycete fish pathogen Saprolegnia parasitica and effect of cellulose biosynthesis inhibitors on enzyme activity and microbial growth. Manuscript. Publication V Liu, Y., Rzeszutek, E., van der Voort, M., Wu, C.-H., Thoen, E., Skaar, I., Bulone, V., Dorrestein, P. C., Raaijmakers, J. M., and De Bruijn, I. (2015). Diversity of aquatic Pseudomonas species and their activity against the fish pathogenic oomycete Saprolegnia. PloS One, 10(8), e0136241. *Both authors contributed equally to the work iii Author’s contribution to the publications Publication I Elzbieta Rzeszutek identified putative Chs genes in the genome of S. parasitica. Publication II Elzbieta Rzeszutek performed the sequence and qPCR analyses of CHSs from S. parasitica, and contributed to the Southern blot experiments. She performed in vitro inhibitory assays, microscope observations, biochemical assays and characterization of the in vitro product. Elzbieta prepared most of the figures and wrote the manuscript with input from all authors. Publication III Elzbieta Rzeszutek performed the expression, purification and biochemical characterization of SpCHS5. She also characterized the in vitro product by enzymatic treatment and glycosidic linkage analysis, and performed cell wall analysis of the yeast S. cerevisiae. Elzbieta prepared the figures and wrote the manuscript together with contributions from the other co-authors. Publication IV Elzbieta Rzeszutek identified putative CesAs genes in S. parasitica and contributed to the qPCR experiment. She performed in vitro inhibitory assays, microscope observations, biochemical assays and in vitro product characterization. Elzbieta also contributed to the preparation of the figures and wrote the manuscript with contributions from all authors. Publication V Elzbieta Rzeszutek contributed to the in vitro inhibitory assays and data analysis. iv List of Abbreviations Bcs Bacterial cellulose synthase CAZy Carbohydrate-active enzymes CesA Cellulose synthase CDB Carbohydrate-binding domain CHS Chitin synthase CI Compound I CLP Cyclic lipopeptides COS Chito-oligosaccharides CWP Cell wall protein DCB 2,6-dichlorobenzonitrile DMS Dimethyl suberimidate GlcNAc N-acetylglucosamine GPI Glycosylphosphatidylinositol GS Glucan synthase GT Glycosyltransferase MIT Microtubule interacting and trafficking domain NZ Nikkomycin Z PH Plekstrin homology domain SuSy Sucrose synthase TC Terminal complex TM Transmembrane domain UDP Uridine
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