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Cell wall biosynthesis in the pathogenic oomycete Saprolegnia parasitica
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Abstract [sv]

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 Chsoch 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 Pseudomonasarter. Denna visade sig ha en starkt inhiberande verkan på S. parasiticatillväxt. 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.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2019. , p. 66
Series
TRITA-CBH-FOU ; 2019:61
Keywords [en]
Cellulose biosynthesis; chitin biosynthesis; cellulose synthase genes; chitin synthase genes; oomycetes; Saprolegnia parasitica
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-263918ISBN: 978-91-7873-370-5 (print)OAI: oai:DiVA.org:kth-263918DiVA, id: diva2:1371170
Public defence
2019-12-16, FB42, Roslagstullsbacken 21, AlbaNova, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 2019-11-19

Available from: 2019-11-19 Created: 2019-11-19 Last updated: 2019-11-19Bibliographically approved
List of papers
1. Distinctive Expansion of Potential Virulence Genes in the Genome of the Oomycete Fish Pathogen Saprolegnia parasitica
Open this publication in new window or tab >>Distinctive Expansion of Potential Virulence Genes in the Genome of the Oomycete Fish Pathogen Saprolegnia parasitica
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2013 (English)In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 9, no 6, p. e1003272-Article in journal (Refereed) Published
Abstract [en]

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

Keywords
Expressed Sequence Tags, Anthocidaris-Crassispina Eggs, Fully Automated Process, Phytophthora-Sojae, Aphanomyces-Euteiches, Plant-Pathogens, Infestans, Cells, Evolution, Reveals
National Category
Genetics
Identifiers
urn:nbn:se:kth:diva-129637 (URN)10.1371/journal.pgen.1003272 (DOI)000321222600001 ()2-s2.0-84879653114 (Scopus ID)
Note

QC 20131003

Available from: 2013-10-03 Created: 2013-10-03 Last updated: 2019-11-19Bibliographically approved
2. Identification and characterization of the chitin synthase genes from the fish pathogen Saprolegnia parasitica
Open this publication in new window or tab >>Identification and characterization of the chitin synthase genes from the fish pathogen Saprolegnia parasitica
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-263916 (URN)
Note

QC 20191125

Available from: 2019-11-19 Created: 2019-11-19 Last updated: 2019-11-25Bibliographically approved
3. Novel insights into chitin biosynthesis through heterologous expression and biochemical characterization of chitin synthase 5 from the pathogenic oomycete Saprolegnia parasitica 
Open this publication in new window or tab >>Novel insights into chitin biosynthesis through heterologous expression and biochemical characterization of chitin synthase 5 from the pathogenic oomycete Saprolegnia parasitica 
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-263864 (URN)
Note

QC 20191125

Available from: 2019-11-18 Created: 2019-11-18 Last updated: 2019-11-25Bibliographically approved
4. 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
Open this publication in new window or tab >>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
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-263865 (URN)
Note

QC 20191121

Available from: 2019-11-18 Created: 2019-11-18 Last updated: 2019-11-21Bibliographically approved
5. Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia
Open this publication in new window or tab >>Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 8, article id e0136241Article in journal (Refereed) Published
Abstract [en]

Emerging fungal and oomycete pathogens are increasingly threatening animals and plants globally. Amongst oomycetes, Saprolegnia species adversely affect wild and cultivated populations of amphibians and fish, leading to substantial reductions in biodiversity and food productivity. With the ban of several chemical control measures, new sustainable methods are needed to mitigate Saprolegnia infections in aquaculture. Here, PhyloChip-based community analyses showed that the Pseudomonadales, particularly Pseudomonas species, represent one of the largest bacterial orders associated with salmon eggs from a commercial hatchery. Among the Pseudomonas species isolated from salmon eggs, significantly more biosurfactant producers were retrieved from healthy salmon eggs than from Saprolegnia-infected eggs. Subsequent in vivo activity bioassays showed that Pseudomonas isolate H6 significantly reduced salmon egg mortality caused by Saprolegnia diclina. Live colony mass spectrometry showed that strain H6 produces a viscosin-like lipopeptide surfactant. This biosurfactant inhibited growth of Saprolegnia in vitro, but no significant protection of salmon eggs against Saprolegniosis was observed. These results indicate that live inocula of aquatic Pseudomonas strains, instead of their bioactive compound, can provide new (micro) biological and sustainable means to mitigate oomycete diseases in aquaculture.

National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-173980 (URN)10.1371/journal.pone.0136241 (DOI)000360299100065 ()26317985 (PubMedID)2-s2.0-84943338927 (Scopus ID)
Note

QC 20151006

Available from: 2015-10-06 Created: 2015-09-24 Last updated: 2019-11-19Bibliographically approved

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