Some oomycetes species are severe pathogens of fish or crops. As such, they are responsible for important losses in the aquaculture industry as well as in agriculture. Saprolegnia parasitica is a major concern in aquaculture as there is currently no method available for controlling the diseases caused by this microorganism. The cell wall is an extracellular matrix composed essentially of polysaccharides, whose integrity is required for oomycete viability. Thus, the enzymes involved in the biosynthesis of cell wall components, such as cellulose and chitin synthases, represent ideal targets for disease control. However, the biochemical properties of these enzymes are poorly understood, which limits our capacity to develop specific inhibitors that can be used for blocking the growth of pathogenic oomycetes. In our work, we have used Saprolegnia monoica as a model species for oomycetes to characterize two types of domains that occur specifically in oomycete carbohydrate synthases: the Pleckstrin Homology (PH) domain of a cellulose synthase and the so-called ‘Microtubule Interacting and Trafficking’ (MIT) domain of chitin synthases. In addition, the chitin synthase activity of the oomycete phytopathogen Aphanomyces euteiches was characterized in vitro using biochemical approaches. The results from our in vitro investigations revealed that the PH domain of the oomycete cellulose synthase binds to phosphoinositides, microtubules and F-actin. In addition, cell biology approaches were used to demonstrate that the PH domain co-localize with F-actin in vivo. The structure of the MIT domain of chitin synthase (CHS) 1 was solved by NMR. In vitro binding assays performed on recombinant MIT domains from CHS 1 and CHS 2 demonstrated that both proteins strongly interact with phosphatidic acid in vitro. These results were further supported by in silico data where biomimetic membranes composed of different phospholipids were designed for interaction studies. The use of a yeast-two-hybrid approach suggested that the MIT domain of CHS 2 interacts with the delta subunit of Adaptor Protein 3, which is involved in protein trafficking. These data support a role of the MIT domains in the cellular targeting of CHS proteins. Our biochemical data on the characterization of the chitin synthase activity of A. euteiches suggest the existence of two distinct enzymes responsible for the formation of water soluble and insoluble chitosaccharides, which is consistent with the existence of two putative CHS genes in the genome of this species. Altogether our data support a role of the PH domain of cellulose synthase and MIT domains of CHS in membrane trafficking and cellular location. / <p>QC 20151014</p>

O aumento da resistência microbiana devido a fatores como uso excessivo e ineficiente de antibióticos convencionais acarreta a necessidade da busca por novos compostos bioativos que atuem por mecanismos de ação diferentes aos fármacos já conhecidos. Na agricultura, o uso intensivo de pesticidas para o combate de microrganismos que comprometem principalmente a parte alimentícia também traz diversos problemas relacionados à resistência antimicrobiana e a riscos ambientais, oriundos do acúmulo dessas substâncias no solo. Dentro deste aspecto, o pseudofungo Pythium aphanidermatum, da classe dos oomicetos, destaca-se por ser uma espécie agressiva e altamente resistente a fungicidas comuns, apodrecendo raízes e frutos de cultivos de tomate, beterraba, pepino, pimentão, etc. A própolis verde, constituída em sua grande parte por material resinoso coletado e processado pela abelha da espécie Apis mellifera tem sido utilizada na medicina tradicional devido ao seu amplo espectro de ações preventivas e tratamentos de doenças, possuindo propriedades anti-inflamatórias, antimicrobianas, anticancerígenas e antioxidantes, tornando-se um produto de grande interesse na busca de novos compostos bioativos. Dentro destes aspectos apresentados, neste trabalho investigamos a ação da própolis verde contra o fitopatógeno P. aphanidermatum e identificamos através da técnica de cromatografia e bioensaios que a Artepillin C (3,5-diprenil-4-ácido-hidroxicinâmico), majoritária na própolis verde, foi o principal composto nesta ação. Os efeitos terapêuticos desta molécula tem sido foco de muitos estudos, porém ainda não há evidência em sua interação com agregados anfifílicos que mimetizam membranas celulares. O caráter anfifílico do composto, elevado pela presença dos grupos prenilados ligados ao ácido cinâmico, favoreceram a sua inserção nas membranas modelo, principalmente em seu estado agregado. Estas conclusões puderam ser inferidas devido às alterações nas propriedades das bicamadas lipídicas na presença da Artepillin C, podendo causar, especificamente para o caso de fitopatógenos como o P. aphanidermatum, perdas funcionais das proteínas de membranas, liberação de eletrólitos intracelulares e desintegração citoplasmática dos micélios e esporos. Ainda, as diferentes composições lipídicas nas vesículas influenciam no modo de interação do composto e consequentes alterações em suas estruturas, principalmente na presença do colesterol, que auxilia na manutenção da permeabilidade da bicamada lipídica, que pode contribuir para a integridade do conteúdo citoplasmático da célula. / The increase in the microbial resistance due to the excessive and inefficient use of conventional antibiotics brings the necessity to search new bioactive compounds which play their mechanism of action differently from the known drugs. In the agriculture, the intensive use of pesticide for the combat of microorganisms which undermine mainly the food portion also brings several issues related to the antimicrobial resistance and environment risks, originated from the high amount of these substances on the soil. In this aspect, the fungus-like Pythium aphanidermatum microorganism, from class Oomycete, stands out for being an aggressive species and highly resistant to common fungicides, rotting roots and fruits of tomato, beet, cucumber, pepper, etc. Green propolis, constituted by resinous material collected and processed by bees of the species Apis mellifera, has been used in the traditional medicine due its wide spectrum of preventive actions and diseases treatments, promoting anti-inflammatory, antimicrobial, anticancer and antioxidant properties, becoming a product of interest for investigation in the research of new bioactive compounds. Under all the aspects showed so far, in this work we investigated the action of the green propolis against the phytopathogen P. aphanidermatum and identified through chromatography and bioassays that Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid), majority in the green propolis, was the main compound in this action. The therapeutic effects of this molecule have been the focus of several studies, but, so far there is no evidence for its interaction with amphiphilic aggregates that mimic cell membranes. The amphiphilic character of the compound, enhanced by the presence of two prenylated groups bounded to the cinnamic acid, favors the insertion of the compound in the model membranes mainly in its aggregation state. These conclusions could be inferred due the alterations in the properties of the lipid bilayer in the presence of Artepillin C, that may cause, specifically in the case of phytopathogens like P. aphanidermatum, functional losses of membrane proteins, releasing of intracellular electrolytes and cytoplasmatic disintegration of mycelium and spores. Moreover, the difference of the lipid composition in the vesicles influence in the action of the compound and consequent alteration in their structures, mainly in the presence of cholesterol, that provides the maintenance of permeability of the lipid bilayer, contributing to the integrity of the cytoplasmic material of the cell.

Afin d’étudier l’écologie microbienne, il est nécessaire, dans un premier temps, de déterminer quels micro-organismes sont présents dans un milieu et à quel instant. Ces informations sont requises pour pouvoir ensuite développer des outils permettant de prédire l’assemblage des communautés et les fonctions que celles-ci peuvent contenir. Cependant, la multitude des processus entrant en jeu dans la structure et la composition des communautés microbiennes, rendent leur étude complexe. Parmi les nombreux processus à étudier, il est notamment question de l’échelle temporelle à prendre en compte pour comprendre l’assemblage des communautés microbiennes. En effet, les événements historiques conditionnent la composition et la biodiversité des futures communautés microbiennes. Pourtant, dans les sols, peu d’études se sont intéressées à l’impact des événements historiques dans l’assemblage des communautés microbiennes. Par conséquent, l’objectif de cette thèse était de quantifier comment les différentes histoires du sol ont influencé la structure et biodiversité des communautés bactériennes et oomycètes associées aux plantes hôtes des Brassicaceae à travers le temps. Les rotations de cultures de Brassicaceae sont de plus en plus courantes dans le monde et ont démontré des avantages pour les cultures concernées, telles que la rétention de l’humidité du sol ou la suppression de certains agents pathogènes des plantes. En revanche, l’impact des rotations de cultures de Brassicaceae sur la structure et biodiversité des communautés microbiennes résidentes est peu connu. Ainsi, des terrains agricoles des prairies canadiennes ayant des expériences de rotations de cultures en cours ont été utilisés pour modéliser l’impact des histoires de sol précédemment établies sur les futures communautés microbiennes. Les communautés microbiennes des racines, de la rhizosphère, et du sol éloigné des racines des Brassicaceae ont été étudiées grâce aux métabarcodes d’ARNr 16S ou ITS. La PCR quantitative et des méthodes phylogénétiques ont été utilisées pour améliorer l’analyse des communautés microbiennes. Cette thèse illustre comment différentes histories de sol établies par les cultures de l’année précédente ont continué à structurer les communautés microbiennes de la rhizosphère tout au long de la saison de croissance, à différents stades de croissance, jusqu’à un an après leur établissement. Cependant, le phénomène de rétroactions entre plantes et micro-organismes a permis de masquer cet héritage dans la rhizosphere de différentes espèces hôtes de Brassicacea pour lesquelles des communautés bactériennes phylogénétiquement similaires ont été retrouvées malgré diverses histoires du sol. Nos résultats montrent également que les différentes espèces hôtes de Brassicacea n’avaient pas d’impact sur la structure des communautés d’oomycètes et que le stress hydrique limitait également cette structuration pour les communautés bactériennes. Dans ces deux cas, l’effet de l’histoire du sol était donc encore visible sur la structure les communautés microbiennes durant l’année subséquente. Les découvertes selon lesquelles différentes histoires de sol persistent jusqu'à un an, même en présence de nouvelles plantes hôtes, et qu’elles peuvent continuer à façonner les communautés microbiennes ont des implications importantes pour la gestion agricole et les recherches futures sur les composants physiques de l'histoire du sol. Comprendre comment l'histoire du sol est impliquée dans la structure et la biodiversité des communautés microbiennes à travers le temps est une limitation de l'écologie microbienne et est nécessaire pour utiliser les technologies microbiennes à l'avenir pour une agriculture durable et dans toute la société. / A fundamental task of microbial ecology is determining which organisms are present, and when, in order to improve the predictive models of community assembly and functions. However, the heterogeneity of community assembly processes that underlie how microbial communities are formed and structured are makes assembly of taxonomic and functional profiles difficult. One reason for this challenge is the compounding effect temporal scales have on microbial communities. For example, historical events have been shown to condition future microbial community composition and biodiversity. Yet, how historical events structure microbial communities in the soil has not been well tested. Therefore, the objective of this thesis was to quantify how different soil histories influenced the structure and biodiversity of bacterial and oomycete communities associated with Brassicaceae host plants through time. Brassicaceae crop rotations are increasingly common globally, and have demonstrated benefits for the crops involved, such as retaining soil moisture, or suppressing certain plant pathogens. In contrast, there is a lack of knowledge surrounding how Brassicaceae crop rotations impact the structure and biodiversity of resident microbial communities. As such, on-going agricultural field experiments with crop rotations on the Canadian prairies were used to model how previously established soil histories impacted future microbial communities. The Brassicaceae microbial communities were inferred from the roots, rhizosphere and bulk soil using 16S rRNA or ITS metabarcodes. Quantitative PCR and phylogenetic methods were used to improve the analysis of the microbial communities. This thesis illustrates how different soil histories established by the previous year’s crops continued to structure the microbial rhizosphere communities throughout the growing season, at various growth stages, and up to a year after being established. However, active plant-soil microbial feedback allowed different Brassicaceae host species to mask the soil history in the rhizosphere and derive phylogenetically similar bacterial communities from these diverse soil histories. Furthermore, host plants were unable to structure the oomycete communities, and lost the ability to structure the bacterial rhizosphere communities under water stress. In both circumstances, the soil history continued to structure the microbial communities. The findings that different soil histories persist for up to a year, even in the presence of new host plants, and can continue to shape microbial communities has important implications for agricultural management and future research on the physical components of soil history. Understanding how soil history is involved in the structure and biodiversity of microbial communities through time is a limitation in microbial ecology and is required for employing microbial technologies in the future for sustainable agriculture and throughout society.

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