Characterization of specific domains of the cellulose and chitin synthases from pathogenic oomycetes

Sammanfattning: 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.