Dataset Information

The MIT domain of chitin synthase 1 from the oomycete Saprolegnia monoica interacts specifically with phosphatidic acid.

ABSTRACT: Chitin synthases are vital for growth in certain oomycetes as chitin is an essential component in the cell wall of these species. In Saprolegnia monoica, two chitin synthases have been found, and both contain a Microtubule Interacting and Trafficking (MIT) domain. The MIT domain has been implicated in lipid interaction, which in turn may be of significance for targeting of chitin synthases to the plasma membrane. In this work we have investigated the lipid interacting properties of the MIT domain from chitin synthase 1 in Saprolegnia monoica. We show by fluorescence spectroscopy techniques that the MIT domain interacts preferentially with phosphatidic acid (PA), while it does not interact with phosphatidylglycerol (PG) or phosphatidylcholine (PC). These results strongly suggest that the specific properties of PA are required for membrane interaction of the MIT domain. PA is negatively charged, binds basic side chains with high affinity and its small headgroup gives rise to membrane packing defects that enable intercalation of hydrophobic amino acids. We propose a mode of lipid interaction that involves a combination of basic amino acid residues and Trp residues that anchor the MIT domain specifically to bilayers that contain PA.

SUBMITTER: Brown C

PROVIDER: S-EPMC8851075 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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The MIT domain of chitin synthase 1 from the oomycete <i>Saprolegnia monoica</i> interacts specifically with phosphatidic acid.

Brown Christian C Patrick Joan J Liebau Jobst J Mäler Lena L

Biochemistry and biophysics reports 20220210

Chitin synthases are vital for growth in certain oomycetes as chitin is an essential component in the cell wall of these species. In <i>Saprolegnia monoica</i>, two chitin synthases have been found, and both contain a Microtubule Interacting and Trafficking (MIT) domain. The MIT domain has been implicated in lipid interaction, which in turn may be of significance for targeting of chitin synthases to the plasma membrane. In this work we have investigated the lipid interacting properties of the MI ...[more]

PMID: 35198741

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Project description:Oomycetes represent some of the most devastating plant and animal pathogens. Typical examples are Phytophthora infestans, which causes potato and tomato late blight, and Saprolegnia parasitica, responsible for fish diseases. Despite the economical and environmental importance of oomycete diseases, their control is difficult, particularly in the aquaculture industry. Carbohydrate synthases are vital for hyphal growth and represent interesting targets for tackling the pathogens. The existence of 2 different chitin synthase genes (SmChs1 and SmChs2) in Saprolegnia monoica was demonstrated using bioinformatics and molecular biology approaches. The function of SmCHS2 was unequivocally demonstrated by showing its catalytic activity in vitro after expression in Pichia pastoris. The recombinant SmCHS1 protein did not exhibit any activity in vitro, suggesting that it requires other partners or effectors to be active, or that it is involved in a different process than chitin biosynthesis. Both proteins contained N-terminal Microtubule Interacting and Trafficking domains, which have never been reported in any other known carbohydrate synthases. These domains are involved in protein recycling by endocytosis. Enzyme kinetics revealed that Saprolegnia chitin synthases are competitively inhibited by nikkomycin Z and quantitative PCR showed that their expression is higher in presence of the inhibitor. The use of nikkomycin Z combined with microscopy showed that chitin synthases are active essentially at the hyphal tips, which burst in the presence of the inhibitor, leading to cell death. S. parasitica was more sensitive to nikkomycin Z than S. monoica. In conclusion, chitin synthases with species-specific characteristics are involved in tip growth in Saprolegnia species and chitin is vital for the micro-organisms despite its very low abundance in the cell walls. Chitin is most likely synthesized transiently at the apex of the cells before cellulose, the major cell wall component in oomycetes. Our results provide important fundamental information on cell wall biogenesis in economically important species, and demonstrate the potential of targeting oomycete chitin synthases for disease control.

Project description:The secondary cysts of the fish pathogen oomycete Saprolegnia parasitica possess bundles of long hooked hairs that are characteristic to this economically important pathogenic species. Few studies have been carried out on elucidating their specific role in the S. parasitica life cycle and the role they may have in the infection process. We show here their function by employing several strategies that focus on descriptive, developmental and predictive approaches. The strength of attachment of the secondary cysts of this pathogen was compared to other closely related species where bundles of long hooked hairs are absent. We found that the attachment of the S. parasitica cysts was around three times stronger than that of other species. The time sequence and influence of selected factors on morphology and the number of the bundles of long hooked hairs conducted by scanning electron microscopy study revealed that these are dynamic structures. They are deployed early after encystment, i.e., within 30 sec of zoospore encystment, and the length, but not the number, of the bundles steadily increased over the encystment period. We also observed that the number and length of the bundles was influenced by the type of substrate and encystment treatment applied, suggesting that these structures can adapt to different substrates (glass or fish scales) and can be modulated by different signals (i.e., protein media, 50 mM CaCl2 concentrations, carbon particles). Immunolocalization studies evidenced the presence of an adhesive extracellular matrix. The bioinformatic analyses of the S. parasitica secreted proteins showed that there is a high expression of genes encoding domains of putative proteins related to the attachment process and cell adhesion (fibronectin and thrombospondin) coinciding with the deployment stage of the bundles of long hooked hairs formation. This suggests that the bundles are structures that might contribute to the adhesion of the cysts to the host because they are composed of these adhesive proteins and/or by increasing the surface of attachment of this extracellular matrix.

Dataset Information

The MIT domain of chitin synthase 1 from the oomycete Saprolegnia monoica interacts specifically with phosphatidic acid.

Publications

The MIT domain of chitin synthase 1 from the oomycete <i>Saprolegnia monoica</i> interacts specifically with phosphatidic acid.

Similar Datasets

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