Mechanism Of Lipid Membrane Damage Induced By NEP1-like Proteins
ARRS project code: J7-1819
Period: 1.7.2019 - 30.6.2022
Head: prof. Anderluh Gregor
Fight against pathogens and antimicrobial resistance is a big and pressing problem of modern society. Novel strategies and targets for development of antimicrobial substances are thus crucially needed in areas as diverse as medicine, production of safe food, etc. The members of the family of necrosis- and ethylene-inducing peptide 1 (NEP1)-like proteins, i.e. NLPs, elicit diverse defence reactions and cell death in eudicot plants but not in monocots. NLPs are widely distributed among taxonomically nonrelated microorganisms like fungi, bacteria and oomycetes. These microorganisms are widespread, they may infect range of different crops, such as potato, tomato, soya and tobacco, and cause enormous economic loss worldwide. It was shown that NLPs function as cytolytic toxins that induce plasma membrane leakage, thus causing cytotoxicity. Based on their crystal structures, NLPs are considered to be distantly related to other pore-forming toxins of animal origin, such as actinoporins from sea anemones.
The mechanism by which NLP induce necrosis is poorly understood. Recently, we have identified glycosylinositol phosphorylceramides (GIPC), a major class of plant sphingolipids, as a target molecule for NLP binding to plant plasma membranes (Lenarčič et al., Science, 2017). GIPCs consist of a polar headgroup bearing variable carbohydrate moieties and inositol phosphorylceramide core. Type and number of terminal hexose groups varies significantly between plant species and plant tissues. Binding of the GIPC terminal hexose moiety induces several conformational changes within the NLP toxin that may precede membrane attachment and host cell lysis. Nothing is known about other steps of membrane damage subsequent to membrane binding and this project aims to clarify steps of membrane damage induced by NLP proteins. We will test the central hypothesis of the project that NLPs damage lipid membranes by a multistep process that lead to pore formation. The main objectives of this project are therefore to (i) determine the molecular mechanism of membrane damage by NLPs, (ii) resolve underlying selectivity of NLPs against eudicot plants at the molecular level and (iii) determine structural features of oligomeric assemblies of NLPs formed at the surface of lipid membranes composed of GIPCs. To achieve objectives of this project proposal we will use state-of-the-art biochemical, biophysical, molecular and structural biology approaches, including X-ray crystallography and cryo-electron microscopy, and molecular modelling.
The results of this project will provide molecular details of NLPs interactions with target cell surface, molecular mechanism of membrane damage and properties of pores formed by NLPs. This will clarify steps in pathogenesis of some of the most pressing pathogens and open avenues for development of strategies for fighting against microbial pathogens.
