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Development of biomaterials through mimesis of plant defensive interfaces to fight wound infections

Periodic Reporting for period 5 - MIMESIS (Development of biomaterials through mimesis of plant defensive interfaces to fight wound infections)

Reporting period: 2021-09-01 to 2022-02-28

Fighting microbial infection of wounds, especially in immunocompromised patients, is a major challenge in the 21st century. The skin barrier is the primary defence against microbial (opportunistic) pathogens. When this barrier is breached even non-pathogenic fungi may cause devastating infections, most of which provoked by crossover fungi able to infect both plant and humans.
My research is driven by the vision of mimicking the functionality of plant polyesters to develop novel and efficient antifungal therapies, specifically wound dressing biomaterials that also show skin regeneration properties.
Land plants have evolved through more than 400 million years, developing ubiquitous defence polyester barriers (lipid-based polymers) that limit pathogen adhesion and invasion. The unique chemical composition and structure of the plant polyester determines its physiological roles. However, conventional methods to extract polyesters from plants result in the loss of both native structure and inherent barrier properties hampering progress in this area. Besides, plant polyesters own an intractable chemistry that remains largely unknown.

To lead discovery, we have developed a biocompatible extraction method that ensures recovery of the native polymer, hence displaying native barrier properties, including potentially broad antimicrobial and anti-biofouling effect. Our major objectives are to elucidate which plant polyester compositions reconstitute ex-situ as stronger antimicrobial films, to then unravel their antimicrobial mechanism of action. Naturally, the challenge is to understand how the chemical structure of the polymer influences the biological activity.
We have redesigned the process for the extraction of polyesters from plants (using as our gold standard suberin from cork) to allow processing greater quantities yet ensuing flexibility to be applied to other plant sources. It consists of a custom-made reactor with controlled mixing and temperature, followed by an air-pressure filtration unit.
We have solved the antimicrobial mode of action of the suberin particles using microscopic-based methods and computational simulations. Suberin particles kill bacteria and yeast cells through contact and inhibit the germination of conidia (asexual spores) while promoting the sexual development of the surviving germinating conidia. The suberin particles retain virtually the same levels of glycerol as those found in planta, suggestive of its near-native configuration. We resolved the molecular structure of suberin using for the first-time solution nuclear magnetic resonance (NMR), specifically dissecting all possible configurations of acylglycerol and quantifying their relative abundances and the relative abundance of linear aliphatic esters, among other functional groups as well. We also analysed suberin in situ, i.e. directly solubilised from cork using cryogenic milling; evidence never reported before. The topography and x-ray scattering pattern of suberin particles were collected, covering different raw materials (including suberin from roots, and woody barks) that show a much larger diversity of compositional signatures that anticipated.
A cell-wall cutin continuum was also isolated and its molecular structure was solved using solution NMR. The cutin materials from both wild type and mutants carrying defective cutin biosynthesis were characterised, showing unexpected features in the cutin of the mutants, especially in the branching of the polymers and crystal organisation. Cutin oligomers are able to induce plant immune responses and activate a unique transcriptional response in Arabidopsis and efficiently kill both Gram+ and Gram-.
Finally, we demonstrated that suberin particles/films are devoid of cytotoxicity and very low concentrations may stimulate the healing of epidermal wounds.
Most of the data so far attained constitute progresses to the state of the art; especially the solution-NMR chemical analysis of suberin and cutin polymers which may be regarded as a breakthrough in the field. The established methodological approach provides us means to characterise fractions of the polymer displaying distinct densities and self-assembling profiles and more relevant also distinct antimicrobial activities.
Representative 2D HSQC spectrum of suberin