Understanding adsorption behavior of antiviral labyrinthopeptin peptides in anion exchange chromatography.

Lantipeptides from bacterial sources are increasingly important as biopharmaceuticals because of their broad range of applications. However, the availability of most lantipeptides is low, and systematic approaches for downstream processing of this group of peptides is still lacking. Model-based development for chromatographic separations has proven to be a useful tool for developing reliable purification processes. One important compound of such a model is the adsorption behavior of the components of interest. In ion-exchange chromatography, the adsorption equilibrium between salt and proteins can be described using the steric mass action (SMA) formalism. Beyond, the model parameters may be related to the lanthipeptides physico-chemical properties. In this study, the antiviral lantipeptides labyrinthopeptin A1 and A2, purified from Actinomadura namibiensis culture broth, were characterized for their adsorption behavior in anion-exchange chromatography in the range from pH 5.0-7.4. The experiments necessary to determine the three SMA parameters were chosen in a way to limit the amount of peptides needed. Linear gradient elution was applied successfully to separate A1 and A2 and to determine the characteristic charge νi and the equilibrium constant [Formula: see text] . Batch adsorption experiments using a robotic workstation for high throughput and accuracy provided non-linear adsorption isotherms and the steric factor σi. Labyrinthopeptin A1 and A2 show a very different adsorption behavior even though the fundamental structure of the two peptides is similar. keq of A1 ranging from 0.18 to 0.88 are approximately one order of magnitude smaller than that of A2 ranging from 3.44 to 9.73 indicating the higher affinity of A2 to the stationary phase. At pH 7.0 σ was 1.12 and 0.60 for A1 and A2, respectively which was expected based on the molecular weight of the peptides. The characteristic charge for both peptides was also theoretically estimated from the amino acids involved in electrostatic interactions which was in good agreement with experimental data. Thereby, this work provides an useful approach to estimate SMA parameters based on simple structural information that can be applied early in chromatographic ion-exchange process development for peptides and may help adapting the processes for future designed lanthipeptides.

An ectomycorrhizal fungus alters sensitivity to jasmonate, salicylate, gibberellin, and ethylene in host roots.

The phytohormones jasmonate, gibberellin, salicylate, and ethylene regulate an interconnected reprogramming network integrating root development with plant responses against microbes. The establishment of mutualistic ectomycorrhizal symbiosis requires the suppression of plant defense responses against fungi as well as the modification of root architecture and cortical cell wall properties. Here, we investigated the contribution of phytohormones and their crosstalk to the ontogenesis of ectomycorrhizae (ECM) between grey poplar (Populus tremula x alba) roots and the fungus Laccaria bicolor. To obtain the hormonal blueprint of developing ECM, we quantified the concentrations of jasmonates, gibberellins, and salicylate via liquid chromatography-tandem mass spectrometry. Subsequently, we assessed root architecture, mycorrhizal morphology, and gene expression levels (RNA sequencing) in phytohormone-treated poplar lateral roots in the presence or absence of L. bicolor. Salicylic acid accumulated in mid-stage ECM. Exogenous phytohormone treatment affected the fungal colonization rate and/or frequency of Hartig net formation. Colonized lateral roots displayed diminished responsiveness to jasmonate but regulated some genes, implicated in defense and cell wall remodelling, that were specifically differentially expressed after jasmonate treatment. Responses to salicylate, gibberellin, and ethylene were enhanced in ECM. The dynamics of phytohormone accumulation and response suggest that jasmonate, gibberellin, salicylate, and ethylene signalling play multifaceted roles in poplar L. bicolor ectomycorrhizal development.