Origin of Secondary Structure Transitions and Peptide Self-Assembly Propensity in Trifluoroethanol-Water Mixtures.

Membrane-peptide interactions are key to the formation of helical intermediates in the early stages of amyloidogenesis. Aqueous solutions of 2,2,2-trifluoroethanol (TFE) provide a membrane-mimetic environment capable of promoting and stabilizing local peptide interactions. Uperin 3.5 (U3.5), a 17-residue and amidated antimicrobial peptide, is unstructured in water but self-assembles into fibrils in the presence of salt. Secondary structure transitions linked to U3.5 self-assembly were investigated in TFE/water mixtures, in both the absence and presence of salt, to assess the role of membrane-peptide interactions on peptide self-assembly and amyloid formation. A 5-to-7-fold increase in fibril yield of U3.5 was observed at low TFE concentrations (10% TFE/water v/v) compared with physiological buffer but only in the presence of salt. No aggregation was observed in salt-free TFE/water mixtures. Circular dichroism spectra showed that partial helical structures, initially stabilized by TFE, transitioned to ß-sheet-rich aggregates in a saline buffer. Molecular dynamics simulations confirmed that TFE and salt act synergistically to enhance peptide-peptide interactions, resulting in ß-sheet-rich U3.5 oligomers at low TFE concentrations. Specifically, TFE stabilized amphipathic, helical intermediates, leading to increased peptide-peptide attraction through hydrophobic interactions. The presence of salt further enhanced the peptide-peptide interactions by screening positively charged residues. Thus, the study revealed the role of a membrane mimic in stabilizing helical intermediates on the pathway to amyloid formation in the antimicrobial U3.5 peptide.

Specific Cationic Antimicrobial Peptides Enhance the Recovery of Low-Load Quiescent Mycobacterium tuberculosis in Routine Diagnostics.

The culture confirmation of Mycobacterium tuberculosis (MTB) remains the gold standard for the diagnosis of Tuberculosis (TB) with culture conversion representing proof of cure. However, over 40% of TB samples fail to isolate MTB even though many patients remain infectious due to the presence of viable non-culturable forms. Previously, we have shown that two short cationic peptides, T14D and TB08L, induce a hormetic response at low concentrations, leading to a stimulation of growth in MTB and the related animal pathogen Mycobacterium bovis (bTB). Here, we examine these peptides showing they can influence the mycobacterial membrane integrity and function through membrane potential reduction. We also show this disruption is associated with an abnormal reduction in transcriptomic signalling from specific mycobacterial membrane sensors that normally monitor the immediate cellular environment and maintain the non-growing phenotype. We observe that exposing MTB or bTB to these peptides at optimal concentrations rapidly represses signalling mechanisms maintaining dormancy phenotypes, which leads to the promotion of aerobic metabolism and conversion into a replicative phenotype. We further show a practical application of these peptides as reagents able to enhance conventional routine culture methods by stimulating mycobacterial growth. We evaluated the ability of a peptide-supplemented sample preparation and culture protocol to isolate the MTB against a gold standard routine method tested in parallel on 255 samples from 155 patients with suspected TB. The peptide enhancement increased the sample positivity rate by 46% and decreased the average time to sample positivity of respiratory/faecal sampling by seven days. The most significant improvements in isolation rates were from sputum smear-negative low-load samples and faeces. The peptide enhancement increased sampling test sensitivity by 19%, recovery in samples from patients with a previously culture-confirmed TB by 20%, and those empirically treated for TB by 21%. We conclude that sample decontamination and culture enhancement with D-enantiomer peptides offer good potential for the much-needed improvement of the culture confirmation of TB.