Expression of Lanthipeptides in Human Cells.

Cyclic peptides represent a burgeoning area of interest in therapeutic and biotechnological research. In opposition to their linear counterparts, cyclic peptides, such as certain ribosomally synthesized and post-translationally modified peptides (RiPPs), are more conformationally constrained and less susceptible to proteolytic degradation. The lanthipeptide RiPP cytolysin L forms a covalently enforced helical structure that may be used to disrupt helical interactions at protein-protein interfaces. Herein, an expression system is reported to produce lanthipeptides and structurally diverse cytolysin L derivatives in mammalian cells. Successful targeting of lanthipeptides to the nucleus is demonstrated. In vivo expression and targeting of such peptides in mammalian cells may allow for screening of lanthipeptide inhibitors of native protein-protein interactions.

Synthesis of Fluorescent Lanthipeptide Cytolysin S Analogues by Late-Stage Sulfamidate Ring Opening.

Nucleophilic ring opening of cyclic sulfamidates derived from amino acids is a common strategy for the synthesis of lanthionine derivatives. In this work, we report the regio-, chemo-, and stereoselective intramolecular S-alkylation of a cysteine residue with N-sulfonyl sulfamidates for the synthesis of cyclic lanthionine-containing peptides. The strategy involves the solid-phase synthesis of sulfamidate-containing peptides followed by late-stage intramolecular cyclization. This protocol allowed for the synthesis of four full-length cytolysin S (CylLS″) analogues, two α-peptides and two hybrid α/ß-peptides. Their conformational preferences and biological activities were assessed and compared with those of wild-type CylLS″.

Structure-Activity Relationships of the Enterococcal Cytolysin.

Enterococcal cytolysin is a hemolytic virulence factor linked to human disease and increased patient mortality. Produced by pathogenic strains of Enterococcus faecalis, cytolysin is made up of two small, post-translationally modified peptides called CylLL" and CylLS". They exhibit a unique toxicity profile where lytic activity is observed for both mammalian cells and Gram-positive bacteria that is dependent on the presence of both peptides. In this study, we performed alanine substitution of all residues in CylLL" and CylLS" and determined the effect on both activities. We identified key residues involved in overall activity and residues that dictate cell type specificity. All (methyl)lanthionines as well as a Gly-rich hinge region were critical for both activities. In addition, we investigated the binding of the two subunits to bacterial cells suggesting that the large subunit CylLL" has stronger affinity for the membrane or a target molecule therein. Genome mining identified other potential two-component lanthipeptides and provided insights into potential evolutionary origins.

New developments in RiPP discovery, enzymology and engineering.

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.

Substrate Recognition by the Class II Lanthipeptide Synthetase HalM2.

Class II lanthipeptides belong to a diverse group of natural products known as ribosomally synthesized and post-translationally modified peptides (RiPPs). Most RiPP precursor peptides contain an N-terminal recognition sequence known as the leader peptide, which is typically recognized by biosynthetic enzymes that catalyze modifications on the C-terminal core peptide. For class II lanthipeptides, these are carried out by a bifunctional lanthipeptide synthetase (LanM) that catalyzes dehydration and cyclization reactions on peptidic substrates to generate thioether-containing, macrocyclic molecules. Some lanthipeptide synthetases are extraordinarily substrate tolerant, making them promising candidates for biotechnological applications such as combinatorial biosynthesis and cyclic peptide library construction. In this study, we characterized the mode of leader peptide recognition by HalM2, the lanthipeptide synthetase responsible for the production of the antimicrobial peptide haloduracin ß. Using NMR spectroscopic techniques, in vitro binding assays, and enzyme activity assays, we identified substrate residues that are important for binding to HalM2 and for post-translational modification of the peptide substrates. Additionally, we provide evidence of the binding site on the enzyme using binding assays with truncated enzyme variants, hydrogen-deuterium exchange mass spectrometry, and photoaffinity labeling. Understanding the mechanism by which lanthipeptide synthetases recognize their substrate will facilitate their use in biotechnology, as well as further our general understanding of how RiPP enzymes recognize their substrates.

Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.