The Role of Key Amino Acids in the Antimicrobial Mechanism of a Bacteriocin Model Revealed by Molecular Simulations.

The AS-48 bacteriocin is a potent antimicrobial polypeptide with enhanced stability due to its circular sequence of peptidic bonds. The mechanism of biological action is still not well understood in spite of both the elucidation of the molecular structure some years ago and several experiments performed that yielded valuable information about the AS-48 bacterial membrane poration activity. In this work, we present a computational study at an atomistic scale to analyze the membrane disruption mechanism. The process is based on the two-stage model: (1) peptide binding to the bilayer surface and (2) membrane poration due to the surface tension exerted by the peptide. Indeed, the induced membrane tension mechanism is able to explain stable formation of pores leading to membrane disruption. The atomistic detail obtained from the simulations allows one to envisage the contribution of the different amino acids during the poration process. Clustering of cationic residues and hydrophobic interactions between peptide and lipids seem to be essential ingredients in the process. GLU amino acids have shown to enhance the membrane disrupting ability of the bacteriocin. TRP24-TRP24 interactions make also an important contribution in the initial stages of the poration mechanism. The detailed atomistic information obtained from the simulations can serve to better understand bacteriocin structural characteristics to design more potent antimicrobial therapies.

Outer-membrane-acting peptides and lipid II-targeting antibiotics cooperatively kill Gram-negative pathogens.

The development and dissemination of antibiotic-resistant bacterial pathogens is a growing global threat to public health. Novel compounds and/or therapeutic strategies are required to face the challenge posed, in particular, by Gram-negative bacteria. Here we assess the combined effect of potent cell-wall synthesis inhibitors with either natural or synthetic peptides that can act on the outer-membrane. Thus, several linear peptides, either alone or combined with vancomycin or nisin, were tested against selected Gram-negative pathogens, and the best one was improved by further engineering. Finally, peptide D-11 and vancomycin displayed a potent antimicrobial activity at low µM concentrations against a panel of relevant Gram-negative pathogens. This combination was highly active in biological fluids like blood, but was non-hemolytic and non-toxic against cell lines. We conclude that vancomycin and D-11 are safe at >50-fold their MICs. Based on the results obtained, and as a proof of concept for the newly observed synergy, a Pseudomonas aeruginosa mouse infection model experiment was also performed, showing a 4 log10 reduction of the pathogen after treatment with the combination. This approach offers a potent alternative strategy to fight (drug-resistant) Gram-negative pathogens in humans and mammals.

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.

Feasability of Introducing a Thioether Ring in Vasopressin by nisBTC Co-expression in Lactococcus lactis.

Introducing one or more intramolecular thioether bridges in a peptide provides a promising approach to create more stable molecules with improved pharmacodynamic properties and especially to protect peptides against proteolytic degradation. Lanthipeptides are compounds that naturally possess thioether bonds in their structure. The model lanthipeptide, nisin, is produced by Lactococcus lactis as a core peptide fused to a leader peptide. The modification machinery responsible for nisin production, including the Ser/Thr-dehydratase NisB and the cyclase NisC, can be applied for introducing a thioether bridge into peptides fused to the nisin leader peptide, e.g., to replace a disulfide bond. Vasopressin plays a key role in water homeostasis in the human body and helps to constrict blood vessels. There are two cysteine residues in the structure of wild type vasopressin, which form a disulfide bridge in the mature peptide. Here, we show it is possible to direct the biosynthesis of vasopressin variants in such a way that the disulfide bridge is replaced by a thioether bridge using the nisin modification machinery NisBTC, albeit at low efficiency. Vasopressin mutants were fused either to the nisin leader peptide directly (Type A), after the first three rings of nisin (Type B/C), or after full nisin (Type D). The type B strategy was optimal for expression. LC-MS/MS data verified the formation of a thioether bridge, which provides proof of principle for this modification in vasopressin. This is a first step prior to the necessary increase of the production yield and further purification of these peptides to finally test their biological activity in tissue and animal models.

Analysis of modular bioengineered antimicrobial lanthipeptides at nanoliter scale.

The rise of antibiotic resistance demands the acceleration of molecular diversification strategies to inspire new chemical entities for antibiotic medicines. We report here on the large-scale engineering of ribosomally synthesized and post-translationally modified antimicrobial peptides carrying the ring-forming amino acid lanthionine. New-to-nature variants featuring distinct properties were obtained by combinatorial shuffling of peptide modules derived from 12 natural antimicrobial lanthipeptides and processing by a promiscuous post-translational modification machinery. For experimental characterization, we developed the nanoFleming, a miniaturized and parallelized high-throughput inhibition assay. On the basis of a hit set of >100 molecules, we identified variants with improved activity against pathogenic bacteria and shifted activity profiles, and extrapolated design guidelines that will simplify the identification of peptide-based anti-infectives in the future.

Major gene-regulatory mechanisms operating in ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthesis.

Post-translationally modified peptides commonly display antimicrobial activity, but can also aid the development of bacterial colonies, giving a competitive advantage in the ecological niche. The production of post-translationally modified peptides by bacteria is a complex and energetically costly process that is strictly orchestrated in the cell. The onset of peptide production is linked to the different enzymes that take part during maturation, the transporters and the immunity determinants (if required). Thus, the population can make optimal use of available resources and obtain the benefits of production at an advantageous moment during growth, avoiding toxicity to itself. The timing and level of expression of the different operons is controlled by diverse (complex) regulatory pathways in response to environmental changes, stress or master regulators during specific growth transition phases. In this review, we highlight the basic principles and mechanisms of regulation of expression of post-translationally modified peptides and the relationship with the overall culture developmental processes and/or cellular differentiation. We also discuss the biotechnological consequences derived from the understanding of regulatory networks involved in the biosynthesis of these natural products.

Posttranslational Peptide-Modification Enzymes in Action: Key Roles for Leaders and Glutamate.

In this issue of Cell Chemical Biology, Ortega et al. (2016) determine the structure of another lantibiotic dehydratase with a tRNA(Glu)-dependent mechanism of modification. Moreover, they identify a common recognition motif involved in leader peptide binding in a number of different peptide-modification enzymes. These findings open up new mining possibilities and allow novel approaches in peptide engineering.

BAGEL3: Automated identification of genes encoding bacteriocins and (non-)bactericidal posttranslationally modified peptides.

Identifying genes encoding bacteriocins and ribosomally synthesized and posttranslationally modified peptides (RiPPs) can be a challenging task. Especially those peptides that do not have strong homology to previously identified peptides can easily be overlooked. Extensive use of BAGEL2 and user feedback has led us to develop BAGEL3. BAGEL3 features genome mining of prokaryotes, which is largely independent of open reading frame (ORF) predictions and has been extended to cover more (novel) classes of posttranslationally modified peptides. BAGEL3 uses an identification approach that combines direct mining for the gene and indirect mining via context genes. Especially for heavily modified peptides like lanthipeptides, sactipeptides, glycocins and others, this genetic context harbors valuable information that is used for mining purposes. The bacteriocin and context protein databases have been updated and it is now easy for users to submit novel bacteriocins or RiPPs. The output has been simplified to allow user-friendly analysis of the results, in particular for large (meta-genomic) datasets. The genetic context of identified candidate genes is fully annotated. As input, BAGEL3 uses FASTA DNA sequences or folders containing multiple FASTA formatted files. BAGEL3 is freely accessible at http://bagel.molgenrug.nl.

Designing and producing modified, new-to-nature peptides with antimicrobial activity by use of a combination of various lantibiotic modification enzymes.

Lanthipeptides are peptides that contain several post-translationally modified amino acid residues and commonly show considerable antimicrobial activity. After translation, the amino acid residues of these peptides are modified by a distinct set of modification enzymes. This process results in peptides containing one or more lanthionine rings and dehydrated Ser and Thr residues. Previously, an in vivo lanthipeptide production system based on the modification machinery of the model lantibiotic nisin was reported. Here, we present the addition of the modification enzymes LtnJ and GdmD to this production system. With these enzymes we can now produce lanthipeptides that contain d-alanines or a C-terminal aminovinyl-cysteine. We show experimentally that the decarboxylase GdmD is responsible for the C-terminal decarboxylation. Our results demonstrate that different lanthipeptide modification enzymes can work together in an in vivo production system. This yields a plug-and-play system that can be used to select different sets of modification enzymes to work on diverse, specifically designed substrates.

Expression of linear permutated variants from circular enterocin AS-48.

To confirm whether the head-to-tail circularization could be involved in the stability and activity of the circular bacteriocin AS-48, two permutated linear structural as-48A genes have been constructed by circular permutation. The absence of the leaderless linear AS(23/24) and AS(48/49) proteins in Escherichia coli, under all the conditions investigated, supports the idea that the circular backbone is important to stabilize their structure and also indicates the significance of a leader peptide. In fact, the approach taken in this study to generate linear permutated proteins fused to an appropriate partner was sufficient to prevent cellular proteolysis. In this case, the high expression levels found favour their intracellular accumulations as inclusion bodies, which after solubilization showed a propensity to aggregate, thus hindering the specific EK cleavage. This could explain the presence of active hybrid tagged proteins identified in this work. The conserved distribution of hydrophobic and hydrophilic surfaces in the hybrid proteins is responsible for the antibacterial activity. In addition, the opening of the AS-48 molecule between the residues G(23) W(24) connecting the α1/α2 helices, confers greater stability, suggesting that the sequence and/or the free amino acid in the polypeptide chain are critical aspects in the design of new variants.