Creating optimized peptide libraries for AMP discovery via PepSAVI-MS.

Antimicrobial peptides (AMPs) are host defense peptides with a range of functions/activities/modes of action that are ubiquitously expressed across all forms of life. Continued discovery of novel AMPs presents exciting opportunities to address evolving resistance to existing treatments in multiple fields, including agricultural pathogens/pests as well as antimicrobial and chemotherapeutics for human health. However, typical discovery methods including bioassay-guided fractionation and genome mining generally lack the capacity for robust AMP discovery in non-model/unsequenced organisms. PepSAVI-MS (Statistically guided bioactive peptides prioritized via mass spectrometry) was developed as an AMP discovery approach that addresses some of the limitations associated with these standard methods. PepSAVI-MS is a multi-pronged pipeline that includes peptide library creation, bioactivity screening, LC-MS analysis, and statistical modeling for putative AMP identification. The original implementation of PepSAVI-MS outlined strategies for the fractionation of plant extracts with strong cation exchange chromatography (SCX). Herein, we elaborate on recent improvements to peptide library creation through the use of orthogonal fractionation methods, specifically crude SCX chromatography and reversed-phase liquid chromatography (RPLC). This optimization of the "peptide library creation" step has demonstrated improvements for processing and AMP identifications via PepSAVI-MS.

Leveraging orthogonal mass spectrometry based strategies for comprehensive sequencing and characterization of ribosomal antimicrobial peptide natural products.

Covering: Up to July 2020Ribosomal antimicrobial peptide (AMP) natural products, also known as ribosomally synthesized and post-translationally modified peptides (RiPPs) or host defense peptides, demonstrate potent bioactivities and impressive complexity that complicate molecular and biological characterization. Tandem mass spectrometry (MS) has rapidly accelerated bioactive peptide sequencing efforts, yet standard workflows insufficiently address intrinsic AMP diversity. Herein, orthogonal approaches to accelerate comprehensive and accurate molecular characterization without the need for prior isolation are reviewed. Chemical derivatization, proteolysis (enzymatic and chemical cleavage), multistage MS fragmentation, and separation (liquid chromatography and ion mobility) strategies can provide complementary amino acid composition and post-translational modification data to constrain sequence solutions. Examination of two complex case studies, gomesin and styelin D, highlights the practical implementation of the proposed approaches. Finally, we emphasize the importance of heterogeneous AMP peptidoforms that confer varying biological function, an area that warrants significant further development.

Implementation of Microfluidics for Antimicrobial Susceptibility Assays: Issues and Optimization Requirements.

Despite the continuous emergence of multi-drug resistant pathogens, the number of new antimicrobials reaching the market is critically low. Natural product peptides are a rich source of bioactive compounds, and advances in mass spectrometry have achieved unprecedented capabilities for the discovery and characterization of novel molecular species. However, traditional bioactivity assay formats hinder the discovery and biochemical characterization of natural product antimicrobial peptides (AMPs), necessitating large sample quantities and significant optimization of experimental parameters to achieve accurate/consistent activity measurements. Microfluidic devices offer a promising alternative to bulk assay systems. Herein, a microfluidics-based bioassay was compared to the traditional 96-well plate format in respective commercially-available hardware. Bioactivity in each assay type was compared using a Viola inconspicua peptide library screened against E. coli ATCC 25922. Brightfield microcopy was used to determine bioactivity in microfluidic channels while both common optical and fluorescence-based measurements of cell viability were critically assessed in plate-based assays. Exhibiting some variation in optical density and fluorescence-based measurements, all plate-based assays conferred bioactivity in late eluting V. inconspicua library fractions. However, significant differences in the bioactivity profiles of plate-based and microfluidic assays were found, and may be derived from the materials comprising each assay device or the growth/assay conditions utilized in each format. While new technologies are necessary to overcome the limitations of traditional bioactivity assays, we demonstrate that off-the-shelf implementation of microfluidic devices is non-trivial and significant method development/optimization is required before conventional use can be realized for sensitive and rapid detection of AMPs in natural product matrices.

Exploring the Diversity of Cysteine-Rich Natural Product Peptides via MS/MS Fingerprint Ions.

Natural product extracts present inherently complex matrices in which the identification of novel bioactive peptide species is challenged by low-abundance masses and significant structural and sequence diversity. Additionally, discovery efforts often result in the re-identification of known compounds, where modifications derived in vivo or during sample handling may obscure true sequence identity. Herein, we identify mass spectral (MS2) "fingerprint" ions characteristic of cyclotides, a diverse and biologically active family of botanical cysteine-rich peptides, based on regions of high sequence homology. We couple mass shift analysis with MS2 spectral fingerprint ions cross referenced with CyBase-a cyclotide database-to discern unique mass species in Viola communis extracts from mass species that are likely already characterized and those with common modifications. The approach is extended to a related class of cysteine-rich peptides, the trypsin inhibitors, using the characterized botanical species Lagenaria siceraria. Coupling the observation of highly abundant MS2 ions with mass shift analysis, we identify a new set of small, highly disulfide-bound cysteine-rich L. siceraria peptides.

Gas-Phase Sequencing of Cyclotides: Introduction of Selective Ring Opening at Dehydroalanine via Ion/Ion Reaction.

The gas-phase linearization of cyclotides via site-selective ring opening at dehydroalanine residues and its application to cyclotide sequencing is presented. This strategy relies on the ability to incorporate dehydroalanine into macrocyclic peptide ions, which is easily accomplished through an ion/ion reaction. Triply protonated cyclotide cations are transformed into radical cations via ion/ion reaction with the sulfate radical anion. Subsequent activation of the cyclotide radical cation generates dehydroalanine at a single cysteine residue, which is easily identified by the odd-electron loss of ·SCH2CONH2. The presence of dehydroalanine in cyclotides provides a site-selective ring-opening pathway that, in turn, generates linear cyclotide analogues in the gas phase. Unlike cyclic variants, product ions derived from the linear peptides provide rich sequence information. The sequencing capability of this strategy is demonstrated with four known cyclotides found in Viola inconspicua, where, in each case, greater than 93% sequence coverage was observed. Furthermore, the utility of this method is highlighted by the partial de novo sequencing of an unknown cyclotide with much greater sequence coverage than that obtained with a conventional Glu-C digestion approach. This method is particularly well-suited for cyclotide species that are not abundant enough to characterize with traditional methods.

PepSAVI-MS Reveals a Proline-rich Antimicrobial Peptide in Amaranthus tricolor.

Traditional medicinal plants are a rich source of antimicrobials; however, the bioactive peptide constituents of most ethnobotanical species remain largely unexplored. Herein, PepSAVI-MS, a mass spectrometry-based peptidomics pipeline, was implemented for antimicrobial peptide (AMP) discovery in the medicinal plant Amaranthus tricolor. This investigation revealed a novel 1.7 kDa AMP with strong activity against Escherichia coli ATCC 25922, deemed Atr-AMP1. Initial efforts to determine the sequence of Atr-AMP1 utilized chemical derivatization and enzymatic digestion to provide information about specific residues and post-translational modifications. EThcD (electron-transfer/higher-energy collision dissociation) produced extensive backbone fragmentation and facilitated de novo sequencing, the results of which were consistent with orthogonal characterization experiments. Additionally, multistage HCD (higher-energy collisional dissociation) facilitated discrimination between isobaric leucine and isoleucine. These results revealed a positively charged proline-rich peptide present in a heterogeneous population of multiple peptidoforms, possessing several post-translational modifications including a disulfide bond, methionine oxidation, and proline hydroxylation. Additional bioactivity screening of a simplified fraction containing Atr-AMP1 revealed activity against Staphylococcus aureus LAC, demonstrating activity against both a Gram-negative and a Gram-positive bacterial species unlike many known short chain proline-rich antimicrobial peptides.

Viola "inconspicua" No More: An Analysis of Antibacterial Cyclotides.

The emergence of rapidly evolving multidrug-resistant pathogens and a deficit of new compounds entering the clinical pipeline necessitate the exploration of alternative sources of antimicrobial therapeutics. Cyclotides revealed in Viola spp. are a class of highly stable, cyclic, and disulfide-bound peptides with diverse intrinsic bioactivities. Herein we have identified a novel complement of 42 putative cyclotide masses in the plant species Viola inconspicua. Cyclotide-containing fractions of a V. inconspicua peptide library revealed potent bioactivities against the Gram-negative bacteria Escherichia coli ATCC 25922 and the highly virulent and multidrug-resistant Klebsiella pneumoniae VK148. As such, six previously uncharacterized cyclotides, cycloviolacins I1-6 (cyI1-cyI6), were prioritized for molecular characterization. Cyclotides cyI3-cyI6 contain a novel "TLNGNPGA" motif in the highly variable loop six region, expanding the already substantial sequence diversity of this peptide class. Library fractions comprised of cyclotides cyI3-cyI6 exhibited MIC values of 18 and 35 µM against E. coli and K. pneumoniae, respectively, whereas isolated cyI3 killed ∼50% of E. coli at 60 µM and isolated cyI4 demonstrated no killing at concentrations >60 µM against both pathogens. This work expands the repertoire of bioactive cyclotides found in Viola spp. and highlights the potential of these antibacterial cyclic peptides.

Correction to: Fungal Secretome Analysis Via PepSAVI-MS: Identification of the Bioactive Peptide KP4 from Ustilago maydis.

In the article "Fungal Secretome Analysis via PepSAVI-MS: Identification of the Bioactive Peptide KP4 from Ustilago maydis", acknowledgement of financial support was inadvertently omitted. The authors apologize for this oversight.

Exploring bioactive peptides from bacterial secretomes using PepSAVI-MS: identification and characterization of Bac-21 from Enterococcus faecalis pPD1.

As current methods for antibiotic drug discovery are being outpaced by the rise of antimicrobial resistance, new methods and innovative technologies are necessary to replenish our dwindling arsenal of antimicrobial agents. To this end, we developed the PepSAVI-MS pipeline to expedite the search for natural product bioactive peptides. Herein we demonstrate expansion of PepSAVI-MS for the discovery of bacterial-sourced bioactive peptides through identification of the bacteriocin Bac-21 from Enterococcus faecalis pPD1. Minor pipeline modifications including implementation of bacteria-infused agar diffusion assays and optional digestion of peptide libraries highlight the versatility and wide adaptability of the PepSAVI-MS pipeline. Additionally, we have experimentally validated the primary protein sequence of the active, mature Bac-21 peptide for the first time and have confirmed its identity with respect to primary sequence and post-translational processing. Successful application of PepSAVI-MS to bacterial secretomes as demonstrated herein establishes proof-of-principle for use in novel microbial bioactive peptide discovery.

PepSAVI-MS reveals anticancer and antifungal cycloviolacins in Viola odorata.

Widespread resistance to antimicrobial and cancer therapeutics is evolving in every country worldwide and has a direct impact on global health, agriculture and the economy. The specificity and selectivity of bioactive peptide natural products present a possible stopgap measure to address the ongoing deficit of new therapeutic compounds. PepSAVI-MS (Statistically-guided bioActive Peptides prioritized VIa Mass Spectrometry) is an adaptable method for the analysis of natural product libraries to rapidly identify bioactive peptides. This pipeline was validated via screening of the cyclotide-rich botanical species Viola odorata and identification of the known antimicrobial and anticancer cyclotide cycloviolacin O2. Herein we present and validate novel bioactivities of the anthelmintic V. odorata cyclotide, cycloviolacin O8 (cyO8), including micromolar anticancer activity against PC-3 prostate, MDA-MB-231 breast, and OVCAR-3 ovarian cancer cell lines and antifungal activity against the agricultural pathogen Fusarium graminearum. A reduction/alkylation strategy in tandem with PepSAVI-MS analysis also revealed several previously uncharacterized putatively bioactive cyclotides. Downstream implementation of ultraviolet photodissociation (UVPD) tandem mass spectrometry is demonstrated for cyO8 as a method to address traditionally difficult-to-sequence cyclotide species. This work emphasizes the therapeutic and agricultural potential of natural product bioactive peptides and the necessity of developing robust analytical tools to deconvolute nature's complexity.

Fungal Secretome Analysis via PepSAVI-MS: Identification of the Bioactive Peptide KP4 from Ustilago maydis.

Fungal secondary metabolites represent a rich and largely untapped source for bioactive molecules, including peptides with substantial structural diversity and pharmacological potential. As methods proceed to take a deep dive into fungal genomes, complimentary methods to identify bioactive components are required to keep pace with the expanding fungal repertoire. We developed PepSAVI-MS to expedite the search for natural product bioactive peptides and herein demonstrate proof-of-principle applicability of the pipeline for the discovery of bioactive peptides from fungal secretomes via identification of the antifungal killer toxin KP4 from Ustilago maydis P4. This work opens the door to investigating microbial secretomes with a new lens, and could have broad applications across human health, agriculture, and food safety. Graphical Abstract.

Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms.

Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity.

A Slow Conformational Switch in the BMAL1 Transactivation Domain Modulates Circadian Rhythms.

The C-terminal transactivation domain (TAD) of BMAL1 (brain and muscle ARNT-like 1) is a regulatory hub for transcriptional coactivators and repressors that compete for binding and, consequently, contributes to period determination of the mammalian circadian clock. Here, we report the discovery of two distinct conformational states that slowly exchange within the dynamic TAD to control timing. This binary switch results from cis/trans isomerization about a highly conserved Trp-Pro imide bond in a region of the TAD that is required for normal circadian timekeeping. Both cis and trans isomers interact with transcriptional regulators, suggesting that isomerization could serve a role in assembling regulatory complexes in vivo. Toward this end, we show that locking the switch into the trans isomer leads to shortened circadian periods. Furthermore, isomerization is regulated by the cyclophilin family of peptidyl-prolyl isomerases, highlighting the potential for regulation of BMAL1 protein dynamics in period determination.

Cryptochrome 1 regulates the circadian clock through dynamic interactions with the BMAL1 C terminus.

The molecular circadian clock in mammals is generated from transcriptional activation by the bHLH-PAS transcription factor CLOCK-BMAL1 and subsequent repression by PERIOD and CRYPTOCHROME (CRY). The mechanism by which CRYs repress CLOCK-BMAL1 to close the negative feedback loop and generate 24-h timing is not known. Here we show that, in mouse fibroblasts, CRY1 competes for binding with coactivators to the intrinsically unstructured C-terminal transactivation domain (TAD) of BMAL1 to establish a functional switch between activation and repression of CLOCK-BMAL1. TAD mutations that alter affinities for co-regulators affect the balance of repression and activation to consequently change the intrinsic circadian period or eliminate cycling altogether. Our results suggest that CRY1 fulfills its role as an essential circadian repressor by sequestering the TAD from coactivators, and they highlight regulation of the BMAL1 TAD as a critical mechanism for establishing circadian timing.