Fluorescence shadow imaging of Hypsibius exemplaris reveals morphological differences between sucrose- and CaCl2-induced osmobiotes.

Tardigrades are renowned for their ability to survive a wide array of environmental stressors. In particular, tardigrades can curl in on themselves while losing a significant proportion of their internal water content to form a structure referred to as a tun. In surviving varying conditions, tardigrades undergo distinct morphological transformations that could indicate different mechanisms of stress sensing and tolerance specific to the stress condition. Methods to effectively distinguish between morphological transformations, including between tuns induced by different stress conditions, are lacking. Herein, an approach for discriminating between tardigrade morphological states is developed and utilized to compare sucrose- and CaCl2-induced tuns, using the model species Hypsibius exemplaris. A novel approach of shadow imaging with confocal laser scanning microscopy enabled production of three-dimensional renderings of Hys. exemplaris in various physiological states resulting in volume measurements. Combining these measurements with qualitative morphological analysis using scanning electron microscopy revealed that sucrose- and CaCl2-induced tuns have distinct morphologies, including differences in the amount of water expelled during tun formation. Further, varying the concentration of the applied stressor did not affect the amount of water lost, pointing towards water expulsion by Hys. exemplaris being a controlled process that is adapted to the specific stressors.

The tardigrade Hypsibius exemplaris dramatically upregulates DNA repair pathway genes in response to ionizing radiation.

Tardigrades can survive remarkable doses of ionizing radiation, up to about 1,000 times the lethal dose for humans. How they do so is incompletely understood. We found that the tardigrade Hypsibius exemplaris suffers DNA damage upon gamma irradiation, but the damage is repaired. We show that this species has a specific and robust response to ionizing radiation: irradiation induces a rapid upregulation of many DNA repair genes. This upregulation is unexpectedly extreme-making some DNA repair transcripts among the most abundant transcripts in the animal. By expressing tardigrade genes in bacteria, we validate that increased expression of some repair genes can suffice to increase radiation tolerance. We show that at least one such gene is important in vivo for tardigrade radiation tolerance. We hypothesize that the tardigrades' ability to sense ionizing radiation and massively upregulate specific DNA repair pathway genes may represent an evolved solution for maintaining DNA integrity.

Cheminformatics-Guided Cell-Free Exploration of Peptide Natural Products.

There have been significant advances in the flexibility and power of in vitro cell-free translation systems. The increasing ability to incorporate noncanonical amino acids and complement translation with recombinant enzymes has enabled cell-free production of peptide-based natural products (NPs) and NP-like molecules. We anticipate that many more such compounds and analogs might be accessed in this way. To assess the peptide NP space that is directly accessible to current cell-free technologies, we developed a peptide parsing algorithm that breaks down peptide NPs into building blocks based on ribosomal translation logic. Using the resultant data set, we broadly analyze the biophysical properties of these privileged compounds and perform a retrobiosynthetic analysis to predict which peptide NPs could be directly synthesized in augmented cell-free translation reactions. We then tested these predictions by preparing a library of highly modified peptide NPs. Two macrocyclases, PatG and PCY1, were used to effect the head-to-tail macrocyclization of candidate NPs. This retrobiosynthetic analysis identified a collection of high-priority building blocks that are enriched throughout peptide NPs, yet they had not previously been tested in cell-free translation. To expand the cell-free toolbox into this space, we established, optimized, and characterized the flexizyme-enabled ribosomal incorporation of piperazic acids. Overall, these results demonstrate the feasibility of cell-free translation for peptide NP total synthesis while expanding the limits of the technology. This work provides a novel computational tool for exploration of peptide NP chemical space, that could be expanded in the future to allow design of ribosomal biosynthetic pathways for NPs and NP-like molecules.

The Greater Celandine: Identification and Characterization of an Antimicrobial Peptide from Chelidonium majus.

Chelidonium majus, known as Greater Celandine, is a latex-bearing plant that has been leveraged for its anticancer and antimicrobial properties. Herein, C. majus aerial tissue is mined for the presence of antimicrobial peptides. A highly abundant cysteine-rich peptide with a length of 25 amino acids, deemed CM-AMP1, is characterized through multiple mass spectrometric approaches. Electron-activated dissociation is leveraged to differentiate between isoleucine and leucine residues and complement conventional collision-induced dissociation to gain full sequence coverage of the full-length peptide. CM-AMP1 shares little sequence similarity with any proteins in publicly available databases, highlighting the novelty of its cysteine landscape and core motif. The presence of three disulfide bonds in the native peptide confers proteolytic stability, and antimicrobial activity is greatly decreased upon the alkylation of the cysteine residues. Synthetic variants of CM-AMP1 are used to confirm the activity of the full-length sequence and the core motif. To assess the biological impact, E. coli was grown in a sublethal concentration of CM-AMP1 and quantitative proteomics was used to identify proteins produced by the bacteria under stress, ultimately suggesting a membrane lytic antimicrobial mechanism of action. This study integrates multiple analytical methods for molecular and biological characterization of a unique antimicrobial peptide identified from C. majus.

Identification and Characterization of CC-AMP1-like and CC-AMP2-like Peptides in Capsicum spp.

Antimicrobial peptides (AMPs) are compounds with a variety of bioactive properties. Especially promising are their antibacterial activities, often toward drug-resistant pathogens. Across different AMP sources, AMPs expressed within plants are relatively underexplored with a limited number of plant AMP families identified. Recently, we identified the novel AMPs CC-AMP1 and CC-AMP2 in ghost pepper plants (Capsicum chinense x frutescens), exerting promising antibacterial activity and not classifying into any known plant AMP family. Herein, AMPs related to CC-AMP1 and CC-AMP2 were identified within both Capsicum annuum and Capsicum baccatum. In silico predictions throughout plants were utilized to illustrate that CC-AMP1-like and CC-AMP2-like peptides belong to two broader AMP families, with three-dimensional structural predictions indicating that CC-AMP1-like peptides comprise a novel subfamily of α-hairpinins. The antibacterial activities of several closely related CC-AMP1-like peptides were compared with a truncated version of CC-AMP1 possessing significantly more activity than the full peptide. This truncated peptide was further characterized to possess broad-spectrum antibacterial activity against clinically relevant ESKAPE pathogens. These findings illustrate the value in continued study of plant AMPs toward characterization of novel AMP families, with CC-AMP1-like peptides possessing promising bioactivity.

Chemobiosis reveals tardigrade tun formation is dependent on reversible cysteine oxidation.

Tardigrades, commonly known as 'waterbears', are eight-legged microscopic invertebrates renowned for their ability to withstand extreme stressors, including high osmotic pressure, freezing temperatures, and complete desiccation. Limb retraction and substantial decreases to their internal water stores results in the tun state, greatly increasing their ability to survive. Emergence from the tun state and/or activity regain follows stress removal, where resumption of life cycle occurs as if stasis never occurred. However, the mechanism(s) through which tardigrades initiate tun formation is yet to be uncovered. Herein, we use chemobiosis to demonstrate that tardigrade tun formation is mediated by reactive oxygen species (ROS). We further reveal that tuns are dependent on reversible cysteine oxidation, and that this reversible cysteine oxidation is facilitated by the release of intracellular reactive oxygen species (ROS). We provide the first empirical evidence of chemobiosis and map the initiation and survival of tardigrades via osmobiosis, chemobiosis, and cryobiosis. In vivo electron paramagnetic spectrometry suggests an intracellular release of reactive oxygen species following stress induction; when this release is quenched through the application of exogenous antioxidants, the tardigrades can no longer survive osmotic stress. Together, this work suggests a conserved dependence of reversible cysteine oxidation across distinct tardigrade cryptobioses.

Recent advances in mass spectrometry-based methods to investigate reversible cysteine oxidation.

The post-translational modification of cysteine to diverse oxidative states is understood as a critical cellular mechanism to combat oxidative stress. To study the role of cysteine oxidation, cysteine enrichments and subsequent analysis via mass spectrometry are necessary. As such, technologies and methods are rapidly developing for sensitive and efficient enrichments of cysteines to further explore its role in signaling pathways. In this review, we analyze recent developments in methods to miniaturize cysteine enrichments, analyze the underexplored disulfide bound redoxome, and quantify site-specific cysteine oxidation. We predict that further development of these methods will improve cysteine coverage across more diverse organisms than those previously studied and elicit novel roles cysteines play in stress response.

Maximizing Depth of PTM Coverage: Generating Robust MS Datasets for Computational Prediction Modeling.

Post-translational modifications (PTMs) regulate complex biological processes through the modulation of protein activity, stability, and localization. Insights into the specific modification type and localization within a protein sequence can help ascertain functional significance. Computational models are increasingly demonstrated to offer a low-cost, high-throughput method for comprehensive PTM predictions. Algorithms are optimized using existing experimental PTM data, thus accurate prediction performance relies on the creation of robust datasets. Herein, advancements in mass spectrometry-based proteomics technologies to maximize PTM coverage are reviewed. Further, requisite experimental validation approaches for PTM predictions are explored to ensure that follow-up mechanistic studies are focused on accurate modification sites.

Abscisic acid-controlled redox proteome of Arabidopsis and its regulation by heterotrimeric Gß protein.

The plant hormone abscisic acid (ABA) plays crucial roles in regulation of stress responses and growth modulation. Heterotrimeric G-proteins are key mediators of ABA responses. Both ABA and G-proteins have also been implicated in intracellular redox regulation; however, the extent to which reversible protein oxidation manipulates ABA and/or G-protein signaling remains uncharacterized. To probe the role of reversible protein oxidation in plant stress response and its dependence on G-proteins, we determined the ABA-dependent reversible redoxome of wild-type and Gß-protein null mutant agb1 of Arabidopsis. We quantified 6891 uniquely oxidized cysteine-containing peptides, 923 of which show significant changes in oxidation following ABA treatment. The majority of these changes required the presence of G-proteins. Divergent pathways including primary metabolism, reactive oxygen species response, translation and photosynthesis exhibited both ABA- and G-protein-dependent redox changes, many of which occurred on proteins not previously linked to them. We report the most comprehensive ABA-dependent plant redoxome and uncover a complex network of reversible oxidations that allow ABA and G-proteins to rapidly adjust cellular signaling to adapt to changing environments. Physiological validation of a subset of these observations suggests that functional G-proteins are required to maintain intracellular redox homeostasis and fully execute plant stress responses.

In silico prediction and mass spectrometric characterization of botanical antimicrobial peptides.

Antimicrobial peptides (AMPs) are promising compounds for the treatment of antibiotic-resistant bacteria and are found across all organisms, including plants. Unlike most antibiotics, AMPs tend to act on more generalized and multiple targets, making development of resistance more difficult. Conventional approaches toward AMP identification include bioactivity-guided fractionation and genome mining. Complementary methods leveraging bioactivity-guided fractionation, cysteine motif-guided in silico AMP prediction, and mass spectrometric approaches can be combined to expand botanical AMP discovery. Herein, we present an integrated workflow which serves to streamline implementation toward a robust botanical AMP discovery pipeline.

Revealing AMP mechanisms of action through resistance evolution and quantitative proteomics.

Antimicrobial resistance (AMR) is a significant public health issue that threatens our ability to treat common infections. AMR often emerges in bacteria through upregulation of proteins that allow a subpopulation of resistant bacteria to proliferate through natural selection. Identifying these proteins is crucial for understanding how AMR develops in bacteria and is essential in developing novel therapeutics to combat the threat of widespread AMR. Mass spectrometry-based proteomics is a powerful tool for understanding the biochemical pathways of biological systems, lending remarkable insight into AMR mechanisms in bacteria through measuring the changing protein abundances as a result of antibiotic treatment. Here, we describe a serial passaging method for evolving resistance in bacteria that implements quantitative proteomics to reveal the differential proteomes of resistant bacteria. The focus herein is on antimicrobial peptides (AMPs), but the approach can be generalized for any antimicrobial compound. Comparative proteomics of sensitive vs. resistance strains in response to AMP treatment reveals mechanisms to survive the bioactive compound and points to the mechanism of action for novel AMPs.

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.

Amaranthus hypochondriacus seeds as a rich source of cysteine rich bioactive peptides.

Amaranthus hypochondriacus is a nutritious alternative grain native to Central and South America. Increased interest in the impact of A. hypochondriacus on the human body has driven characterization of bioactive secondary metabolites. The seeds are known to contain bioactive small molecules but little is known regarding endogenous peptides. Cysteine-rich peptides (CRPs) in foodstuffs are particularly relevant because they are stabilized by disulfide bonds enhancing resistance to digestion. Here, in silico predictions, proteomics, and simulated gastrointestinal digestions are leveraged to identify digestion resistant CRPs within A. hypochondriacus seeds. Thirteen in silico predicted CRPs were detected in a seed extract providing evidence for the translation of five CRP families. Mature forms of six CRPs were characterized via top-down proteomics revealing multiple post-translational modifications. All six peptides demonstrated resistance to simulated gastrointestinal digestion, suggesting that A. hypochondriacus CRPs may exhibit bioactivity after consumption and should be prioritized for further characterization.

Mass Spectrometric Identification of Antimicrobial Peptides from Medicinal Seeds.

Traditional medicinal plants contain a variety of bioactive natural products including cysteine-rich (Cys-rich) antimicrobial peptides (AMPs). Cys-rich AMPs are often crosslinked by multiple disulfide bonds which increase their resistance to chemical and enzymatic degradation. However, this class of molecules is relatively underexplored. Herein, in silico analysis predicted 80-100 Cys-rich AMPs per species from three edible traditional medicinal plants: Linum usitatissimum (flax), Trifolium pratense (red clover), and Sesamum indicum (sesame). Bottom-up proteomic analysis of seed peptide extracts revealed direct evidence for the translation of 3-10 Cys-rich AMPs per species, including lipid transfer proteins, defensins, α-hairpinins, and snakins. Negative activity revealed by antibacterial screening highlights the importance of employing a multi-pronged approach for AMP discovery. Further, this study demonstrates that flax, red clover, and sesame are promising sources for further AMP discovery and characterization.

IreK-Mediated, Cell Wall-Protective Phosphorylation in Enterococcus faecalis.

Enterococcus faecalis is a Gram-positive bacterium that is a major cause of hospital-acquired infections due, in part, to its intrinsic resistance to cell wall-active antimicrobials. One critical determinant of this resistance is the transmembrane kinase IreK, which belongs to the penicillin-binding protein and serine/threonine kinase-associated kinase family of bacterial signaling proteins involved with the regulation of cell wall homeostasis. The activity of IreK is enhanced in response to cell wall stress, but direct substrates of IreK phosphorylation, leading to antimicrobial resistance, are largely unknown. To better understand stress-modulated phosphorylation events contributing to antimicrobial resistance, wild type E. faecalis cells treated with cell wall-active antimicrobials, chlorhexidine or ceftriaxone, were examined via phosphoproteomics. Among the most prominent changes was increased phosphorylation of divisome components after both treatments, suggesting that E. faecalis modulates cell division in response to cell wall stress. Phosphorylation mediated by IreK was then determined via a similar analysis with a E. faecalis ΔireK mutant strain, revealing potential IreK substrates involved with the regulation of peptidoglycan biosynthesis and within the E. faecalis CroS/R two-component system, another signal transduction pathway that promotes antimicrobial resistance. These results reveal critical insights into the biological functions of IreK.

Crosslinking mass spectrometry unveils novel interactions and structural distinctions in the model green alga Chlamydomonas reinhardtii.

Interactomics is an emerging field that seeks to identify both transient and complex-bound protein interactions that are essential for metabolic functions. Crosslinking mass spectrometry (XL-MS) has enabled untargeted global analysis of these protein networks, permitting largescale simultaneous analysis of protein structure and interactions. Increased commercial availability of highly specific, cell permeable crosslinkers has propelled the study of these critical interactions forward, with the development of MS-cleavable crosslinkers further increasing confidence in identifications. Herein, the global interactome of the unicellular alga Chlamydomonas reinhardtii was analyzed via XL-MS by implementing the MS-cleavable disuccinimidyl sulfoxide (DSSO) crosslinker and enriching for crosslinks using strong cation exchange chromatography. Gentle lysis via repeated freeze-thaw cycles facilitated in vitro analysis of 157 protein-protein crosslinks (interlinks) and 612 peptides linked to peptides of the same protein (intralinks) at 1% FDR throughout the C. reinhardtii proteome. The interlinks confirmed known protein relationships across the cytosol and chloroplast, including coverage on 42% and 38% of the small and large ribosomal subunits, respectively. Of the 157 identified interlinks, 92% represent the first empirical evidence of interaction observed in C. reinhardtii. Several of these crosslinks point to novel associations between proteins, including the identification of a previously uncharacterized Mg-chelatase associated protein (Cre11.g477733.t1.2) bound to seven distinct lysines on Mg-chelatase (Cre06.g306300.t1.2). Additionally, the observed intralinks facilitated characterization of novel protein structures across the C. reinhardtii proteome. Together, these data establish a framework of protein-protein interactions that can be further explored to facilitate understanding of the dynamic protein landscape in C. reinhardtii.

Inositol polyphosphates and target of rapamycin kinase signalling govern photosystem II protein phosphorylation and photosynthetic function under light stress in Chlamydomonas.

Stress and nutrient availability influence cell proliferation through complex intracellular signalling networks. In a previous study it was found that pyro-inositol polyphosphates (InsP7 and InsP8 ) produced by VIP1 kinase, and target of rapamycin (TOR) kinase signalling interacted synergistically to control cell growth and lipid metabolism in the green alga Chlamydomonas reinhardtii. However, the relationship between InsPs and TOR was not completely elucidated. We used an in vivo assay for TOR activity together with global proteomic and phosphoproteomic analyses to assess differences between wild-type and vip1-1 in the presence and absence of rapamycin. We found that TOR signalling is more severely affected by the inhibitor rapamycin in a vip1-1 mutant compared with wild-type, indicating that InsP7 and InsP8 produced by VIP1 act independently but also coordinately with TOR. Additionally, among hundreds of differentially phosphorylated peptides detected, an enrichment for photosynthesis-related proteins was observed, particularly photosystem II proteins. The significance of these results was underscored by the finding that vip1-1 strains show multiple defects in photosynthetic physiology that were exacerbated under high light conditions. These results suggest a novel role for inositol pyrophosphates and TOR signalling in coordinating photosystem phosphorylation patterns in Chlamydomonas cells in response to light stress and possibly other stresses.

Too Hot to Handle: Antibacterial Peptides Identified in Ghost Pepper.

Capsicum spp. (hot peppers) demonstrate a range of interesting bioactive properties spanning anti-inflammatory, antioxidant, and antimicrobial activities. While several species within the genus are known to produce antimicrobial peptides (AMPs), AMP sequence mining of genomic data indicates this space remains largely unexplored. Herein, in silico AMP predictions were paired with peptidomics to identify novel AMPs from the interspecific hybrid ghost pepper (Capsicum chinense × frutescens). AMP prediction algorithms revealed 115 putative AMPs within the Capsicum chinense genome, of which 14 were identified in the aerial tissue peptidome. PepSAVI-MS, de novo sequencing, and complementary approaches were used to fully molecularly characterize two novel AMPs, CC-AMP1 and CC-AMP2, including elucidation of a pyroglutamic acid post-translational modification of CC-AMP1 and disulfide bond connectivity of both. Both CC-AMP1 and CC-AMP2 have little homology with known AMPs and exhibited low µM antimicrobial activity against Gram-negative bacteria, including Escherichia coli. These findings demonstrate the complementary nature of peptidomics, bioactivity-guided discovery, and bioinformatics-based investigations to characterize plant AMP profiles.

Evolution of Polymyxin Resistance Regulates Colibactin Production in Escherichia coli.

The complex reservoir of metabolite-producing bacteria in the gastrointestinal tract contributes tremendously to human health and disease. Bacterial composition, and by extension gut metabolomic composition, is undoubtably influenced by the use of modern antibiotics. Herein, we demonstrate that polymyxin B, a last resort antibiotic, influences the production of the genotoxic metabolite colibactin from adherent-invasive Escherichia coli (AIEC) NC101. Colibactin can promote colorectal cancer through DNA double stranded breaks and interstrand cross-links. While the structure and biosynthesis of colibactin have been elucidated, chemical-induced regulation of its biosynthetic gene cluster and subsequent production of the genotoxin by E. coli are largely unexplored. Using a multiomic approach, we identified that polymyxin B stress enhances the abundance of colibactin biosynthesis proteins (Clb's) in multiple pks+ E. coli strains, including pro-carcinogenic AIEC, NC101; the probiotic strain, Nissle 1917; and the antibiotic testing strain, ATCC 25922. Expression analysis via qPCR revealed that increased transcription of clb genes likely contributes to elevated Clb protein levels in NC101. Enhanced production of Clb's by NC101 under polymyxin stress matched an increased production of the colibactin prodrug motif, a proxy for the mature genotoxic metabolite. Furthermore, E. coli with a heightened tolerance for polymyxin induced greater mammalian DNA damage, assessed by quantification of γH2AX staining in cultured intestinal epithelial cells. This study establishes a key link between the polymyxin B stress response and colibactin production in pks+ E. coli. Ultimately, our findings will inform future studies investigating colibactin regulation and the ability of seemingly innocuous commensal microbes to induce host disease.

A deep learning based approach for prediction of Chlamydomonas reinhardtii phosphorylation sites.

Protein phosphorylation, which is one of the most important post-translational modifications (PTMs), is involved in regulating myriad cellular processes. Herein, we present a novel deep learning based approach for organism-specific protein phosphorylation site prediction in Chlamydomonas reinhardtii, a model algal phototroph. An ensemble model combining convolutional neural networks and long short-term memory (LSTM) achieves the best performance in predicting phosphorylation sites in C. reinhardtii. Deemed Chlamy-EnPhosSite, the measured best AUC and MCC are 0.90 and 0.64 respectively for a combined dataset of serine (S) and threonine (T) in independent testing higher than those measures for other predictors. When applied to the entire C. reinhardtii proteome (totaling 1,809,304 S and T sites), Chlamy-EnPhosSite yielded 499,411 phosphorylated sites with a cut-off value of 0.5 and 237,949 phosphorylated sites with a cut-off value of 0.7. These predictions were compared to an experimental dataset of phosphosites identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a blinded study and approximately 89.69% of 2,663 C. reinhardtii S and T phosphorylation sites were successfully predicted by Chlamy-EnPhosSite at a probability cut-off of 0.5 and 76.83% of sites were successfully identified at a more stringent 0.7 cut-off. Interestingly, Chlamy-EnPhosSite also successfully predicted experimentally confirmed phosphorylation sites in a protein sequence (e.g., RPS6 S245) which did not appear in the training dataset, highlighting prediction accuracy and the power of leveraging predictions to identify biologically relevant PTM sites. These results demonstrate that our method represents a robust and complementary technique for high-throughput phosphorylation site prediction in C. reinhardtii. It has potential to serve as a useful tool to the community. Chlamy-EnPhosSite will contribute to the understanding of how protein phosphorylation influences various biological processes in this important model microalga.