, known as Greater Celandine, is a latex-bearing plant that has been leveraged for its anticancer and antimicrobial properties. Herein,
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,
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
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
. 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
. Cyclotide-containing fractions of a
peptide library revealed potent bioactivities against the Gram-negative bacteria
ATCC 25922 and the highly virulent and multidrug-resistant
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
and
, respectively, whereas isolated cyI3 killed ∼50% of
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
. and highlights the potential of these antibacterial cyclic peptides.
There have been significant advances in the flexibility and power of
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.
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.
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
(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
.
are largely unexplored. Using a multiomic approach, we identified that polymyxin B stress enhances the abundance of colibactin biosynthesis proteins (Clb's) in multiple
.
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
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,
.
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
.
. Ultimately, our findings will inform future studies investigating colibactin regulation and the ability of seemingly innocuous commensal microbes to induce host disease.
Bacteria control the length of their polysaccharides, which can control cell viability, physiology, virulence, and immune evasion. Polysaccharide chain length affects immunomodulation, but its impact ...on bacterial physiology and antibiotic susceptibility was unclear. We probed the consequences of truncating the mycobacterial galactan, an essential linear polysaccharide of about 30 residues. Galactan covalently bridges cell envelope layers, with the outermost cell wall linkage point occurring at residue 12. Reducing galactan chain length by approximately half compromises fitness, alters cell morphology, and increases the potency of hydrophobic antibiotics. Systematic variation of the galactan chain length revealed that it determines periplasm size. Thus, glycan chain length can directly affect cellular physiology and antibiotic activity, and mycobacterial glycans, not proteins, regulate periplasm size.
Abstract only
Despite their ubiquity of polysaccharides, we lack detailed mechanistic understanding toward how carbohydrate polymers are assembled. Unlike the predictable linkage patterns used in ...other major molecules, polysaccharides can draw upon a large bevy of diverse glycosidic linkages to form complex polymers. To better understand polysaccharide assembly, our research group has turned toward using the mycobacterial galactan as a model. Composed of galactofuranose (Gal
f
) residues, this essential polymer serves as a covalent connector between the mycolic acids and peptidoglycan of mycolated bacteria. The processive glycosyltransferase GlfT2 uses the activated form of galactofuranose, UDP‐Gal
f
, to continuously add residues onto the galactan until reaching a mature length.
Due to its virulence, one of the most extensively studied mycobacteria is
Mycobacterium tuberculosis
(
M. tb
).
M. tb
GlfT2 makes alternating
β
‐(1→5) and
β
‐(1→6) linkages in the galactan biosynthesis. Previous work from our research group sought to evaluate whether
M. tb
GlfT2 strictly adheres to this linkage pattern. When given UDP‐Gal
f
with a floro‐ substituted 5 or 6 position, the enzyme is unable to elongate the polymer past two units.
1
This suggests that the alternating
β
‐(1→5),
β
‐(1→6) linkage pattern is maintained with high fidelity. Curiously, linkage patterns within galactan isolated from other mycolated bacterium, such as
Nocardia
and
Rhodococcus
species differ from those in
Mycobacterium.
Data suggests that
Nocardia brasiliensis (N. bras)
only synthesizes
β
‐(1→5) linkages and
Rhodococcus equi
(
R. equi
) synthesizes
β
‐(1→3),
β
‐(1→5), and
β
‐(1→6) linkages.
2
Since GlfT2 is responsible for much of galactan biosynthesis, we propose that GlfT2 from these organisms contain differences from
M. tb
GlfT2, allowing them to synthesize galactan with different glycosidic linkages.
Our research group is keenly interested in gaining mechanistic insight toward what variation in the active site of GlfT2 leads to control over glycosidic linkage formation. We are specifically interested in understanding differences and similarities to the mechanism used by
M. tb
GlfT2. Since our research group has demonstrated two ‘DXD’ motifs are critical to GlfT2 catalysis, we seek to examine the active site of
Nocardia brasiliensis
and
Rhodococcus equi
GlfT2 by mutating these motifs. We hope that these studies will better illuminate which features of glycosyltransferases guide glycosidic linkage formation.
Bacteria control the length of their polysaccharides, which can control cell viability, physiology, virulence, and immune evasion. Polysaccharide chain length affects immunomodulation, but its impact ...on bacterial physiology and antibiotic susceptibility was unclear. We probed the consequences of truncating the mycobacterial galactan, an essential linear polysaccharide of about 30 residues. Galactan covalently bridges cell envelope layers, with the outermost cell wall linkage point occurring at residue 12. Reducing galactan chain length by approximately half compromises fitness, alters cell morphology, and increases the potency of hydrophobic antibiotics. Systematic variation of the galactan chain length revealed that it determines periplasm size. Thus, glycan chain length can directly affect cellular physiology and antibiotic activity, and mycobacterial glycans, not proteins, regulate periplasm size.
PDF
