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Precursor-centric genome-mining approach for lasso peptide discovery.

ABSTRACT: Lasso peptides are a class of ribosomally synthesized posttranslationally modified natural products found in bacteria. Currently known lasso peptides have a diverse set of pharmacologically relevant activities, including inhibition of bacterial growth, receptor antagonism, and enzyme inhibition. The biosynthesis of lasso peptides is specified by a cluster of three genes encoding a precursor protein and two enzymes. Here we develop a unique genome-mining algorithm to identify lasso peptide gene clusters in prokaryotes. Our approach involves pattern matching to a small number of conserved amino acids in precursor proteins, and thus allows for a more global survey of lasso peptide gene clusters than does homology-based genome mining. Of more than 3,000 currently sequenced prokaryotic genomes, we found 76 organisms that are putative lasso peptide producers. These organisms span nine bacterial phyla and an archaeal phylum. To provide validation of the genome-mining method, we focused on a single lasso peptide predicted to be produced by the freshwater bacterium Asticcacaulis excentricus. Heterologous expression of an engineered, minimal gene cluster in Escherichia coli led to the production of a unique lasso peptide, astexin-1. At 23 aa, astexin-1 is the largest lasso peptide isolated to date. It is also highly polar, in contrast to many lasso peptides that are primarily hydrophobic. Astexin-1 has modest antimicrobial activity against its phylogenetic relative Caulobacter crescentus. The solution structure of astexin-1 was determined revealing a unique topology that is stabilized by hydrogen bonding between segments of the peptide.

SUBMITTER: Maksimov MO

PROVIDER: S-EPMC3458324 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Precursor-centric genome-mining approach for lasso peptide discovery.

Maksimov Mikhail O MO Pelczer István I Link A James AJ

Proceedings of the National Academy of Sciences of the United States of America 20120904 38

Lasso peptides are a class of ribosomally synthesized posttranslationally modified natural products found in bacteria. Currently known lasso peptides have a diverse set of pharmacologically relevant activities, including inhibition of bacterial growth, receptor antagonism, and enzyme inhibition. The biosynthesis of lasso peptides is specified by a cluster of three genes encoding a precursor protein and two enzymes. Here we develop a unique genome-mining algorithm to identify lasso peptide gene c ...[more]

PMID: 22949633

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Project description:BackgroundProteolysis regulation allows gut microbes to respond rapidly to dynamic intestinal environments by fast degradation of misfolded proteins and activation of regulatory proteins. However, alterations of gut microbial proteolytic signatures under complex disease status such as inflammatory bowel disease (IBD, including Crohn's disease (CD) and ulcerative colitis (UC)), have not been investigated. Metaproteomics holds the potential to investigate gut microbial proteolysis because semi-tryptic peptides mainly derive from endogenous proteolysis.ResultsWe have developed a semi-tryptic peptide centric metaproteomic mining approach to obtain a snapshot of human gut microbial proteolysis signatures. This approach employed a comprehensive meta-database, two-step multiengine database search, and datasets with high-resolution fragmentation spectra to increase the confidence of semi-tryptic peptide identification. The approach was validated by discovering altered proteolysis signatures of Escherichia coli heat shock response. Utilizing two published large-scale metaproteomics datasets containing 623 metaproteomes from 447 fecal and 176 mucosal luminal interface (MLI) samples from IBD patients and healthy individuals, we obtain potential signatures of altered gut microbial proteolysis at taxonomic, functional, and cleavage site motif levels. The functional alterations mainly involved microbial carbohydrate transport and metabolism, oxidative stress, cell motility, protein synthesis, and maturation. Altered microbial proteolysis signatures of CD and UC mainly occurred in terminal ileum and descending colon, respectively. Microbial proteolysis patterns exhibited low correlations with β-diversity and moderate correlations with microbial protease and chaperones levels, respectively. Human protease inhibitors and immunoglobulins were mainly negatively associated with microbial proteolysis patterns, probably because of the inhibitory effects of these host factors on gut microbial proteolysis events.ConclusionsThis semi-tryptic peptide centric mining strategy offers a label-free approach to discover signatures of in vivo gut microbial proteolysis events if experimental conditions are well controlled. It can also capture in vitro proteolysis signatures to facilitate the evaluation and optimization of experimental conditions. Our findings highlight the complex and diverse proteolytic events of gut microbiome, providing a unique layer of information beyond taxonomic and proteomic abundance. Video abstract.

Project description:Antimicrobial peptides (AMPs), as an important part of the innate immune system of an organism, is a kind of promising drug candidate for novel antibiotics due to their unique antibacterial mechanism. However, the discovery of novel AMPs is facing a great challenge due to the complexity of systematic experiments and the poor predictability of antimicrobial activity. Here, a novel and comprehensive screening system, the Multiple Descriptor Multiple Strategy (MultiDS), was proposed based on 59 physicochemical and structural parameters, three strategies, and four algorithms for the mining of α-helical AMPs. This approach was applied to mine the encrypted peptide antibiotics from the global human genome, including introns and exons. A library of approximately 70 billion peptides with 15-25 amino acid residues was screened by the MultiDS system and generated a list of peptides with the Multiple Descriptor Index (MD index) scores, which was the core part of the MultiDS system. Sixty peptides with top MD scores were chemically synthesized and experimentally tested their antimicrobial activity against 10 kinds of Gram-positive bacteria, Gram-negative bacteria (including drug-resistant pathogens). A total of fifty-nine out of 60 (98.3%) peptides exhibited antimicrobial activity (MIC ≤ 64 μg/mL), and 24 out of 60 (40%) peptides showed high activity (MIC ≤ 2 μg/mL), validating the MultiDS system was an effective and predictive screening tool with high hit rate and superior antimicrobial activity. For further investigation, AMPs S1, S2, and S3 with the highest MD scores were used to treat the skin infection mouse models in vivo caused by Escherichia coli, drug-resistance Escherichia coli, and Staphylococcus aureus, respectively. All of S1, S2, and S3 showed comparable therapeutic effects on promoting infection healing to or even better than the positive drug levofloxacin. A mechanism study discovered that rapid bactericidal action was caused by cell membrane disruption and content leakage. The MultiDS system not only provides a high-throughput approach that allows for the mining of candidate AMPs from the global genome sequence but also opens up a new route to accelerate the discovery of peptide antibiotics.

Dataset Information

Precursor-centric genome-mining approach for lasso peptide discovery.

Publications

Precursor-centric genome-mining approach for lasso peptide discovery.

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