A systematic analysis of biosynthetic gene clusters in the human microbiome reveals a common family of antibiotics

Abstract

In complex biological systems, small molecules often mediate microbe-microbe and microbe-host interactions. Using a systematic approach, we identified 3,118 small-molecule biosynthetic gene clusters (BGCs) in genomes of human-associated bacteria and studied their representation in 752 metagenomic samples from the NIH Human Microbiome Project. Remarkably, we discovered that BGCs for a class of antibiotics in clinical trials, thiopeptides, are widely distributed in genomes and metagenomes of the human microbiota. We purified and solved the structure of a thiopeptide antibiotic, lactocillin, from a prominent member of the vaginal microbiota. We demonstrate that lactocillin has potent antibacterial activity against a range of Gram-positive vaginal pathogens, and we show that lactocillin and other thiopeptide BGCs are expressed in vivo by analyzing human metatranscriptomic sequencing data. Our findings illustrate the widespread distribution of small-molecule-encoding BGCs in the human microbiome, and they demonstrate the bacterial production of drug-like molecules in humans. PAPERCLIP:

Figure 1. Overview of BGCs in the human microbiome

A) Computational and experimental workflow for the identification of BGCs from human-associated bacteria. B) A bar graph showing the top 30 families by average BGC abundance from the human microbiome, and the average number and type of BGCs discovered in isolates of each genus using the workflow in A. See also Figure S1 and Supplemental Data File 1 for the full data set of predicted BGCs.

Figure 2. A selected subset of BGCs from the human microbiota

A) 25 selected BGCs from the human microbiota, spanning each of the body sites (gut, vagina, airways and skin, and oral cavity), BGC types (PKS, NRPS, RiPPs, terpenes, NI siderophores and saccharides) and prevalent bacterial phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria). The label of each gene cluster indicates its source organism, body site of origin, and the percentage of HMP samples harboring this cluster in its body site of origin. All but two of these BGCs are present in more than 10% of the samples from their body site of origin, indicating that they are widely distributed among healthy subjects. B) Heat map showing the representation of BGCs from A in a subset of 60 selected HMP metagenomic samples from four body sites. The color of the cells in the heat map represents an abundance score ranging from 10 (blue) to 1000 (red) (key shown to the right, see Extended Experimental Procedures for calculation of abundance scores, and see also Figure S2 and Table S1).

Figure 3. An abundant family of NRPS BGCs is found exclusively in gut isolates and stool metagenomes

Left, a phylogenetic tree (Maximum Parsimony, MEGA5) based on the main NRPS gene of 28 clusters from human bacterial gut isolates, two from bovine rumen archaeal isolates, and four from environmental isolates (see Experimental Procedures). The numbers next to the branches represent the percentage of replicate trees in which this topology was reached in a bootstrap test of 1000 replicates. Middle, schematic of each BGC (see also Figure S3 for a full heat map showing the prevalence and abundance of each member of this NRPS family in HMP stool samples). Right, domain organization of the NRPS genes of each cluster (A, adenylation domain; C, condensation domain; T, thiolation domain; R, terminal reductase domain).

Figure 4. A family of complex PKS BGCs is prevalent in the human oral cavity

A) Related PKS BGCs in human oral actinobacteria P. propionicum F0230a and A. timonensis DSM 23838 and the marine actinobacterium Streptomyces sp. A7248. The label of each BGC indicates its source organism. B) Domain organization of the three BGCs shown in A (AT, acyltransferase domain; KS, ketosynthase domain; MT, methyltransferase domain; Ox, oxidation domain; ECH, enoyl-CoA hydratase domain; DH, dehydratase domain; KR, ketoreductase domain; ACL, acyl-CoA ligase domain; T, thiolation domain, HCS, HMG-CoA synthase domain; FKB, FkbH-like protein. Note that the domain architecture is remarkably conserved among the three pathways, despite low amino acid sequence identity (~40%). C) Structures of SIA7248, the product of the Streptomyces sp. A7248 BGC shown in A, and the related molecule marinomycin A.

Figure 5. Thiopeptide BGCs are widespread in isolates and metagenomes of all main human body sites

A) Five thiopeptide BGCs from human isolates, eight thiopeptide BGCs from human metagenomes, and one thiopeptide BGC from a pig isolate are shown. The label of each BGC indicates its body site of origin. B) Precursor peptides corresponding to the thiopeptide BGCs shown in A. Note that the precursor peptides fall into six subgroups of nearly identical sequences. The structural portion of the precursor peptide is shown in red (see also Figure S5 for a phylogenetic analysis of thiopeptide BGCs). C) A heat map showing the representation and abundance of six oral thiopeptide BGCs in HMP metagenomic oral samples (see also Table S2 for the quantification of all thiopeptide BGCs in all HMP samples). Note that although each BGC shown in the heat map is well represented in the oral cavity, most samples harbor only one thiopeptide BGC.

Figure 6. Characterization of a new antibiotic, lactocillin, from a human vaginal isolate

A) HPLC analysis of organic extracts of cell pellets from wild-type (red) and lclD insertional mutant (blue) strains of L. gasseri JV-V03, monitored at 350 nm. An asterisk indicates the HPLC peak corresponding to lactocillin, B) Planar structure of lactocillin (see Experimental Procedures and see also Figure S6 and Supplemental Data File 2 for details about its purification and structural elucidation), the Bacillus cereus antibiotic thiocillin, and the clinical candidate LFF571 (Phase II, Novartis). Note the structural similarities among the three thiopeptides. C) Plasmid harboring the lactocillin BGC (see Experimental Procedures for details of the experimental closure of the circular plasmid). The lactocillin gene cluster (red) occupies ~30% of the plasmid; other elements on the plasmid include plasmid replication and maintenance genes (blue), transporters (black), transcription regulators (brown) and transposases and phage integrases (white). D) Raw oral metatranscriptomic reads were recruited to the lcl cluster using blastn and aligned using Geneious. Each black bar represents one read. Note that the precursor peptide lclB is amongst the most deeply covered genes in the lcl cluster, as anticipated for a RiPP pathway (see Experimental Procedures and see also Figure S7 for metatranscriptomic analysis of the whole lcl plasmid, and of bgc65).