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. 2017 Jan 26;168(3):517-526.e18.
doi: 10.1016/j.cell.2016.12.021. Epub 2017 Jan 19.

Discovery of Reactive Microbiota-Derived Metabolites that Inhibit Host Proteases

Chun-Jun Guo  1 , Fang-Yuan Chang  2 , Thomas P Wyche  3 , Keriann M Backus  4 , Timothy M Acker  5 , Masanori Funabashi  1 , Mao Taketani  1 , Mohamed S Donia  6 , Stephen Nayfach  7 , Katherine S Pollard  7 , Charles S Craik  5 , Benjamin F Cravatt  4 , Jon Clardy  3 , Christopher A Voigt  2 , Michael A Fischbach  8
Affiliations

Discovery of Reactive Microbiota-Derived Metabolites that Inhibit Host Proteases

Chun-Jun Guo et al. Cell. .

Abstract

The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals.

Keywords: biosynthetic gene cluster; metagenomics; microbiome; natural products; peptide aldehyde; protease inhibitor; synthetic biology.

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Figures

Figure 1
Figure 1. Phylogenetic analysis of a family of NRPS BGCs found exclusively in gut isolates
Shown on the left is a phylogenetic tree (maximum parsimony, MEGA6) based on the large NRPS gene of the 47 BGCs in the family. Numbers next to the branches represent the percentage of replicate trees in which this topology was reached using a bootstrap test of 1000 replicates. The names of BGCs characterized experimentally are colored red (products obtained) or blue (no products observed). The domain organization of the NRPS enzyme(s) are shown to the right of each cluster (A, adenylation domain; C, condensation domain: T, thiolation domain; R, reductase domain). BGCs without an index number were discovered from non-human (e.g., rumen) gut bacterial isolates. See also Supplemental Figure S1, Figure S4, Figure S7, Table S1, and Table S2.
Figure 2
Figure 2. Chemical and biochemical analysis of the gut NRPS BGCs
(A) Chemical structures of the small molecule products of the gut NRPS gene clusters. (B) HPLC or LC-MS profiles showing the production of each molecule in Escherichia coli or Bacillus subtilis. From top left, E. coli DH10β (Ec); and Ec expressing bgc34, bgc35, and bgc52, as detected by UV absorption at 300 nm. B. subtilis 168 sfp+ (Bs); and Bs expressing bgc38 and bgc39, as detected by UV absorption at 360 nm. E. coli BAP1 (Ec) and Ec expressing bgc33; extracted ion chromatograms for the indicated masses are shown. (C) In vitro reconstitution of the bgc35 NRPS. From top right, HPLC profiles of organic extracts of the reaction without adding the enzyme (negative control) and the complete in vitro reaction (bgc35 iv), as detected by UV absorption at 300 nm. Below are authentic standards of compounds 4 and 2, and extracted ion chromatograms at the indicated masses showing production of compounds 4 and 2 in the reaction. The numbering of the peaks in (B) and (C) corresponds to the small molecules shown in (A). See also Supplemental Figure S1, Figure S2, Figure S3, Figure S7, Table S1, Table S2, Table S4, and Table S5.
Figure 3
Figure 3. Functional analyses of the gut NRPS cluster and its small molecule products
(A) Seven of eight publicly available gut metatranscriptomic data sets harbored an actively transcribed gene cluster from the gut NRPS family. Typical cluster tiling is shown for bgc52; a sample with unusually robust transcription of bgc71 is also shown (see Figure S4 for the remaining sample tilings). (B) A biosynthetic scheme for the pathways encoded by the gut NRPS clusters. A C-terminal reductase (R) domain catalyzes nicotinamide-dependent reduction of the thioester, releasing a free dipeptide aldehyde that has a half-life of hours under physiological conditions, and exists in equilibrium with the cyclic imine. In the presence of oxygen, this dihydropyrazinone oxidizes irreversibly to the pyrazinone. The chemical structure of the proteasome inhibitor bortezomib, which has a scaffold derived from dipeptide aldehydes, is shown in the box. (C) Results from a panel of in vitro protease inhibition assays using free-amino and N-acylated dipeptide aldehydes discovered in this study. IC50 values are shown in μM. Cat = cathepsin; N/O = no inhibition observed. Data for the corresponding N-Boc protected dipeptide aldehydes and pyrazinones are shown in Figure S5. (D and E) Competitive isoTOP-ABPP identifies CTSL as a target of the bgc35 product Phe-Phe-H. The heat map (D) shows all cathepsin cysteines, including both catalytic and non-catalytic detected in isoTOP-ABPP experiments where the THP-1 membrane fraction was subjected to the indicated concentrations of the Phe-Phe-H aldehyde. Note that cysteines on the same tryptic peptide cannot be differentiated and are indicated together, e.g. C135/138. The graphs (E) show the MS1 chromatographic peak ratios (R values) for all peptides identified from the THP1 membrane fraction treated with 25 μM Phe-Phe-H. The red dots indicate cysteines with R values >5 and the red line indicates the R value >5 threshold. See also Supplemental Figure S4, Figure S5, Figure S6, and Table S1.
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