Background & Aims

Digestive diseases, including inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), are often characterized by visceral pain. Although dysbiosis is a feature of IBD and IBS, the mechanisms by which colonic bacteria cause pain are largely unknown. Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor that is expressed in colonocytes, enteric neurons, and nociceptors. Mammalian serine and cysteine proteases from inflammatory cells (mast cell tryptase, neutrophil elastase, macrophage cathepsin S, legumain) and epithelial cells (trypsin) can activate PAR2 by diverse mechanisms and cause PAR2-dependent pain, which can contribute to IBD and IBS pain1. Thus PAR2 is a potential therapeutic target for visceral pain. We developed a strategy to identify proteases secreted from human colonic bacteria that can cleave the extracellular PAR2 N-terminal domain (NTD) and induce PAR2-dependent colonic pain.

Methods

A library of commensal and pathobiont bacteria (Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria, Actinobacteria, Verrucomicrobia) was studied. A substrate screening assay was used to identify secreted proteases that cleave the PAR2 NTD. Proteases were characterized by inhibitor susceptibility and identified using activity-based probes and mass spectrometry. Prioritized proteases were cloned, expressed in E. coli, and purified. Wild-type (WT) and knockout (KO) protease strains were generated. The ability of proteases to cleave and activate PAR2 was determined in HEK-PAR2 cells. The nociceptive actions of proteases were examined by measuring the excitability of nociceptors and colonic nociceptive responses in WT, Par2-/- and Par2-/-Nav1.8 mice. Par2-gfp knockin mice were used to assess whether proteases cause PAR2 activation and endocytosis.

Results

A screen of supernatant from 250 bacterial strains identified 52 with PAR2 NTD cleavage activity. Serine proteases from B. fragilis, P johnsonii, P distasonis and C. cadaveris and cysteine proteases from B. intestinalis and B. cellulosilyticus cleaved PAR2 NTD. Supernatants cleaved PAR2 in HEK-PAR2 cells and stimulated endocytosis and Ca2+ signaling. Serine hydrolase activity-based probes and LC-MS/MS analysis identified the serine protease Q5LDF9 from B. fragilis. Cloned and expressed Q5LDF9 retained PAR2-cleavage activity. B. fragilis supernatant containing Q5LDF9 induced excitability of mouse dorsal root ganglion nociceptors. When introduced into the mouse colon, B. fragilis supernatant caused abdominal mechanical allodynia and Par2-gfp endocytosis. These effects were absent in PAR2 KO mice. Supernatant from ?Q5LDF9 KO B. fragilis was inactive. Engraftment with WT but not DeltaQ5LDF9 B. fragilis in antibiotic-treated mice decreased caused mechanical allodynia

Conclusions

We have developed and validated a strategy to identify proteases that are secreted by human colonic bacteria that cleave PAR2 and cause PAR2-dependent excitation of nociceptors and colonic pain. Colonic bacteria secrete serine and cysteine proteases that can cleave and activate PAR2. Serine proteases from B. fragilis, notably Q5LDF9, cause PAR2-dependent colonic pain. The finding that bacterial proteases activate PAR2 to cause colonic pain has implications for development of drugs to treat painful digestive diseases associated with dysbiosis. Mammalian protease inhibitors lack selectivity, which impedes drug development. Bacterial proteases have low homology to mammalian proteases and are tractable drug targets. Since bacteria reside in the lumen and mucosa, luminally or mucosally-restricted protease inhibitors could be effective, minimizing side effects from systemic exposure. The detection of bacterial proteases in feces using activity-based probes may identify therapeutic targets.

References

1 Nestor N. Jimenez-Vargas et al. Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome. PNAS 2018

Presenting Author

Rocco Latorre

Poster Authors

Rocco Latorre

PhD

NYU-Pain Research Center

Lead Author

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Markus Lakemeyer

Friedrich Schiller University Jena, Institute of Organic Chemistry & Macromolecular Chemistry (IOMC)

Lead Author

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Kristyna Blazkova

Stanford University School of Medicine Department of Pathology

Lead Author

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Dane Jensen

NYU College of Dentistry

Lead Author

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Yatendra Mulpuri

New York University

Lead Author

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Chloe Peach

Lead Author

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Matthew Bogyo

Stanford University School of Medicine Department of Pathology

Lead Author

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Nigel Bunnett

Department of Molecular Pathobiology, Pain Research Center, NYU

Lead Author

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Topics

  • Models: Chronic Pain - Inflammatory