Signals from the Stool

Proteases are enzymes that act to cleave proteins. Among them are a group of proteases called cathepsins that were first identified in the lysosome, but are found to be active in other cellular areas as well. Cathepsins are known to prefer acidic environments and many are known to degrade various parts of the extra cellular matrix like collagen, elastin, and fibronectin. Furthermore, cathepsins are known to play a role in various immune responses like antigen presentation in intestinal cells1.

Gut microbiota are bacteria that live within a hosts gut and act to digest starches that cannot be digested by the hosts natural enzymes. In humans, the gut microbiota is key for converting consumed plant and animal glycans into short chain fatty acids that can be further utilized by the body. Imbalances in the gut microbiota have been linked to a variety of diseases in the host like obesity, inflammatory bowel disease and even colon cancer. Changes in the gut microbiota have also been known to compound over time impacting future generations2. These implications have been fairly recent, so little is known about molecular signaling between the microbiota and the host. In their paper, Guo et al. propose a novel small molecule product of the microbiota that may play a role in signaling via protease inhibition.

Guo et al. focused on a family of nonribosomal peptide synthetase (NRPS) gene clusters in their studies. Using a BLAST search, they were able to identify a total of 47 clusters. Among these 47 clusters, almost all are known to reside in isolates the human or other mammal gut, while not typically found in free-living organisms, implying a role relevant to the human gut. Guo et al. chose 14 clusters that were representative of the 47. They confirmed that these 14 NRPS clusters were widely present in the human population using an optimized BLAST algorithm and found that the clusters were present in more than 88% of the 149 stool samples collected from humans and 96% of the 1,267 publicly available stool samples. They also used RNA-sequencing data from the publicly available stool samples to confirm that the NRPS clusters were actively transcribed in humans.

Of the 14 clusters, 3 were isolates from Escheria coli and Bacillus subtilis, so they were easily put into these hosts. The other 11 were unable to be traced back to an original organism or the original organism was unable to be obtained for largescale testing, so they were synthesized using optimized codons and cloned into E. coli or B. subtilis.

After cultivation, the researchers used liquid chromatography-mass spectrometry (LC-MS) to analyze the extract for small molecule products of NRPS. The evidence indicated that these extracts contained new peaks corresponding to a family of pyrazinones and dihydropyrazinones. They confirmed these findings using UV-Vis spectroscopy and NMR. Overall, 32 total compounds were isolated from 7 of the 14 clusters. The authors confirmed that these were not a result of expression in E. coli and B. subtilis by cultivating two other bacteria known to harbor two of NRPS clusters being tested and observed these peaks were still present. Guo et al. suggested that these products may be the end result of the release of a C-terminal aldehyde, as NRPSs are known to have a C-terminal reductase domain that catalyzes this reaction (Fig 1). This aldehyde is known to be stable in physiological conditions and can act as a cell-permeable protease inhibitor.

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