Biology subjects

Keller, L. J.

Publications and source records attributed to Keller, L. J..

4 recordsLinked to original sources

bioRxiv2025

Identification of a secreted protease from Bacteroides fragilis that induces intestinal pain and inflammation by cleavage of PAR2

Protease-activated receptor 2 (PAR2) is a central regulator of intestinal barrier function, inflammation and pain. Upregulated intestinal proteolysis and PAR2-signaling are implicated in inflammatory bowel diseases (IBDs) and irritable bowel syndrome (IBS). To identify potential bacterial regulators of PAR2 activity, we developed a functional assay for PAR2 processing and used it to screen conditioned media from a library of diverse gut commensal microbes. We found that multiple bacteria secrete proteases that cleave host PAR2. Using chemoproteomic profiling with a covalent irreversible inhibitor, we identified a previously uncharacterized Bacteroides fragilis serine protease Bfp1, and showed that it cleaves and activates PAR2 in multicellular and murine models. PAR2 cleavage by Bfp1 disrupts the intestinal barrier, sensitizes nociceptors, and triggers colonic inflammation and abdominal pain. Collectively, our findings uncover Bfp1-mediated PAR2-processing as a new axis of host-commensal-interaction in the gut that has the potential to be targeted for therapeutic intervention in IBD or IBS.

microbiology↗

bioRxiv2022

Chemoproteomic identification of a dipeptidyl peptidase 4 (DPP4) homolog in Bacteroides thetaiotaomicron important for envelope integrity and fitness

Serine hydrolases play important roles in signaling and human metabolism, yet little is known about the functions of these enzymes in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human protease dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Functional studies reveal that BT4193 is a true homolog of hDPP4 while the other is misannotated and is a proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and is inhibited by FDA-approved type 2 diabetes drugs that target hDPP4. Loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. Taken together, our findings suggest that serine hydrolases contribute to gut microbiota dynamics and may be off-targets for existing drugs that could cause unintended impact on the microbiota.

microbiology↗

bioRxiv2021

A screen of covalent inhibitors in Mycobacterium tuberculosis identifies serine hydrolases involved in lipid metabolism as potential therapeutic targets

The increasing incidence of antibiotic-resistant Mycobacterium tuberculosis infections is a global health threat necessitating the development of new antibiotics. Serine hydrolases (SHs) are a promising class of targets because of their importance for the synthesis of the mycobacterial cell envelope. We screened a library of small molecules containing serine-reactive electrophiles and identified narrow spectrum inhibitors of M. tuberculous growth. Using these lead molecules, we performed competitive activity-based protein profiling and identified multiple SH targets, including enzymes with uncharacterized functions. Lipidomic analyses of compound-treated cultures revealed an accumulation of free lipids and a substantial decrease in lipooligosaccharides, linking SH inhibition to defects in cell envelope biogenesis. Mutant analysis revealed a path to resistance via the synthesis of mycocerates, but not through mutations to target enzymes. Our results suggest that simultaneous inhibition of multiple SH enzymes is likely to be an effective therapeutic strategy for the treatment of M. tuberculosis infections.

microbiology↗

bioRxiv2020

A chemiluminescent protease probe for rapid, sensitive, and inexpensive detection of live Mycobacterium tuberculosis

Tuberculosis (TB) is a top-ten cause of death worldwide. Successful treatment is often limited by insufficient diagnostic capabilities, especially at the point of care in low-resource settings. The ideal diagnostic must be fast, cheap, and require minimal clinical resources while providing high sensitivity, selectivity, and the ability to differentiate live from dead bacteria. We describe here the development of a Fast, Luminescent, and Affordable Sensor of Hip1 (FLASH) for the diagnosis and monitoring of drug sensitivity of Mycobacterium tuberculosis (Mtb). FLASH is a selective chemiluminescent substrate for the Mtb protease Hip1 that when processed, produces visible light that can be measured with a high signal to noise ratio using inexpensive sensors. FLASH is sensitive to fmol of recombinant Hip1 enzyme in vitro and can detect as few as thousands of Mtb cells in culture or in human sputum samples within minutes. The probe is highly selective for Mtb compared to other non-tuberculous mycobacteria and can distinguish live from dead cells. Importantly, FLASH can be used to measure antibiotic killing of Mtb in culture with greatly accelerated timelines compared to traditional protocols. Overall, FLASH has the potential to enhance both TB diagnostics and drug resistance monitoring in resource-limited settings. One Sentence SummaryA luminescent probe enables sensitive detection of Mycobacterium tuberculosis for diagnostics, treatment monitoring, and drug susceptibility testing.

microbiology↗