Dipeptidyl peptidases constitute a class of non-classical serine proteases that regulate an array of biological functions, making them pharmacologically attractive enzymes. With this work, we identified and characterized a dipeptidyl peptidase from Mycobacterium tuberculosis (MtDPP) displaying a strong preference for proline residues at the P1 substrate position and an unexpectedly high thermal stability. MtDPP was also characterized with alanine replacements of residues of its active site that yielded, for the most part, loss of catalysis. We show that MtDPP catalytic activity is inhibited by well-known human DPP4 inhibitors. Using MALDI-TOF mass spectrometry we also describe that in vitro, MtDPP mediates the truncation of the C-X-C motif chemokine ligand 10, indicating a plausible role in immune modulation for this mycobacterial enzyme.

Dipeptidyl peptidases constitute a class of non-classical serine proteases that regulate an array of biological functions, making them pharmacologically attractive enzymes. With this work, we identified and characterized a dipeptidyl peptidase from Mycobacterium tuberculosis (MtDPP) displaying a strong preference for proline residues at the P1 substrate position and an unexpectedly high thermal stability. MtDPP was also characterized with alanine replacements of residues of its active site that yielded, for the most part, loss of catalysis. We show that MtDPP catalytic activity is inhibited by well-known human DPP4 inhibitors. Using MALDI-TOF mass spectrometry we also describe that in vitro, MtDPP mediates the truncation of the C-X-C motif chemokine ligand 10, indicating a plausible role in immune modulation for this mycobacterial enzyme.

Antimicrobial Resistance is challenging healthcare and food security and is driven by antibiotic overuse and environmental pollution. Longitudinal monitoring of antibiotic residue would help to track pollution sources and assess the effect of interventions. Here we propose a novel high-throughput monitoring method. We mutagenized E. coli MG1655 and identified and characterized 12 antibiotic-sensitive biosensors (ABSBs) with different genotypes by a newly developed method called Antibiotic Response determined by Euclidean Distance (ARED), based on growth (OD600) and metabolic activity (resazurin). The ABSBs contain nonsense or frameshift mutations in expected genes, like those coding for efflux pumps and lipopolysaccharide biosynthesis enzymes, as well as mutations in genes lesser or not known to be important for antibiotic sensitivity. Resazurin-based ARED can achieve antibiotic detection in the ng/mL range for most antibiotics tested in aqueous solutions. Our study shows that mutagenesis of E. coli can generate a tremendous shift in antibiotic sensitivity and that quantification of metabolic activity with ARED is a straightforward manner to monitor antibiotic activity in aqueous solutions. We propose that this method can be adopted for any collection of cell strains that possess differential antibiotic sensitivity and thus can be implemented for widespread monitoring of samples with unknown antibiotic complexion and origin.