Abstract
Aggregation behavior provides bacteria protection from harsh environments and threats to survival. Two uncharacterized proteases, LapX and Lap, are important for Vibrio cholerae liquid-based aggregation. Here, we determined that LapX is a serine protease with a preference for cleavage after glutamate and glutamine residues in the P1 position, which processes a physiologically based peptide substrate with a catalytic efficiency of 180 ± 80 M-1s-1. The activity with a LapX substrate identified by a multiplex substrate profiling by mass spectrometry screen was 590 ± 20 M-1s-1. Lap shares high sequence identity with an aminopeptidase (termed VpAP) from Vibrio proteolyticus and contains an inhibitory bacterial prepeptidase C-terminal domain that, when eliminated, increases catalytic efficiency on leucine p-nitroanilide nearly four-fold from 5.4 ± 4.1 × 104 M−1s−1 to 20.3 ± 4.3 × 104 M−1s−1. We demonstrate that LapX processes Lap to its mature form and thus amplifies Lap activity. The increase is approximately eighteen-fold for full-length Lap (95.7 ± 5.6 × 104 M−1s−1) and six-fold for Lap lacking the prepeptidase C-terminal domain (11.3 ± 1.9 × 105 M−1s−1). In addition, substrate profiling reveals preferences for these two proteases that could inform in vivo function. Furthermore, purified LapX and Lap restore the timing of the V. cholerae aggregation program to a mutant lacking the lapX and lap genes. Both proteases must be present to restore WT timing, and thus they appear to act sequentially: LapX acts on Lap, and Lap acts on the substrate involved in aggregation.
Original language | English (US) |
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Article number | 105386 |
Journal | Journal of Biological Chemistry |
Volume | 299 |
Issue number | 12 |
DOIs | |
State | Published - Dec 2023 |
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Biochemistry
- Cell Biology
Keywords
- Vibrio cholerae
- aggregation
- biofilm
- proteolysis