Proteases influence colony aggregation behavior in Vibrio cholerae

J Biol Chem. 2023 Dec;299(12):105386. doi: 10.1016/j.jbc.2023.105386. Epub 2023 Oct 26.


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.

Keywords: Vibrio cholerae; aggregation; biofilm; proteolysis.

MeSH terms

  • Bacterial Proteins* / chemistry
  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / physiology
  • Catalysis
  • Leucyl Aminopeptidase* / chemistry
  • Leucyl Aminopeptidase* / genetics
  • Leucyl Aminopeptidase* / physiology
  • Peptides
  • Serine Proteases* / chemistry
  • Serine Proteases* / genetics
  • Serine Proteases* / physiology
  • Substrate Specificity
  • Vibrio cholerae* / enzymology
  • Vibrio cholerae* / genetics
  • Vibrio cholerae* / physiology


  • Bacterial Proteins
  • Leucyl Aminopeptidase
  • Peptides
  • Serine Proteases