Proteases influence colony aggregation behavior in Vibrio cholerae

Tyler C Detomasi; Allison E Batka; Julie S Valastyan; Molly A Hydorn; Charles S Craik; Bonnie L Bassler; Michael A Marletta

doi:10.1016/j.jbc.2023.105386

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.

Authors

Tyler C Detomasi¹, Allison E Batka², Julie S Valastyan³, Molly A Hydorn⁴, Charles S Craik⁵, Bonnie L Bassler³, Michael A Marletta⁶

Affiliations

¹ Department of Chemistry, University of California, Berkeley, Berkeley, California, USA; Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA.
² Department of Chemistry, University of California, Berkeley, Berkeley, California, USA.
³ Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA; The Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
⁴ Department of Chemistry, University of California, Berkeley, Berkeley, California, USA; Department of Microbiology and Immunology, College of Physicians and Surgeons, Columbia University, New York, New York, USA.
⁵ Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA.
⁶ Department of Chemistry, University of California, Berkeley, Berkeley, California, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, California, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA. Electronic address: marletta@berkeley.edu.

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.

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

Substances

Bacterial Proteins
Leucyl Aminopeptidase
Peptides
Serine Proteases

Grants and funding

U19 AI171110/AI/NIAID NIH HHS/United States