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Proteases influence colony aggregation behavior in Vibrio cholerae.

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.

SUBMITTER: Detomasi TC

PROVIDER: S-EPMC10709122 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Proteases influence colony aggregation behavior in Vibrio cholerae.

Detomasi Tyler C TC Batka Allison E AE Valastyan Julie S JS Hydorn Molly A MA Craik Charles S CS Bassler Bonnie L BL Marletta Michael A MA

The Journal of biological chemistry 20231026 12

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<sup>-1</sup>s<sup>-1</sup>. The activity with a LapX su ...[more]

PMID: 37898401

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Project description:Rhomboid proteases are universally conserved and facilitate the proteolysis of peptide bonds within or adjacent to cell membranes. While eukaryotic rhomboid proteases have been demonstrated to harbor unique cellular roles, prokaryotic members have been far less characterized. For the first time, we demonstrate that Vibrio cholerae expresses two active rhomboid proteases that cleave a shared substrate at distinct sites, resulting in differential localization of the processed protein. The rhomboid protease rhombosortase (RssP) was previously found to process a novel C-terminal domain called GlyGly-CTERM, as demonstrated by its effect on the extracellular serine protease VesB during its transport through the V. cholerae cell envelope. Here, we characterize the substrate specificity of RssP and GlpG, the universally conserved bacterial rhomboid proteases. We show that RssP has distinct cleavage specificity from GlpG, and specific residues within the GlyGly-CTERM of VesB target it to RssP over GlpG, allowing for efficient proteolysis. RssP cleaves VesB within its transmembrane domain, whereas GlpG cleaves outside the membrane in a disordered loop that precedes the GlyGly-CTERM. Cleavage of VesB by RssP initially targets VesB to the bacterial cell surface and, subsequently, to outer membrane vesicles, while GlpG cleavage results in secreted, fully soluble VesB. Collectively, this work builds on the molecular understanding of rhomboid proteolysis and provides the basis for additional rhomboid substrate recognition while also demonstrating a unique role of RssP in the maturation of proteins containing a GlyGly-CTERM.ImportanceDespite a great deal of insight into the eukaryotic homologs, bacterial rhomboid proteases have been relatively understudied. Our research aims to understand the function of two rhomboid proteases in Vibrio cholerae. This work is significant because it will help us better understand the catalytic mechanism of rhomboid proteases as a whole and assign a specific role to a unique subfamily whose function is to process a subset of effector molecules secreted by V. cholerae and other pathogenic bacteria.

Project description:Motility is required for many bacterial pathogens to reach and colonize target sites. Vibrio cholerae traverses a thick mucus barrier coating the small intestine to reach the underlying epithelium. We screened a transposon library in motility medium containing mucin to identify factors that influence mucus transit. Lesions in structural genes of the type VI secretion system (T6SS) were among those recovered. Two-dimensional (2D) and 3D single-cell tracking was used to compare the motility behaviors of wild-type cells and a mutant that collectively lacked three essential T6SS structural genes (T6SS-). In the absence of mucin, wild-type and T6SS- cells exhibited similar speeds and run-reverse-flick (RRF) swimming patterns, in which forward-moving cells briefly backtrack before stochastically reorienting (flicking) in a new direction upon resuming forward movement. We show that mucin induced T6SS expression and activity in wild-type bacteria but significantly decreased their swimming speed and flicking, yielding curvilinear or near-surface circular traces for many cells. Conversely, mucin slowed T6SS- cells to a lesser extent, and many continued to flick and produce RRF-like traces. ΔcheY3 cells, which exclusively swim in the forward direction and thus cannot flick, also produced curvilinear traces with or without mucin present and, on occasion, near-surface circular traces in the presence of mucin. The dependence of flicking on swimming speed suggested that mucin-induced T6SS activity further decreased V. cholerae motility and thereby reduced flicking probability during reverse-to-forward transitions. We propose that this encourages cells to continue on their current trajectory rather than reorienting, which may benefit those tracking toward the epithelial surface.IMPORTANCEV. cholerae deploys an arsenal of virulence factors as it attempts to traverse a protective mucus layer and reach the epithelial surface of the distal small intestine. The T6SS used to cull bacterial competition during infection is induced by mucus. We show that this activity may serve an additional purpose by further decreasing motility in the presence of mucin, thereby reducing the probability of speed-dependent, near-perpendicular directional changes. We posit that this encourages cells to maintain course rather than change direction, which may aid those attempting to reach and colonize the epithelial surface.

Dataset Information

Proteases influence colony aggregation behavior in Vibrio cholerae.

Publications

Proteases influence colony aggregation behavior in Vibrio cholerae.

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