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bioRxiv · 10.64898/2026.02.05.703148

The GlyGly-CTERM domain functions as an independent motif that targets proteins to rhombosortase in Vibrio cholerae

Roberts, C. S.·Kannatey-Asibu, K.·Potocek, E.·Sandkvist, M.
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Abstract

Vibrio cholerae secretes a variety of effector proteins that are freely released into the extracellular space via its Type II Secretion System (T2SS) including cholera toxin, the causative agent of the disease cholera. In contrast to cholera toxin, a growing number of T2SS effectors is increasingly understood to remain associated with the cell surface. The serine protease VesB from V. cholerae is a surface protein that is produced with a short C-terminal motif, called GlyGly-CTERM. This motif is linked to the rest of VesB via a predicted unstructured linker. In addition to VesB, V. cholerae encodes five additional GlyGly-CTERM proteins including the serine proteases VesA and VesC, a putative metalloprotease VCA0065, the DNase Xds, and VC1485, a protein of unknown function. Proteins with a GlyGly-CTERM are co-distributed in bacteria with a specific rhomboid protease called rhombosortase (RssP), and it has been demonstrated that VesB requires processing by RssP for surface localization and activation. Here, we investigate the intrinsic function of the GlyGly-CTERM by proteomics, enzyme assays and heterologous expression of alternative motifs on model protein VesB as well as on unrelated periplasmic and extracellular proteins. We show that the GlyGly-CTERM and processing by RssP is sufficient for membrane association, but a secondary secretion signal is required for outer membrane translocation. Unexpectedly, VesC is released from the cells through autoproteolytic processing at a site within the unstructured linker. We propose that the GlyGly-CTERM facilitates efficient secretion of proteins via its intrinsic ability to target them to RssP resulting in membrane association. ImportanceVibrio cholerae is responsible for the disease cholera. Without treatment, V. cholerae causes massive dehydration with high mortality rates (1). It utilizes the Type II Secretion System to export the causative agent of disease, cholera toxin, as well as a suite of additional effector proteins that are involved in pathogenesis. Here, we investigate the unique transport mechanism of a subset of effectors secreted by this pathogen that are targeted to the cell surface.

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Roberts, C. S., Kannatey-Asibu, K., Potocek, E., Sandkvist, M.. 2026-02-05. The GlyGly-CTERM domain functions as an independent motif that targets proteins to rhombosortase in Vibrio cholerae. https://doi.org/10.64898/2026.02.05.703148

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