Research Article: C-terminal processing of GlyGly-CTERM containing proteins by rhombosortase in Vibrio cholerae

Date Published: October 23, 2018

Publisher: Public Library of Science

Author(s): Shilpa Gadwal, Tanya L. Johnson, Henriette Remmer, Maria Sandkvist, Karla J. F. Satchell.

http://doi.org/10.1371/journal.ppat.1007341

Abstract

Vibrio cholerae and a subset of other Gram-negative bacteria, including Acinetobacter baumannii, express proteins with a C-terminal tripartite domain called GlyGly-CTERM, which consists of a motif rich in glycines and serines, followed by a hydrophobic region and positively charged residues. Here we show that VesB, a V. cholerae serine protease, requires the GlyGly-CTERM domain, the intramembrane rhomboid-like protease rhombosortase, and the type II secretion system (T2SS) for localization at the cell surface. VesB is cleaved by rhombosortase to expose the second glycine residue of the GlyGly-CTERM motif, which is then conjugated to a glycerophosphoethanolamine-containing moiety prior to engagement with the T2SS and outer membrane translocation. In support of this, VesB accumulates intracellularly in the absence of the T2SS, and surface-associated VesB activity is no longer detected when the rhombosortase gene is inactivated. In turn, when VesB is expressed without an intact GlyGly-CTERM domain, VesB is released to the extracellular milieu by the T2SS and does not accumulate on the cell surface. Collectively, our findings suggest that the posttranslational modification of the GlyGly-CTERM domain is essential for cell surface localization of VesB and other proteins expressed with this tripartite extension.

Partial Text

The type II secretion system (T2SS) is a multi-protein complex used by many Gram-negative bacteria to secrete extracellular proteins [1–4]. Most notably, Vibrio cholerae, the causative agent of cholera, uses the T2SS to secrete cholera toxin [5, 6]. Cholera toxin and other T2SS substrates are secreted in a two-step process. First, proteins translocate across the inner membrane via recognition of their signal peptide by the Sec or Tat systems. Then, the secretion intermediates fold, engage with the T2SS and traverse the channel formed by the outer membrane embedded secretin [7–9]. These substrates are then free to diffuse away from the cell.

In this study, we used the trypsin-like serine protease VesB as a model protein to determine the relationship between the GlyGly-CTERM domain, rhombosortase, and the T2SS in V. cholerae. T2S substrates translocate through the outer membrane and are either released into the extracellular milieu or retained on the cell surface through a variety of mechanisms. We show here that the majority of VesB is surface-associated; however, depending on growth conditions, various amounts of the surface-localized VesB are released to the extracellular space. Rhombosortase cleaves off the GlyGly-CTERM domain and the newly generated C-terminus is further modified to retain VesB on the cell surface once transported through the T2SS. When rhombosortase is absent, full-length VesB is largely subjected to degradation, but a small fraction is cleaved by GlpG. However, there is likely no additional posttranslational modification of GlpG-cleaved VesB and, therefore, GlpG-cleaved VesB is released to the extracellular milieu. Additionally, rhombosortase-mediated C-terminal processing leads to subsequent VesB auto-activation, while cleavage by GlpG in the absence of rhombosortase results in an inactive VesB, indicating that either correct C-terminal processing of VesB and/or its localization to the outer membrane are required for its auto-activation.

 

Source:

http://doi.org/10.1371/journal.ppat.1007341

 

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