# Molecular Mechanisms of Protein Sorting by the Type II Secretion System

> **NIH NIH R01** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2020 · $393,892

## Abstract

Cholera, an acute diarrheal disease, remains a global burden to human health. The key factor chiefly responsible
for this devastating disease is cholera toxin, an AB5 toxin that is produced and secreted by Vibrio cholerae. Its
extracellular secretion is dependent on the type II secretion system (T2SS), which is also responsible for the
outer membrane translocation of proteases, lipases, nucleases and chitinases. Common to many Gram-negative
pathogens, it uniquely transports these factors from the periplasmic compartment to the extracellular
environment in their fully folded conformations. Despite a dramatic increase in structural knowledge of the T2SS
and its individual components in recent years, the mechanism by which T2S substrates are recognized and
sorted for outer membrane translocation remains to be determined. A lack of significant sequence and structural
similarity between these proteins complicates the identification of a common secretion signal. Other confounding
factors include the findings that the T2SS supports the extracellular transport of both soluble proteins and
lipoproteins and that there are differences in the final destination among T2S substrates. Some T2S substrates
such as cholera toxin are released to the extracellular space once transported through the outer membrane;
however, others remain surface associated or may reattach to the bacterial cell surface following extracellular
release. The trypsin-like protease VesB is an example of a T2S substrate that is primarily retained on the cell
surface. VesB belongs to a unique class of extracellular enzymes that have a C-terminal extension consisting of
two prominent glycines and a hydrophobic helix followed by positively charged residues (GlyGly-CTERM
domain). Rhombosortase, a newly discovered member of the intramembrane rhomboid protease family, cleaves
off the GlyGly-CTERM domain during transit of VesB through the cell envelope, and the posttranslationally
modified VesB is localized to the cell surface.
 The experiments described in this proposal are designed to test the hypothesis that the T2S system, in
collaboration with rhombosortase, mediates the maturation and surface localization of GlyGly-CTERM containing
proteins. Specifically, this proposal will determine the mechanism of surface anchoring of proteins produced with
GlyGly-CTERM extensions, decipher how GlyGly-CTERM proteins are differentially recognized and secreted by
the T2SS, and assess the T2SS/rhombosortase system for surface localization of GlyGly-CTERM-tagged
heterologous proteins. The findings will facilitate understanding of the function and specificity of rhombosortase
as well as the broader class of medically relevant rhomboid proteases and may identify ways to manipulate the
T2S/rhombosortase system for preventative use.

## Key facts

- **NIH application ID:** 9983565
- **Project number:** 5R01AI137085-03
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Maria B Sandkvist
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $393,892
- **Award type:** 5
- **Project period:** 2018-09-24 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9983565

## Citation

> US National Institutes of Health, RePORTER application 9983565, Molecular Mechanisms of Protein Sorting by the Type II Secretion System (5R01AI137085-03). Retrieved via AI Analytics 2026-06-10 from https://api.ai-analytics.org/grant/nih/9983565. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*