# Post-translational modification of GlyGly-Cterm Proteins

> **NIH NIH F32** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2024 · $79,984

## Abstract

Abstract
Cholera continues to be a global burden manifesting as acute diarrheal disease that impacts impoverished and
destabilized regions. Bacteria including Vibrio cholerae utilize the Type II Secretion System (T2SS) for the
secretion of a diverse array of effector proteins and toxins to adapt to environmental changes, notably cholera
toxin the causative agent of cholera symptoms. While a variety of T2SS substrates including cholera toxin are
fully secreted, a subset is retained on the cell surface. A recently identified group of diverse substrates found in
some gram-negative bacteria including V. cholerae contains a homologous C-terminal domain, called the GlyGly-
Cterm, that targets its passenger proteins to the cell surface. This newly identified domain is processed by
rhombosortase, a subfamily of rhomboid protease, before T2SS transport. It remains unclear what sequence of
the GlyGly-Cterm is required for surface localization, what, if any, enzymes in addition to rhombosortase are
responsible for GlyGly-Cterm protein maturation, and importantly, why V. cholerae have this distinct surface-
retention system. Model protein, VesB from V. cholerae, will be used to probe the molecular requirements of the
GlyGly-Cterm domain by employing fluorescence microscopy, cell fractionation, and activity assays. Additional
genes coding for putative enzymes possibly involved in processing of GlyGly-Cterm proteins have been identified
in an ordered V. cholerae transposon library screen. This will be followed up on by generating clean gene
deletions and assessing VesB surface localization. Quantitative and spatial proteomics as well as enzymatic
assays will be used to characterize the role of these gene products. The importance of the GlyGly-Cterm will be
probed by expressing GlyGly-Cterm proteins with and without this extension. Currently characterized cell-surface
associated bacterial enzymes are almost exclusively retained by N-terminal lipidation, while the GlyGly-Cterm
system represents a novel C-terminal cell-surface anchoring mechanism. The enzymes containing a
GlyGly-Cterm are involved in a variety of functions contributing to V. cholerae environmental persistence and
pathogenesis including nutrient acquisition, biofilm formation, and potentially maintenance of membrane
integrity. The importance of GlyGly-Cterm proteins is demonstrated by disruption of rhombosortase, which
results in impaired cell growth, reduced biofilm formation, and sensitivity to membrane stress. Characterization
of this system has the potential to explain why some bacteria have evolved this unique cell-surface retention
system and may provide insight into conserved mechanisms of C-terminal membrane anchoring in higher
organisms.

## Key facts

- **NIH application ID:** 10906663
- **Project number:** 5F32AI178852-02
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Cameron Roberts
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $79,984
- **Award type:** 5
- **Project period:** 2023-07-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10906663

## Citation

> US National Institutes of Health, RePORTER application 10906663, Post-translational modification of GlyGly-Cterm Proteins (5F32AI178852-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10906663. Licensed CC0.

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