# ABC transporter-mediated secretion of capsular polysaccharides

> **NIH NIH R21** · UNIVERSITY OF VIRGINIA · 2021 · $225,215

## Abstract

Essentially all living systems produce cell surface structures to rigidify cells, form protective coats, or facilitate
cell adhesion and migration. Microbial ‘cell walls’ usually perform protective functions for survival under
detrimental conditions, to reduce the efficacy of their host’s innate immune response, or to form 3-dimensional
meshworks, called biofilms. Common building materials for these extracellular structures are polysaccharides
that either function on their own or are integrated with other polymers into elaborate composite materials.
 Capsular polysaccharides (CPS) are abundant among Gram-negative and –positive bacteria. The
polymers form dense extracellular structures that limit diffusion, aid in osmoregulation, and form thick
protective coats around the cell. Some CPS mimic host glycans, thereby disguising potent pathogens under an
immunologically invisible coat. The polymers are synthesized and deposited on the cell surface by two
fundamentally different pathways. One assembles the polymer in the periplasm from short lipid-linked
precursors and translocates it across the outer membrane (OM) concomitantly. In the ABC transporter-
dependent pathway, however, the CPS is synthesized intracellularly on a lipid anchor and transported after its
completion through a secretion system that spans the inner and the OM. The molecular and mechanistic
mechanisms of both pathways remain poorly understood. To aid the development of novel antibiotic strategies,
we seek to establish a detail structure-function analysis of the abundant ABC transporter-dependent CPS
biosynthesis pathway.
 Our approach is two-pronged. First, we seek to establish a robust genetically tractable model system
for CPS secretion (Aim 1A and B). Second, we will complement our functional analyses with detailed structural
insights into the CPS ABC transporter (Aim 2), thereby providing the molecular basis for substrate recognition,
CPS translocation, as well as interaction with periplasmic and OM transporter components.
 To this end, we engineered a standard E. coli laboratory strain to produce a polysaccharide capsule
from plasmid-encoded components. The expressed operon contains 9 genes and each can be removed from
its expression plasmid by standard restriction enzyme digestion. Further, we also developed a molecular probe
enabling the detection of the synthesized capsule on the cell surface, thereby correlating CPS production with
the expression of the biosynthetic machinery.
 To integrate our functional analyses with a 3D structure of the CPS ABC transporter, we purified a
stable transporter in complex with its periplasmic subunit that likely stabilizes interactions with the OM pore.
We will use cryo electron microscopy to determine the transporter’s structure in different nucleotide-bound
states. Combined, our proposed research will provide the molecular basis for CPS secretion and lay the
foundation for structure-guided drug development.

## Key facts

- **NIH application ID:** 10287699
- **Project number:** 1R21AI164216-01
- **Recipient organization:** UNIVERSITY OF VIRGINIA
- **Principal Investigator:** Jochen Zimmer
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $225,215
- **Award type:** 1
- **Project period:** 2021-06-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10287699

## Citation

> US National Institutes of Health, RePORTER application 10287699, ABC transporter-mediated secretion of capsular polysaccharides (1R21AI164216-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10287699. Licensed CC0.

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