# N-terminal acylation of Lipoproteins in Firmicutes

> **NIH NIH R01** · PENNSYLVANIA STATE UNIVERSITY, THE · 2021 · $314,133

## Abstract

Project Summary
 The long term goal of the application is to understand why and how lipoproteins from Firmicutes
undergo modifications. Lipoproteins are membrane associated globular proteins anchored to the bacterial
membrane surface through a lipidated N-terminal cysteine residue. They are ubiquitous cell envelope
structures found in both gram positive and negative bacteria, accounting for 1-5% of all genes in a typical
bacterial genome. Lipoproteins play roles in nearly every aspect of bacterial cell envelope physiology, from
nutrient acquisition to mediating cellular contacts. Lipoproteins are also important during the detection and
mounting of initial immune responses to clear bacteria infections. Due to their multiple essential cellular roles,
abundance, universal distribution, and their unique and highly conserved structure, innate immunity detects the
presence of bacteria through binding lipoproteins using Toll-like receptor 2 (TLR-2) complexes. TLR2 binds
lipoproteins through forming heterodimers, using either TLR-1 or TLR-6 depending on the state of N-acylation.
As such, understanding how and why certain bacteria utilize lipoprotein structural variations is pertinent to both
fundamental bacterial physiology and infection biology.
 In particular, this application studies the role of N-acylation in Enterococcus faecalis. There is emerging
evidence that lipoproteins are differentially N-acylated amongst bacteria in the Firmicutes phylum. A class of
integral membrane N-acylating proteins named Lit was identified in E. faecalis that makes lyso- form
lipoproteins in this organism. The lyso- form of lipoprotein has a unique acyl chain distribution pattern.
Whereas the diacylglyceryl form has both acyl chains on the glyceryl residue, and the triacyl form has these
two acyl chains plus a third N-terminal acyl chain connected through an amide bond to the α-amino cysteine
group, the lyso-form has a single acyl chain on both the N-terminus and glyceryl unit. The lyso- structure
suggests an acyl chain is removed from the diacylglyceryl unit and transferred to the N-terminus to form the
lyso- structure or that there are lipoprotein esterases working concert with Lit. The application aims to
reconstitute Lit activity by recombinant expression so as to test the proposed intramolecular transferase using
specifically labeled diacylglyceryl lipoprotein substrates. The products will be characterized by MALDI mass
spectrometry to determine acyl chain donor origin. A second aim is to test environmental stress condition in
order to gain insight into the biological role for N-acylation using the E. faecalis/Δlit isogenic paired strain set.
The third aim is to probe how Lit affects TLR-2 signaling, as preliminary data has suggested lyso-form
lipoproteins bind in more complex ways to TLR2 than their triacyl- and diacyl-glycerol congeners. The final aim
is to develop functional assays to uncover other lipoprotein N-terminal modifying enzymes.

## Key facts

- **NIH application ID:** 10187587
- **Project number:** 5R01GM127482-04
- **Recipient organization:** PENNSYLVANIA STATE UNIVERSITY, THE
- **Principal Investigator:** Timothy C. Meredith
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $314,133
- **Award type:** 5
- **Project period:** 2018-05-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10187587

## Citation

> US National Institutes of Health, RePORTER application 10187587, N-terminal acylation of Lipoproteins in Firmicutes (5R01GM127482-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10187587. Licensed CC0.

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