# 8-azido-3,8-dideoxy-D-manno-octulosonic acid-mediated metabolic oligosaccharide engineering for specific labeling of diverse Enterobacterales

## TL;DR

This paper introduces a new sugar-based labeling method to track specific gut bacteria in real-time, enabling detailed in vivo studies of bacterial interactions.

## Contribution

A novel metabolic labeling strategy using Kdo-N3 enables species-specific in vivo imaging of diverse Enterobacterales.

## Key findings

- Kdo-N3 enables specific labeling of Gram-negative bacteria's LPS core across multiple Enterobacterales species.
- In vivo imaging in the murine gastrointestinal tract demonstrates species-specific labeling with Kdo-N3.
- LPS profiling reveals Kdo-N3 incorporation and O-antigen-dependent shielding effects.

## Abstract

Current microbiome studies lack real-time or spatial information, often requiring large cohorts for longitudinal investigations. Real-time, in vivo molecular imaging would alleviate these shortcomings but is hampered by the limited labeling performance of current strategies. Here, we use the azido-modified sugar 8-Azido-3,8-dideoxy-D-manno-octulosonic acid (Kdo-N3) to metabolically engineer the core oligosaccharide of Gram negative lipopolysaccharide, enabling conjugation with DBCO-fluorophores. We extend this approach by systematically screening multiple Enterobacteriaceae strains, optimizing labeling conditions, validating core-oligosaccharide-specific labeling using LPS knockout mutants, and demonstrating in vivo species-specific MOE-BCC labeling with Kdo-N3 inside the murine gastrointestinal tract and subsequent in vivo imaging. Beyond Salmonella Typhimurium and Escherichia coli Nissle 1917, a broad range of Enterobacterales species were amenable to Kdo-N3 sugar incorporation, potentially unlocking species-specific studies of bacterial interaction in vitro and in vivo.

•LPS O-antigen fluorescent labeling shows limited cross-species applicability•LPS core labeling is achieved specifically by Kdo-N3 in various Enterobacterales•In situ Kdo-N3 click labeling enables species specific in vivo bacteria imaging•LPS profiling shows Kdo-N3 incorporation and O-antigen dependent shielding

LPS O-antigen fluorescent labeling shows limited cross-species applicability

LPS core labeling is achieved specifically by Kdo-N3 in various Enterobacterales

In situ Kdo-N3 click labeling enables species specific in vivo bacteria imaging

LPS profiling shows Kdo-N3 incorporation and O-antigen dependent shielding

Molecular biology; Microbiology; Bioengineering