# Beyond serology: saccharide profiling enables identification of antigenically similar Leptospira and prompts re-evaluation of bacterial lipopolysaccharide evolution

## TL;DR

A new method using sugar profiling of bacterial structures helps identify Leptospira strains and reveals insights into their evolution.

## Contribution

A novel, non-animal-based method for identifying Leptospira serovars through LPS sugar profiling is introduced.

## Key findings

- LPS sugar profiling can distinguish antigenically similar Leptospira serovars without animal-derived reagents.
- LPS structures of Leptospira interrogans and Leptospira borgpetersenii from the same serovar Hardjo differ despite similarities.
- Phylogenetic analysis identifies glycosyltransferases linked to LPS structure evolution and potential horizontal gene transfer.

## Abstract

Leptospirosis is a zoonotic infectious disease of growing importance in both human and veterinary medicine. Gram-negative spirochetes of Leptospira are traditionally classified into serovars based on their antigenic identity, which must be ascertained to design effective treatment procedures for humans and appropriate vaccination strategies in pets and livestock. Unfortunately, identifying Leptospira serovars is challenging and currently requires access to a wide panel of reference strains, animal-derived antisera, or monoclonal antibodies. Here, we describe a new method for the identification of Leptospira serovars that is based on monosaccharide composition analysis of the polysaccharide part of bacterial lipopolysaccharide (LPS) structures. Our approach requires no animal sacrifice and can be implemented in any laboratory equipped for chromatographic analysis. An LPS sugar fingerprint that is specific to each bacterial isolate that we studied can be generated. Importantly, sugar profiling of LPS enables distinguishing Leptospira serovars that are antigenically very similar. Using our new approach, we discover that the LPS structures of two cattle pathogens belonging to two different species: Leptospira interrogans and Leptospira borgpetersenii, and to one serovar: Hardjo, can be distinguished despite sharing major similarities. Through extensive phylogenetic analysis, we reveal which specific glycosyltransferases of the LPS biosynthesis rfb locus likely drove the emergence of these similarities and identify a single glycosyltransferase that might have contributed to the formation of saccharide differences in the LPS structure. Our findings have implications for future work on the evolution of bacterial polysaccharide synthesis and highlight the importance of preventing horizontal gene transfer between pathogenic bacteria.