Beyond Serology: Saccharide profiling enables identification of antigenically similar Leptospira and prompts re-evaluation of bacterial lipopolysaccharide evolution

Aleksandra Lewicka^1*Jan Lyczakowski²Pardyak Laura¹Dubniewicz Klaudia¹Dariusz Latowski²Arent Zbigniew¹

• ¹University of Agriculture in Krakow, Krakow, Poland
• ²Jagiellonian University, Kraków, Lesser Poland, Poland

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 that needs to 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 identification of Leptospira serovars that is based on monosaccharide composition analysis of the polysaccharide part of bacterial LPS. Our approach requires no animal sacrifice and can be implemented in any laboratory equipped for chromatographic analysis. A sugar fingerprint of LPS 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 LPS 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 which might have contributed to formation of saccharide differences in LPS structure. Our findings have implications for future work on evolution of bacterial polysaccharide synthesis and highlight the importance of preventing horizontal gene transfer between pathogenic bacteria.