CRISPR-based mutagenesis of lipopolysaccharide biosynthesis genes in Leptospira interrogans reveals gene essentiality and confirms the role of an O-antigen polymerase

Authors: FERNANDES, LUIS - Oak Ridge Institute For Science And Education (ORISE); Nally, Jarlath

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/6/2026
Publication Date: 3/13/2026
Citation: Fernandes, L.G., Nally, J.E. 2026. CRISPR-based mutagenesis of lipopolysaccharide biosynthesis genes in Leptospira interrogans reveals gene essentiality and confirms the role of an O-antigen polymerase. Scientific Reports. https://doi.org/10.1038/s41598-026-43869-y.
DOI: https://doi.org/10.1038/s41598-026-43869-y

Interpretive Summary: Pathogenic Leptospira species are the causative agent of leptospirosis, a bacterial zoonosis of global significance. Surface lipopolysaccharide (LPS) plays a major role in the virulence of Gram-negative bacteria, and their respective host interactions. However, little is known about the biogenesis of LPS in Leptospira. Here, we used advanced genetic tools to disrupt specific genes involved in the production of LPS. Among the targets for mutagenesis, we showed that frameshift deletions were never possible in several genes suggesting that they are essential for viability. Notably, one mutant strain with a truncated LPS due to mutation of an O-antigen polymerase was unable to trigger acute illness in animals. These findings help clarify how Leptospira cause infection and may guide the development of new bacterin vaccines to protect against different serovars.

Technical Abstract: Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira. Lipopolysaccharide (LPS) is an immunodominant and protective antigen for Leptospira, but its biosynthesis remains poorly understood. In this study, we employed CRISPR/Cas9-non-homologous end-joining and CRISPR-Prime Editing to mutate key genes within the rfb locus of L. interrogans, including those involved in core oligosaccharide assembly, and the biogenesis, polymerization, and ligation of O-antigen. Mutants were successfully generated in LIC11312 (waaF, heptosyltransferase II) and LIC12137 (wcaJ, undecaprenyl-phosphate glycosyltransferase) but yielded only in-frame deletions suggesting their essentiality. Mutants were also successfully generated in LIC12143, a putative O-antigen polymerase, which exhibited truncated LPS that failed to induce acute leptospirosis in hamsters but retained the ability to colonize kidneys. Mutation of LIC_RS09320, an O-antigen ligase, did not display a change in LPS phenotype. Bacterins prepared with either control wild-type or LIC12143 mutant cells conferred complete homologous protection with sterile immunity, though failed to protect against heterologous challenge. These findings confirm LIC12143 as a functional O-antigen polymerase and underscore the challenges in generating knockout mutants to understand LPS biosynthesis in leptospires.