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

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.