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

Abstract

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.