LipL41 and LigA/LigB Gene Silencing on a LipL32 Knockout Leptospira interrogans Reveals the Impact of Multiple Mutations on Virulence

Leptospirosis is a global zoonosis caused by pathogenic bacteria of the genus Leptospira. The application CRISPR/Cas9 system has facilitated generation mutants and subsequent evaluation phenotypes. Since DNA breaks induced RNA-guided Cas9 nuclease are lethal to Leptospira, different methodologies were implemented overcome this limitation. Initially, CRISPR interference (CRISPRi) was employed create knockdown mutants, utilizing catalytically inactive (dCas9). Subsequently, co-expression repair from Mycobacterium smegmatis enabled scarless knockout mutants. We eliminated plasmids lipL32 L. interrogans strain further achieved multiple gene mutations via silencing in background. Strains lacking both LipL41 LipL32 LigA, LigB, LipL32, evaluated. absence proteins had no effect on leptospiral virulence. On other hand, unable cause acute disease. expanded apparatus for genetic manipulation leptospires allowed upon This work shows that not required disease consolidates LigA LigB as virulence factors.