Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37C

Authors: FERNANDES, LUIS - Butantan Institute; Hornsby, Richard; NASCIMENTO, ANA - Butantan Institute; Nally, Jarlath

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/4/2021
Publication Date: 1/19/2021
Citation: Fernandes, L.G., Hornsby, R.L., Nascimento, A.L., Nally, J.E. 2021. Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37C. Scientific Reports. 11. https://doi.org/10.1038/s41598-021-81400-7.
DOI: https://doi.org/10.1038/s41598-021-81400-7

Interpretive Summary: Leptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira. Pathogenic mechanisms of leptospirosis remain poorly understood and despite the recent advances for genetic manipulation of many microbial species, target mutations or gene silencing in pathogenic Leptospira continues to be inherently inefficient, laborious, costly and difficult to implement. In this work, we demonstrate the application of CRISPR interference (CRISPRi) to block transcription of genes encoding major outer membrane proteins. We also optimized conjugation and mutant recovery in a newly described growth medium for pathogenic leptospires. The ease in generating constructs for gene-specific silencing, combined with the rapid recovery of mutants in modified HAN solid media is a significant milestone in leptospirosis research that will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.

Technical Abstract: Leptospirosis is a neglected widespread zoonosis caused by pathogenic species of the genus Leptospira. Manifestations of disease can range from fever, chills, headache, and myalgia, to severe icteric Weil’s disease, causing up to 60,000 deaths per year. Pathogenic mechanisms of leptospirosis are poorly understood, in part because of a lack of effective, affordable and specific genetic tools for gene silencing. In addition, pathogenic leptospires are highly fastidious and the selection of mutants on solid agar media can take up to 6 weeks. The catalytically inactive Cas9 ("dead”, dCas9) is an RNA-guided DNA-binding protein derived from the type II CRISPR/Cas system from Streptococcus pyogenes. dCas9 can be directed towards specific coding regions, thus hampering transcription and causing gene silencing, in a strategy termed CRISPR interference (CRISPRi), and as recently applied to the saprophytic L. biflexa. In the present work, dCas9 and single guide-RNA (sgRNA) expressing plasmids were employed to silence genes encoding outer membrane proteins of pathogenic L. interrogans. Conjugation protocols were optimized using the newly described HAN media modified for rapid mutant recovery at 37 degrees C in 3 percent CO2 within 8 days. Complete silencing of LipL32 was achieved using CRISPRi to provide a stable phenotype. CRISPRi was also applied to demonstrate concomitant and complete silencing of both LigA and LigB outer membrane proteins; evaluation of the mutant phenotype revealed for the first time that these proteins are involved in pathogenic Leptospira serum resistance. Gene silencing in pathogenic leptospires will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.