Evaluation of LipL32 and LigA/LigB knockdown mutants in Leptospira interrogans Serovar Copenhageni: impacts to proteome and virulence

Authors: Putz, Ellie; FERNANDES, LUIS - Butantan Institute; Stasko, Judith; Lippolis, John; NASCIMENTO, ANNA - Butantan Institute; Nally, Jarlath

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/17/2021
Publication Date: 2/2/2022
Citation: Fernandes, L.G., Putz, E.J., Stasko, J.A., Lippolis, J.D., Nascimento, A.L., Nally, J.E. 2022. Evaluation of LipL32 and LigA/LigB knockdown mutants in Leptospira interrogans Serovar Copenhageni: impacts to proteome and virulence. Frontiers in Microbiology. 12. Article 799012. https://doi.org/10.3389/fmicb.2021.799012.
DOI: https://doi.org/10.3389/fmicb.2021.799012

Interpretive Summary: Leptospirosis is a devastating zoonotic bacterial disease that affects humans, companion animals, and all major livestock species. Symptoms vary greatly depending on host species and infecting Leptospira, and can range from completely asymptomatic to multi-organ failure and death in severe cases. Leptospires can colonize the kidneys of infected hosts where bacteria can then be subsequently shed in the urine. This contributes greatly to the persistence of leptospirosis as it can be contracted from direct contact with an infected host, or indirectly from the environment. Leptospirosis is considered a neglected tropical disease and although vaccines exist, they largely suffer from a lack of cross protection between serovars. In the lab, leptospires are slow growing, extremely fastidious, and highly sensitive to media and culture conditions. This has contributed to characterization limitations in Leptospira species. Recently, Clustered Regularly Interspaced Short Palindromic Repeat interference (CRISPRi) has been used to successfully create silenced virulence factor mutant Leptospira strains. Established strains include silenced LipL32, which is an important outer membrane protein used to identify pathogenic leptospires, as well as a combined silencing of LigA and LigB (LigAB) proteins which are established virulence factors. This study further characterizes the virulence of LipL32 and LigAB mutant strains, both by looking at disease presentation in the hamster model of leptospirosis and as well as examining the total protein changes with proteomic analysis. When hamsters were challenged with the LipL32 mutant strain, animals developed severe clinical symptoms of disease. Animals infected with the LigAB mutant by contrast, developed no clinical symptoms. However, upon necropsy it was discovered that the kidneys of LigAB challenged hamsters still contained leptospires suggesting chronic disease. Proteomic analysis revealed that there were 46 significantly differentially expressed (DE) proteins between LigAB mutants and control leptospires. In contrast, LipL32 mutant strains had 243 DE proteins, suggesting the loss of surface LipL32 has substantial changes on the surface protein profile of pathogenic leptospires. Collectively, this work emphasizes the advantages using CRISPRi technology to evaluate and characterize virulence factors of leptospires and their respective host-pathogen interactions in animal models of leptospirosis.

Technical Abstract: Leptospirosis is a worldwide zoonosis caused by pathogenic species of the genus Leptospira, accounting for more than one million cases and nearly 60,000 deaths per year. Recently, the application of CRISPR interference (CRISPRi) technology to Leptospira has resulted in targeted gene silencing and ushered in a new era of molecular characterization and mode of investigation for the leptospirosis field. CRISPRi relies on the episomal and constitutive expression of two components: a DNA binding protein, catalytically “dead” Cas9 (dCas9), and a single-guide RNA (sgRNA), responsible for target specificity. Previously our group generated a LipL32 and a double LigA/LigB (LigAB) mutant, which in the current study, were validated and further characterized by whole-cell proteomics in comparison to control leptospires harboring plasmid expressing dCas9 alone. Comparison of control and LigAB mutant leptospires identified 46 significantly differentially expressed (DE) proteins, whereas comparison of control and LipL32 mutant leptospires resulted in 243 DE proteins, including increased amounts of the known virulence impactors and surface membrane receptors LipL45, LipL31, LigB and LipL41. We also evaluated the virulence of LipL32 and LigAB mutants in the hamster model of leptospirosis. Hamster challenge experiments showed that the LigAB mutant was unable to cause acute disease, but mutant leptospires could still be recovered from target organs, indicating attenuation of virulence and a shift to chronic disease. Notably, the LipL32 mutant displayed augmented virulence, supported by consistent acute manifestations and early onset of clinical symptoms compared to control animals challenged with leptospires expressing only dCas9. Evaluation of LipL32 and LigAB mutants recovered from liver and kidney revealed high plasmid stability, and by extension, gene silencing in vivo. Collectively, this work emphasizes the advantages and feasibility of using CRISPRi technology to evaluate and characterize virulence factors of leptospires and their respective host-pathogen interactions in animal models of leptospirosis. Importantly, it also provides insight into the requirements of LigA and LigB for acute disease, and explores the impact of silencing expression of lipL32 which induced substantial proteomic changes including changes in amounts of outer membrane proteins.