Control of Gene Expression in Leptospira spp. by Transcription Activator-Like Effectors Demonstrates a Potential Role for LigA and LigB in Leptospira interrogans Virulence

Abstract

Leptospirosis is a zoonotic disease that affects ∼1 million people annually, with a mortality rate of >10%. Currently, there is an absence of effective genetic manipulation tools for targeted mutagenesis in pathogenic leptospires. Transcription activator-like effectors (TALEs) are a recently described group of repressors that modify transcriptional activity in prokaryotic and eukaryotic cells by directly binding to a targeted sequence within the host genome. To determine the applicability of TALEs within Leptospira spp., two TALE constructs were designed. First, a constitutively expressed TALE gene specific for the lacO-like region upstream of bgaL was trans inserted in the saprophyte Leptospira biflexa (the TALEβgal strain). Reverse transcriptase PCR (RT-PCR) analysis and enzymatic assays demonstrated that BgaL was not expressed in the TALEβgal strain. Second, to study the role of LigA and LigB in pathogenesis, a constitutively expressed TALE gene with specificity for the homologous promoter regions of ligA and ligB was cis inserted into the pathogen Leptospira interrogans (TALElig). LigA and LigB expression was studied by using three independent clones: TALElig1, TALElig2, and TALElig3. Immunoblot analysis of osmotically induced TALElig clones demonstrated 2- to 9-fold reductions in the expression levels of LigA and LigB, with the highest reductions being noted for TALElig1 and TALElig2, which were avirulent in vivo and nonrecoverable from animal tissues. This study reconfirms galactosidase activity in the saprophyte and suggests a role for LigA and LigB in pathogenesis. Collectively, this study demonstrates that TALEs are effective at reducing the expression of targeted genes within saprophytic and pathogenic strains of Leptospira spp., providing an additional genetic manipulation tool for this genus.

FIG 1

The TALE_βgal gene represses β-galactosidase activity in L. biflexa. (A) RT-PCR of L. biflexa wild-type (WT) and TALE_βgal strains. (Left) Image showing that the TALE_βgal strain transcribes the TALE_βgal gene. (Right) Image showing that the wild type transcribes bgaL, while the TALE_βgal strain does not. (B) Wild-type, natural Patoc β-galactosidase (β-gal) mutant, and TALE_βgal cells were grown on solid-phase EMJH medium and exposed to X-gal for 16 h. The wild type was able to cleave X-gal to galactose and 5-bromo-4-chloro-3-hydroxyindole, creating a blue color. Natural Patoc βgal mutant and TALE_βgal cells were unable to cleave X-gal; thus, the colonies remained colorless.

FIG 2

TALE_lig represses expression of LigA and LigB in L. interrogans. (A) Representative immunoblot of the L. interrogans wild type and 3 biologically distinct TALE_lig transformants (TALE_lig1, TALE_lig2, and TALE_lig3). A total of 3.5 μg of protein extract/sample was reacted with antisera specific for LigA/B (top) or FlaA2 (bottom). Analysis revealed a 3.1- ± 1.2-fold reduction in the LigA level (P < 0.05) and a 6.6- ± 1.2-fold reduction in the LigB level (P < 0.05) in the TALE_lig1 strain compared to the wild type, a 5.3- ± 1.3-fold reduction in the LigA level (P < 0.05) and a 9.1- ± 3.8-fold reduction in the LigB level (P < 0.05) in the TALE_lig2 strain compared to the wild type, and a 2.1- ± 0.4-fold reduction in the LigA level (P < 0.05) and a 4.6- ± 1.6-fold reduction in the LigB level (P < 0.05) in the TALE_lig3 strain compared to wild type. (B) Survival analysis of intraperitoneal inoculation of 10⁶ cells of the wild-type, TALE_lig1, TALE_lig2, or TALE_lig3 strain in hamsters shows reduced virulence of the TALE_lig3 transformant compared to that of the wild type (P = 0.09) and a significant attenuation of virulence in the TALE_lig1 (P < 0.01) and TALE_lig2 (P < 0.01) transformants compared to that of the wild type.