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

<jats:title>ABSTRACT</jats:title> <jats:p> 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 <jats:named-content content-type="genus-species">Leptospira</jats:named-content> spp., two TALE constructs were designed. First, a constitutively expressed TALE gene specific for the <jats:italic>lacO</jats:italic> -like region upstream of <jats:italic>bgaL</jats:italic> was <jats:italic>trans</jats:italic> inserted in the saprophyte <jats:named-content content-type="genus-species">Leptospira biflexa</jats:named-content> (the TALE <jats:sub>βgal</jats:sub> strain). Reverse transcriptase PCR (RT-PCR) analysis and enzymatic assays demonstrated that BgaL was not expressed in the TALE <jats:sub>βgal</jats:sub> 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 <jats:italic>ligA</jats:italic> and <jats:italic>ligB</jats:italic> was <jats:italic>cis</jats:italic> inserted into the pathogen <jats:named-content content-type="genus-species">Leptospira interrogans</jats:named-content> (TALE <jats:sub> <jats:italic>lig</jats:italic> </jats:sub> ). LigA and LigB expression was studied by using three independent clones: TALE <jats:sub> <jats:italic>lig1</jats:italic> </jats:sub> , TALE <jats:sub> <jats:italic>lig2</jats:italic> </jats:sub> , and TALE <jats:sub> <jats:italic>lig3</jats:italic> </jats:sub> . Immunoblot analysis of osmotically induced TALE <jats:sub> <jats:italic>lig</jats:italic> </jats:sub> clones demonstrated 2- to 9-fold reductions in the expression levels of LigA and LigB, with the highest reductions being noted for TALE <jats:sub> <jats:italic>lig1</jats:italic> </jats:sub> and TALE <jats:sub> <jats:italic>lig2</jats:italic> </jats:sub> , which were avirulent <jats:italic>in vivo</jats:italic> 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 <jats:named-content content-type="genus-species">Leptospira</jats:named-content> spp., providing an additional genetic manipulation tool for this genus. </jats:p>