Control of Gene Expression in Leptospira spp. by Transcription Activator-Like Effectors Demonstrates a Potential Role for LigA and LigB in Leptospira interrogans Virulence.
Appl Environ Microbiol
; 81(22): 7888-92, 2015 Nov.
Article
em En
| MEDLINE
| ID: mdl-26341206
ABSTRACT
Leptospirosis is a zoonotic disease that affects â¼1 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 TALEßgal strain). Reverse transcriptase PCR (RT-PCR) analysis and enzymatic assays demonstrated that BgaL was not expressed in the TALEßgal 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.
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Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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Virulência
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Regulação Bacteriana da Expressão Gênica
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Leptospira
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Leptospira interrogans
Idioma:
En
Revista:
Appl Environ Microbiol
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Estados Unidos