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

Autor(es)

Fernandes, L. G. V. ^{[1, 2]} ; Hornsby, R. L. ^[2] ; Nascimento, A. L. T. O. ^[1] ; Nally, J. E. ^[2]

Afiliação do(s) autor(es)

^[1] Inst Butantan, Lab Desenvolvimento Vacinas, BR-05503900 Sao Paulo - Brazil

^[2] ARS, Infect Bacterial Dis Res Unit, Natl Anim Dis Ctr, USDA, Ames, IA 50011 - USA

• Tipo de documento: Artigo Científico
• Fonte: SCIENTIFIC REPORTS; v. 11, n. 1 JAN 19 2021.
• Citações Web of Science: 2

Resumo

Leptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira, and is responsible for 60,000 deaths per year. Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic Leptospira continues to be inherently inefficient, laborious, costly and difficult to implement. 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 (dCas9) is an RNA-guided DNA-binding protein from the Streptococcus pyogenes CRISPR/Cas system and can be used for gene silencing, in a strategy termed CRISPR interference (CRISPRi). Here, this technique was employed to silence genes encoding major 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% CO2 within 8 days. Complete silencing of LipL32 and concomitant and complete silencing of both LigA and LigB outer membrane proteins were achieved, revealing for the first time that Lig proteins are involved in pathogenic Leptospira serum resistance. Gene silencing in pathogenic leptospires and rapid mutant recovery will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis. (AU)

• Processo FAPESP: 14/50981-0 - Busca de proteínas de superfície nas sequências do genoma da Leptospira interrogans: caracterização funcional e imunológica para o entendimento de mecanismos envolvidos na patogênese de bactéria
• Beneficiário: Ana Lucia Tabet Oller do Nascimento
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 17/06731-8 - Aplicação de CRISPR-interferência na elucidação da patogênese da leptospirose e desenvolvimento de novas estratégias para obtenção de mutantes knockout
• Beneficiário: Luis Guilherme Virgílio Fernandes
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 19/20302-8 - Silenciamento de RNA não-codificador (ncRNA) mediado por interferência CRISPR (CRISPRi) em cepas saprofíticas e patogênicas de Leptospira
• Beneficiário: Luis Guilherme Virgílio Fernandes
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Pós-Doutorado