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Development of new strategies for genetic manipulation ín Leptospira spp.: antisense RNA ánd CRISPR/Cas9

Grant number: 17/06731-8
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): July 01, 2017
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal Investigator:Ana Lucia Tabet Oller Do Nascimento
Grantee:Luis Guilherme Virgílio Fernandes
Home Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
Associated research grant:14/50981-0 - Search for surface proteins among the genome sequences of Leptospira interrogans: functional and immunological characterization to understanding mechanisms involved in the bacterial pathogenesis, AP.TEM
Associated scholarship(s):19/20302-8 - CRISPR interference (CRISPRi)-mediated silencing of non-coding RNA (ncRNA) in saprophyte and pathogenic strains of Leptospira, BE.EP.PD


Leptospirosis is a zoonosis of global importance recently included in the list of Neglected Tropical Diseases. In urban areas, rodents are the main reservoirs of the disease by allowing the colonization of leptospires in their proximal renal tubules and excreting them alive in the urine. According to the World Health Organization, the number of annual clinical cases worldwide remains underestimated. A better understanding of the bacterial pathogenesis is a crucial point in the development of an effective vaccine. The generation of Leptospira mutants is a key part in understanding the virulence factors and the basic biology of the bacteria. Although there have been advances in the development of genetic tools for manipulation of Leptospira spp. - especially with the development of replicative vectors - directional mutagenesis and gene silencing currently rely exclusively on homologous recombination with a resistance marker. However, the frequency of knock out mutants recovered is extremely low, suggesting an inefficient capacity for double recombination events, especially for pathogenic strains. In addition, the homologous recombination methodology makes the study of essential genes impossible. The use of antisense RNA technology may favor gene silencing, especially by reducing the translation of proteins by obstruction of the ribosome binding sites in messenger RNA, and the use of inducible promoters can enable the study of essential genes. The use of the CRISPR / Cas9 system may favor gene inactivation events, using the guiding RNA for cleavage of target genes. The validation and implementation of these methodologies will bring great contribution in the field of study of the pathogenesis of leptospirosis.

Scientific publications (5)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
TEIXEIRA, ALINE F.; FERNANDES, V, LUIS G.; CAVENAGUE, MARIA F.; TAKAHASHI, MARIA B.; SANTOS, JADEMILSON C.; PASSALIA, FELIPE J.; DAROZ, BRENDA B.; KOCHI, LEANDRO T.; VIEIRA, MONICA L.; NASCIMENTO, ANA L. T. O. Adjuvanted leptospiral vaccines: Challenges and future development of new leptospirosis vaccines. Vaccine, v. 37, n. 30, p. 3961-3973, JUL 9 2019. Web of Science Citations: 1.
FERNANDES, L. G. V.; GUAMAN, L. P.; VASCONCELLOS, S. A.; HEINEMANN, MARCOS B.; PICARDEAU, M.; NASCIMENTO, A. L. T. O. Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira. SCIENTIFIC REPORTS, v. 9, FEB 12 2019. Web of Science Citations: 0.
KOCHI, LEANDRO T.; FERNANDES, V, LUIS G.; SOUZA, GISELE O.; VASCONCELLOS, SILVIO A.; HEINEMANN, MARCOS B.; ROMERO, ELIETE C.; KIRCHGATTER, KARIN; NASCIMENTO, ANA L. T. O. The interaction of two novel putative proteins of Leptospira interrogans with E-cadherin, plasminogen and complement components with potential role in bacterial infection. VIRULENCE, v. 10, n. 1, p. 734-753, JAN 1 2019. Web of Science Citations: 0.
NASCIMENTO FILHO, EDSON G.; VIEIRA, MONICA L.; TEIXEIR, ALINE F.; SANTOS, JADEMILSON C.; FERNANDES, LUIS G. V.; PASSALIA, FELIPE J.; DAROZ, BRENDA B.; ROSSINI, AMANDA; KOCHI, LEANDRO T.; CAVENAGUE, MARIA F.; PIMENTA, DANIEL C.; NASCIMENTO, ANA L. T. O. Proteomics as a tool to understand Leptospira physiology and virulence: Recent advances, challenges and clinical implications. JOURNAL OF PROTEOMICS, v. 180, n. SI, p. 80-87, MAY 30 2018. Web of Science Citations: 1.
TEIXEIRA, ALINE F.; FERNANDES, LUIS G. V.; SOUZA FILHO, ANTONIO; SOUZA, GISELE O.; VASCONCELLOS, SILVIO A.; HEINEMANN, MARCOS B.; NASCIMENTO, ANA L. T. O. Evaluation of Lsa46 and Lsa77 Leptospiral Proteins for Their Immunoprotective Activities in Hamster Model of Leptospirosis. BIOMED RESEARCH INTERNATIONAL, 2018. Web of Science Citations: 2.

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