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Application of CRISPR-interference for elucidating leptospirosis pathogenesis and development of novel strategies for knockout mutant's obtainment

Grant number: 17/06731-8
Support Opportunities:Scholarships in Brazil - Post-Doctoral
Effective date (Start): July 01, 2017
Effective date (End): October 31, 2022
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal Investigator:Ana Lucia Tabet Oller do Nascimento
Grantee:Luis Guilherme Virgílio Fernandes
Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
Associated research grant:19/17488-2 - Advancing the understanding of pathogenesis and virulence of Leptospira interrogans through proteomics, structural, mutagenesis and immunological analyses, 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

Abstract

Leptospirosis is a zoonosis of global importance recently included in the List of Neglected Tropical Diseases. In urban areas, rodents play the role of the main reservoirs of the disease by allowing the colonization of leptospires in proximal tubes and excreting them alive via urine. According to the World Health Organization, the number of annual clinical cases worldwide remains underestimated. A better understanding of the pathogenesis of the bacteria is crucial for the development of an effective vaccine. The generation of Leptospira mutants is a key step in the elucidation of virulence factors and the basic biology of the bacterium. During the development of the Postdoctoral Project (2017/06731-8 and BEPE 2019/20302-8), the CRISPR-interference tool for epissomal and complete gene silencing in saprophytic and pathogenic strains was developed, and for the first time in the literature, species besides L. interrogans were manipulated. The CRISPRi system requires the constitutive expression of dCas9 ("dead" Cas9 protein) and a guide RNA (sgRNA), which directs the protein to the targets of interest, blocking transcription. This tool has been shown to be extremely useful in vitro and in vivo for elucidation of the role of Leptospira spp. proteins. In this context, our group is working with numerous potential genes for silencing by this technique, and with this additional period, it is intended to apply and to disseminate the CRISPRi technique in our laboratory, as well as to explore these potential targets. It is also intended to develop new plasmids for genetic knockout, through the use of the enzyme Cas9 and co-expression of Mycobacterium tuberculosis proteins that act in the repair of genomic breakdown induced by the enzyme.

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Scientific publications (19)
(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)
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.; et al. 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, . (17/01411-5, 17/06731-8, 14/50981-0, 17/01102-2, 16/11541-0, 16/04295-3, 16/01384-5)
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, . (16/11541-0, 14/50981-0, 17/06731-8)
KOCHI, LEANDRO TOSHIO; FERNANDES, LUIS GUILHERME VIRGILIO; NASCIMENTO, ANA LUCIA TABET OLLER. Heterologous Expression of the Pathogen-Specific LIC11711 Gene in the Saprophyte L. biflexa Increases Bacterial Binding to Laminin and Plasminogen. PATHOGENS, v. 9, n. 8, . (18/09652-4, 17/06731-8, 14/50981-0)
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, . (17/01411-5, 17/06731-8, 17/25167-6, 14/50981-0, 17/01102-2, 16/11541-0, 17/26223-7, 17/00236-5, 16/01384-5)
FERNANDES, L. G. V.; HORNSBY, R. L.; NASCIMENTO, A. L. T. O.; NALLY, J. E.. Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 degrees C. SCIENTIFIC REPORTS, v. 11, n. 1, . (14/50981-0, 17/06731-8, 19/20302-8)
DAROZ, BRENDA BEVILAQUA; VIRGILIO FERNANDES, LUIS GUILHERME; TEIXEIRA, ALINE FLORENCIO; TABET OLLER NASCIMENTO, ANA LUCIA. In Silico Structural and Functional Characterization of HtrA Proteins of Leptospira spp.: Possible Implications in Pathogenesis. TROPICAL MEDICINE AND INFECTIOUS DISEASE, v. 5, n. 4, . (18/21959-8, 17/06731-8, 16/11541-0, 14/50981-0)
CAVENAGUE, MARIA F. F.; TEIXEIRA, ALINE F. F.; FERNANDES, LUIS G. V.; NASCIMENTO, ANA L. T. O.. LIC12254 Is a Leptospiral Protein That Interacts with Integrins via the RGD Motif. TROPICAL MEDICINE AND INFECTIOUS DISEASE, v. 8, n. 5, p. 15-pg., . (18/08131-0, 19/17488-2, 16/11541-0, 17/06731-8)
FERNANDES, L. G., V; HORNSBY, R. L.; NASCIMENTO, A. L. T. O.; NALLY, J. E.. Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, v. N/A, n. 174, p. 14-pg., . (17/06731-8, 14/50981-0, 19/20302-8)
STONE, NATHAN E.; HALL, CARINA M.; ORTIZ, MARIELISA; HUTTON, SHELBY M.; SANTANA-PROPPER, ELLA; CELONA, KIMBERLY R.; WILLIAMSON, CHARLES H. D.; BRATSCH, NICOLE; FERNANDES, LUIS G. V.; BUSCH, JOSEPH D.; et al. Diverse lineages of pathogenic Leptospira species are widespread in the environment in Puerto Rico, USA. PLoS Neglected Tropical Diseases, v. 16, n. 5, p. 27-pg., . (19/20302-8, 17/06731-8)
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, v. 2018, p. 13-pg., . (14/50981-0, 16/11541-0, 17/06731-8)
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.; et al. Proteomics as a tool to understand Leptospira physiology and virulence: Recent advances, challenges and clinical implications. JOURNAL OF PROTEOMICS, v. 180, p. 8-pg., . (14/50981-0, 17/01411-5, 17/01102-2, 16/01384-5, 16/04295-3, 16/11541-0, 17/06731-8)
FERNANDES, LUIS G., V; AVELAR, KATIA E. S.; ROMERO, ELIETE C.; HEINEMANN, MARCOS B.; KIRCHGATTER, KARIN; NASCIMENTO, ANA L. T. O.. A New Recombinant Multiepitope Chimeric Protein of Leptospira interrogans Is a Promising Marker for the Serodiagnosis of Leptospirosis. TROPICAL MEDICINE AND INFECTIOUS DISEASE, v. 7, n. 11, p. 14-pg., . (17/06731-8, 19/17488-2)
DAROZ, BRENDA B.; FERNANDES, LUIS G. V.; CAVENAGUE, MARIA F.; KOCHI, LEANDRO T.; PASSALIA, FELIPE J.; TAKAHASHI, MARIA B.; NASCIMENTO FILHO, EDSON G.; TEIXEIRA, ALINE F.; NASCIMENTO, ANA L. T. O.. A Review on Host-Leptospira Interactions: What We Know and Future Expectations. FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY, v. 11, p. 13-pg., . (18/06201-1, 18/08131-0, 17/01102-2, 17/26223-7, 16/11541-0, 18/09652-4, 18/21959-8, 19/17488-2, 17/06731-8)
SANTOS, JADEMILSON C.; HANDA, SUMIT; FERNANDES, LUIS G. V.; BLEICHER, LUCAS; GANDIN, CESAR A.; DE OLIVEIRA-NETO, MARIO; GHOSH, PARTHO; NASCIMENTO, ANA LUCIA T. O.. Structural and biochemical characterization of Leptospira interrogans Lsa45 reveals a penicillin-binding protein with esterase activity. Process Biochemistry, v. 125, p. 13-pg., . (14/50981-0, 17/25167-6, 18/20321-0, 19/17488-2, 17/06731-8)
TEIXEIRA, ALINE F.; CAVENAGUE, MARIA F.; KOCHI, LEANDRO T.; FERNANDES, LUIS G.; SOUZA, GISELE O.; DE SOUZA FILHO, ANTONIO FRANCISCO; VASCONCELLOS, SILVIO A.; HEINEMANN, MARCOS BRYAN; NASCIMENTO, ANA L. T. O.. Immunoprotective Activity Induced by Leptospiral Outer Membrane Proteins in Hamster Model of Acute Leptospirosis. FRONTIERS IN IMMUNOLOGY, v. 11, . (14/50981-0, 17/06731-8, 18/09652-4, 18/08131-0, 16/11541-0)
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, . (16/01384-5, 17/06731-8, 14/50981-0)
FERNANDES, LUIS G. V.; PUTZ, ELLIE J.; STASKO, JUDITH; LIPPOLIS, JOHN D.; NASCIMENTO, ANA L. T. O.; NALLY, JARLATH E.. valuation of LipL32 and LigA/LigB Knockdown Mutants in Leptospira interrogans Serovar Copenhageni: Impacts to Proteome and Virulenc. FRONTIERS IN MICROBIOLOGY, v. 12, . (17/06731-8, 19/20302-8, 19/17488-2)
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, . (17/06731-8, 14/50981-0)
FERNANDES, LUIS G., V; STONE, NATHAN E.; ROE, CHANDLER C.; GORIS, MARGA G. A.; VAN DER LINDEN, HANS; SAHL, JASON W.; WAGNER, DAVID M.; NALLY, JARLATH E.. Leptospira sanjuanensis sp. nov., a pathogenic species of the genus Leptospira isolated from soil in Puerto Rico. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, v. 72, n. 10, p. 9-pg., . (19/20302-8, 17/06731-8)

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