Generation of T. cruzi (CL Brener) strains knockout for IP6K and evaluation of homologous recombination (HR) repair capacity

• Grant number: 20/16480-5
• Support Opportunities: Scholarships in Brazil - Master
• Effective date (Start): March 01, 2021
• Effective date (End): July 31, 2023
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Marcelo Santos da Silva
• Grantee: Bryan Etindi Abuchery
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated research grant: 19/10753-2 - Investigation on the role of inositol pyrophosphates (PP-IPs) in DNA repair pathways and telomere dynamics using trypanosomatids as a model, AP.JP
• Associated scholarship(s): 22/04595-8 - Application of the CRISPR/Cas9-riboswitch-based method to downregulate the inositol polyphosphate kinase (IP6K) expression in Trypanosoma cruzi, BE.EP.MS

Abstract

In model eukaryotes, inositol pyrophosphates (PP-IPs) - mainly PP-IP4, IP7, and IP8 - are involved in a wide range of processes, such as regulation of telomere length, and homologous recombination (HR). However, the mechanism of action of PP-IPs in pathways related to DNA metabolism (especially homologous recombination repair) is not fully understood. PP-IP4, IP7, and IP8 are synthesized by pathways involving the participation of IP6K and PP-IP5K kinases. Trypanosomatids has an ortholog gene for IP6K, but apparently does not have orthologs for PP-IP5K, i.e., they do not synthesize IP8, which make them excellent models for the study of PP-IP4 e IP7. Thus, this MSc research plan consists of generating T. cruzi (CL Brener) strains KO to IP6K (IP6K-/+ e IP6K-/-) using the CRISPR/Cas9 system, challenge the strains generated by ionizing radiation (IR), and evaluate the HR repair capacity relative to WT using flow cytometry, growth curves, and protein recruitment kinetics (western blot - WB and immunofluorescence - IFA). Briefly, analysis of DNA content by flow cytometry will be done to identify possible cell cycle arrests and to measure the period required to overcome it. Growth curves will be performed to observe the proliferation pattern after IR, and comparative recruitment kinetics could identify subtle differences in the recruitment pattern of some key HR pathway components. It is worth emphasizing that this approach will be of fundamental importance to answer the essential questions raised by the main project. (AU)

Scientific publications

(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)

ABUCHERY, BRYAN E.; BLACK, JENNIFER A.; DA SILVA, MARCELO S.. Spotlight transcriptomics reveals hidden information in trypanosomatids. Trends in Parasitology, v. 38, n. 1, p. 3-pg., 2021-12-15. (20/01883-7, 20/16480-5, 20/10277-3, 19/10753-2)

ASSIS, LUIZ H. C.; ANDRADE-SILVA, DEBORA; SHIBURAH, MARK E.; DE OLIVEIRA, BEATRIZ C. D.; PAIVA, STEPHANY C.; ABUCHERY, BRYAN E.; FERRI, YETE G.; FONTES, VERONICA S.; DE OLIVEIRA, LEILANE S.; DA SILVA, MARCELO S.; et al. Cell Cycle, Telomeres, and Telomerase in Leishmania spp.: What Do We Know So Far?. CELLS, v. 10, n. 11, NOV 2021. (19/10753-2, 20/08162-3, 21/04253-7, 20/00316-1, 20/16465-6, 18/04375-2, 21/05523-8, 20/10277-3, 19/25985-6, 20/16480-5)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

ABUCHERY, Bryan Etindi. Generation of T. cruzi strains knock-out for IP6K and evaluation of the resulting phenotypes. 2023. Master's Dissertation - Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu Botucatu.