Generation of T. cruzi strains knock-out for IP6K and evaluation of the resulting phenotypes

Author(s)

Bryan Etindi Abuchery

• Document type: Master's Dissertation
• Press: Botucatu. 2023-07-17.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2023-07-07
• Advisor: Marcelo Santos da Silva

Abstract

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. There are no effective vaccines and the available treatment options are effective only in the acute phase of the disease but can cause serious side effects on the patients. Thus, the search for the elucidation of molecular pathways that may provide potential targets for drug development is of paramount importance. Inositol pyrophosphates (PP-IPs) – mainly IP7, and IP8 – are involved in a wide range of processes in eukaryotes. However, the mechanism of action of these metabolites is not yet fully understood. IP7 and IP8 are synthesized by pathways involving the participation of IP6K and PP-IP5K kinases, respectively. Trypanosomatids have an ortholog gene for IP6K but do not have orthologs for PP-IP5K, making them excellent models for studying IP7. Here, using CRISPR/Cas9 approach, we disrupted single and double alleles of IP6K, generating IP6K-/+ and IP6K-/- lineages, respectively. IP6K inactivation causes several morphological effects, such as rounding and wrinkling of the cell body, increased number of glycosomes, and mitochondrial enlargement. Notably, IP6K-/- lineage (double-null) was unable to proliferate for more than 7 days, suggesting that this kinase is essential for this organism. Interestingly, IP6K-/+ lineage (single-null) showed a slight cell cycle arrest at G0/G1 phase with no DNA damage (quiescent cells). Moreover, the IP6K-/+ lineage showed a high proliferative capacity within the host mammalian cell, suggesting that the quiescent cells observed may be metacyclic forms. Together, our findings suggest that the total loss of IP6K has harmful consequences for T. cruzi. However, paradoxically, the disruption of a single allele of IP6K leads to T. cruzi being prone to multiplying further inside the host cell, generating a conundrum that we are still trying to understand. (AU)

• FAPESP's process: 20/16480-5 - Generation of T. cruzi (CL Brener) strains knockout for IP6K and evaluation of homologous recombination (HR) repair capacity
• Grantee: Bryan Etindi Abuchery
• Support Opportunities: Scholarships in Brazil - Master