Palmitoylation in Toxoplasma gondii: acyltransferases and their role in apical organelle secretion and redox balance

• Grant number: 23/01428-6
• Support Opportunities: Scholarships abroad - Research
• Start date: August 13, 2023
• End date: August 12, 2024
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Ana Patricia Yatsuda Natsui
• Grantee: Ana Patricia Yatsuda Natsui
• Host Investigator: Matthew Andrew Child
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: Imperial College London, England

Abstract

Fatty acylated proteins are critical players in signalling in both normal cells and disease states, including infectious diseases. S-acylation (S-palmitoylation) is an abundant and relevant Post Translation Modification (PTM) in apicomplexan parasites. This PTM is added by Protein S-AcylTransferases (DHHC-PATs), transmembrane proteins with a catalytic amino acid motif DHHC (Asp-His-His-Cys) within a cysteine-rich domain (CRD). In Toxoplasma gondii, there are 18 DHHC-PATs, where five were shown to be essential (TgDHHC 2, 6, 7, 9 and 14), localized at the Golgi, Inner Membrane Complex (IMC),plasma membrane, Endoplasmic Reticulum (ER) and rhoptries. N-acylation and O-acylation also occur, but differently from S-acylation, are considered to be irreversible. This PTM is catalysed by enzymes of the Membrane-Bound O-acyltransferases (MBOAT) family, multispanning transmembrane enzymes, with more than 08 transmembrane domains that contain the active site. T. gondii has three predicted MBOAT genes, but no study has ever been done with these enzymes. S-acylation occurs in many Apical Organelle associate Proteins (AOPs) such as AMA-1, with strong indications that this PTM is an important regulator of apical organelle secretion. T. gondii must deal with oxidative pressure within the host intracellular environment, activating its antioxidant network. Human DHHC3 has a strong role in inhibiting the antioxidant response in cancer and in decreasing the efficacy of drug-induced apoptosis, favouring the tumour development. So far, no publication is available to establish a correlation between DHHCs and regulation of the redox system in T. gondii and no study has ever been made with the MBOATs in the Apicomplexa phylum. Therefore, the general aim of this project is to investigate the correlation between T.gondii DHHC-PATs and TgMBOAT enzymes in the survival, the redox balance and the AOPs secretion dynamics of T. gondii. A technique developed by Prof. Matthew Child, named CRISPR-based Oligo Recombineering (CORe), will be employed. CRISPR CORe plasmids will be used to replace the cysteine and/or histidine of the catalytic domains (DHHC) from the 18 DHHC-PATs of T. gondii or to replace the histidine and/or aspartic acid of the catalytic domains from the three MBOATs of T. gondii. The catalytic amino acids from the DHHC-PATs and MBOATs will be switched for other amino acids in parallel to their recodonized switch controls. The viability of T.gondii will be analysed in each of the mutants of the TgDHHCs and TgMBOATs using the essentiality assay employing the fitness scores developed by Child´s Lab. Inducible knockout strains of at least one TgDHHC and one TgMBOAT will be obtained to evaluate the effects in the antioxidant capacity of each strain and in the secretion of TgAOPs. This project will bring new knowledge to the so far undescribed TgMBOATs and will start to investigate a possible correlation between acyltransferases of T.gondii and the redox balance and secretion dynamics of the AOPs. (AU)