A Study on Protein Lactylation and Metabolic Regulation Reveals New Areas of Toxoplasma gondii Biology

Background: Toxoplasma gondii is a globally distributed protozoan parasite that infects over 30% of the world’s population. It causes toxoplasmosis, a widespread zoonotic disease. The parasite has a complex life cycle, including sexual and asexual stages, mainly existing in the tissues of definitive hosts (felines) and intermediate hosts (warm-blooded animals). T. gondii can cause severe or even fatal symptoms in immunocompromised individuals and developing fetuses. Although existing treatments such as azithromycin, pyrimethamine, and spiramycin can inhibit certain activities of T. gondii, they are generally only effective against rapidly replicating tachyzoites, with little effect on slowly proliferating bradyzoites, and often come with host cell toxicity. Therefore, further understanding of T. gondii’s biological characteristics can help identify new drug targets to improve treatment efficacy.

Research Source: This research paper was written by a team from Shenyang Agricultural University, including the Key Laboratory of Animal Infectious Diseases of the Ministry of Education and the Pathogenic Mechanism Research Unit of Zoonotic Parasitic Diseases, Chinese Academy of Medical Sciences. The paper was published in “Genomics, Proteomics & Bioinformatics” in 2023.

Research Content and Methods:

1. Research Methods:

   • To understand protein lactylation in T. gondii, the research team first analyzed whole-cell lysates of rapidly proliferating tachyzoites using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to validate lactylated proteins.
   • Immunofluorescence analysis (IFA) and Western blotting were used to verify the distribution of lactylated proteins in T. gondii RH and ME49 strains.
   • A total of 1964 lactylation sites on 955 proteins were identified across three experiments, validating the accuracy and reproducibility of the mass spectrometry data.
   • Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and protein subcellular localization analysis were performed on lactylated proteins.

2. Main Experimental Results:

   • Lactylated proteins were widely distributed across multiple subcellular compartments in T. gondii, mainly concentrated in the nucleus, cytoplasm, and mitochondria, involved in various biological processes such as cellular metabolism, protein binding, and signal transduction.
   • KEGG pathway enrichment analysis showed that lactylated proteins were mainly enriched in central metabolic pathways, including spliceosome, aminoacyl-tRNA biosynthesis, glycolysis, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation.
   • Motif analysis of lactylated protein sequences indicated that methionine (M), alanine (A), and lysine (K) were often found upstream of lactylation sites, while alanine (A), glycine (G), and lysine (K) were enriched downstream.

3. In-depth Research and Analysis:

   • To further understand the biological function of lactylation, the research team explored the role of lactylated proteins in gene transcription and expression regulation processes. Particular attention was paid to the relationship between lactylated proteins and rhoptry proteins, the Actin-Myosin complex, and key enzymes such as phosphofructokinase II (PFKII) and lactate dehydrogenase (LDH).
   • Chromatin immunoprecipitation sequencing (ChIP-Seq) results using specific antibodies against lactylated histones revealed the roles of Histone H4k12la and H3k14la in Toxoplasma gene regulation.
   • In epigenetic regulation studies, the research demonstrated 17 lactylation sites in Histones H2a.z, H2b.z, H3, and H4.

4. Research Significance and Value:

   • This study mapped the lactylome of T. gondii for the first time, revealing the central role of lactylation in the parasite’s biology and energy metabolism regulation.
   • Lactylated proteins are widely involved in multiple key biological processes, including energy metabolism, signal transduction, and RNA processing, providing an important basis for discovering new anti-parasitic drug targets.
   • The research suggests that lactylation may have profound effects on various aspects of T. gondii’s growth, development, and metabolic regulation, particularly significantly affecting the parasite’s energy metabolism under specific physiological conditions.

Summary: Through comprehensive analysis, this study found that lactylation is widespread in T. gondii proteins, participating in multiple important metabolic processes and gene transcription regulation. The results provide a new perspective for the treatment of toxoplasmosis and lay a solid foundation for subsequent research. In the future, by further understanding the lactylation regulatory mechanism, it is hoped that new, efficient anti-toxoplasmosis drugs can be developed.