In vivo identification of Toxoplasma gondii antigenic proteins and in silico study of their polymorphism.

AbstractThis study aims to identify novel candidates for the development of serotyping assays for Toxoplasma gondii (T. gondii). We employed an in vivo approach, utilizing co-immunoprecipitation (Co-IP) with sera from infected mice to identify antigenic proteins, followed by detection via LC-MS/MS. Additionally, an in silico analysis was conducted to explore the genetic polymorphism of the genes encoding these proteins and to determine mutations associated with specific genotypes.MethodologyProteomicsCo-immunoprecipitation reactions were performed using sera from mice hyperimmunized against toxoplasmosis in combination with lysates of T. gondii tachyzoites. Three strains were utilized: Fou (Africa 1), Me49 (type II), and VEG (type III). Proteins in the Co-IP eluates were identified by LC-MS/MS through MASCOT, with comparisons made to the reference strain ME49. The mass spectrometry identification data, including accession numbers, protein coverage, and the minimum number of peptides required for identification, are available in the mass_spec document.Samples and SequencingWe examined genetic polymorphism across 117 T. gondii strains, leveraging NGS sequences from studies by Galal et al. (2022) and Lorenzi et al., 2016. The strains are categorized as follows: 15 type Africa 1, 48 type II, 19 type III, 15 Amazonian, 3 type I, 3 type 12, 4 type Africa 3, 3 type Africa 4, and 7 Caribbean. References for these strains can be found in the Strains_metadata document.Data ProcessingComplete genomes obtained through NGS sequencing were downloaded from the ENA platform (https://www.ebi.ac.uk/ena/browser/home). Sequences were cleaned and aligned to the Me49 reference genome (GCA_000006565.2, 2018) using BWA-MEM (v0.7.17.2). Alignments were sorted with Samtools (v2.0.4), and duplicates were marked with MarkDuplicate (Picard tools, v2.18.2.3). Alignments with a mapping quality below 20 and coverage below 5 were excluded.SNP IdentificationSingle nucleotide polymorphisms (SNPs) in the genes coding for antigenic proteins were identified using FreeBayes (v1.3.6). The identified variants were annotated with SNpEff (v4.3+T.) based on annotations from the Me49 reference genome. Annotated variant calls were merged, focusing on gene coordinates identified with bcftools merge (v1.15.1). Gene coordinates are detailed in the Gene_List document.Variant FilteringThe raw data underwent two stages of filtering using VCF Filter: first, SNP variants were selected, followed by variants meeting the criteria of QUAL > 20, DPB > 3, MQMR > 20, and MQR > 20. The filtered data is presented in the Filtered_Variants document.Annotation of Filtered DataThe Filtered_Variants file was annotated using the Python script MutationRateCalculation.py to distinguish between silent and non-silent SNPs and to calculate the mutation rate of each SNP across different T. gondii strain types. The annotated data are summarized in the Annotated_variant document, while the analyzed data can be found in the Analized_variant document.Available Datamass_spec: Mass spectrometric identification data for antigenic proteins obtained via Co-IP.Strains_metadata: References for the strains used.Gene_List: Coordinates of the genes.File 1: Merged raw data from annotated variant call analyses.Filtered_Variants: Data for filtered variants.Annotated_variant: Annotated data for identified variants.MutationRateCalculation.py: Python script for annotating the filtered data.Analized_variant: Formatted data for variants of interest.ConclusionThis study establishes a robust foundation for identifying potential antigenic targets for T. gondii serotyping, paving the way for future research into associated genotypes and mutations.