Uncovering Biomarkers for Chronic Toxoplasmosis Detection Highlights Alternative Pathways Shaping Parasite Dormancy

Toxoplasma gondii, a protozoan parasite, causes toxoplasmosis, a widespread zoonotic and food-borne infection that poses significant risks, particularly in opportunistic and congenital cases. This obligate intracellular parasite is highly prevalent worldwide, largely due to its versatile life cycle, involving multiple hosts and transmission routes, and its ability to establish chronic infections. The presence of this neurotropic parasite in the brain poses a reactivation risk in immunocompromised individuals and might be associated with a higher likelihood of developing mental disorders. However, the role of the dormant bradyzoite stage in the pathophysiology of the disease is underexplored, mainly due to the lack of non-invasive detection methods and serologic tests targeting bradyzoite- or cyst-specific antigens. In this study, we performed an unbiased screening of the bradyzoite proteome and identified the Bradyzoite Serological Marker (BSM) as an additional serological biomarker, alongside the cyst-associated BCLA, to detect chronic stages in vivo. BSM and BCLA show high sensitivity and specificity in identifying cyst-bearing mice. However, in humans, these markers exhibit only moderate concordance, with a 30% positivity rate among individuals with prior immunity, suggesting variability in immune responses and complexities in diagnosing chronic toxoplasmosis. Importantly, bradyzoite serology helps differentiate recent from past infections by the teratogenic parasite Toxoplasma gondii, with BCLA improving the accuracy of pergestational infection diagnosis. Exploring the regulatory mechanisms controlling the expression of these markers revealed that the chromatin modifiers MORC and HDAC3 exert epistatic control over BFD1, a key regulator of bradyzoite development. While BFD1 governs a specific subset of bradyzoite markers, including BCLA, a sub-transcriptome comprising BSM relies on MORC/HDAC3 independently of BFD1. This complex gene regulation highlights the challenge in understanding Toxoplasma persistence, while offering new opportunities for improved serological diagnosis of congenital and chronic toxoplasmosis, particularly in individuals with mental health conditions or a risk of toxoplasmic reactivation. Overall design: We report transcriptome of Toxoplasma gondii tachyzoites using Illumina RNA sequencing using the following methods: Total RNAs were extracted and purified using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (Qiagen). RNA quantity and quality were measured by NanoDrop 2000 (Thermo Scientific). For each condition, RNAs were prepared from three biological replicates. RNA integrity was assessed by standard non-denaturing 1.2% TBE agarose gel electrophoresis. RNA sequencing was performed following standard Illumina protocols, by Novogene (Cambridge, United Kingdom). Briefly, RNA quantity, integrity, and purity were determined using the Agilent 5400 Fragment Analyzer System (Agilent Technologies, Palo Alto, California, USA). The RQN ranged from 7.8 to 10 for all samples, which was considered sufficient. Messenger RNAs (mRNA) were purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. Then the second strand cDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount. The samples were sequenced on the Illumina NovaSeq platform (2 x 150 bp, strand-specific sequencing) and generated ~40 million paired-end reads for each sample. The quality of the raw sequencing reads was assessed using FastQC (www.bioinformatics. babraham.ac.uk/projects/fastqc/) and MultiQC. For the expression data quantification and normalization, the FASTQ reads were aligned to the ToxoDB-49 build of the T. gondii ME49 genome using Subread version 2.0.1 with the following options 'subread-align -d 50 -D 600 --sortReadsByCoordinates'. Read counts for each gene were calculated using featureCounts from the Subread package.