Proteotyping reactivant toxoplasmic encephalitis reveals virulence-associated dense granule protein GRA5 polymorphisms

A fatal case of toxoplasmic encephalitis, and others like it, has caused microbiologists and clinicians to question whether different strains of T. gondii have more pathogenic potential than others. This raises significant concern, as T. gondii is a widely spread parasitic organism that is presumed to lie dormant in a third of the worlds general population. In this study, we expand on a previously published proteomicanalysis reactivated toxoplasmic encephalitis and have been able to identify T. gondii-specific peptides in the cerebrospinal fluid (CSF) of this patient and two additional cases of toxoplasmic reactivation. Multilocus PCR-restriction fragment-length polymorphism (PCR-RFLP) was used to genetically identify the T. gondii strain that resulted in this fatal case, which belonged to ToxoDB PCR-RFLP genotype #7. Using other T. gondii strains of the same genotype, we performed bioassays to compare the pathogenicity of this genotype with that of a clinically relevant strain, ME49. Both of the tested genotype #7 strains appear to have a greater pathogenic potential, although through likely different mechanisms. Of the most abundant T. gondii-specific CSF peptides across multiple patients, we identified a polymorphic region of the dense granule protein GRA5 that appears to have strain specificity. This approach could represent a "proteotype" that allows for T. gondii strain risk stratification within clinical samples. Ultimately spinal fluid could be a valuable tool in distinguishing between T. gondii exposed individuals with no cyst forms in the brain versus those exposed individuals that harbor "clinically silent" but viable brain cysts. SIGNIFICANCEA third of the worlds population is exposed to the parasite Toxoplasma gondii, residing in a dormant, encysted stage within neurons. T. gondii is a diverse microorganism with some strains having greater reactivation potential. There is no means of identifying which individuals are at risk of reactivation. Proteomic analysis of cerebrospinal fluid from patients with reactivated toxoplasmosis demonstrated a consistent pattern of T. gondii peptides, including GRA5, a secreted virulence factor. Postmortem analysis identified a ToxoDB PCR-RFLP genotype #7 strain associated with a fatality. Cerebrospinal fluid could provide clues to persistent brain infection and may identify strains able to reactivate.