Toxoplasma gondii induces robust humoral immune response against cyst wall antigens in chronically infected animals and humans.

Toxoplasma gondii differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission. Strong humoral immune response has been reported against tachyzoite antigens, however, antibody-mediated response towards bradyzoite antigens is poorly characterized. This work aimed to study the humoral immune response towards bradyzoite and associated cyst wall antigens particularly CST1. The immunoreactivity of 404 goats, 88 sheep and 92 human sera to recombinant (CST1 and SRS9) and native proteins of encysted bradyzoite along with well-established tachyzoite antigens (SAG1 and GRA7) was determined using ELISA, Western blot and immunofluorescence analysis (IFA). ELISA results revealed nearly 50% of sera contain T. gondii specific antibodies. Results were further validated using Western blot and IFA. T. gondii positive sera predominantly recognized the cyst wall besides the known tachyzoite surface antigens. The presence of CST1 antibodies in seropositive samples were in line with the staining patterns which were consistent with CST localization. Notably, T. gondii IgM- IgG+ sera recognize the cyst wall whereas IgM + IgG-sera recognize tachyzoite antigens indicating acute infection consistent with presence of parasite DNA. The study demonstrates a strong humoral response against bradyzoite associated cyst wall antigens across naturally infected animals and humans. CST1 emerged as a key immunomodulatory antigen which may have direct implications for clinical immunodiagnostics.

Characterization of Toxoplasma gondii Spt5 like transcription elongation factor.

Elongation has emerged as a highly regulated step in the multistage process of transcription. Control of gene expression mediated through transcription elongation remains an unexplored area of study in Toxoplasma gondii where the demands of complex lifecycle necessitate a regulated transcription program. Here, we elucidate the central role of Spt5 homolog in T. gondii mRNA transcription. We demonstrate that TgSpt5 functions in conjunction with a small zinc finger protein TgSpt4. TgSpt5 interacts with TgRpb1, the largest subunit of RNA polymerase II and associates with actively transcribed genes. Enrichment of TgSpt5 towards the 3' end of genes coinciding with P-Ser2 form of RNAPII, a marker of active elongation further underscores its pivotal role in transcription. TgSpt5 undergoes phosphorylation mediated through Toxoplasma Cdk9 homolog, TgCrk9, which appears crucial for its function. Inhibition of TgCrk9, which also regulates RNAPII by differential phosphorylation of its C terminal domain, results in loss of TgSpt5 enrichment at 3' sites of the genes and an overall repressive effect on parasite progression. TgSpt5 along with TgSpt4 could successfully complement the loss of function mutations in yeast counterparts emphasizing its functional significance. Together, the results highlight the possible role of TgSpt5 in transcript elongation regulated through phosphorylation by TgCrk9.

Cdk-related kinase 9 regulates RNA polymerase II mediated transcription in Toxoplasma gondii.

Cyclin-dependent kinases are an essential part of eukaryotic transcriptional machinery. In Apicomplexan parasites, the role and relevance of the kinases in the multistep process of transcription seeks more attention given the absence of full repertoire of canonical Cdks and cognate cyclin partners. In this study, we functionally characterize T. gondii Cdk-related kinase 9 (TgCrk9) showing maximal homology to eukaryotic Cdk9. An uncanonical cyclin, TgCyclin L, colocalizes with TgCrk9 in the parasite nucleus and co-immunoprecipitate, could activate the kinase in-vitro. We identify two threonines in conserved T-loop domain of TgCrk9 that are important for its activity. The activated TgCrk9 phosphorylates C-terminal domain (CTD) of TgRpb1, the largest subunit of RNA polymerase II highlighting its role in transcription. Selective chemical inhibition of TgCrk9 affected serine 2 phosphorylation in the heptapeptide repeats of TgRpb1-CTD towards 3' end of genes consistent with a possible role in transcription elongation. Interestingly, TgCrk9 kinase activity is regulated by the upstream TgCrk7 based CAK complex. TgCrk9 was found to functionally complement the role of its yeast counterpart Bur1 establishing its role as an important transcriptional kinase. In this study, we provide robust evidence that TgCrk9 is an important part of transcription machinery regulating gene expression in T. gondii.