Evidence for nuclear localisation of two stage-specific isoenzymes of enolase in Toxoplasma gondii correlates with active parasite replication.

The protozoan parasite Toxoplasma gondii has a complex life cycle involving the developmental transition between the asexual exo-enteric stages (tachyzoites and bradyzoites) and the coccidian (sexual and asexual) forms (schizonts, macrogametes and microgametes). Previous work has established the stage-specific expression of certain proteins including two glycolytic isoenzymes of enolase and lactate dehydrogenase in T. gondii. Here we describe the expression and subcellular localisation of the two isoforms of enolase (ENO1 and ENO2) and lactate dehydrogenase (LDH1 and LDH2) in vivo using immunocytochemistry. In mice, proliferating parasites in the lung expressed ENO2 and LDH1 and were characterised as tachyzoites by the presence of a tachyzoite specific surface antigen (SAG1). In contrast, ENO1 and LDH2 were expressed by bradyzoites present in tissue cysts in the brain characterised by the presence of the bradyzoite specific antigen (BAG1). During stage conversion (tachyzoite/bradyzoite), the isoenzyme changes occur at an early stage when the bradyzoites are actively proliferating and thus may not simply be reflecting reduced metabolic needs. When the coccidian stages were examined in the cat intestine, they were negative for SAG1, BAG1, LDH2 and ENO1 but were similar to the tachyzoite in strongly expressing LDH1 and ENO2. The isoenzymes LDH1 and LDH2 were exclusively expressed in the cytoplasm. In contrast, it was observed that the strongest labelling for both ENO1 and ENO2 was observed in the nucleus with less intense but specific cytoplasmic staining. Immunoelectron microscopy confirmed the cytoplasmic location of LDH and the predominantly nuclear location of enolase. During early intracellular proliferation and development, all stages of the life cycle (tachyzoite, bradyzoite and coccidian stages) exhibited very strong nuclear labelling for enolase but this was markedly reduced in mature parasites to levels below that seen in the cytoplasm. The specific nuclear localisation of enolases appears to be associated with nuclear activity (transcription and/or division) and may play some part in the control of gene regulation during parasite proliferation and differentiation in addition to its role in glycolysis.