HAD2A

HAD2a is a haloacid dehalogenase (HAD)-family phosphatase in Toxoplasma gondii that provides phosphoregulatory control over basal complex contraction and daughter cell separation during endodyogeny (PMID:26840427). It possesses demonstrable HAD phosphatase activity in vitro (PMID:26840427) and physically associates with MORN1, the major scaffolding protein of the basal complex, as identified by yeast two-hybrid screening (PMID:26840427); consistent with this partnership it localizes to nascent daughter buds and co-localizes with MORN1 at the contracting basal complex (PMID:26840427). Conditional loss of HAD2a disrupts basal complex assembly and causes incomplete cytokinesis with conjoined daughters and severely impaired proliferation, establishing it as essential for daughter bud separation (PMID:26840427). Independent knockout in the type I RH strain confirms these budding and replication defects and shows HAD2a is required for virulence in mice (PMID:38392859). Beyond this basal-complex role, the physiological substrates of HAD2a and the phosphosites governing basal complex contraction have not been characterized in the available corpus.

1. 2016 Medium

   Whether a phosphatase acts at the apicomplexan basal complex was unknown; identifying a MORN1 interactor placed HAD2a within the basal complex scaffold machinery.

   Evidence Yeast two-hybrid screen against TgMORN1 with co-localization by fluorescence microscopy in Toxoplasma gondii

   PMID:26840427

   Open questions at the time

   • No reciprocal Co-IP or pulldown reported
   • Direct vs. bridged interaction with MORN1 not resolved
2. 2016 Medium

   To establish HAD2a as an enzyme rather than a passive scaffold component, its catalytic activity was tested, confirming it is a functional HAD-family phosphatase.

   Evidence In vitro phosphatase assay

   PMID:26840427

   Open questions at the time

   • No catalytic-dead mutant to link activity to phenotype
   • Physiological substrate unidentified
3. 2016 Medium

   Whether HAD2a acts at the site and time of basal complex contraction was addressed by localization, tying it spatially and temporally to endodyogeny.

   Evidence Fluorescence microscopy of subcellular localization during daughter budding in Toxoplasma gondii

   PMID:26840427

   Open questions at the time

   • Recruitment mechanism to the basal complex unknown
   • Dependence of localization on MORN1 not tested
4. 2016 High

   The functional requirement for HAD2a was tested by conditional knockout, establishing it as essential for basal complex assembly and daughter bud separation.

   Evidence Conditional knockout with phenotypic readouts of basal complex assembly, cytokinesis, and proliferation in Toxoplasma gondii

   PMID:26840427

   Open questions at the time

   • Whether the phenotype depends on phosphatase activity not demonstrated
   • Direct substrates driving contraction not identified
5. 2016 Low

   To test conservation of HAD2a function across apicomplexan budding, the Plasmodium ortholog was localized, showing basal complex targeting late in schizogony.

   Evidence Fluorescence microscopy of the HAD2a ortholog in Plasmodium

   PMID:26840427

   Open questions at the time

   • No functional validation in Plasmodium reported
   • Interaction with a MORN1 ortholog not tested
6. 2024 Medium

   Whether HAD2a is required in a virulent strain and contributes to infection was tested, confirming its essentiality for replication and identifying it as a virulence determinant.

   Evidence Gene knockout, in vitro replication assay, and mouse virulence model in the type I RH strain

   PMID:38392859

   Open questions at the time

   • Molecular basis of virulence attenuation beyond replication defect not defined
   • Single lab; substrate-level mechanism still open

• The physiological substrate(s) of HAD2a and the phosphorylation events it reverses to drive basal complex contraction remain unidentified, as does whether its catalytic activity is required for daughter bud separation.
• No phosphoproteomic substrate mapping
• No catalytic-dead rescue experiment
• Mechanism of MORN1-dependent recruitment unresolved