Toxoplasma gondii infection damages the perineuronal nets in a murine model

BACKGROUND Behavioral and neurochemical alterations associated with toxoplasmosis may be influenced by the persistence of tissue cysts and activation of an immune response in the brain of Toxoplasma gondii-infected hosts. The cerebral extracellular matrix is organised as perineuronal nets (PNNs) that are both released and ensheath by some neurons and glial cells. There is evidences to suggest that PNNs impairment is a pathophysiological mechanism associated with neuropsychiatric conditions. However, there is a lack of information regarding the impact of parasitic infections on the PNNs integrity and how this could affect the host's behavior. OBJECTIVES In this context, we aimed to analyse the impact of T. gondii infection on cyst burden, PNNs integrity, and possible effects in the locomotor activity of chronically infected mice. METHODS We infected mice with T. gondii ME-49 strain. After thirty days, we assessed locomotor performance of animals using the open field test, followed by evaluation of cysts burden and PNNs integrity in four brain regions (primary and secondary motor cortices, prefrontal and somesthetic cortex) to assess the PNNs integrity using Wisteria floribunda agglutinin (WFA) labeling by immunohistochemical analyses. FINDINGS AND MAIN CONCLUSIONS Our findings revealed a random distribution of cysts in the brain, the disruption of PNNs surrounding neurons in four areas of the cerebral cortex and hyperlocomotor behavior in T. gondii-infected mice. These results can contribute to elucidate the link toxoplasmosis with the establishment of neuroinflammatory response in neuropsychiatric disorders and to raise a discussion about the mechanisms related to changes in brain connectivity, with possible behavioral repercussions during chronic T. gondii infection.

Interaction between Parasitophorous Vacuolar Membraneassociated GRA3 and Calcium Modulating Ligand of Host Cell Endoplasmic Reticulum in the Parasitism of Toxoplasma gondii

A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5'-RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.