Toxoplasma gondii chitinase induces macrophage activation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 degrees C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathoge1012112FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNQP - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2013/10741-8]2013/10741-8SEM INFORMAÇÃOThis study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant number 2013/10741-8). Additional financial help was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Fundação de Apoio ao Ensino, Pe

Toxoplasma gondii Chitinase Induces Macrophage Activation.

Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 °C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection

Tg_chitinase subcellular localization.

<p>(A) <i>T</i>. <i>gondii</i> tachyzoite DNA was stained with DAPI (blue), and (B) immunostained for Tg_chitinase with an anti-chitinase antibody, which was biotinylated with an antibody conjugated to anti-streptavidin-FITC (green). Superimposition of images stained for Tg_chitinase and DAPI (Merge and phase microscopy, C and D, respectively). Tg_chitinase is localized throughout the cytosol of <i>T</i>. <i>gondii</i> tachyzoites. Scale bar = 2.5 μm.</p

Chitinase activity from <i>T</i>. <i>gondii</i>.

<p>Soluble <i>T</i>. <i>gondii</i> antigens were purified by affinity with an IgY-Sepharose 4B column, and the chitinase activity of the delayed fractions was measured. (A) Electrophoresis analysis of purified chitinase from <i>T</i>. <i>gondii</i>. First lane—MW (Molecular Weight Ladder), second lane—purified chitinase in 12% SDS-PAGE. (B) Crude extract and purified chitinase activity from <i>T</i>. <i>gondii</i> was measured. Error bars represent standard errors calculated from three replicates.</p

Amino acid sequence and predicted 3D structure of Tg_chitinase from <i>T</i>. <i>gondii</i>.

<p>Tg_chitinase from <i>T</i>. <i>gondii</i> was identified by mass spectrometry using the amino acid sequences of tryptic peptides listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144507#pone.0144507.t002" target="_blank">Table 2</a> (indicated in bold). (A) The amino acid sequence obtained from analysis of the peptide (SEIYQGDSVR) identified by BLASTP <i>T</i>. <i>gondii</i> GT1 (TGGT1_286465). (B) Predicted 3D structure of Tg_chitinase using I-TASSER.</p