Molecular cloning of a Trypanosoma cruzi cell surface casein kinase II substrate, Tc-1, involved in cellular infection

Abstract

In this work, we report the cloning and characterization of the first cell surface casein kinase II (CKII) substrate (Tc-1) of Trypanosoma cruzi, the causative agent of Chagas' disease. Analysis of the gene sequence revealed a 1,653-bp open reading frame coding for 550 amino acid residues. Northern blot analysis showed a 4.5-kb transcript that is expressed in invasive trypomastigotes but not in noninvasive epimastigote forms of T. cruzi. Southern blot analysis indicates that Tc-1 is a single-copy gene. At the amino acid level, Tc-1 displayed 95% and 99% identity to two hypothetical proteins recently reported by the T. cruzi genome project. Analysis of the translated amino acid sequence indicates that the Tc-1 gene has a putative transmembrane domain with multiple cytoplasmic and extracellular CKII phosphosites. Exogenous human CKII was able to phosphorylate serine residues on both recombinant Tc-1 and Tc-1 of intact trypomastigotes. This phosphorylation was inhibited by the CKII inhibitors heparin and 4,5,6,7,-tetrabromo-2-azabenzimidazole. Immunoblots of solubilized trypomastigotes, epimastigotes, and amastigotes probed with anti-recombinant Tc-1 immunoglobulin G revealed a 62-kDa protein that is expressed only in infective trypomastigotes. Immunoprecipitation of labeled surface proteins of trypomastigotes indicated that the 62-kDa protein is a surface protein, and we found that the protein is uniformly distributed on the surface of trypomastigotes by direct immunofluorescence. Antibodies to Tc-1 effectively blocked trypomastigote invasion of host cells and consequently reduced parasite load. Preincubation of either trypomastigotes or myoblasts with CKII inhibitors blocked T. cruzi infection. Thus, for the first time, we describe a cell surface CKII substrate of a protozoan parasite that is phosphorylated by human CKII and that is involved in cellular infection.

FIG. 1.

Alignment of the deduced amino acid sequences of the Tc-1 gene (GenBank accession no. AY167028) with those of two genes coding for two hypothetical proteins (GenBank accession no. EAN95234 and EAN90929), which were reported as a result of the completion of the T. cruzi genome project (4, 5). BLASTp analysis revealed that Tc-1 had 99% and 95% identity to two hypothetical proteins of T. cruzi. Identical amino acid residues are indicated with an asterisk (*).

FIG. 2.

Hydrobobicity profile of Tc-1 showing a single transmembrane region and the predicted model of the Tc-1 protein structure showing its phosphorylation sites. (A) Hydrophobicity plot of the Tc-1 protein predicts a single transmembrane region between amino acids 344 and 364 using Toppred protein analysis (7). (B) Envisage model of the Tc-1 protein structure showing its phosphorylation sites. Prosite and Scansite software analysis of Tc-1 showed 16 CKII phosphorylation sites, 4 PKC phosphorylation sites, a tyrosine phosphorylation site, and 2 cAMP sites.

FIG. 3.

Tc-1 is a single-copy gene that is expressed in invasive trypomastigotes but not in noninvasive epimastigotes. (A). Southern blot analysis of T. cruzi indicates that Tc-1 is a single-copy gene. T. cruzi DNA (5 μg) was digested with several restriction enzymes, separated on an agarose gel, blotted onto nylon membranes, and probed with ³²P-labeled Tc-1 as indicated in Materials and Methods. (B) Northern blot analysis indicates that the Tc-1 gene shows a 4.5-kb transcript that is expressed only in trypomastigotes. Total RNA (20 μg) of trypomastigotes and epimastigotes was separated on an agarose gel, blotted onto nylon membranes, and probed with ³²P-labeled Tc-1. The bottom of panel B represents ethidium bromide staining of the gel as RNA loading controls. The results presented in panels A and B are representative of one experiment of three performed with similar results.

FIG. 4.

T. cruzi trypomastigotes but not epimastigotes or amastigotes express Tc-1 on their surface. (A) Immunoblotting analysis indicates that invasive trypomastigotes but not noninvasive epimastigotes or amastigotes express Tc-1. The same concentration of solubilized trypomastigotes, epimastigotes, and amastigotes was separated by SDS-PAGE, transferred onto nitrocellulose membranes, probed with Tc-1 IgG, and developed by ECL as indicated in Materials and Methods. Blots were stripped and probed with β-actin antibody as protein loading controls. (B) Immunoprecipitation of biotin-labeled surface proteins of T. cruzi indicates that Tc-1 is expressed as a surface protein in trypomastigotes. The same numbers of trypomastigotes, epimastigotes, and amastigotes were biotinylated and solubilized, and the same concentration of protein was immunoprecipitated with Tc-1 IgG supplemented with protein G. The immunoprecipitated material was separated by SDS-PAGE, blotted onto nitrocellulose, probed with Tc-1 IgG, and developed by ECL as described in Materials and Methods. Blots presented in panels A and B are representative of one experiment of three performed with similar results.

FIG. 5.

FITC-labeled Tc-1 IgG specifically binds to the surface of trypomastigotes. Trypomastigotes were washed with Hanks' balanced salt solution, fixed with 1% paraformaldehyde, and incubated with FITC-labeled Tc-1 IgG, FITC-labeled Tc-1 IgG in the presence of a 100× excess of unlabeled Tc-1 IgG, or FITC-labeled preimmune IgG. (A) FITC-labeled IgG to Tc-1 binds uniformly to the surface of trypomastigotes. (B) Binding of FITC-labeled IgG to Tc-1 is abolished by a 100× excess of unlabeled Tc-1 IgG. (C) FITC-labeled preimmune IgG does not bind to trypomastigotes. A′, B′, and C′ show that trypanosomes were present in the preparations shown in A, B, and C, as seen in their respective light fields. This is a representative experiment of three independent experiments performed with similar results.

FIG. 6.

Human exogenous CKII phosphorylates recombinant Tc-1 or Tc-1 of intact T. cruzi trypomastigotes on serine residues, and the phosphorylation is inhibited by the CKII inhibitor heparin or TBB. (A) Phosphorylation of recombinant Tc-1 (r-Tc-1) by exogenous CKII. Recombinant Tc-1 (40 μg) was incubated with 20 ng of human CKII, with phosphorylation buffer in place of CKII, or with 20 ng CKII in the presence of heparin, a CKII inhibitor, for 10 min at 30°C. The same amount of each sample was separated by SDS-PAGE, blotted onto nitrocellulose membranes, probed with anti-phosphoserine antibodies (Anti-P-serine abs), and developed by ECL as described in Materials and Methods. Lane 1 shows the phosphorylation of serine residues on Tc-1 by CKII. Lane 2 (control) shows that Tc-1 is not phosphorylated in the presence of reaction buffer alone. Lane 3 shows that heparin inhibits the phosphorylation of Tc-1 by CKII. This is a representative experiment of three experiments performed with similar results. (B) Trypomastigotes (7 × 10⁷) were starved in DMEM and incubated with 20 ng of human CKII, with phosphorylation buffer in place of CKII, or with 20 ng CKII in the presence of TBB, a CKII inhibitor, for 10 min at 30°C. The same protein concentration of solubilized samples was immunoprecipitated (IP) with anti-Tc-1 IgG, separated by SDS-PAGE, blotted onto nitrocellulose membranes, probed with anti-phosphoserine antibodies, and developed by ECL. Lane 1 (control) shows endogenous phosphorylation of serine residues on Tc-1 of trypomastigotes in the absence of exogenous CKII. Lane 2 shows the phosphorylation of serine residues on Tc-1 of intact T. cruzi by exogenous CKII. Lane 3 shows that TBB inhibits the phosphorylation of Tc-1 on intact T. cruzi by exogenous CKII. This is a representative experiment of three experiments performed with similar results. WB, Western blot.

FIG. 7.

Tc-1 IgG and CKII inhibitors prevent the binding of trypomastigotes to heart myoblasts at 2 h, causing a significant reduction in the parasite load at 72 h. (A) Tc-1 IgG inhibits trypomastigote binding to heart myoblasts in a concentration-dependent and saturable manner, whereas preimmune IgG has no effect. Trypomastigotes were preincubated with increasing concentrations of either Tc-1 IgG or preimmune IgG and then exposed to heart myoblast monolayers at a ratio of 5 parasites per cell for 2 h, and trypanosome binding was evaluated as described in Materials and Methods. (B) Reduction of trypanosome binding caused by Tc-1 IgG reduced parasite multiplication in heart myoblasts. Trypomastigotes were preincubated with either Tc-1 IgG or preimmune IgG and then exposed to myoblast monolayers for 2 h as described in panel A. After unbound trypanosomes were removed, the cell monolayers received fresh complete medium and were postincubated for 72 h, where T. cruzi multiplication was evaluated. (C) Pretreatment of trypomastigotes with CKII inhibitors inhibits trypomastigote binding to myoblasts at 2 h (left panel), and the reduction of parasite binding caused by CKII inhibitors decreased parasite load at 72 h (right panel). DMSO, dimethyl sulfoxide. (D) Pretreatment of myoblasts with CKII inhibitors at the concentration of 40 μM inhibited parasite binding at 2 h (left panel) and multiplication at 72 h (right panel). Parasite binding and multiplication assays were performed as described above and as described in Materials and Methods. Bars represent the means ± 1 standard deviation of results from triplicate samples in one representative experiment (±1 standard deviation) selected from three experiments with similar results. *, significant difference compared to control values (P < 0.05).