Molecular cloning of a Trypanosoma cruzi cell surface casein kinase II substrate, Tc-1, involved in cellular infection
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Published on: November 5, 2021
Swinburne A J Augustine1, Yuliya Y Kleshchenko, Pius N Nde
1Division of Microbial Pathogenesis and Immune Response, Department of Biomedical Sciences, School of Medicine, Meharry Medical College, 1005 Dr. D. B. Todd Jr. Blvd., Nashville, TN 37208, USA.
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.
Insights
We identified Tc-1, a cell surface protein of Trypanosoma cruzi, as the first casein kinase II (CKII) substrate in this parasite. This protein is crucial for parasite invasion and infection, making it a potential therapeutic target for Chagas' disease.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Chagas' disease, caused by Trypanosoma cruzi, remains a significant global health concern.
- Understanding parasite surface proteins and their roles in infection is critical for developing new treatments.
Purpose of the Study:
- To clone and characterize the first cell surface casein kinase II (CKII) substrate (Tc-1) from Trypanosoma cruzi.
- To investigate the role of Tc-1 phosphorylation in parasite invasion and infection.
Main Methods:
- Gene cloning and sequence analysis of Tc-1.
- Northern and Southern blot analyses to determine gene expression and copy number.
- In vitro phosphorylation assays using human CKII and CKII inhibitors.
- Immunoblotting, immunoprecipitation, and immunofluorescence to characterize Tc-1 expression and localization.
- Functional assays to assess the impact of anti-Tc-1 antibodies and CKII inhibitors on parasite invasion.
Main Results:
- Tc-1 is a 1,653-bp gene encoding a 550-amino acid protein with a transmembrane domain and multiple CKII phosphosites.
- Tc-1 is expressed as a 62-kDa protein on the surface of invasive trypomastigotes, but not in other life stages.
- Human CKII phosphorylates Tc-1, and this process is inhibited by specific CKII inhibitors.
- Antibodies against Tc-1 block trypomastigote invasion of host cells, reducing parasite load.
- CKII inhibitors also effectively block Trypanosoma cruzi infection.
Conclusions:
- Tc-1 is the first identified cell surface CKII substrate in Trypanosoma cruzi.
- Phosphorylation of Tc-1 by human CKII is essential for parasite infectivity.
- Tc-1 represents a promising new target for therapeutic intervention against Chagas' disease.
