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Infection and Immunity, July 2006, p. 3922-3929, Vol. 74, No. 7
0019-9567/06/$08.00+0     doi:10.1128/IAI.00045-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Cloning of a Trypanosoma cruzi Cell Surface Casein Kinase II Substrate, Tc-1, Involved in Cellular Infection

Swinburne A. J. Augustine,{dagger} Yuliya Y. Kleshchenko,{dagger} Pius N. Nde, Siddharth Pratap,{ddagger} Edward A. Ager,§ James M. Burns Jr, Maria F. Lima, and Fernando Villalta*

Division of Microbial Pathogenesis and Immune Response, Department of Biomedical Sciences, School of Medicine, Meharry Medical College, Nashville, Tennessee

Received 10 January 2006/ Returned for modification 20 February 2006/ Accepted 4 April 2006

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.

* Corresponding author. Mailing address: Division 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. Phone: (615) 327-6173. Fax: (615) 327-6072. E-mail: fvillalta{at}mmc.edu.

Editor: W. A. Petri, Jr.

{dagger} S.A.J.A. and Y.Y.K. contributed equally to this work.

{ddagger} Present address: Department of Biomedical Bioinformatics, Vanderbilt University School of Medicine, 2209 Garland Ave., Nashville, TN 37232.

§ Present address: Center for Molecular Diagnostics, Madigan Army Medical Center, Ft. Lewis, WA 98431.

Present address: Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129.

Infection and Immunity, July 2006, p. 3922-3929, Vol. 74, No. 7
0019-9567/06/$08.00+0     doi:10.1128/IAI.00045-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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