Molecular cloning of a Trypanosoma cruzi cell surface casein kinase II substrate, Tc-1, involved in cellular infection.
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.
Augustine
Swinburne A J
SA
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, USA.
Kleshchenko
Yuliya Y
YY
Nde
Pius N
PN
Pratap
Siddharth
S
Ager
Edward A
EA
Burns
James M
JM
Lima
Maria F
MF
Villalta
Fernando
F
D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D032386:Myoblasts, Cardiac / Q000201:enzymology / Q000469:parasitology*; D010766:Phosphorylation; D015800:Protozoan Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity; D037521:Virulence Factors / Q000235:genetics / Q000302:isolation & purification
D008565:Membrane Proteins; D015800:Protozoan Proteins; D037521:Virulence Factors; D047390:Casein Kinase II; C512597:Tc-1 protein, Trypanosoma cruzi