Trypanosoma cruzi GP63

Trypanosoma cruzi is a flagellated protozoan parasite that causes Chagas disease.  This disease is a major health concern in Latin America, with as many as 16-18 million people chronically infected with this parasite.  T. cruzi can infect a multitude of vertebrate hosts, including, humans, dogs, cats, and rodents. The parasite life cycle consists of three developmental stages that allows the cell to proliferate in both an insect vector and a vertebrate host.  The parasite divides within the midgut of the reduviid bug as an extracellular epimastigote, eventually moving to the hindgut where it differentiates into an infective metacyclic trypomastigote.  The infective form is excreted in the feces as the insect takes a blood meal from the host, entering the host through the bite wound or through the mucous membranes of the host.  The trypomastigotes invade host cells, and differentiate into amastigotes.  This intracellular form of the parasite in turn divides within the cytoplasm of the cell.  After several rounds of division and just prior to the cell rupturing, the parasites again differentiate back into trypomastigotes.  Upon cell rupture, parasites are released and infection of neighboring cells and/or dissemination to specific tissues occurs.  Parasites that can establish and maintain an infection in a mammalian host utilize various methods to avoid the harmful effects of the host immune responses. Leishmania uses the major surface protease, GP63, as a means to survive host immune mechanisms and aid in cell invasion.  Leishmania GP63 functions as a zinc metalloprotease, is located on the cell surface, and provides resistance to complement-mediated lysis.

In all Leishmania species studied thus far, GP63 proteins are encoded by multiple genes. Leishmania differentially regulates this multigene family in a life cycle stage dependent manner through elements within the 3’ untranslated region (UTR) of the genes.  Specifically, one Leishmania GP63 gene class is constitutively expressed throughout the life cycle, and another GP63 gene class is expressed at higher levels when the parasite differentiates into an infective form. 

Homologues of the mRNA for the zinc metalloprotease, GP63, have now been identified in Trypanosoma cruzi.  This surface protein is best characterized in Leishmania, where it provides the parasite with resistance to complement, facilitate attachment to macrophages, and promote the intracellular survival of phagocytosed parasites within the macrophage.  Eight unique T. cruzi GP63 cDNAs were cloned from two life cycle stage specific cDNA libraries.  The eight genes fall into two categories based on overall sequence similarities and 3’ UTR sequences.  One gene family codes for a 2.4-kb transcript primarily expressed in the amastigote life cycle stage, while the other encodes for a smaller 2.1-kb transcript.  Northern blot analysis of cultured epimastigote, amastigote, and trypomastigote parasites shows an 8-10 fold increase in expression of the 2.4-kb transcript during the intracellular amastigote stage and constitutive expression of the 2.1-kb transcript in all three life cycle stages.  The predicted T. cruzi GP63 protein sequences share a high degree of similarity with one another and are 37% and 43% identical to GP63 proteins in Leishmania guyanensis and Trypanosoma brucei, respectively.  Complete conservation of the zinc binding domain and high homology to core macrophage binding domains of Leishmania GP63 suggest a conserved role for T. cruzi GP63 proteins, while the differential expression of GP63 mRNA in the intracellular amastigote stage may reflect additional functions for T. cruzi GP63 in the life cycle of the parasite.  The identification of multiple GP63 genes in T. cruzi provides an opportunity to further study the potential roles of this protein in the survival and infectivity of this parasite.  It may also permit a fundamental study of the role UTRs have in differential expression of genes in T. cruzi.