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Title: The glycosylphosphatidylinositol-anchored protein repertoire of babesia bovis and its significance for erythrocyte invasion

Author
item RODRIGUEZ, A - National Institute Of Agricultural Technology(INTA)
item FLORIN-CHRISTENSEN, M - National Institute Of Agricultural Technology(INTA)
item FLORES, D - National Institute Of Agricultural Technology(INTA)
item Suarez, Carlos
item SCHNITTGER, L - National Institute Of Agricultural Technology(INTA)

Submitted to: Ticks and Tick Borne Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/31/2013
Publication Date: 4/5/2014
Citation: Rodriguez, A., Florin-Christensen, M., Flores, D., Suarez, C.E., Schnittger, L. 2014. The glycosylphosphatidylinositol-anchored protein repertoire of babesia bovis and its significance for erythrocyte invasion. Ticks and Tick Borne Diseases. 5(3):343-348.

Interpretive Summary: Glycosylphosphatidyl-anchored proteins are particularly abundant on the surface of pathogenic protozoans and might play an important role for parasite survival. In the present work the relevance of GPI-anchored proteins for erythrocyte invasion of Babesia bovis, one of the tick-transmitted causative agents of bovine babesiosis, was studied. We show that enzymatic removal of the GPI-anchored proteins completely abolished invasion of erythrocytes, thus demonstrating the importance of this class of molecules for parasite propagation. In addition, we defined the repertoire of GPI-anchored proteins of B. bovis using bioinformatic genomic tools. Altogether 16 GPI-anchored proteins were identified of which five represent the Variable Merozoite Surface Antigens (VMSAs) that have been intensively studied as vaccine candidates and diagnostic antigens. The remaining eleven have not been studied so far and their function is unknown. Fifteen of the identified GPI-anchored proteins contain 2 to 16 amino acid repeats indicating that they are likely involved in functions of recognition, adhesion, or transport. The evolutionary relation of the identified GPI-anchored proteins was assessed by analysis of the genome location of their encoding genes and a reverse BLASTp test. Apart from the five paralogous merozoite surface antigen genes that have been reported to be clustered in a single genomic region, all other eleven genes encoding GPI-anchored proteins were found to be rather dispersed in the genome. While nine of these represent orphan genes, two were found to be each part of gene families containing four and three members that encode non GPI-anchored proteins, respectively. The majority of the GPI-anchored antigens reported in this work has remained unnoticed so far but may represent potential vaccine candidates for the development of subunit vaccines.

Technical Abstract: Glycosylphosphatidyl-anchored proteins are particularly abundant on the surface of pathogenic protozoans and might play an important role for parasite survival. In the present work the relevance of GPI-anchored proteins for erythrocyte invasion of Babesia bovis, one of the tick-transmitted causative agents of bovine babesiosis, was studied. We show that cleavage of GPI-anchored antigens from the merozoite parasite stage by phosphatidylinositol-specific phospholipase C completely abolished invasion of erythrocytes demonstrating the importance of this class of molecules for parasite propagation. In addition, the repertoire of GPI-anchored proteins of B. bovis was defined with high fidelity by searching its genome with available web-based bioinformatic tools, such as “big-PI”, “Pred-GPI”, “GPI-SOM”, and “FragAnchor”. Candidates predicted with these algorithms were eliminated (a) if they lacked a signal peptide or a C-terminal transmembrane region as the existence of both is a necessary prerequisite for GPI-anchor attachment, and (b) when multiple transmembrane regions were detected, as this feature regularly excludes the presence of a GPI-anchor attachment site. Altogether 16 GPI-anchored proteins were identified of which five represent the Variable Merozoite Surface Antigens (VMSAs) that have been intensively studied as vaccine candidates and diagnostic antigens. The remaining eleven have not been studied so far and their function is unknown. Fifteen of the identified GPI-anchored proteins contain 2 to 16 amino acid repeats indicating that they are likely involved in functions of recognition, adhesion, or transport. In comparison to non-repetitive regions, repeats were found to contain a highly significant increased frequency of proline, indicative of unstructured regions. Repeats were also estimated to be 2.27 times more hydrophilic than non-repeat regions. In accordance with the “smoke screen hypothesis”, the property of being unstructured and highly hydrophilic may suggest that repeats represent eminent antibody epitopes. The evolutionary relation of the identified GPI-anchored proteins was assessed by analysis of the genome location of their encoding genes and a reverse BLASTp test. Apart from the five paralogous merozoite surface antigen genes that have been reported to be clustered in a single genomic region, all other eleven genes encoding GPI-anchored proteins were found to be rather dispersed in the genome. While nine of these represent orphan genes, two were found to be each part of gene families containing four and three members that encode non GPI-anchored proteins, respectively. The majority of the GPI-anchored antigens reported in this work has remained unnoticed so far but may represent potential vaccine candidates for the development of subunit vaccines.