Submitted to: Infection and Immunity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/27/2011
Publication Date: 5/16/2011
Citation: Solomon, R.S., Schneider, D.A., Brayton, K.A., Ueti, M.W., Graca, T., Futse, J.E., Noh, S.M., Baszler, T.V., Palmer, G.H. 2011. Expression of Anaplasma marginale ankyrin repeat-containing proteins during infection of the mammalian host and tick vector. Infection and Immunity. 79(7):2847-2855. Interpretive Summary: Ankyrin repeats are a common motif linked to protein-protein interaction. The protein AnkA of the obligate intracellular bacterial pathogen, Anaplasma phagocytophilum, is an ankyrin repeat-containing protein that is translocated to the nucleus of the host cell where it interacts with nuclear proteins and alters gene expression, perhaps as a mechanism to enhance its intracellular survival. Such pathogen:host protein interactions may represent key targets for improved pathogen control. In the present study we identified three ankyrin repeat-containing proteins expressed by a related pathogen, Anaplasma marginale: AnkA (AM705, a homologue of AnkA from A. phagocytophilum), AnkB (AM926) and AnkC (AM638). Since A. marginale infects both non-nucleated erythrocytes of the mammalian host as well as the nucleated cells of its tick vector, we hypothesized that these ankyrin repeat-containing proteins would be upregulated during infection of nucleated (tick) cells in a manner analogous to observations with AnkA from A. phagocytophilum. Although each of these A. marginale proteins was expressed in tick cells, expression of AnkA and AnkB were respectively higher or similar to that in tick cells, contrary to our hypothesis. Furthermore, translocation of these A. marginale ankyin repeat-containing proteins to the nucleus of the tick cells was not evident. We conclude that the apparent retention of several ankyrin repeat-containing proteins may indicate their importance in mediating protein:protein interactions, but that the AnkA, AnkB and AnkC proteins of A. marginale lack a necessary transport mechanism for any functional interaction with tick nuclear proteins.
Technical Abstract: Using searches of the NCBI conserved domain database and SMART genomic architecture analysis, we identified three ankyrin repeat-containing genes in Anaplasma marginale: AM705, AM926 and AM638. Recombinant protein was used to immunize mice and generate fusion hybridomas secreting protein-specific monoclonal antibodies, specificity confirmed by western blot analysis. In situ(authentic) protein expression in the salivary gland acinar cells of the tick vector was confirmed for all three by immunohistochemistry. These expressed, ankyrin repeat-containing proteins of A. marginale were named: AnkA (Am705), AnkB (AM926) and AnkC (AM638). Comparative analysis of translated amino acid sequences demonstrated high conservation of AnkB and AnkC, but less so for AnkA, amongst sensu stricto strains of A. marginale. Quantitative western blotting was used to compare relative protein expression during A. marginale St. Maries infection of nucleated ISE6 tick cells versus infection of the non-nucleated erythrocytes of the mammalian host. All three were expressed during infection of ISE6 cells. The relative expressions of AnkA and AnkB during infection of erythrocytes were, respectively, greater and similar to expression levels in ISE6 cells, whereas AnkC expression was not detected. Using dual fluorescenc microscopy, translocation of these A. marginale ankyrin repeat-containing proteins to the DAPI-stained nucleus of infected ISE6 cells was not detected.