Identification of novel surface proteins of Anaplasma phagocytophilum by affinity purification and proteomics.
Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA), one of the major tick-borne zoonoses in the United States. The surface of A. phagocytophilum plays a crucial role in subverting the hostile host cell environment. However, except for the P44/Msp2 outer membrane protein family, the surface components of A. phagocytophilum are largely unknown. To identify the major surface proteins of A. phagocytophilum, a membrane-impermeable, cleavable biotin reagent, sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin), was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin agarose affinity purification and then separated by electrophoresis, followed by capillary liquid chromatography-nanospray tandem mass spectrometry analysis. Among the major proteins captured by affinity purification were five A. phagocytophilum proteins, Omp85, hypothetical proteins APH_0404 (designated Asp62) and APH_0405 (designated Asp55), P44 family proteins, and Omp-1A. The surface exposure of Asp62 and Asp55 was verified by immunofluorescence microscopy. Recombinant Asp62 and Asp55 proteins were recognized by an HGA patient serum. Anti-Asp62 and anti-Asp55 peptide sera partially neutralized A. phagocytophilum infection of HL-60 cells in vitro. We found that the Asp62 and Asp55 genes were cotranscribed and conserved among members of the family Anaplasmataceae. With the exception of P44-18, all of the proteins were newly revealed major surface-exposed proteins whose study should facilitate understanding the interaction between A. phagocytophilum and the host. These proteins may serve as targets for development of chemotherapy, diagnostics, and vaccines.
Ge
Yan
Y
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210, USA.
Rikihisa
Yasuko
Y
D041081:Anaplasma phagocytophilum / Q000235:genetics* / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000032:analysis*; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D008051:Lipid Bilayers; D008563:Membrane Lipids / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis*; D040901:Proteomics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001426:Bacterial Proteins; D017931:DNA Primers; D008051:Lipid Bilayers; D008563:Membrane Lipids; D008565:Membrane Proteins