Identification of novel surface proteins of Anaplasma phagocytophilum by affinity purification and proteomics

Abstract

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.

FIG. 1.

Streptavidin affinity purification of Sulfo-NHS-SS-Biotin-labeled A. phagocytophilum surface proteins. In lane 1, Sulfo-NHS-SS-Biotin-labeled A. phagocytophilum surface proteins were separated by 10% SDS-PAGE and stained with GelCode blue. Bands 1 to 7 were subjected to Nano-LC/MS/MS analysis. The marker lane contained Precision Plus prestained protein standards (Bio-Rad).

FIG. 2.

Two-dimensional structure of A. phagocytophilum Asp62 and Asp55 with respect to the outer membrane lipid bilayer predicted using the Posterior Decoding method available in PRED-TMBB: graphical representations of the predicted topology with respect to the outer membrane lipid bilayers of Asp62 (A) and Asp55 (B).

FIG. 3.

Schematic diagram of the organization of genes encoding the APH_0404 (Asp62), APH_0405 (Asp55), APH_0406, and APH_0407 proteins in A. phagocytophilum HZ and the orthologous genes in A. marginale strain St. Maries, E. chaffeensis Arkansas, E. canis Jake, and E. ruminantium Welgevonden. Open reading frames are represented by open arrows that indicate their orientations. Orthologs are indicated by dashed lines at the ends of each open reading frame. The number of amino acid (aa) residues for each open reading frame is shown. The E value cutoff is e⁻²².

FIG. 4.

Cotranscriptional analysis of asp62 and asp55 by RT-PCR. Total RNA was isolated from A. phagocytophilum-infected HL-60 cells. Lane M, marker (1Kb Plus DNA ladder; Invitrogen); lane 1, cotranscript of asp62 and asp55, including the 65-nt intergenic region; lane 3, asp62; lane 5, asp55; lane 2, asp62 and asp55 control reaction without reverse transcriptase; lane 4, asp62 control reaction without reverse transcriptase; lane 6, asp55 control reaction without reverse transcriptase. The amplicon sizes were in agreement with predicted amplicon lengths (i.e., 197 bp for asp62, 340 bp for asp55, and 795 bp for the cotranscript of asp62 and asp55).

FIG. 5.

Surface localization of A. phagocytophilum Asp62 and Asp55 as determined by an immunofluorescence assay. Host cell-free A. phagocytophilum bacteria were fixed in paraformaldehyde, incubated with rabbit serum against the Asp62 C-terminal peptide (amino acids 534 to 552) or the Asp55 C-terminal peptide (amino acids 501 to 514), stained with Alexa Fluor 488 goat anti-rabbit IgG, and visualized by fluorescence microscopy. (a) Mottled ring-like bacterial surface staining of Asp62. (b) Mottled ring-like bacterial surface staining of Asp55. (c and d) Bacteria treated with pronase E and then incubated with rabbit anti-Asp62 peptide serum (c) or anti-Asp55 peptide serum (d). (e) Bacteria incubated with rabbit anti-irrelevant peptide serum. Scale bar, 1 μm.

FIG. 6.

(A) Expression of rAsp62 and rAsp55 in E. coli BL21(DE3) cells. Proteins were separated by 12% SDS-PAGE and stained with GelCode blue. Lane 1, rAsp62 expressed in E. coli BL21(DE3) with a predicted molecular mass of 35.9 kDa; lane 2, rAsp55 expressed in E. coli BL21(DE3) with a predicted molecular mass of 63 kDa; lane 3, pET33b vector expressed in E. coli BL21(DE3). Lane M contained markers (Precision Plus prestained protein standards; Bio-Rad). (B) Western immunoblot analysis of immunogenicity of rAsp62 and rAsp55 in an HGA patient. rAsp62, rAsp55, and the pET33b empty vector expressed in E. coli BL21(DE3) cells were used. The sera used in this study included rabbit preimmune sera for rAsp62 (Pre) and rAsp55 (Pre′), rabbit anti-Asp62 peptide serum (anti-Asp62), rabbit anti-Asp55 peptide serum (anti-Asp55), and HGA patient (HGA) and uninfected human (uninfect) sera.

FIG. 7.

Neutralization of A. phagocytophilum infection of HL-60 cells by Asp62 and Asp55 rabbit peptide antisera. After A. phagocytophilum pretreated with Asp62 or Asp55 rabbit peptide antiserum or preimmune sera was added, HL-60 cells were cultured for 2 to 3 days. The numbers of A. phagocytophilum bacteria were counted after Diff-Quik staining, using 100 cells per well and triplicate wells. The values are the means and standard deviations (n = 3). An asterisk indicates that there is a significant difference between the peptide antiserum and the preimmune serum (P < 0.05). The data are representative of three independent experiments.