Anaplasma phagocytophilum MSP2(P44)-18 predominates and is modified into multiple isoforms in human myeloid cells

Abstract

Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis. MSP2(P44), the bacterium's major surface protein, is encoded by a paralogous gene family and has been implicated in a variety of pathobiological processes, including antigenic variation, host adaptation, adhesion, porin activity, and structural integrity. The consensus among several studies performed at the DNA and RNA levels is that a heterogeneous mix of a limited number of msp2(p44) transcripts is expressed by A. phagocytophilum during in vitro cultivation. Such analyses have yet to be extended to the protein level. In this study, we used proteomic and molecular approaches to determine that MSP2(P44)-18 is the predominant if not the only paralog expressed and is modified into multiple 42- to 44-kDa isoforms by A. phagocytophilum strain HGE1 during infection of HL-60 cells. The msp2(p44) expression profile was homogeneous for msp2(p44)-18. Thus, MSP2(P44)-18 may have a fitness advantage in HL-60 cell culture in the absence of selective immune pressure. Several novel 22- to 27-kDa MSP2 isoforms lacking most of the N-terminal conserved region were also identified. A. phagocytophilum MSP2(P44) orthologs expressed by other pathogens in the family Anaplasmataceae are glycosylated. Gas chromatography revealed that recombinant MSP2(P44)-18 is modified by glucose, galactose, xylose, mannose, and trace amounts of other glycosyl residues. These data are the first to confirm differential modification of any A. phagocytophilum MSP2(P44) paralog and the first to provide evidence for expression of truncated versions of such proteins.

FIG. 1.

MSP2(P44) MAb 20B4-screened Western blot of A. phagocytophilum HGE1 whole-cell lysate and Triton X-114-partitioned hydrophobic and hydrophilic liquid fractions. Ten micrograms each of A. phagocytophilum HGE1 whole-cell lysate and the hydrophobic liquid and hydrophilic liquid fractions obtained after Triton X-114 phase partitioning were screened via Western blot analysis using anti-MSP2(P44) MAb 20B4.

FIG. 2.

2DE and Western blot analyses of A. phagocytophilum HGE1 MSP2(P44) proteins present in the hydrophobic pellet. Hydrophobic pellet fractions enriched for A. phagocytophilum outer membrane proteins were isoelectrically focused in IPG strips (pH 5.0 to 8.0), followed by resolution in the second dimension in gradient SDS-polyacrylamide (4 to 20%) gels. (A) Silver-stained gel. MSP2 proteins of ∼42 to 44 kDa in size that were excised for LC-MS/MS identification or detected by anti-MSP2 antibodies are denoted by a solid box. A dotted box denotes a series of higher-molecular-mass proteins that are recognized by MAb 2H8. A dashed box denotes a series of proteins of ∼22 to 27 kDa that were recognized by rabbit polyclonal anti-rMSP2(P44). (B) An enlarged view of the region of the silver-stained gel in panel A marked by a solid box. Numbered spots were excised and subjected to LC-MS/MS identification. (C to E) Spots in the predicted size range for full-length MSP2(P44) proteins were recognized by MAbs 2H8 (C), 20B4 (D), and 3G10 (E). (F) List of numbered spots denoted in panel B that were detected by each anti-MSP2(P44) MAb.

FIG. 3.

MSP2(P44)-18 protein sequence. The full-length protein sequence of MSP2(P44)-18 is provided. Bold peptides were recovered from the hydrophobic pellet. Underlined peptides were recovered from the hydrophobic liquid fraction. The asparagine residue denoted by black shading and white text is predicted by NetNGlyc 1.0 (www.cbs.dtu.dk/services/NetNGlyc/) to be a potential N-linked glycosylation site. Amino acids shaded in gray correspond to those identified by NetOGlyc 3.1 (www.cbs.dtu.dk/services/NetOGlyc/) (24) and Support Vector Machines models (www.biosino.org/Oglyc) (25) as being potential O-linked glycosylation sites.

FIG. 4.

2DE and Western blot analyses of A. phagocytophilum MSP2(P25) proteins present in the hydrophobic liquid fraction. Hydrophobic liquid fractions enriched for A. phagocytophilum outer membrane proteins were isoelectrically focused in IPG strips (pH 5.0 to 8.0), followed by resolution in the second dimension in gradient SDS-polyacrylamide (4 to 20%) gels. (A) Silver-stained gel. MSP2 proteins of ∼22 to 27 kDa in size that were excised for LC-MS/MS identification or detected by anti-MSP2 antibodies are denoted by a solid box. (B) An enlarged view of the region of the silver-stained gel in panel A marked by a solid box. Numbered spots were excised and subjected to LC-MS/MS identification. (C) Spots in the predicted size range for the N-terminally truncated MSP2(P25) proteins were recognized by MAb 20B4.

FIG. 5.

Expression and Western blot analysis of rMSP2(P44)-18. (A) Coomassie blue-stained gel of whole-cell lysates of uninduced and IPTG-induced E. coli transformed to express rMSP2(P44)-18 and purified rMSP2(P44)-18. (B) Western blot of rMSP2(P44)-18 and uninfected and A. phagocytophilum HGE1-infected HL-60 cells screened with anti-MSP2(P44) MAb 20B4.