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.

Differential Expression and Glycosylation of Anaplasma phagocytophilum Major Surface Protein 2 Paralogs during Cultivation in Sialyl Lewis x-Deficient Host Cells

“…As we have reported previously for A. phagocytophilum HGE1 Msp2(P44)-18 (38) and as has been observed for major outer membrane proteins of other Anaplasmataceae pathogens (41,42), the multiple Msp2(P44) isoforms observed for NCH-1 and NCH-1A likely result from glycosylation or other posttranslational modifications. Because glycosylation of Msp2(P44) paralogs may contribute to their pathobiological functions, we performed in silico analyses (22,23) to identify possible N-and O-linked glycosylation sites in the amino acid sequences predicted for the HVR of each expressed paralog.…”

“…4). Twenty-nine different Msp2(P44) peptides were identified from 11 of the spots ( Table 2 (38), a series of MAb 20B4-immunoreactive full-length 44-kDa and truncated 25-to 27-kDa proteins were present in the NCH-1A hydrophobic liquid fraction, and the truncated 25-to 27-kDa proteins were more abundant (Fig. 5).…”

“…phagocytophilum NCH-1 and NCH-1A express full-length and N-terminally truncated Msp2 proteins. We previously determined that A. phagocytophilum HGE1 expresses full-length 42-to 44-kDa Msp2 proteins, as well as 18-to 27-kDa isoforms that we proposed to be N-terminally truncated (38). Screening whole-cell lysates with MAb 20B4 (33), which recognizes both Msp2 isoforms, demonstrated that NCH-1 and NCH-1A also express both full-length and truncated isoforms (Fig.…”

“…Certain Msp2(P44) paralogs (3,20,21,27,45,46), as well as paralogs of other Anaplasmataceae pathogens' outer membrane protein families (41,42), exhibit exclusive expression patterns in tick or mammalian environments, which suggests that they may have host cell-specific roles. Indeed, msp2(p44)-18 transcription is specific to and dominates the msp2(p44) expression profile during A. phagocytophilum HZ, HGE1, and HGE2 infection of HL-60 cells (3,21,38,46), and Msp2(P44)-18 is the predominant, if not the only, paralog expressed by HGE1 during growth in HL-60 cells (38). HGE1 also expresses 18-to 27-kDa Msp2 proteins [Msp2(P25)], and both Msp2(P44) and Msp2(P25) are posttranslationally modified into multiple isoforms.…”

“…Three hundred fifty micrograms of an outer membrane-enriched hydrophobic pellet fraction was resolved by 2DE on five 4 to 20% polyacrylamide gradient gels (70 g per gel; Bio-Rad). The spots corresponding to Msp2(P44) were excised from each gel and were processed for and subjected to gas chromatography-mass spectroscopy analyses exactly as described previously (38). Monosaccharide analysis was performed at the University of Georgia Complex Carbohydrate Research Center, Athens.…”