Mitogenic component in polar lipid-enriched Anaplasma phagocytophilum membranes

Abstract

Human granulocytic anaplasmosis is an emerging tick-borne disease caused by Anaplasma phagocytophilum. A. phagocytophilum cells activate Toll-like receptor 2 signaling and possess mitogenic activity, and A. phagocytophilum infection in vivo activates NKT cells unrelated to major surface protein 2 (Msp2) hypervariable region expression. Thus, we hypothesized that lipoprotein or glycolipid components of A. phagocytophilum membranes could be important triggers of the innate immune response and immunopathology. A. phagocytophilum membranes depleted of Msp2 and protein antigens enhanced the proliferation of naïve mouse splenocytes beyond that of untreated membranes. Protein-depleted and polar lipid-enriched membranes from low-passage A. phagocytophilum cultures enhanced naïve splenocyte lymphoproliferation to a much greater degree than did these fractions from high-passage cultures of bacterial membranes (1.8- to 3.7-fold for protein-depleted fractions and 4.8- to > or =17.7-fold for polar lipid-enriched fractions). These results support the hypothesis that components that are enriched among polar lipids in the A. phagocytophilum membrane stimulate innate immune cell proliferation, possibly activating NKT cells that link innate and adaptive immunity, and immunopathology.

FIG. 1.

Relative distribution of proteins, antigens, and Msp2 in membrane and cytosolic fractions of A. phagocytophilum. Lanes 1 and 2 and 3 and 4 represent membrane fractions 1 to 9 (f1-9) and 10 to 15 (f10-15), respectively, from separate preparations, and lane 5 represents the pooled cytoplasmic fractions from these preparations. The top panel shows a total protein Coomassie blue stain illustrating the overall similar protein contents but dissimilar protein profiles between membranes and bacterial cytosol. The middle panel is a protein immunoblot reacted with rabbit polyclonal antibody prepared for whole, purified A. phagocytophilum strain Webster cells, again depicting the 4.1- to 5.3-fold-greater distribution of antigens into the membrane fraction. The bottom panel shows protein immunoblotting with MAb 20B4 to A. phagocytophilum Msp2 and depicts even greater (15.5- to 30.1-fold) partitioning of Msp2 into bacterial membranes than into bacterial cytosol. Molecular weights (mw) (in thousands) are shown on the left side of each panel.

FIG. 2.

Methanol-chloroform extraction of polar lipids (Ap lipid) and protease treatment (Ap PrK) of purified A. phagocytophilum membranes from low-passage (Ap memb p7) and high-passage (Ap memb p22) bacteria removes detectable Msp2 by protein immunoblotting using MAb 20B4. Note that the typical monomeric and oligomeric Msp2 bands present without treatment are absent after treatment. mw, molecular weight (in thousands).

FIG. 3.

Proliferation of naïve mouse splenic lymphocytes as reflected by the optical density (o.d.) of BrdU incorporation after exposure to A. phagocytophilum purified membranes (Ap membranes), cytosol (Ap cytosol), whole bacterial cells (Ap cells), ConA, and medium only. Significantly more proliferation in response to purified membranes than in response to either whole cells or cytosol was observed, suggesting the enrichment or availability of a membrane-associated mitogenic factor.

FIG. 4.

Splenocyte proliferation with exposure to untreated low- and high-passage A. phagocytophilum membranes (A), protease-treated bacterial membranes (B), and membranes enriched for polar lipids (C). Although proliferations in response to untreated membranes are similar when splenocytes are stimulated with membranes from low- and high-passage bacterial cultures, depletion of proteins enhances proliferation more, and enrichment for polar lipids retains more proliferative activity for low-passage than for high-passage bacterial cultures. D illustrates the lymphoproliferative activity of splenocytes exposed to medium only or ConA for experiments that used low- or high-passage A. phagocytophilum-derived antigens, although bacterial antigens were not used in the control cultures.