Essential Domains of Anaplasma phagocytophilum Invasins Utilized to Infect Mammalian Host Cells

Abstract

Anaplasma phagocytophilum causes granulocytic anaplasmosis, an emerging disease of humans and domestic animals. The obligate intracellular bacterium uses its invasins OmpA, Asp14, and AipA to infect myeloid and non-phagocytic cells. Identifying the domains of these proteins that mediate binding and entry, and determining the molecular basis of their interactions with host cell receptors would significantly advance understanding of A. phagocytophilum infection. Here, we identified the OmpA binding domain as residues 59 to 74. Polyclonal antibody generated against a peptide spanning OmpA residues 59 to 74 inhibited A. phagocytophilum infection of host cells and binding to its receptor, sialyl Lewis x (sLex-capped P-selectin glycoprotein ligand 1. Molecular docking analyses predicted that OmpA residues G61 and K64 interact with the two sLex sugars that are important for infection, α2,3-sialic acid and α1,3-fucose. Amino acid substitution analyses demonstrated that K64 was necessary, and G61 was contributory, for recombinant OmpA to bind to host cells and competitively inhibit A. phagocytophilum infection. Adherence of OmpA to RF/6A endothelial cells, which express little to no sLex but express the structurally similar glycan, 6-sulfo-sLex, required α2,3-sialic acid and α1,3-fucose and was antagonized by 6-sulfo-sLex antibody. Binding and uptake of OmpA-coated latex beads by myeloid cells was sensitive to sialidase, fucosidase, and sLex antibody. The Asp14 binding domain was also defined, as antibody specific for residues 113 to 124 inhibited infection. Because OmpA, Asp14, and AipA each contribute to the infection process, it was rationalized that the most effective blocking approach would target all three. An antibody cocktail targeting the OmpA, Asp14, and AipA binding domains neutralized A. phagocytophilum binding and infection of host cells. This study dissects OmpA-receptor interactions and demonstrates the effectiveness of binding domain-specific antibodies for blocking A. phagocytophilum infection.

Anaplasma marginale Outer Membrane Protein A Is an Adhesin That Recognizes Sialylated and Fucosylated Glycans and Functionally Depends on an Essential Binding Domain

“…Recombinant ApOmpA binds to host cells, confers adhesiveness and invasiveness to inert beads, and acts as a competitive agonist to inhibit A. phagocytophilum infection in vitro (17,18), confirming that it alone is sufficient to mediate binding and uptake. ApOmpA functionally depends on a lysine and a glycine in its essential linear binding domain that interacts with ␣2,3-sialic acid and ␣1,3-fucose of the Lewis antigen receptor, sialyl Lewis x (sLe x ; NeuAc␣2,3Gal␤1,4[Fuc␣1,3]GlcNac), on myeloid cells and 6-sulfo-sialyl Lewis x (6-sulfo-sLe x ; NeuAc␣2,3Gal␤1-4[Fuc␣1,3] HSO 3 3,6GlcNac) on endothelial cells (17,18). Antibodies raised against full-length ApOmpA or its 16-residue binding domain inhibit A. phagocytophilum infection of host cells (18).…”

“…Indeed, we discovered that A. phagocytophilum OmpA (ApOmpA) is one of a trio of adhesins that cooperatively function to mediate optimal bacterial binding to and invasion of host cells (17,18,20,21). Recombinant ApOmpA binds to host cells, confers adhesiveness and invasiveness to inert beads, and acts as a competitive agonist to inhibit A. phagocytophilum infection in vitro (17,18), confirming that it alone is sufficient to mediate binding and uptake. ApOmpA functionally depends on a lysine and a glycine in its essential linear binding domain that interacts with ␣2,3-sialic acid and ␣1,3-fucose of the Lewis antigen receptor, sialyl Lewis x (sLe x ; NeuAc␣2,3Gal␤1,4[Fuc␣1,3]GlcNac), on myeloid cells and 6-sulfo-sialyl Lewis x (6-sulfo-sLe x ; NeuAc␣2,3Gal␤1-4[Fuc␣1,3] HSO 3 3,6GlcNac) on endothelial cells (17,18).…”

“…OmpA is highly conserved among A. marginale sensu stricto strains and isolates, exhibiting 99.6 to 100% identity (14). Clues pertaining to the role of A. marginale OmpA (AmOmpA) are provided by recent studies demonstrating the importance of OmpA proteins to cellular invasion by A. phagocytophilum and Ehrlichia chaffeensis, two Anaplasmataceae members that cause potentially fatal infections of humans and animals (17)(18)(19). Indeed, we discovered that A. phagocytophilum OmpA (ApOmpA) is one of a trio of adhesins that cooperatively function to mediate optimal bacterial binding to and invasion of host cells (17,18,20,21).…”

“…Clues pertaining to the role of A. marginale OmpA (AmOmpA) are provided by recent studies demonstrating the importance of OmpA proteins to cellular invasion by A. phagocytophilum and Ehrlichia chaffeensis, two Anaplasmataceae members that cause potentially fatal infections of humans and animals (17)(18)(19). Indeed, we discovered that A. phagocytophilum OmpA (ApOmpA) is one of a trio of adhesins that cooperatively function to mediate optimal bacterial binding to and invasion of host cells (17,18,20,21). Recombinant ApOmpA binds to host cells, confers adhesiveness and invasiveness to inert beads, and acts as a competitive agonist to inhibit A. phagocytophilum infection in vitro (17,18), confirming that it alone is sufficient to mediate binding and uptake.…”

“…ApOmpA functionally depends on a lysine and a glycine in its essential linear binding domain that interacts with ␣2,3-sialic acid and ␣1,3-fucose of the Lewis antigen receptor, sialyl Lewis x (sLe x ; NeuAc␣2,3Gal␤1,4[Fuc␣1,3]GlcNac), on myeloid cells and 6-sulfo-sialyl Lewis x (6-sulfo-sLe x ; NeuAc␣2,3Gal␤1-4[Fuc␣1,3] HSO 3 3,6GlcNac) on endothelial cells (17,18). Antibodies raised against full-length ApOmpA or its 16-residue binding domain inhibit A. phagocytophilum infection of host cells (18). Likewise, antibodies against E. chaffeensis OmpA inhibit ehrlichial infection in vitro (19).…”