title: Anaplasma phagocytophilum PSGL-1-independent infection does not require Syk and leads to less efficient AnkA delivery.
abstract: Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to cause granulocytic anaplasmosis in humans and mammals. P-selectin glycoprotein ligand-1 (PSGL-1) and the tetrasaccharide sialyl Lewis x (sLe(x)), which caps the PSGL-1 N-terminus, are confirmed A. phagocytophilum receptors. A. phagocytophilum is capable of sLe(x)-modified PSGL-1-dependent and -independent infection. PSGL-1 N-terminus-mediated entry is dependent on spleen tyrosine kinase (Syk). Here, we determined that PSGL-1-independent entry does not alter bacterial replication and investigated whether it involves Syk using NCH-1A2, an enriched subpopulation of A. phagocytophilum NCH-1 obtained through cultivation in a sLe(x)-deficient HL-60 cell line, HL-60 A2. Pharmacological inhibition of Syk nearly abolishes NCH-1 infection, but does not alter NCH-1A2 invasion and only marginally reduces NCH-1A2 propagation. This phenomenon was confirmed by a competitive infection assay using PSGL-1-dependent and -independent A. phagocytophilum organisms transformed to express mCherry or green fluorescent protein respectively. We also assayed for delivery and tyrosine phosphorylation of the A. phagocytophilum effector, AnkA, following NCH-1or NCH-1A2 incubation with HL-60 or HL-60 A2 cells in the presence of PSGL-1 blocking antibody. PSGL-1 N-terminus recognition promotes optimal AnkA delivery while binding to sLe(x) or the unknown receptor is comparably less important for this process.
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, College of Medicine, University of Kentucky, Lexington, KY, USA.
authors.forename: Dexter V
authors.initials: DV
authors.lastname: Reneer
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Troese
authors.forename: Bernice
authors.initials: B
authors.lastname: Huang
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Kearns
authors.forename: Jason A
authors.initials: JA
authors.lastname: Carlyon
mesh_terms: D041081:Anaplasma phagocytophilum / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000378:metabolism*; D018395:CA-19-9 Antigen; D016873:Ehrlichiosis / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018395:CA-19-9 Antigen; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; C084882:P-selectin ligand protein; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase