Anaplasma phagocytophilum PSGL-1-independent infection does not require Syk and leads to less efficient AnkA delivery.
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.
Reneer
Dexter V
DV
Department of Microbiology, Immunology, and Molecular Genetics, College of Medicine, University of Kentucky, Lexington, KY, USA.
Troese
Matthew J
MJ
Huang
Bernice
B
Kearns
Sarah A
SA
Carlyon
Jason A
JA
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
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