Cellular Microbiology (2006) 8(12), 1972–1984 doi:10.1111/j.1462-5822.2006.00764.x First published online 25 July 2006 © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd Blackwell Publishing LtdOxford, UKCMICellular Microbiology1462-5814© 2006 The Authors; Journal compilation © 2006 Blackwell Publishing Ltd ? 200681219721984Original ArticleNovel A. phagocytophilum adhesin activityD. V. Reneer et al. Received 24 April, 2006; revised 31 May, 2006; accepted 13 June, 2006. *For correspondence. E-mail jason.carlyon@uky.edu; Tel. (+1) 859 323 3873; Fax (+1) 859 257 8994. † Both authors contributed equally to this study. Characterization of a sialic acid- and P-selectin glycoprotein ligand-1-independent adhesin activity in the granulocytotropic bacterium Anaplasma phagocytophilum Dexter V. Reneer, 1† Sarah A. Kearns, 1† Tadayuki Yago, 2 Jonathan Sims, 1 Richard D. Cummings, 3 Rodger P. McEver 2,3 and Jason A. Carlyon 1 * 1 Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY, USA. 2 Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation and 3 Department of Biochemistry and Molecular Biology and Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. Summary Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils and neutrophil precursors. The granulocytotropic bacte- rium uses multiple adhesins that cooperatively bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1) and to sialyl Lewis x (sLe x ) expressed on myeloid cell surfaces. Recognition of sLe x occurs through interactions with a2,3-sialic acid and a1,3-fucose. It is unknown whether other bacte- ria–host cell interactions are involved. In this study, we have enriched for A. phagocytophilum organisms that do not rely on sialic acid for cellular adhesion and entry by maintaining strain NCH-1 in HL-60 cells that are severely undersialylated. The selected bacteria, termed NCH-1A, also exhibit lessened dependencies on PSGL-1 and a1,3-fucose. Optimal adhesion and invasion by NCH-1A require interactions with the known determinants (sialic acid, PSGL-1 and a1,3- fucose), but none of them is absolutely necessary. NCH-1A binding to sLe x -modified PSGL-1 requires recognition of the known determinants in the same manners as other A. phagocytophilum strains. These data suggest that A. phagocytophilum expresses a separate adhesin from those targeting sialic acid, a1,3-fucose and the N-terminal region of PSGL-1. We propose that NCH-1A upregulates expression of this adhesin. Introduction Human granulocytic anaplasmsosis (HGA; formerly human granulocytic ehrlichiosis) is an emerging tick- transmitted disease caused by the obligate intracellular bacterium Anaplasma phagocytophilum (Bakken et al., 1994; Chen et al., 1994; Dumler et al., 2001; Carlyon and Fikrig, 2004; Goodman, 2005). In the United States, HGA occurs primarily in the north-eastern and Midwestern states and California, which are endemic for ticks of the Ixodes persulcatus complex, the vectors for the disease. A. phagocytophilum zoonotically cycles between its arthropod vector and small rodent reservoirs; human infection is inadvertent. HGA is an acute febrile illness, the symptoms of which can range from subclinical infec- tion to severe, even fatal, disease. Clinical manifestations associated with HGA include fever, headache, myalgia, leucopenia, thrombocytopenia and impaired host defence mechanisms (Carlyon and Fikrig, 2004; Goodman, 2005). While recent evidence has hinted that capillary endothelial cells are subject to infection (Munderloh et al., 2004; Herron et al., 2005), the pathological hall- mark of HGA is the intracytoplasmic colonization of neu- trophils (Carlyon and Fikrig, 2004; Goodman, 2005), which highlights the organism’s unique granulocytotropic nature. Host–pathogen interactions preceding bacterial coloni- zation are mediated by adhesins (Karlsson, 2001; Boyle and Finlay, 2003; Niemann et al., 2004; Remaut and Waksman, 2004), which recognize protein (Boyle and Fin- lay, 2003; Niemann et al., 2004; Remaut and Waksman, 2004) and carbohydrate (Karlsson, 2001) determinants expressed on eukaryotic cell surfaces. Adhesins have spe- cific structural requirements for targeting optimal receptors that result in the bacterium occupying a particular niche (Boyle and Finlay, 2003; Niemann et al., 2004). The ability of A. phagocytophilum to bind and invade human neutro- phils and HL-60 cells is linked to the host cells’ surface