Cellular Microbiology (2004) 6(8), 743–751 doi:10.1111/j.1462-5822.2004.00400.x © 2004 Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKCMICellular Microbiology 1462-5814Blackwell Publishing Ltd, 20046 8743751Original ArticleJ. Park et al.AnkA binds to host DNA and nuclear proteins Received 22 December, 2003; revised 27 February, 2004; accepted 2 March, 2004. *For correspondence. E-mail sdumler@jhmi.edu; Tel. (+1) 410 955 8654; Fax (+1) 443 287 3665. †Present address: Department of Veterinary Internal Medicine, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561–756, Korea. Anaplasma phagocytophilum AnkA binds to granulocyte DNA and nuclear proteins Jinho Park, 1† Kee Jun Kim, 2 Kyoung-seong Choi, 1 Dennis J. Grab 2 and J. Stephen Dumler 1 * 1 Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Ross Research Building, 720 Rutland Avenue, Baltimore, Maryland, USA. 2 Division of Infectious Diseases, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Summary Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. The bacterium infects, survives, propagates in, and alters neutrophil phenotype, indi- cating unique survival mechanisms. AnkA is the only known A. phagocytophilum component that gains access beyond neutrophil vacuoles and is trans- ported to the infected host cell nucleus. The ability of native and recombinant AnkA to bind DNA and nuclear proteins from host HL-60 cells was assessed by the use of immunoprecipitation after cis- diamminedichloroplatinum (cis-DDP) DNA-protein crosslinking, by probing uninfected HL-60 cell nuclear lysates for AnkA binding, and by recovery and sequence analysis of immunoprecipitated DNA. AnkA binds HL-60 cell DNA as well as nuclear proteins of approximately 86, 53 and 25 kDa, whereas recombi- nant A. phagocytophilum Msp2 or control proteins do not. DNA immunoprecipitation reveals AnkA binding to a variety of target genes in the human genome, including genes that encode proteins with ATPase, tyrosine phosphatase and NADH dehydrogenase-like functions. These data indicate that AnkA could exert some effect on cells through binding to protein:DNA complexes in neutrophil nuclei. Whether AnkA bind- ing leads to neutrophil functional alterations, and how such alterations might occur will depend upon defin- itive identification of binding partners and associated metabolic and biochemical pathways. Introduction Anaplasma phagocytophilum is a tick-borne, obligate intracellular bacterium that infects neutrophils of mam- mals, including man (Dumler et al., 2001). The unique niche of this bacterium implies that it has developed spe- cialized systems for survival and propagation. It is now well documented that A. phagocytophilum alters neutro- phil function by inducing an ‘activated-deactivated’ pheno- type, wherein infected neutrophils are incapable of generating phagocyte oxidase, are defective at adhering to and transmigrating across activated endothelium, and are defective at phagocytosis and microbicidal activity. Yet, infection activates cells for production of proinflammatory responses with chemokine secretion, degranulation and release of metalloproteases (Woldehiwet, 1987; Banerjee et al., 2000; Klein et al., 2000; Choi et al., 2003; Park et al., 2003a). The mechanisms that underlie control of these various cellular processes by the bacterium are increasingly studied and appear to involve regulation at the level of host cell mRNA, via signal transduction, and perhaps by effects upon transcription factors (Banerjee et al., 2000; Carlyon et al., 2002; Kim and Rikihisa, 2002; Lin and Rikihisa, 2003). AnkA (also known as Ank) is a unique protein of A. phagocytophilum that is high molecular weight (160 kDa), not associated with the bacterial membrane, but is found localized within nuclei of infected granulocyte hosts, pre- sumably after secretion and transport through at least three distinct membranes (Caturegli et al., 2000). Although its close ultrastructural association with con- densed chromatin and the presence of multiple ankyrin repeat domains tempts speculation about a role in affect- ing gene transcription or transcription factors, the actual binding targets in the nucleus are not known. We hypoth- esized that AnkA would bind to DNA or DNA–protein targets of granulocyte nuclei, and tested this by analysing these binding properties in vitro. The results of these analyses provide strong evidence of a DNA binding role for AnkA, with a potential contribution by protein, and lead to further speculation about the functional nature of AnkA–host cell DNA interactions.