title: The Anaplasma phagocytophilum PleC histidine kinase and PleD diguanylate cyclase two-component system and role of cyclic Di-GMP in host cell infection.
abstract: Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis (HGA), has genes predicted to encode three sensor kinases, one of which is annotated PleC, and three response regulators, one of which is PleD. Prior to this study, the roles of PleC and PleD in the obligatory intracellular parasitism of A. phagocytophilum and their biochemical activities were unknown. The present study illustrates the relevance of these factors by demonstrating that both pleC and pleD were expressed in an HGA patient. During A. phagocytophilum development in human promyelocytic HL-60 cells, PleC and PleD were synchronously upregulated at the exponential growth stage and downregulated prior to extracellular release. A recombinant PleC kinase domain (rPleCHKD) has histidine kinase activity; no activity was observed when the conserved site of phosphorylation was replaced with alanine. A recombinant PleD (rPleD) has autokinase activity using phosphorylated rPleCHKD as the phosphoryl donor but not with two other recombinant histidine kinases. rPleCHKD could not serve as the phosphoryl donor for a mutant rPleD (with a conserved aspartic acid, the site of phosphorylation, replaced by alanine) or two other A. phagocytophilum recombinant response regulators. rPleD had diguanylate cyclase activity to generate cyclic (c) di-GMP from GTP in vitro. UV cross-linking of A. phagocytophilum lysate with c-di-[(32)P]GMP detected an approximately 47-kDa endogenous protein, presumably c-di-GMP downstream receptor. A new hydrophobic c-di-GMP derivative, 2'-O-di(tert-butyldimethylsilyl)-c-di-GMP, inhibited A. phagocytophilum infection in HL-60 cells. Our results suggest that the two-component PleC-PleD system is a diguanylate cyclase and that a c-di-GMP-receptor complex regulates A. phagocytophilum intracellular infection.
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210-1093, USA.
authors.forename: Tzung-Huei
authors.initials: TH
authors.lastname: Lai
authors.forename: Yumi
authors.initials: Y
authors.lastname: Kumagai
authors.forename: Mamoru
authors.initials: M
authors.lastname: Hyodo
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Hayakawa
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Rikihisa
mesh_terms: D041081:Anaplasma phagocytophilum / Q000201:enzymology*; D000712:Anaplasmosis / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D015153:Blotting, Western; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D016873:Ehrlichiosis / Q000201:enzymology; D029968:Escherichia coli Proteins; D018922:HL-60 Cells; D000071677:Histidine Kinase; D006801:Humans; D008957:Models, Genetic; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D029968:Escherichia coli Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D011494:Protein Kinases; D000071677:Histidine Kinase; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP