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Journal of Bacteriology, February 2009, p. 693-700, Vol. 191, No. 3
0021-9193/09/$08.00+0     doi:10.1128/JB.01218-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Anaplasma phagocytophilum PleC Histidine Kinase and PleD Diguanylate Cyclase Two-Component System and Role of Cyclic Di-GMP in Host Cell Infection{triangledown}

Tzung-Huei Lai,1 Yumi Kumagai,1 Mamoru Hyodo,2 Yoshihiro Hayakawa,2 and Yasuko Rikihisa1*

Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, Ohio 43210,1 Laboratory of Bioorganic Chemistry, Graduate School of Information Science, Nagoya University, Chikusa, Nagoya 464-8601, Japan2

Received 29 August 2008/ Accepted 22 October 2008

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-[32P]GMP detected an ~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.

* Corresponding author. Mailing address: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210-1093. Phone: (614) 292-9677. Fax: (614) 292-6473. E-mail: rikihisa.1{at}osu.edu

{triangledown} Published ahead of print on 31 October 2008.

Journal of Bacteriology, February 2009, p. 693-700, Vol. 191, No. 3
0021-9193/09/$08.00+0     doi:10.1128/JB.01218-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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Cyclic Di-GMP (c-Di-GMP) Goes in1593to Host Cells—c-Di-GMP Signaling in the Obligate Intracellular Pathogen Anaplasma phagocytophilum
Ute Römling
JB 2009 191: 683-686. [Full Text]  



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