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.