title: Biochemical activities of three pairs of Ehrlichia chaffeensis two-component regulatory system proteins involved in inhibition of lysosomal fusion.
abstract: Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, replicates in early endosomes by avoiding lysosomal fusion in monocytes and macrophages. In E. chaffeensis we predicted three pairs of putative two-component regulatory systems (TCSs) designated PleC-PleD, NtrY-NtrX, and CckA-CtrA based on amino acid sequence homology. In the present study to determine biochemical pairs and specificities of the TCSs, the recombinant proteins of the three putative histidine kinase (HK) kinase domains (rPleCHKD, rNtrYHKD, and MBP-rCckAHKD) and the full-length forms of three putative response regulators (RRs) (rPleD, rNtrX, and rCtrA) as well as the respective mutant recombinant proteins (rPleCHKDH244A, rNtrYHKDH498A, MBP-rCckAHKDH449A, rPleDD53A, rNtrXD59A, and rCtrAD53A) were expressed and purified as soluble proteins. The in vitro HK activity, the specific His residue-dependent autophosphorylation of the kinase domain, was demonstrated in the three HKs. The specific Asp residue-dependent in vitro phosphotransfer from the kinase domain to the putative cognate RR was demonstrated in each of the three RRs. Western blot analysis of E. chaffeensis membrane and soluble fractions using antibodies specific for each recombinant protein detected PleC and CckA in the membrane fraction, whereas it detected NtrY, NtrX, and PleD in the soluble fraction. CtrA was found in the two fractions at similar levels. E. chaffeensis was sensitive to closantel, an HK inhibitor. Closantel treatment induced lysosomal fusion of the E. chaffeensis inclusion in a human monocytic leukemia cell line, THP-1 cells, implying that functional TCSs are essential in preventing lysosomal fusion of the E. chaffeensis inclusion compartment.
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210-1093, USA.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Kumagai
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Cheng
authors.forename: Mingqun
authors.initials: M
authors.lastname: Lin
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Rikihisa
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D018469:Ehrlichia chaffeensis / Q000187:drug effects / Q000378:metabolism / Q000472:pathogenicity*; D016873:Ehrlichiosis / Q000276:immunology; D015870:Gene Expression; D005798:Genes, Bacterial; D000071677:Histidine Kinase; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000382:microbiology*; D008561:Membrane Fusion / Q000187:drug effects; D009000:Monocytes / Q000737:chemistry / Q000276:immunology / Q000382:microbiology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012458:Salicylanilides / Q000494:pharmacology; D015398:Signal Transduction; D015534:Trans-Activators / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D012458:Salicylanilides; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; D011494:Protein Kinases; D000071677:Histidine Kinase; C023342:closantel