期刊
INFECTION AND IMMUNITY
卷 74, 期 9, 页码 5014-5022出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/IAI.00735-06
关键词
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资金
- NIAID NIH HHS [R01 AI047885, R01 AI054476, R01 AI 054476, R01 AI 47885] Funding Source: Medline
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R01AI047885, R01AI054476] Funding Source: NIH RePORTER
Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, replicates in early endosomes by avoiding lysosomal fusion in monocytes and macrophages. In E. chafeensis 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. chafeensis inclusion in a human monocytic leukemia cell line, THP-1 cells, implying that functional TCSs are essential in preventing lysosomal fusion of the E. chafeensis inclusion compartment.
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