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. 2006 Sep;74(9):5014-22.
doi: 10.1128/IAI.00735-06.

VSports注册入口 - Biochemical activities of three pairs of Ehrlichia chaffeensis two-component regulatory system proteins involved in inhibition of lysosomal fusion

Yumi Kumagai  1 , Zhihui Cheng, Mingqun Lin, Yasuko Rikihisa
Affiliations

Biochemical activities of three pairs of Ehrlichia chaffeensis two-component regulatory system proteins involved in inhibition of lysosomal fusion (VSports注册入口)

Yumi Kumagai et al. Infect Immun. 2006 Sep.

"V体育官网" Abstract

Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, replicates in early endosomes by avoiding lysosomal fusion in monocytes and macrophages. In E VSports手机版. 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. .

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"V体育官网入口" Figures

FIG. 1.
FIG. 1.
Schematic representation of the domain structures of HKs and RRs in E. chaffeensis and reference bacteria. The numbers on each protein are amino acid residues. The conserved histidine (H) in HKs and the conserved aspartate (D) in RRs, GenBank accession numbers, and predicted molecular masses are shown. The bars above HKs and RRs are regions cloned for biochemical analysis. The percentages in the parentheses between the two aligned proteins indicate amino acid identity. HTH, helix-turn-helix.
FIG. 2.
FIG. 2.
mRNA expression of six genes by E. chaffeensis in human peripheral blood monocytes. Total RNA was prepared from E. chaffeensis-infected human blood monocytes, and RT-PCR was performed using specific primers. D, positive control (chromosomal DNA was used as a template); + and − indicate the presence and absence of reverse transcriptase, respectively. Genes and base pair sizes of amplified products are indicated.
FIG. 3.
FIG. 3.
Intracellular localization of HKs and RRs in E. chaffeensis. Shown are results of Western blot analysis with rabbit antibodies to PleC, NtrY, CckA, PleD, NtrX, CtrA, and E. chaffeensis membrane protein control P28. Ten micrograms (anti-PleC, -PleD, -NtrX, and -CtrA) or 20 μg (anti-NtrY and -CckA) each of E. chaffeensis (Ec) lysate and soluble and membrane fractions derived from the same number of E. chaffeensis cells as the lysate and 10 μg THP-1 cell lysate were loaded on a 12% (anti-PleC, -PleD, -NtrX, and -CtrA) or 8% (anti-NtrY and -CckA) SDS-polyacrylamide gel. Numbers on the left are molecular masses in kilodaltons.
FIG. 4.
FIG. 4.
His kinase and phosphotransfer activities. (A) Purity of soluble recombinant HKDs and RRs used for biochemical assays. Purified protein samples were loaded onto a 12% SDS-polyacrylamide gel, and the gel was stained with Coomassie brilliant blue. The asterisk indicates a CtrA degradation product. Numbers on the bottom are calculated molecular masses in kilodaltons. (B) Autoradiogram showing the specific histidine residue-dependent autokinase activity of recombinant HKDs. rPleCHKD, rPleCHKDH244A, rNtrYHKD, and rNtrYHKDH498A (2 μg each) and MBP-rCckAHKD and MBP-rCckAHKDH449A (10 μg each) were incubated with [γ-32P]ATP. Only wild-type (wt) HKDs were 32P phosphorylated. (C) Autoradiogram showing phosphotransfer of 32P from rPleCHKD, rNtrYHKD, and MBP-rCckAHKD (arrowheads) to recombinant RRs (arrows). rHKDs (10 μg each) were incubated with [γ-32P]ATP, followed by incubation with wild-type (rPleD, rNtrX, or rCtrA) and mutant (rPleDD53A, rNtrXD59A, or rCtrAD53A) RRs (10 μg each). rCtrA + Ca2+: rCtrA (30 μg) was incubated with 32P-phosphorylated MBP-rCckAHKD in the presence of CaCl2 and the phosphatase inhibitor cocktail. Numbers on the left of each panel are molecular masses in kilodaltons.
FIG. 5.
FIG. 5.
Closantel treatment induced lysosomal fusion with E. chaffeensis inclusions. E. chaffeensis-infected THP-1 cells were treated with 100 μM closantel or DMSO (as a control) for 1 h. (A) Double immunofluorescence staining with dog anti-E. chaffeensis and mouse anti-LAMP-1 antibodies followed by Alexa Fluor 555-conjugated goat anti-mouse and fluorescein isothiocyanate-conjugated goat anti-dog secondary antibodies. Asterisks and arrows indicate noncolocalization and colocalization, respectively. Bar, 5 μm. (B) E. chaffeensis inclusions colocalized and noncolocalized with LAMP-1 were counted. The bars represent mean percentages of 100 morulae in triplicate cultures.
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"V体育官网入口" References

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