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Aggregate-reactivation activity of the molecular chaperone ClpB from Ehrlichia chaffeensis.

Zhang T, Kedzierska-Mieszkowska S, Liu H, Cheng C, Ganta RR, Zolkiewski M - PLoS ONE (2013)

Bottom Line: Unlike EcClpB, which requires the co-chaperones for aggregate reactivation, EhClpB reactivates G6PDH even in the absence of KJE.Moreover, EhClpB is functionally distinct from EcClpB as evidenced by its failure to rescue a temperature-sensitive phenotype of the clpB- E. coli.This study sets the stage for assessing the importance of the chaperone activity of ClpB for E. chaffeensis growth within the mammalian and tick hosts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, United States of America.

ABSTRACT
Rickettsiale diseases, including human monocytic ehrlichiosis caused by Ehrlichia chaffeensis, are the second leading cause of the tick-borne infections in the USA and a growing health concern. Little is known about how E. chaffeensis survives the host-induced stress in vertebrate and tick hosts. A molecular chaperone ClpB from several microorganisms has been reported to reactivate aggregated proteins in cooperation with the co-chaperones DnaK/DnaJ/GrpE (KJE). In this study, we performed the first biochemical characterization of ClpB from E. chaffeensis. The transcript of E. chaffeensis ClpB (EhClpB) is strongly upregulated after infection of cultured macrophages and its level remains high during the Ehrlichia replicative stage. EhClpB forms ATP-dependent oligomers and catalyzes the ATP hydrolysis, similar to E. coli ClpB (EcClpB), but its ATPase activity is insensitive to the EcClpB activators, casein and poly-lysine. EhClpB in the presence of E. coli KJE efficiently reactivates the aggregated glucose-6-phosphate dehydrogenase (G6PDH) and firefly luciferase. Unlike EcClpB, which requires the co-chaperones for aggregate reactivation, EhClpB reactivates G6PDH even in the absence of KJE. Moreover, EhClpB is functionally distinct from EcClpB as evidenced by its failure to rescue a temperature-sensitive phenotype of the clpB- E. coli. The clpB expression pattern during the E. chaffeensis infection progression correlates with the pathogen's replicating stage inside host cells and suggests an essential role of the disaggregase activity of ClpB in the pathogen's response to the host-induced stress. This study sets the stage for assessing the importance of the chaperone activity of ClpB for E. chaffeensis growth within the mammalian and tick hosts.

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Sedimentation velocity of ClpB from E. chaffeensis.Shown are the apparent distributions of the sedimentation coefficient for 0.3 mg/ml EhClpB in the absence of nucleotides (A) and in the presence of 2 mM ATPγS (B).
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pone-0062454-g002: Sedimentation velocity of ClpB from E. chaffeensis.Shown are the apparent distributions of the sedimentation coefficient for 0.3 mg/ml EhClpB in the absence of nucleotides (A) and in the presence of 2 mM ATPγS (B).

Mentions: Since the oligomerization of Hsp100 proteins is linked to their chaperone activity [46], we tested the self-association properties of the recombinant EhClpB using sedimentation velocity. As shown in Fig. 2A, EhClpB in the absence of nucleotides sedimented as a single species with the apparent sedimentation coefficient of ∼4.5 S, which agreed with the previously determined sedimentation coefficient of the monomeric E. coli ClpB (EcClpB) [46]. The addition of a non-hydrolyzable ATP analog, ATPγS induced self-association of EhClpB into ∼14.7-S particles (see Fig. 2B), which approximates the sedimentation coefficient of the hexameric EcClpB [47]. We next investigated the ATPase activity of EhClpB. As shown in Fig. 3, the basal ATPase of EhClpB was similar to that of the purified EcClpB. However, while the ATPase of EcClpB was efficiently activated in the presence of casein or poly-lysine, the ATPase of EhClpB did not respond to the putative activators.


Aggregate-reactivation activity of the molecular chaperone ClpB from Ehrlichia chaffeensis.

Zhang T, Kedzierska-Mieszkowska S, Liu H, Cheng C, Ganta RR, Zolkiewski M - PLoS ONE (2013)

Sedimentation velocity of ClpB from E. chaffeensis.Shown are the apparent distributions of the sedimentation coefficient for 0.3 mg/ml EhClpB in the absence of nucleotides (A) and in the presence of 2 mM ATPγS (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646808&req=5

pone-0062454-g002: Sedimentation velocity of ClpB from E. chaffeensis.Shown are the apparent distributions of the sedimentation coefficient for 0.3 mg/ml EhClpB in the absence of nucleotides (A) and in the presence of 2 mM ATPγS (B).
Mentions: Since the oligomerization of Hsp100 proteins is linked to their chaperone activity [46], we tested the self-association properties of the recombinant EhClpB using sedimentation velocity. As shown in Fig. 2A, EhClpB in the absence of nucleotides sedimented as a single species with the apparent sedimentation coefficient of ∼4.5 S, which agreed with the previously determined sedimentation coefficient of the monomeric E. coli ClpB (EcClpB) [46]. The addition of a non-hydrolyzable ATP analog, ATPγS induced self-association of EhClpB into ∼14.7-S particles (see Fig. 2B), which approximates the sedimentation coefficient of the hexameric EcClpB [47]. We next investigated the ATPase activity of EhClpB. As shown in Fig. 3, the basal ATPase of EhClpB was similar to that of the purified EcClpB. However, while the ATPase of EcClpB was efficiently activated in the presence of casein or poly-lysine, the ATPase of EhClpB did not respond to the putative activators.

Bottom Line: Unlike EcClpB, which requires the co-chaperones for aggregate reactivation, EhClpB reactivates G6PDH even in the absence of KJE.Moreover, EhClpB is functionally distinct from EcClpB as evidenced by its failure to rescue a temperature-sensitive phenotype of the clpB- E. coli.This study sets the stage for assessing the importance of the chaperone activity of ClpB for E. chaffeensis growth within the mammalian and tick hosts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, United States of America.

ABSTRACT
Rickettsiale diseases, including human monocytic ehrlichiosis caused by Ehrlichia chaffeensis, are the second leading cause of the tick-borne infections in the USA and a growing health concern. Little is known about how E. chaffeensis survives the host-induced stress in vertebrate and tick hosts. A molecular chaperone ClpB from several microorganisms has been reported to reactivate aggregated proteins in cooperation with the co-chaperones DnaK/DnaJ/GrpE (KJE). In this study, we performed the first biochemical characterization of ClpB from E. chaffeensis. The transcript of E. chaffeensis ClpB (EhClpB) is strongly upregulated after infection of cultured macrophages and its level remains high during the Ehrlichia replicative stage. EhClpB forms ATP-dependent oligomers and catalyzes the ATP hydrolysis, similar to E. coli ClpB (EcClpB), but its ATPase activity is insensitive to the EcClpB activators, casein and poly-lysine. EhClpB in the presence of E. coli KJE efficiently reactivates the aggregated glucose-6-phosphate dehydrogenase (G6PDH) and firefly luciferase. Unlike EcClpB, which requires the co-chaperones for aggregate reactivation, EhClpB reactivates G6PDH even in the absence of KJE. Moreover, EhClpB is functionally distinct from EcClpB as evidenced by its failure to rescue a temperature-sensitive phenotype of the clpB- E. coli. The clpB expression pattern during the E. chaffeensis infection progression correlates with the pathogen's replicating stage inside host cells and suggests an essential role of the disaggregase activity of ClpB in the pathogen's response to the host-induced stress. This study sets the stage for assessing the importance of the chaperone activity of ClpB for E. chaffeensis growth within the mammalian and tick hosts.

Show MeSH
Related in: MedlinePlus