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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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Reactivation of aggregated glucose-6-phosphate dehydrogenase in the presence of ClpB from E. chaffeensis and E. coli.(A) A representative time-course of the reactivation of aggregated G6PDH in the presence of DnaK/DnaJ/GrpE from E. coli without ClpB and with EcClpB or EhClpB. (B) A representative time-course of the reactivation of aggregated G6PDH in the absence of chaperones (control) and in the presence of EcClpB or EhClpB. (C) Initial rates of G6PDH reactivation (from the linear slopes of the data in (A) and (B)). The average values from three independent experiments are shown with the standard deviations. T-test scores: **, p<0.01; ***, p<0.001.
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pone-0062454-g004: Reactivation of aggregated glucose-6-phosphate dehydrogenase in the presence of ClpB from E. chaffeensis and E. coli.(A) A representative time-course of the reactivation of aggregated G6PDH in the presence of DnaK/DnaJ/GrpE from E. coli without ClpB and with EcClpB or EhClpB. (B) A representative time-course of the reactivation of aggregated G6PDH in the absence of chaperones (control) and in the presence of EcClpB or EhClpB. (C) Initial rates of G6PDH reactivation (from the linear slopes of the data in (A) and (B)). The average values from three independent experiments are shown with the standard deviations. T-test scores: **, p<0.01; ***, p<0.001.

Mentions: We tested the reactivation of two previously investigated in vitro substrates of Hsp100 chaperones: large aggregates produced from chemically denatured glucose-6-phosphate dehydrogenase (G6PDH) [37], [48] and thermally aggregated firefly luciferase [46]. As shown in Fig. 4A, EhClpB reactivated aggregated G6PDH in the presence of the E. coli co-chaperones DnaK/DnaJ/GrpE (KJE). The apparent rate of the G6PDH disaggregation was ∼5-fold higher for EhClpB than for EcClpB (see Fig. 4A, C). Unexpectedly, we found that EhClpB reactivated aggregated G6PDH even in the absence of KJE (Fig. 4B) with the reactivation rate close to that found for EcClpB in the presence of the co-chaperones (Fig. 4C).


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)

Reactivation of aggregated glucose-6-phosphate dehydrogenase in the presence of ClpB from E. chaffeensis and E. coli.(A) A representative time-course of the reactivation of aggregated G6PDH in the presence of DnaK/DnaJ/GrpE from E. coli without ClpB and with EcClpB or EhClpB. (B) A representative time-course of the reactivation of aggregated G6PDH in the absence of chaperones (control) and in the presence of EcClpB or EhClpB. (C) Initial rates of G6PDH reactivation (from the linear slopes of the data in (A) and (B)). The average values from three independent experiments are shown with the standard deviations. T-test scores: **, p<0.01; ***, p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0062454-g004: Reactivation of aggregated glucose-6-phosphate dehydrogenase in the presence of ClpB from E. chaffeensis and E. coli.(A) A representative time-course of the reactivation of aggregated G6PDH in the presence of DnaK/DnaJ/GrpE from E. coli without ClpB and with EcClpB or EhClpB. (B) A representative time-course of the reactivation of aggregated G6PDH in the absence of chaperones (control) and in the presence of EcClpB or EhClpB. (C) Initial rates of G6PDH reactivation (from the linear slopes of the data in (A) and (B)). The average values from three independent experiments are shown with the standard deviations. T-test scores: **, p<0.01; ***, p<0.001.
Mentions: We tested the reactivation of two previously investigated in vitro substrates of Hsp100 chaperones: large aggregates produced from chemically denatured glucose-6-phosphate dehydrogenase (G6PDH) [37], [48] and thermally aggregated firefly luciferase [46]. As shown in Fig. 4A, EhClpB reactivated aggregated G6PDH in the presence of the E. coli co-chaperones DnaK/DnaJ/GrpE (KJE). The apparent rate of the G6PDH disaggregation was ∼5-fold higher for EhClpB than for EcClpB (see Fig. 4A, C). Unexpectedly, we found that EhClpB reactivated aggregated G6PDH even in the absence of KJE (Fig. 4B) with the reactivation rate close to that found for EcClpB in the presence of the co-chaperones (Fig. 4C).

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