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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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Effect of the production of ClpB from E. coli and E. chaffeensis on the growth and survival of E. coli under heat shock.(A) Autoradiography of the E. coli cell lysates obtained from the clpB- strain transformed with pGB2, or with pGB2-EhClpB, or with pGB2-EcClpB, grown at 30°C or 45°C (heat shock conditions, HS). (B) Growth curves of the clpB- strain transformed with pGB2, pGB2-EhClpB, or pGB2-EcClpB at 45°C. (C) Survival of the clpB- strain transformed with pGB2, pGB2-EhClpB, or pGB2-EcClpB, 50°C. The average values from two experiments are shown in (B) and (C).
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pone-0062454-g007: Effect of the production of ClpB from E. coli and E. chaffeensis on the growth and survival of E. coli under heat shock.(A) Autoradiography of the E. coli cell lysates obtained from the clpB- strain transformed with pGB2, or with pGB2-EhClpB, or with pGB2-EcClpB, grown at 30°C or 45°C (heat shock conditions, HS). (B) Growth curves of the clpB- strain transformed with pGB2, pGB2-EhClpB, or pGB2-EcClpB at 45°C. (C) Survival of the clpB- strain transformed with pGB2, pGB2-EhClpB, or pGB2-EcClpB, 50°C. The average values from two experiments are shown in (B) and (C).

Mentions: In E. coli, ClpB is dispensable for growth under normal conditions, but becomes essential for survival during heat-shock [26]. We tested if EcClpB can be functionally substituted in E. coli with EhClpB. We subcloned the EhClpB DNA sequence into a low-copy pGB2 plasmid and placed it under a control of the native EcClpB heat-shock promoter. Autoradiography of the E. coli lysates in Fig. 7A showed that the heat-shock conditions (45°C) induced a prominent expression of a discrete set of proteins, which likely represented the major heat-shock protein families. The Hsp100 signal was absent in the clpB- strain transformed with pGB2, but was present in that strain transformed with pGB2 containing either the EhClpB or EcClpB coding sequence. As reported earlier [26], the lack of a functional ClpB decreased the growth rate of E. coli at 45°C (Fig. 7B) and inhibited survival at 50°C (Fig. 7C). Interestingly, the heat-shock-inducible production of EhClpB (Fig. 7A) did not rescue the temperature-sensitive phenotypes in the clpB- E. coli (Fig. 7B, C).


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)

Effect of the production of ClpB from E. coli and E. chaffeensis on the growth and survival of E. coli under heat shock.(A) Autoradiography of the E. coli cell lysates obtained from the clpB- strain transformed with pGB2, or with pGB2-EhClpB, or with pGB2-EcClpB, grown at 30°C or 45°C (heat shock conditions, HS). (B) Growth curves of the clpB- strain transformed with pGB2, pGB2-EhClpB, or pGB2-EcClpB at 45°C. (C) Survival of the clpB- strain transformed with pGB2, pGB2-EhClpB, or pGB2-EcClpB, 50°C. The average values from two experiments are shown in (B) and (C).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646808&req=5

pone-0062454-g007: Effect of the production of ClpB from E. coli and E. chaffeensis on the growth and survival of E. coli under heat shock.(A) Autoradiography of the E. coli cell lysates obtained from the clpB- strain transformed with pGB2, or with pGB2-EhClpB, or with pGB2-EcClpB, grown at 30°C or 45°C (heat shock conditions, HS). (B) Growth curves of the clpB- strain transformed with pGB2, pGB2-EhClpB, or pGB2-EcClpB at 45°C. (C) Survival of the clpB- strain transformed with pGB2, pGB2-EhClpB, or pGB2-EcClpB, 50°C. The average values from two experiments are shown in (B) and (C).
Mentions: In E. coli, ClpB is dispensable for growth under normal conditions, but becomes essential for survival during heat-shock [26]. We tested if EcClpB can be functionally substituted in E. coli with EhClpB. We subcloned the EhClpB DNA sequence into a low-copy pGB2 plasmid and placed it under a control of the native EcClpB heat-shock promoter. Autoradiography of the E. coli lysates in Fig. 7A showed that the heat-shock conditions (45°C) induced a prominent expression of a discrete set of proteins, which likely represented the major heat-shock protein families. The Hsp100 signal was absent in the clpB- strain transformed with pGB2, but was present in that strain transformed with pGB2 containing either the EhClpB or EcClpB coding sequence. As reported earlier [26], the lack of a functional ClpB decreased the growth rate of E. coli at 45°C (Fig. 7B) and inhibited survival at 50°C (Fig. 7C). Interestingly, the heat-shock-inducible production of EhClpB (Fig. 7A) did not rescue the temperature-sensitive phenotypes in the clpB- E. coli (Fig. 7B, C).

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