Research Article: Characterization of the molecular chaperone ClpB from the pathogenic spirochaete Leptospira interrogans

Date Published: July 10, 2017

Publisher: Public Library of Science

Author(s): Joanna Krajewska, Anna Modrak-Wójcik, Zbigniew J. Arent, Daniel Więckowski, Michal Zolkiewski, Agnieszka Bzowska, Sabina Kędzierska-Mieszkowska, Jeffrey L. Brodsky.


Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that an AAA+ chaperone ClpB (a member of the Hsp100 family) from L. interrogans (ClpBLi) is not only essential for survival of Leptospira under the thermal and oxidative stresses, but also during infection of a host. The aim of this study was to provide further insight into the role of ClpB in the pathogenic spirochaetes and explore its biochemical properties. We found that a non-hydrolysable ATP analogue, ATPγS, but not AMP-PNP induces the formation of ClpBLi hexamers and stabilizes the associated form of the chaperone. ADP also induces structural changes in ClpBLi and promotes its self-assembly, but does not produce full association into the hexamers. We also demonstrated that ClpBLi exhibits a weak ATPase activity that is stimulated by κ-casein and poly-lysine, and may mediate protein disaggregation independently from the DnaK chaperone system. Unexpectedly, the presence of E. coli DnaK/DnaJ/GrpE did not significantly affect the disaggregation activity of ClpBLi and ClpBLi did not substitute for the ClpBEc function in the clpB-null E. coli strain. This result underscores the species-specificity of the ClpB cooperation with the co-chaperones and is most likely due to a loss of interactions between the ClpBLi middle domain and the E. coli DnaK. We also found that ClpBLi interacts more efficiently with the aggregated G6PDH in the presence of ATPγS rather than ATP. Our results indicate that ClpB’s importance during infection might be due to its role as a molecular chaperone involved in reactivation of protein aggregates.

Partial Text

Bacterial ClpB is a molecular chaperone belonging to the Hsp100 subfamily of AAA+ ATPases (ATPases associated with a variety of cellular activities) that cooperates with the DnaK chaperone system in solubilization and reactivation of aggregated proteins [1–4]. There are a number of observations indicating that the cooperation of ClpB and DnaK in protein disaggregation is species-specific [5–8].

To date, little is known about the structure and biological role of molecular chaperones from Leptospira spp., including ClpB. It has been demonstrated that the L. interrogans ClpB is essential for the pathogen survival under stress conditions and also during infection of the host [18]. Moreover, a recently reported immunoreactivity of ClpBLi with the sera collected from Leptospira-infected animals [24] and the fact that clpBLi is up-regulated in leptospiral cells [18] suggest that the ClpB activity may be important for pathogenicity of Leptospira. In this work, we described the biochemical and structural properties of ClpB from L. interrogans.

Our studies revealed several crucial structural and biochemical properties of the molecular chaperone ClpB from Leptospira, which may support its role in pathogenicity of spirochaetes. We showed that ClpBLi forms hexameric assemblies that are stabilized and interact with protein aggregates in the ATP-bound state. Moreover, ClpBLi exhibits a protein disaggregase activity that may contribute to the survival of L. interrogans under the host-induced proteotoxic stress. Our studies make a novel contribution to the largely uncharacterized biology of Leptospira and suggest that the L. interrogans and E. coli chaperones evolved differently to respond to the different nature of stress that the proteomes of either bacteria can be exposed to.




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