Abstract
Bacterial ClpB is an ATP-dependent Hsp100 chaperone that reactivates aggregated proteins in cooperation with the DnaK chaperone system and promotes survival of bacteria under stress conditions. A large number of publications also indicate that ClpB supports the virulence of bacteria, including a pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in both animals and humans. However, the exact role of ClpB in bacterial pathogenicity remains poorly characterized. It can be assumed that ClpB, due to its role as the molecular chaperone, mediates refolding of essential bacterial proteins, including the known virulence factors, which may become prone to aggregation under infection-induced stresses. In this study, we identified putative substrates of ClpB from L. interrogans (ClpBLi). For this purpose, we used a proteomic approach combining the ClpB-Trap affinity pull-down assays, Liquid chromatography-tandem mass spectrometry (LC-MS-MS/MS), and bioinformatics analyses. Most of the identified proteins were enzymes predominantly associated with major metabolic pathways like the tricarboxylic acid (TCA) cycle, glycolysis–gluconeogenesis and amino acid and fatty acid metabolism. Based on our proteomic study, we suggest that ClpB can support the virulence of L. interrogans by protecting the conformational integrity and catalytic activity of multiple metabolic enzymes, thus maintaining energy homeostasis in pathogen cells.
Highlights
Bacterial ClpB is a molecular chaperone belonging to the Hsp100 subfamily of the AAA+ ATPases that cooperates with the DnaK chaperone system in solubilization and reactivation of aggregated proteins [1,2,3,4,5,6,7]
We have demonstrated that the recombinant ClpB from L. interrogans (ClpBLi) displays the aggregate-reactivation activity that may support the survival of L. interrogans under host-induced stress, which is likely to cause denaturation and aggregation of pathogen proteins [35]
To reveal the underlying mechanism by which the ClpB chaperone may influence virulence traits in L. interrogans, we have attempted for the first time to identify the Leptospira proteins that can be recognized and potentially reactivated by ClpBLi in cells under environmental stress, including changes in temperature
Summary
Bacterial ClpB is a molecular chaperone belonging to the Hsp100 subfamily of the AAA+ ATPases that cooperates with the DnaK chaperone system in solubilization and reactivation of aggregated proteins [1,2,3,4,5,6,7]. ClpB plays a crucial role in the survival of bacteria under stressful conditions [7,13], and in supporting the virulence of some bacterial pathogens, including a pathogenic spirochete Leptospira interrogans [14,15,16,17,18] responsible for leptospirosis affecting animals and humans worldwide. Several virulence factors have been described in Leptospira, including the ompA-like surface lipoprotein, Loa22 [25], proteins involved in spirochete motility: FliY [26], FlaA2 [27] and LB139 [28], a heme oxygenase, HemO, which is essential for heme-iron utilization [29]; a catalase, KatE, required for resistance to extracellular oxidative stress [30]; phospholipase C, associated with Leptospira-induced macrophage death [31] and HtpG, the highly conserved molecular chaperone from the Hsp family [32]. The remaining ClpB-interacting proteins were associated with other essential cellular processes like transcription, protein synthesis, cell wall and membrane biogenesis, spirochete motility, and chemotaxis
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