AAA+ Molecular Chaperone ClpB in Leptospira interrogans: Its Role and Significance in Leptospiral Virulence and Pathogenesis of Leptospirosis

Bacterial ClpB is an ATP-dependent disaggregase that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and cooperates with the DnaK chaperone system in the reactivation of aggregated proteins, as well as promotes bacterial survival under adverse environmental conditions, including thermal and oxidative stresses. In addition, extensive evidence indicates that ClpB supports the virulence of numerous bacteria, including pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in animals and humans. However, the specific function of ClpB in leptospiral virulence still remains to be fully elucidated. Interestingly, ClpB was predicted as one of the L. interrogans hub proteins interacting with human proteins, and pathogen–host protein interactions are fundamental for successful invasion of the host immune system by bacteria. The aim of this review is to discuss the most important aspects of ClpB’s function in L. interrogans, including contribution of ClpB to leptospiral virulence and pathogenesis of leptospirosis, a zoonotic disease with a significant impact on public health worldwide

Immunoreactivity of a Putative ECF σ Factor, LIC_10559, from <i>Leptospira interrogans</i> with Sera from <i>Leptospira</i>-Infected Animals

L. interrogans belongs to highly invasive spirochaetes causing leptospirosis in mammals, including humans. During infection, this pathogen is exposed to various stressors, and therefore, it must reprogram its gene expression to survive in the host and establish infection in a short duration of time. Host adaptation is possible thanks to molecular responses where appropriate regulators and signal transduction systems participate. Among the bacterial regulators, there are σ factors, including ECF (extracytoplasmic function) σ factors. The L. interrogans genome encodes 11 putative ECF σE-type factors. Currently, none of them has been characterized biochemically, and their functions are still unknown. One of them, LIC_10559, is the most likely to be active during infection because it is only found in the highly pathogenic Leptospira. The aim of this study was to achieve LIC_10559 overexpression to answer the question whether it may be a target of the humoral immune response during leptospiral infections. The immunoreactivity of the recombinant LIC_10559 was evaluated by SDS-PAGE, ECL Western blotting and ELISA assay using sera collected from Leptospira-infected animals and uninfected healthy controls. We found that LIC_10559 was recognized by IgG antibodies from the sera of infected animals and is, therefore, able to induce the host’s immune response to pathogenic Leptospira. This result suggests the involvement of LIC_10559 in the pathogenesis of leptospirosis

Identification of σE-Dependent Promoter Upstream of clpB from the Pathogenic Spirochaete Leptospira interrogans by Applying an E. coli Two-Plasmid System

There is limited information on gene expression in the pathogenic spirochaete Leptospira interrogans and genetic mechanisms controlling its virulence. Transcription is the first step in gene expression that is often determined by environmental effects, including infection-induced stresses. Alterations in the environment result in significant changes in the transcription of many genes, allowing effective adaptation of Leptospira to mammalian hosts. Thus, promoter and transcriptional start site identification are crucial for determining gene expression regulation and for the understanding of genetic regulatory mechanisms existing in Leptospira. Here, we characterized the promoter region of the L. interrogans clpB gene (clpBLi) encoding an AAA+ molecular chaperone ClpB essential for the survival of this spirochaete under thermal and oxidative stresses, and also during infection of the host. Primer extension analysis demonstrated that transcription of clpB in L. interrogans initiates at a cytidine located 41 bp upstream of the ATG initiation codon, and, to a lesser extent, at an adenine located 2 bp downstream of the identified site. Transcription of both transcripts was heat-inducible. Determination of clpBLi transcription start site, combined with promoter transcriptional activity assays using a modified two-plasmid system in E. coli, revealed that clpBLi transcription is controlled by the ECF &sigma;E factor. Of the ten L. interrogans ECF &sigma; factors, the factor encoded by LIC_12757 (LA0876) is most likely to be the key regulator of clpB gene expression in Leptospira cells, especially under thermal stress. Furthermore, clpB expression may be mediated by ppGpp in Leptospira

Isolation and Identification of Putative Protein Substrates of the AAA+ Molecular Chaperone ClpB from the Pathogenic Spirochaete Leptospira interrogans

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&ndash;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

Characterization of the molecular chaperone ClpB from the pathogenic spirochaete Leptospira interrogans.

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

Summary of sedimentation coefficients for ClpB_Li.

<p>Summary of sedimentation coefficients for ClpB_Li.</p

Effect of the <i>clpB</i>_Li gene expression on the growth and survival of <i>E</i>. <i>coli</i> <i>Δ</i><i>clpB</i> mutant under heat shock.

<p>(A) Immunodetection of ClpB_Li with specific antibodies in <i>E</i>. <i>coliΔclpB</i> cells grown at 30°C and after 2h of heat shock at 45°C. An asterisk indicates ClpB_Li. The position of ClpB_Ec (control of heat-inducible expression) was marked by a circle. (B) Growth curves of <i>E</i>. <i>coliΔclpB</i> cells carrying empty pGB2 (control 1), pGB2-ClpB_Ec (control 2) or pGB2-ClpB_Li exposed to a mild heat shock at 45°C for the indicated times. (C) Survival of the same bacterial strains as in (B) after exposure to a severe heat shock at 50°C for the indicated times. The average values from three independent experiments are shown in (B) and (C).</p

Structural characteristics of ClpB_Li used in this study.

<p>(A) Comparison of the domain organization of ClpB from <i>L</i>. <i>interrogans</i> and <i>E</i>. <i>coli</i>. Bacterial ClpB proteins are composed of the following domains: N-terminal domain (ND), nucleotide binding domain 1 (NBD1), middle coiled-coil domain (MD), and nucleotide binding domain 2 (NBD2). The functions of the domains are indicated at the top. The amino acid residue numbers are shown for each chaperone and the amino acid sequence identity between ClpB_Ec and ClpB_Li is indicated for each domain. (B) CD spectra of ClpB_Li at 20°C (folded form) and 75°C (unfolded form) are shown. The CD signal was expressed as mean molar residue ellipticity (θ). (C) Temperature-induced changes in the CD signal at 222 nm for ClpB_Li.</p