Leptospira interrogans genome is predicted to encode multiple isoforms of caseinolytic proteases (ClpP1 and ClpP2). The ClpP proteins with the aid of its ATPase chaperone are known to be involved in establishing cellular proteostasis and have emerged as a target for developing new antibiotics. We report the molecular characterization of recombinant ClpP1 (rClpP1) and rClpP2 of Leptospira along with its ATPase chaperone rClpX. The two isoforms of rClpPs when coupled together in an equivalent concentration exhibit optimum activity on small fluorogenic peptide substrates, whereas the pure rClpP isoforms are enzymatically inactive. Isothermal titration calorimetry analysis suggests that the two rClpP isoforms bind each other moderately in a 1:1 stoichiometry with a dissociation constant of 2.02 ± 0.1 μM at 37 °C and is thermodynamically favored. Size exclusion chromatography fractionates the majority of pure rClpP1 at ≥308 kDa (14–21-mer) and the pure rClpP2 at 308 kDa (tetradecamer), whereas the functionally active rClpP isoform mixture fractionates as a tetradecamer. The distinct and unprecedented oligomeric form of rClpP1 was also evident through native-gel and dynamic light scattering. Moreover, the rClpP isoform mixture formed after the site-directed mutation of either or both the isoforms at one of the catalytic triad residues (Ser 98/97 to Ala 98/97) resulted in the complete loss of protease activity. The rClpP isoform mixture gets stimulated to degrade the casein substrate in the presence of rClpX and in an energy-dependent manner. On the contrary, pure rClpP1 or the rClpP2 isoform in association with rClpX are incapable of forming operative protease. The reported finding suggests that in Leptospira , the enzymatic activity of the rClpP protease complex in the presence or absence of cochaperone is performed solely by the tetradecamer structure which is hypothesized to be composed of 2-stacked ClpP heptameric rings, wherein each ring is a homo-oligomer of ClpP1 and ClpP2 subunits. Understanding the activities and regulation principle of multi-isoforms of ClpP in pathogenic bacteria may aid in intervening disease outcomes particularly to the co-evolving antibiotic resistance strains.

Leptospirosis is one of the most widespread zoonoses caused by pathogenic Leptospira spp. In this study, we report that the LIC11966/ErpY-like lipoprotein is a surface-exposed outer membrane protein exclusively present in pathogenic species of Leptospira. The recombinant ErpY (rErpY)-like protein is recognized by the immunoglobulins of confirmed leptospirosis sera of diverse hosts (human, bovine, and canine), suggesting the expression of the native leptospiral surface protein during infection. Circular dichroism of pure rErpY-like protein showed the secondary structural integrity to be uncompromised during the purification process. Analysis of the rErpY-like protein by native polyacrylamide gel electrophoresis, chemical cross-linking, dynamic light scattering, and field emission transmission electron microscopy demonstrated it undergoes supramolecular assembly. The rErpY-like protein can bind to diverse host extracellular matrices, and it presented a saturable and strong binding affinity (dissociation constant [KD] of 70.45 ± 4.13 nM) to fibrinogen, a central host plasma component involved in blood clotting. In the presence of the rErpY-like supramolecule, thrombin-catalyzed fibrin clot formation is inhibited up to 7%, implying its role in inhibiting blood coagulation during Leptospira infection. In addition, binding of the rErpY-like supramolecule to complement factors H and I suggests the protein also contributes to Leptospira evading innate host defense during infection by inactivating alternative complement pathways. This study reveals that rErpY-like protein is functionally active in the supramolecular state and performs moonlighting activity under the given in vitro conditions.

No abstract

The caseinolytic protease (ClpP) complex in Leptospira interrogans is unusual in its functional activation. The genus Leptospira has two ClpPs, ClpP1 and ClpP2, which transcribes independently, regardless it couples to form the active tetradecamer. Acyldepsipeptide (ADEP) antibiotic hampers the growth of numerous bacterial species by activating the target protein ClpP and dysregulating the physiological proteostasis within the cell. In vitro culture of the L. interrogans fortified with the ADEP impeded the spirochete growth accompanied by a more elongated morphology. The chemoactivation of the ClpP is conditional on the duration of the self-compartmentalization of each of the ClpP isoforms. The small extent (10 min) self-assembled ClpP1P2 revealed inhibition in the peptidase activity (7-fold) in the presence of the ADEP due to the self-cleavage of the ClpP subunits. On supplementation of the β-casein or bovine serum albumin, the peptidase activity of the ClpP1P2 (short-incubated) got enhanced by the ADEP, while the ClpP1P2 (long-incubated) activity was retained to the same level. ADEP can also switch on the ClpP1P2 from a strict peptidase into proteolytic machinery that discerns and degrades the unfolded protein substrates autonomous of the cognate chaperone ClpX. In consensus to the most prokaryotes with the multi ClpP variants, the computational prototype of the ClpP1P2 tertiary structure infers that the hydrophobic pocket wherein the ADEPs predominantly docks are present in the ClpP2 heptamer. Additionally, the dynamic light scattering and the site-directed mutagenesis of a catalytic serine residue in either of the ClpP isoforms proposes a second interaction site for the ADEP.

The genomic analysis of Leptospira reveals a trigger factor (TF) encoding gene (tig) to be colocalized along with the clpP1 and clpX. The TF is a crouching dragon-like protein known to be a ribosomeassociated chaperone that is involved in cotranslational protein folding in bacteria in an ATP-independent mode. In Leptospira, tig is localized upstream of the clpP1 with a short (4 bp) overlap. In the present study, we document the distinctive role of Leptospira TF (LinTF) in the caseinolytic protease (ClpP) system. The recombinant LinTF (rLinTF) was found to improve the peptidase or protease activity of the ClpP1P2 heterocomplex and ClpXP1P2 complex, respectively, on model substrates. In addition, on supplementation of rLinTF to rClpP1P2 bound to its physiological ATPase chaperone ClpX or the antibiotic analogue acyldepsipeptide (ADEP), an augmentation in the activity of ClpP1P2 was observed. These studies underscore the novel role of LinTF in aiding the caseinolytic protease activity of Leptospira. Supplementation of rLinTF to a peptidase assay of rClpP1P2 conditionally in the presence of a salt (sodium citrate) with high Hofmeister strength led us to speculate that rLinTF may have a role in the assembly of multimeric proteins. The deletion of one of the arms (arm-2) of the LinTF structure from the carboxy terminal domain indicated a reduction in its capacity to stimulate rClpP1P2 activity. Thus, the Cterminal domain of LinTF may have a role in the assembly of multimeric ClpP protein, leading to enhancement of ClpP activity.