Leptospira ClpP mutant variants in association with the ClpX, acyldepsipeptide, and the trigger factor displays unprecedented gain-of-function.

The functionally active ClpP (LinClpP) of Leptospira interrogans is composed of two different isoforms (LinClpP1 and LinClpP2). In this study, five mutants of LinClpP (LinClpP1E170D, LinClpP1N172D, LinClpP2IG_del, LinClpP2S40AK41N, LinClpP2Y62A) targeting its critical hotspot residues were generated. The functional activity of pure LinClpP mutant variants or its heterocomplex and its effect when associated with a chaperone (LinClpX)/antibiotic acyldepsipeptide (ADEP1)/trigger factor (LinTF) was examined. The two mutants (LinClpP2S40AK41N and LinClpP2Y62A) displayed gain-of-function (GOF) in peptidase activity. The ADEP1-bound heterocomplex (LinClpP1P2S40AK41N and LinClpP1P2Y62A) measured 1.7 and 1.5-fold higher protease activity than ADEP-bound LinClpP1P2. The dynamic light scattering analysis of ADEP1-bound GOF mutants displayed increased hydrodynamic diameter. In the presence of LinTF, the heterocomplex (LinClpP1P2S40AK41N and LinClpP1P2Y62A) exhibited a 3-fold surge in peptidase activity. The deletion mutant (LinClpP2IG_del) or its heterocomplex (LinClpP1P2IG_del) displayed no activity. Similarly, the pure LinClpP1E170D and LinClpP1N172D could not cleave a model dipeptide. However, its heterocomplex (LinClpP1E170DP2 and LinClpP1N172DP2) showed 0.5-fold lower peptidase activity than the LinClpP1P2. Collectively, two mutants (LinClpP2S40AK41N and LinClpP2Y62A) have GOF and can degrade model dipeptide substrate without the aid of LinClpP1 isoform and thus provide new insights into unprecedented LinClpP activation.

Acyldepsipeptide activated ClpP1P2 macromolecule of Leptospira, an ideal Achilles' heel to hamper the cell survival and deregulate ClpP proteolytic activity.

Antibiotic acyldepsipeptide (ADEP) targets the bacterial ClpP serine protease and can inhibit the growth of numerous bacterial species by activating/dysregulating the protease activity within the cell. The spirochete Leptospira interrogans harbors two ClpP isoforms (LepClpP1 and LepClpP2). Supplementation of ADEP in the Leptospira growth medium resulted in the inhibition of bacterial growth. The ADEP mediated activation of the LepClpP mixture was dependent on the time allowed for the self-assembly of LepClpP1 and LepClpP2. The dynamic light scattering of the LepClpP mixture in the presence of the ADEP indicated a conformational transformation of the LepClpP machinery. Serine 98, a catalytic triad residue of the LepClpP1 in the LepClpP1P2 heterocomplex, was critical for the ADEP mediated activation. The computational prototype of the LepClpP1P2 structure suggested that the hydrophobic pockets wherein the ADEPs or the physiological chaperone ClpX predominantly dock are exclusively present in the LepClpP2 heptamer. Using the ADEP as a tool, this investigation provides an insight into the molecular function of the LepClpP1P2 in a coalition with its ATPase chaperone LepClpX. The shreds of the evidence illustrated in this investigation verify that ADEP1 possesses the ability to control the LepClpP system in an unconventional approach than the other organisms.

Trigger Factor in Association with the ClpP1P2 Heterocomplex of Leptospira Promotes Protease/Peptidase Activity.

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 ribosome-associated 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 C-terminal domain of LinTF may have a role in the assembly of multimeric ClpP protein, leading to enhancement of ClpP activity.

Role of Supramolecule ErpY-Like Lipoprotein of Leptospira in Thrombin-Catalyzed Fibrin Clot Inhibition and Binding to Complement Factors H and I, and Its Diagnostic Potential.

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.

Insights to the Assembly of a Functionally Active Leptospiral ClpP1P2 Protease Complex along with Its ATPase Chaperone ClpX.

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.

Production enhancement and characterization of the polyhydroxyalkanoate produced by Natrinema ajinwuensis (as synonym) ≡ Natrinema altunense strain RM-G10.

Application of halophiles can decrease the cost of polyhydroxyalkanoate (PHA) production or bioplastic which are an alternative to the petroleum-derived plastic. Extremely halophilic archaeon, Natrinema ajinwuensis RM-G10 accumulated 61.02±0.68% PHA of its cell dry mass at 72h in repeated batch cultures yielding 0.210±0.001gL-1h-1 volumetric productivity after selection of the best cultivation conditions. Transmission electron microscopy showed the presence of PHA granules inside the archaeal cells. Characterization by gas chromatographic analysis, gas chromatographic- mass spectrophotometric analysis, thermogravimetric analysis, differential scanning calorimetric analysis, X-ray diffraction analysis, Fourier transform infra red spectroscopy and nuclear magnetic resonance spectroscopy revealed the polymer to be poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with 13.93mol% 3-hydroxyvalerate content and having 35.45% crystallinity, -12.3°C glass transition temperature, 143°C and 157.5°C melting temperatures and 284°C degradation temperature. This is the first report on production enhancement (on a small scale) and characterization of the polyhydroxyalkanoate produced by Natrinema ajinwuensis (as synonym) ≡ Natrinema altunense strain RM-G10 and the Natrinema genus in general.