Olga Kandror

Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures

Trehalose accumulates dramatically in microorganisms during heat shock and osmotic stress and helps protect cells against thermal injury and oxygen radicals. Here we demonstrate an important role of this sugar in cold-adaptation of bacteria. A mutant Escherichia coli strain unable to produce trehalose died much faster than the wild type at 4°C. Transformation of the mutant with the otsA/otsB genes, responsible for trehalose synthesis, restored trehalose content and cell viability at 4°C. After temperature downshift from 37°C to 16°C (“cold shock”), trehalose levels in wild-type cells increased up to 8-fold. Although this accumulation of trehalose did not influence growth at 16°C, it enhanced cell viability when the temperature fell further to 4°C. Before the trehalose build-up, levels of mRNA encoding OtsA/OtsB increased markedly. This induction required the σ factor, RpoS, but was independent of the major cold-shock protein, CspA. otsA/B mRNA was much more stable at 16°C than at 37°C and contained a “downstream box,” characteristic of cold-inducible mRNAs. Thus, otsA/otsB induction and trehalose synthesis are activated during cold shock (as well as during heat shock) and play an important role in resistance of E. coli (and probably other organisms) to low temperatures.

Yeast Adapt to Near-Freezing Temperatures by STRE/Msn2,4-Dependent Induction of Trehalose Synthesis and Certain Molecular Chaperones

Virtually nothing is known about the biochemical adaptations in eukaryotic cells that may enhance survival at low temperatures or upon freezing. Here we demonstrate an adaptive response in yeast that is activated below 10 degrees C and increases tolerance to low temperatures and freezing. This response involves a dramatic accumulation of the chemical chaperone trehalose and induction of trehalose-synthesizing enzymes (Tps1, Tps2) and certain heat shock proteins (Hsp104, Hsp42, Hsp12, Ssa4). mRNAs for these proteins increase dramatically below 10 degrees C and even at 0 degrees C. Their expression requires Msn2,4 transcription factors but also involves marked mRNA stabilization. Upon return to 30 degrees C, TPS1, TPS2, and HSP104 mRNAs, trehalose levels and tolerance to freezing fall dramatically within minutes. Mutants lacking trehalose or Msn2,4 die more rapidly at 0 degrees C and upon freezing. Thus, below 10 degrees C, yeast show an adaptive response that sustains viability at low or freezing temperatures, which are commonly encountered in natural environments and laboratory refrigerators.

Trigger factor is involved in GroEL-dependent protein degradation in Escherichia coli and promotes binding of GroEL to unfolded proteins.

In Escherichia coli, the molecular chaperones of hsp60/hsp10 (GroEL/GroES) families are required not only for protein folding but also for the rapid degradation of certain abnormal proteins. The rate‐limiting step in the degradation of the fusion protein CRAG by protease ClpP appears to be the formation of a complex with GroEL. We have isolated these complexes and found that each GroEL 14mer contained a short‐lived fragment of CRAG plus a 50 kDa polypeptide, which we identified by sequencing and immunological methods as Trigger Factor (TF). Upon ATP addition, GroEL and TF dissociated together from CRAG but remained tightly associated with each other even upon gel filtration. TF was originally proposed to function in protein translocation across membranes but altering cellular content of TF did not affect this process in vivo. By contrast, low levels of TF expression markedly reduced CRAG degradation, while an overproduction of TF greatly stimulated this process. Furthermore, in extracts of cells expressing high levels of TF, the capacity of GroEL to bind to CRAG is greatly increased. Overproduction of TF also stimulated GroELs ability to bind to other unfolded proteins (fetuin and histone). Thus, TF is a rate‐limiting factor for CRAG degradation; it appears to regulate GroEL function and to promote the formation of TF‐GroEL‐CRAG complexes which are critical for proteolysis.

Mycobacterium tuberculosis ClpP1 and ClpP2 Function Together in Protein Degradation and Are Required for Viability in vitro and During Infection

In most bacteria, Clp protease is a conserved, non-essential serine protease that regulates the response to various stresses. Mycobacteria, including Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis, unlike most well studied prokaryotes, encode two ClpP homologs, ClpP1 and ClpP2, in a single operon. Here we demonstrate that the two proteins form a mixed complex (ClpP1P2) in mycobacteria. Using two different approaches, promoter replacement, and a novel system of inducible protein degradation, leading to inducible expression of clpP1 and clpP2, we demonstrate that both genes are essential for growth and that a marked depletion of either one results in rapid bacterial death. ClpP1P2 protease appears important in degrading missense and prematurely terminated peptides, as partial depletion of ClpP2 reduced growth specifically in the presence of antibiotics that increase errors in translation. We further show that the ClpP1P2 protease is required for the degradation of proteins tagged with the SsrA motif, a tag co-translationally added to incomplete protein products. Using active site mutants of ClpP1 and ClpP2, we show that the activity of each subunit is required for proteolysis, for normal growth of Mtb in vitro and during infection of mice. These observations suggest that the Clp protease plays an unusual and essential role in Mtb and may serve as an ideal target for antimycobacterial therapy.

Trigger Factor Associates with GroEL in Vivo and Promotes Its Binding to Certain Polypeptides

Trigger factor (TF) is a putative molecular chaperone recently found to function together with GroEL in the degradation of the fusion protein, CRAG. TF overproduction enhanced the ability of GroEL to form complexes with CRAG, as well as fetuin or histone. To define further this effect on GroEL binding, affinity columns containing a variety of denatured proteins were used. When cell extracts were applied onto a fetuin column, both TF and GroEL bound but not GroES. Upon ATP addition, TF and GroEL were eluted together and remained tightly associated (even in presence of GroES) in complexes containing one TF per GroEL 14-mer. Overproduction of TF enhanced the capacity of GroEL to bind to many denatured proteins. Moreover, GroEL-TF complexes isolated from such cells showed much greater binding capacity than GroEL from TF-deficient cells. Furthermore, the addition of pure TF to pure GroEL also enhanced markedly its binding capacity. The affinity of GroEL for CRAG also rises during heat shock due to GroEL phosphorylation. TF expression, however, did not promote GroEL phosphorylation. Moreover, heat shock and TF overproduction affected GroEL binding to other denatured polypeptides in distinct ways; only TF promoted binding to certain polypeptides, whereas only phosphorylation increased binding to others. Thus, association with TF and phosphorylation are independent regulators of GroEL function. This enhanced affinity of TF-GroEL complexes for unfolded proteins may also be important in protein folding, because TF has prolyl isomerase activity and associates with nascent polypeptides.

The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring

Mycobacterium tuberculosis (Mtb) contains two clpP genes, both of which are essential for viability. We expressed and purified Mtb ClpP1 and ClpP2 separately. Although each formed a tetradecameric structure and was processed, they lacked proteolytic activity. We could, however, reconstitute an active, mixed ClpP1P2 complex after identifying N‐blocked dipeptides that stimulate dramatically (>1000‐fold) ClpP1P2 activity against certain peptides and proteins. These activators function cooperatively to induce the dissociation of ClpP1 and ClpP2 tetradecamers into heptameric rings, which then re‐associate to form the active ClpP1P2 2‐ring mixed complex. No analogous small molecule‐induced enzyme activation mechanism involving dissociation and re‐association of multimeric rings has been described. ClpP1P2 possesses chymotrypsin and caspase‐like activities, and ClpP1 and ClpP2 differ in cleavage preferences. The regulatory ATPase ClpC1 was purified and shown to increase hydrolysis of proteins by ClpP1P2, but not peptides. ClpC1 did not activate ClpP1 or ClpP2 homotetradecamers and stimulated ClpP1P2 only when both ATP and a dipeptide activator were present. ClpP1P2 activity, its unusual activation mechanism and ClpC1 ATPase represent attractive drug targets to combat tuberculosis.

The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

ABSTRACT Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development.

Rapid degradation of an abnormal protein in Escherichia coli proceeds through repeated cycles of association with GroEL.

Molecular chaperones are necessary for the breakdown of many abnormal proteins, but their functions in this process have remained obscure. The rapid degradation of the abnormal fusion protein CRAG in Escherichia coli requires the molecular chaperones GroEL, GroES, and trigger factor and proceeds through the formation of a CRAG-GroEL-trigger factor complex. Also associated with GroEL are smaller discrete fragments of CRAG. Pulse-chase experiments showed that these fragments were short-lived intermediates in CRAG degradation formed by C-terminal cleavages. Thus, CRAG degradation is not highly processive. In cells lacking the ClpP protease, the generation of these fragments and their subsequent degradation were much slower than in the wild type. Dissociation of CRAG from GroEL was necessary for its digestion by the ClpP protease, because in a groES temperature-sensitive mutant, CRAG was stable and accumulated on GroEL. Furthermore, the expression of a dominant GroEL mutant defective in substrate dissociation slowed degradation of both CRAG and the fragments. Therefore, we suggest that CRAG degradation proceeds through multiple rounds of substrate binding to GroEL, followed by their GroES-dependent dissociation, which allows further digestion by the protease. In this multistep process, GroEL and GroES function repeatedly, apparently to allow further degradation of CRAG and its fragments by the protease.

Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease.

The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance and activation mechanism. Whilst most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram‐positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl‐leucyl‐leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP‐activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP‐dependent protein degradation.

Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity

Background: ClpP1P2 is a novel protease complex essential for viability of Mycobacterium tuberculosis. Results: Cleavage preferences of ClpP1P2 were defined, which allowed us to design potent substrate-based boronate inhibitors showing anti-mycobacterial activity. Conclusion: Excellent new fluorogenic peptide substrates of ClpP1P2 were obtained, and novel enzyme properties were identified. Significance: Selective inhibition of ClpP1P2 activity is a promising approach for drug development. The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.