Disruption of nucleoid expanded conformation by toxic aberrant proteins synthesized in Escherichia coli

Aminoglycoside antibiotics interfere with selection of cognate tRNAs during translation, resulting in the production of aberrant proteins that are the ultimate cause of the antibiotic bactericidal effect. To determine if these aberrant proteins are recognized as substrates by the cell’s protein quality control machinery, we studied whether the heat shock (HS) response was activated following exposure of Escherichia coli to the aminoglycoside kanamycin (Kan). Levels of the HS transcription factor σ32 increased about 10-fold after exposure to Kan, indicating that at least some aberrant proteins were recognized as substrates by the molecular chaperone DnaK. To investigate whether toxic aberrant proteins therefore might escape detection by the QC machinery, we studied model aberrant proteins that had a bactericidal effect when expressed in E. coli from cloned genes. As occurred following exposure to Kan, levels of σ32 were permanently elevated following expression of an acutely toxic 48-residue protein (ARF48), indicating that toxic activity and recognition by the QC machinery are not mutually exclusive properties of aberrant proteins, and that the HS response was blocked in these cells at some step downstream of σ32 stabilization. This block could result from halting of protein synthesis or from radial condensation of nucleoids, both of which occurred rapidly following ARF48 induction. Nucleoids were similarly condensed following expression of toxic aberrant secretory proteins, suggesting that transertion of inner membrane proteins, a process that expands nucleoids into an open conformation that promotes growth and gene expression, was disrupted in these cells. The 48-residue ARF48 protein would be well-suited for structural studies to further investigate the toxic mechanism of aberrant proteins.

A scaffolded approach to unearth potential antibacterial components from epicarp of Malaysian Nephelium lappaceum L.

The emergence and spread of antimicrobial resistance have been of serious concern to human health and the management of bacterial infectious diseases. Effective treatment of these diseases requires the development of novel therapeutics, preferably free of side effects. In this regard, natural products are frequently conceived to be potential alternative sources for novel antibacterial compounds. Herein, we have evaluated the antibacterial activity of the epicarp extracts of the Malaysian cultivar of yellow rambutan fruit (Nephelium lappaceum L.) against six pathogens namely, Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella enterica. Among a series of solvent extracts, fractions of ethyl acetate and acetone have revealed significant activity towards all tested strains. Chemical profiling of these fractions, via HPLC, LC–MS and GC–MS, has generated a library of potentially bioactive compounds. Downstream virtual screening, pharmacological prediction, and receptor-ligand molecular dynamics simulation have eventually unveiled novel potential antibacterial compounds, which can be extracted for medicinal use. We report compounds like catechin, eplerenone and oritin-4-beta-ol to be computationally inhibiting the ATP-binding domain of the chaperone, DnaK of P. aeruginosa and MRSA. Thus, our work follows the objective to propose new antimicrobials capable of perforating the barrier of resistance posed by both the gram positives and the negatives.

Chaperone-Mediated Stress Sensing in Mycobacterium tuberculosis Enables Fast Activation and Sustained Response

ABSTRACT Dynamical properties of gene regulatory networks are tuned to ensure bacterial survival. In mycobacteria, the MprAB-σE network responds to the presence of stressors, such as surfactants that cause surface stress. Positive feedback loops in this network were previously predicted to cause hysteresis, i.e., different responses to identical stressor levels for prestressed and unstressed cells. Here, we show that hysteresis does not occur in nonpathogenic Mycobacterium smegmatis but does occur in Mycobacterium tuberculosis. However, the observed rapid temporal response in M. tuberculosis is inconsistent with the model predictions. To reconcile these observations, we implement a recently proposed mechanism for stress sensing, namely, the release of MprB from the inhibitory complex with the chaperone DnaK upon the stress exposure. Using modeling and parameter fitting, we demonstrate that this mechanism can accurately describe the experimental observations. Furthermore, we predict perturbations in DnaK expression that can strongly affect dynamical properties. Experiments with these perturbations agree with model predictions, confirming the role of DnaK in fast and sustained response. IMPORTANCE Gene regulatory networks controlling stress response in mycobacterial species have been linked to persistence switches that enable bacterial dormancy within a host. However, the mechanistic basis of switching and stress sensing is not fully understood. In this paper, combining quantitative experiments and mathematical modeling, we uncover how interactions between two master regulators of stress response—the MprAB two-component system (TCS) and the alternative sigma factor σE—shape the dynamical properties of the surface stress network. The result show hysteresis (history dependence) in the response of the pathogenic bacterium M. tuberculosis to surface stress and lack of hysteresis in nonpathogenic M. smegmatis. Furthermore, to resolve the apparent contradiction between the existence of hysteresis and fast activation of the response, we utilize a recently proposed role of chaperone DnaK in stress sensing. These result leads to a novel system-level understanding of bacterial stress response dynamics.

The Disordered C-Terminus of the Chaperone DnaK Increases the Competitive Fitness of Pseudomonas putida and Facilitates the Toxicity of GraT

Chaperone proteins are crucial for proper protein folding and quality control, especially when cells encounter stress caused by non-optimal temperatures. DnaK is one of such essential chaperones in bacteria. Although DnaK has been well characterized, the function of its intrinsically disordered C-terminus has remained enigmatic as the deletion of this region has been shown to either enhance or reduce its protein folding ability. We have shown previously that DnaK interacts with toxin GraT of the GraTA toxin-antitoxin system in Pseudomonas putida. Interestingly, the C-terminal truncation of DnaK was shown to alleviate GraT-caused growth defects. Here, we aim to clarify the importance of DnaK in GraT activity. We show that DnaK increases GraT toxicity, and particularly important is the negatively charged motif in the DnaK C-terminus. Given that GraT has an intrinsically disordered N-terminus, the assistance of DnaK is probably needed for re-modelling the toxin structure. We also demonstrate that the DnaK C-terminal negatively charged motif contributes to the competitive fitness of P. putida at both high and optimal growth temperatures. Thus, our data suggest that the disordered C-terminal end of DnaK enhances the chaperone functionality.

Chaperone-mediated stress-sensing in Mycobacterium tuberculosis enables fast activation and sustained response

ABSTRACTDynamical properties of gene-regulatory networks are tuned to ensure bacterial survival. In mycobacteria, MprAB-σE network responds to the presence of stressors, such as surfactants causing surface stress. Positive feedback loops in this network were previously predicted to cause hysteresis, i.e. different responses to identical stressor levels for pre-stressed and unstressed cells. Here we show that hysteresis does not occur in non-pathogenic Mycobacterium smegmatis but occurs in Mycobacterium tuberculosis. However, the observed rapid temporal response in M. tuberculosis is inconsistent with the model predictions. To reconcile these observations, we implement a recently proposed mechanism for stress-sensing: the release of MprB from the inhibitory complex with chaperone DnaK upon the stress exposure. Using modeling and parameter fitting, we demonstrate that this mechanism can accurately describe the experimental observations. Furthermore, we predict perturbations in DnaK expression that can strongly affect dynamical properties. Experiments with these perturbations agree with model predictions, confirming the role of DnaK in fast and sustained response.IMPORTANCEGene-regulatory networks controlling stress response in mycobacterial species have been linked to persistence switches enabling the bacterial dormancy within a host. However, the mechanistic basis of switching and stress sensing is not fully understood. In this paper, combining quantitative experiments and mathematical modeling, we uncover how interactions between two master regulators of stress response -- MprAB two-component system and alternative sigma factor σE – shape the dynamical properties of surface-stress network. The result show hysteresis (history dependence) in the response of pathogenic bacteria M. tuberculosis to surface stress and lack of the hysteresis in non-pathogenic M. smegmatis. Furthermore, to resolve the apparent contradiction between the existence of hysteresis and fast activation of the response, we utilize a recently proposed role of chaperone DnaK in stress-sensing. The results leads to a novel system-level understanding of bacterial stress-response dynamics.

Growth-driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells inCaulobacter crescentus

All living cells must deal with protein aggregation, which can occur as a result of experiencing stress. In the bacteriaEscherichia coliandMycobacterium smegmatis, aggregates collect at the cell poles and are retained over consecutive cell divisions only in the daughter cell that inherits the old pole, resulting in aggregation-free progeny within a few generations. Here we have studied thein vivokinetics of aggregate formation and clearance following heat and antibiotic stress inCaulobacter crescentus, which divides by a pre-programmed asymmetric cell cycle. Unexpectedly, we find that aggregates do not preferentially collect at the cell poles, but form as multiple distributed foci throughout the cell volume. Time-lapse microscopy revealed that under moderate stress, the majority of protein aggregates are short-lived and rapidly dissolved by the major chaperone DnaK and the disaggregase ClpB. Severe stress or genetic perturbation of the protein quality machinery results in long-lived protein aggregates, which individual cells can only clear by passing on to their progeny. Importantly, these persistent aggregates are neither collected at the old pole over multiple generations nor inherited exclusively by the old pole-inheriting stalked cell, but instead are partitioned between both daughter cells during successive division events in the same ratio. Our data indicate that this symmetric mode of aggregate inheritance is driven by the elongation and division of the growing mother cell. In conclusion, our study revealed a new pattern of aggregate inheritance in bacteria.

The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication ofVibrio choleraeChromosome 2

ABSTRACTReplication ofVibrio choleraechromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations inrctBthat reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.IMPORTANCEThe capacity of proteins to undergo remodeling provides opportunities to control their function. However, remodeling remains a poorly understood aspect of the structure-function paradigm due to its dynamic nature. Here we have studied remodeling of the initiator of replication ofVibrio choleraeChr2 by the molecular chaperone, DnaK. We show that DnaK binds to a site on the Chr2 initiator (RctB) that promotes initiation by reducing the initiator’s propensity to dimerize. Dimerization of the initiator of the putative plasmid progenitor of Chr2 is also reduced by DnaK, which promotes initiation. Paradoxically, the DnaK binding also promotes replication inhibition by reducing an autoinhibitory activity of RctB. In the plasmid-to-chromosome transition, it appears that the initiator has acquired an autoinhibitory activity and along with it a new chaperone activity that apparently helps to control replication inhibition independently of replication promotion.

Reconstitution of aMycobacterium tuberculosisproteostasis network highlights essential cofactor interactions with chaperone DnaK

During host infection,Mycobacterium tuberculosis(Mtb) encounters several types of stress that impair protein integrity, including reactive oxygen and nitrogen species and chemotherapy. The resulting protein aggregates can be resolved or degraded by molecular machinery conserved from bacteria to eukaryotes. Eukaryotic Hsp104/Hsp70 and their bacterial homologs ClpB/DnaK are ATP-powered chaperones that restore toxic protein aggregates to a native folded state. DnaK is essential inMycobacterium smegmatis, and ClpB is involved in asymmetrically distributing damaged proteins during cell division as a mechanism of survival in Mtb, commending both proteins as potential drug targets. However, their molecular partners in protein reactivation have not been characterized in mycobacteria. Here, we reconstituted the activities of the Mtb ClpB/DnaK bichaperone system with the cofactors DnaJ1, DnaJ2, and GrpE and the small heat shock protein Hsp20. We found that DnaJ1 and DnaJ2 activate the ATPase activity of DnaK differently. A point mutation in the highly conserved HPD motif of the DnaJ proteins abrogates their ability to activate DnaK, although the DnaJ2 mutant still binds to DnaK. The purified Mtb ClpB/DnaK system reactivated a heat-denatured model substrate, but the DnaJ HPD mutants inhibited the reaction. Finally, either DnaJ1 or DnaJ2 is required for mycobacterial viability, as is the DnaK-activating activity of a DnaJ protein. These studies lay the groundwork for strategies to target essential chaperone–protein interactions in Mtb, the leading cause of death from a bacterial infection.