Biochemical characterization of ClpB and DnaK from Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an intracellular tick-transmitted bacterial pathogen that infects neutrophils in mammals and causes granulocytic anaplasmosis. In this study, we investigated the molecular chaperones ClpB and DnaK from A. phagocytophilum. In Escherichia coli, ClpB cooperates with DnaK and its co-chaperones DnaJ and GrpE in ATP-dependent reactivation of aggregated proteins. Since ClpB is not produced in metazoans, it is a promising target for developing antimicrobial therapies, which generates interest in studies on that chaperone's role in pathogenic bacteria. We found that ClpB and DnaK are transcriptionally upregulated in A. phagocytophilum 3-5 days after infection of human HL-60 and tick ISE6 cells, which suggests an essential role of the chaperones in supporting the pathogen's intracellular life cycle. Multiple sequence alignments show that A. phagocytophilum ClpB and DnaK contain all structural domains that were identified in their previously studied orthologs from other bacteria. Both A. phagocytophilum ClpB and DnaK display ATPase activity, which is consistent with their participation in the ATP-dependent protein disaggregation system. However, despite a significant sequence similarity between the chaperones from A. phagocytophilum and those from E. coli, the former were not as effective as their E. coli orthologs during reactivation of aggregated proteins in vitro and in supporting the survival of E. coli cells under heat stress. We conclude that the A. phagocytophilum chaperones might have evolved with distinct biochemical properties to maintain the integrity of pathogenic proteins under unique stress conditions of an intracellular environment of host cells.

Identification of ClpB, a molecular chaperone involved in the stress tolerance and virulence of Streptococcus agalactiae.

Bacterial ClpB is an ATP-dependent disaggregate that belongs to the Hsp100/Clp family and facilitates bacterial survival under hostile environmental conditions. Streptococcus agalactiae, which is regarded as the major bacterial pathogen of farmed Nile tilapia (Oreochromis niloticus), is known to cause high mortality and large economic losses. Here, we report a ClpB homologue of S. agalactiae and explore its functionality. S. agalactiae with a clpB deletion mutant (∆clpB) exhibited defective tolerance against heat and acidic stress, without affecting growth or morphology under optimal conditions. Moreover, the ΔclpB mutant exhibited reduced intracellular survival in RAW264.7 cells, diminished adherence to the brain cells of tilapia, increased sensitivity to leukocytes from the head kidney of tilapia and whole blood killing, and reduced mortality and bacterial loads in a tilapia infection assay. Furthermore, the reduced virulence of the ∆clpB mutant was investigated by transcriptome analysis, which revealed that deletion of clpB altered the expression levels of multiple genes that contribute to the stress response as well as certain metabolic pathways. Collectively, our findings demonstrated that ClpB, a molecular chaperone, plays critical roles in heat and acid stress resistance and virulence in S. agalactiae. This finding provides an enhanced understanding of the functionality of this ClpB homologue in gram-positive bacteria and the survival strategy of S. agalactiae against immune clearance during infection.

The prevalence of dental agenesis among children with cleft lip and palate patients in Lahore, Pakistan.

Objective: This retrospective, cross-sectional analytical study investigated the incidence of tooth agenesis in cleft lip and palate (CLP) patients. Cone Beam Computed Tomography (CBCT) radiographs of the CLP children were examined for congenitally missing teeth. Method: This study was conducted at three radiology centers in Lahore, namely, the Pakistan Jinnah MRI and Body Scan Centre, the University of Lahore Radiology Centres, and Fatima Memorial Hospital, from September 2021 to August 2022. The CLP patients were divided into four groups based on the location of the cleft: Cleft Lip and Palate Right (CLPR), Cleft Lip and Palate Left (CLPL), Bilateral Cleft (CLPB), and Midline Cleft (CLPM), inside and outside the cleft region. Two-way ANOVA was employed to compare the means of agenesis. Tukey's test was utilized to ascertain where the difference lies. The significance level was set at p ≤ 0.05. Results: Moreover, a significant number of missing teeth were found inside the cleft. This study observed the CLPL (42.3%) and CLPR (13.6%) types more in number. Maxillary first premolars were found more missing outside the cleft region in CLPL and CLPB types. Although CLPB and CLPM types revealed a pattern of missing teeth, only a few cases were found in this study. Moreover, mean tooth agenesis was highest (4.5 SD.71) in the CLPM group, followed up by CLPB (2.75 SD 2.49), CLPR (1.23 SD 1.27), and CLPL Group (1.15 SD 1.12). Conclusions: Unilateral cleft lip and palate patients reported significant agenesis patttern compared to bilateral and median cleft cases.

A review on oligomeric polydispersity and oligomers-dependent holding chaperone activity of the small heat-shock protein IbpB of Escherichia coli.

Inclusion body-associated proteins IbpA and IbpB of MW 16 KDa are the two small heat-shock proteins (sHSPs) of Escherichia coli, and they have only holding, but not folding, chaperone activity. In vitro holdase activity of IbpB is more than that of IbpA, and in combination, they synergise. Both IbpA and IbpB monomers first form homodimers, which as building blocks subsequently oligomerize to make heavy oligomers with MW of MDa range; for IbpB, the MW range of heavy oligomers is 2.0-3.0 MDa, whereas for IbpA oligomers, the values in MDa are not so specified/reported. By temperature upshift, such large oligomers of IbpB, but not of IbpA, dissociate to make relatively small oligomeric assemblies of MW around 600-700KDa. The larger oligomers of IbpB are assumed to be inactive storage form, which on facing heat or oxidative stress dissociate into smaller oligomers of ATP-independent holding chaperone activity. These smaller oligomers bind with stress-induced partially denatured/unfolded and thereby going to be aggregated proteins, to give them protection against permanent damage and aggregation. On withdrawal of stress, IbpB transfers the bound substrate protein to the ATP-dependent bi-chaperone system DnaKJE-ClpB, having both holdase and foldase properties, to finally refold the protein. Of the two sHSPs IbpA and IbpB of E. coli, this review covers the recent advances in research on IbpB only.

Knock-sideways by inducible ER retrieval enables a unique approach for studying Plasmodium-secreted proteins.

Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting Plasmodium-secreted protein functions are limited. We report knockER, a unique DiCre-mediated knock-sideways approach to sequester secreted proteins in the ER by inducible fusion with a KDEL ER-retrieval sequence. We show conditional ER sequestration of diverse proteins is not generally toxic, enabling loss-of-function studies. We employed knockER in multiple Plasmodium species to interrogate the trafficking, topology, and function of an assortment of proteins that traverse the secretory pathway to diverse compartments including the apicoplast (ClpB1), rhoptries (RON6), dense granules, and parasitophorous vacuole (EXP2, PTEX150, HSP101). Taking advantage of the unique ability to redistribute secreted proteins from their terminal destination to the ER, we reveal that vacuolar levels of the PTEX translocon component HSP101 but not PTEX150 are maintained in excess of what is required to sustain effector protein export into the erythrocyte. Intriguingly, vacuole depletion of HSP101 hypersensitized parasites to a destabilization tag that inhibits HSP101-PTEX complex formation but not to translational knockdown of the entire HSP101 pool, illustrating how redistribution of a target protein by knockER can be used to query function in a compartment-specific manner. Collectively, our results establish knockER as a unique tool for dissecting secreted protein function with subcompartmental resolution that should be widely amenable to genetically tractable eukaryotes.

Protein Extract of a Probiotic Strain of Hafnia alvei and Bacterial ClpB Protein Improve Glucose Tolerance in Mice.

A commercial strain of Hafnia alvei (H. alvei) 4597 bacteria was shown to reduce food intake and promote weight loss, effects possibly induced by the bacterial protein ClpB, an antigen-mimetic of the anorexigenic α-melanocyte-stimulating hormone. A decrease in the basal plasma glucose levels was also observed in overweight fasted humans and mice receiving H. alvei. However, it is not known whether H. alvei influences sweet taste preference and whether its protein extract or ClpB are sufficient to increase glucose tolerance; these are the objectives tested in the present study. C57BL/6J male mice were kept under standard diet and were gavaged daily for 17 days with a suspension of H. alvei (4.5 × 107 CFU/animal) or with H. alvei total protein extract (5 µg/animal) or saline as a control. Sweet taste preference was analyzed via a brief-access licking test with sucrose solution. Glucose tolerance tests (GTT) were performed after the intraperitoneal (IP) or intragastric (IG) glucose administration at the 9th and 15th days of gavage, respectively. The expression of regulatory peptides' mRNA levels was assayed in the hypothalamus. In another experiment performed in non-treated C57BL/6J male mice, effects of acute IP administration of recombinant ClpB protein on glucose tolerance were studied by both IP- and IG-GTT. Mice treated with the H. alvei protein extract showed an improved glucose tolerance in IP-GTT but not in IG-GTT. Both groups treated with H. alvei bacteria or protein extract showed a reduction of pancreatic tissue weight but without significant changes to basal plasma insulin. No significant effects of H. alvei bacteria or its total protein extract administration were observed on the sweet taste preference, insulin tolerance and expression of regulatory peptides' mRNA in the hypothalamus. Acute administration of ClpB in non-treated mice increased glucose tolerance during the IP-GTT but not the IG-GTT, and reduced basal plasma glucose levels. We conclude that both the H. alvei protein extract introduced orally and the ClpB protein administered via IP improve glucose tolerance probably by acting at the glucose postabsorptive level. Moreover, H. alvei probiotic does not seem to influence the sweet taste preference. These results justify future testing of both the H. alvei protein extract and ClpB protein in animal models of diabetes.

Fast dynamics shape the function of the AAA+ machine ClpB: lessons from single-molecule FRET spectroscopy.

It has been recently shown that in some proteins, tertiary-structure dynamics occur surprisingly fast, that is on the microsecond or sub-millisecond time scales. In this State of the Art Review, we discuss how such ultrafast domain motions relate to the function of caseinolytic peptidase B (ClpB), a AAA+ disaggregation machine. ClpB is a large hexameric protein that collaborates with cellular chaperone machinery to rescue protein chains from aggregates. We used single-molecule FRET spectroscopy to capture the dynamics of essential structural elements within this machine. It was found that the middle domain of ClpB, known to act as its activator, toggles between two states much faster than the overall activity cycle of the protein, suggesting a novel mode of continuous, tunable switching. Motions of the N-terminal domain were observed to restrict the conformational space of the M domain in the absence of a substrate protein, thereby preventing it from tilting and spuriously activating ClpB. Finally, microsecond dynamics of pore loops responsible for substrate pulling through ClpB's central channel, together with their response to specific perturbations, point to a Brownian-ratchet mechanism for protein translocation. Based on our findings, we propose a two-time-scale model for the activity of ClpB, in which fast conformational dynamics affect slower functional steps, determined by ATP hydrolysis time. Future work on this and other proteins is likely to shed further light on the role of ultrafast dynamics on protein function.

Premature Ovarian Insufficiency in CLPB Deficiency: Transcriptomic, Proteomic and Phenotypic Insights.

CONTEXT: Premature ovarian insufficiency (POI) is a common form of female infertility that usually presents as an isolated condition but can be part of various genetic syndromes. Early diagnosis and treatment of POI can minimize comorbidity and improve health outcomes. OBJECTIVE: We aimed to determine the genetic cause of syndromic POI, intellectual disability, neutropenia, and cataracts. METHODS: We performed whole-exome sequencing (WES) followed by functional validation via RT-PCR, RNAseq, and quantitative proteomics, as well as clinical update of previously reported patients with variants in the caseinolytic peptidase B (CLPB) gene. RESULTS: We identified causative variants in CLPB, encoding a mitochondrial disaggregase. Variants in this gene are known to cause an autosomal recessive syndrome involving 3-methylglutaconic aciduria, neurological dysfunction, cataracts, and neutropenia that is often fatal in childhood; however, there is likely a reporting bias toward severe cases. Using RNAseq and quantitative proteomics we validated causation and gained insight into genotype:phenotype correlation. Clinical follow-up of patients with CLPB deficiency who survived to adulthood identified POI and infertility as a common postpubertal ailment. CONCLUSION: A novel splicing variant is associated with CLPB deficiency in an individual who survived to adulthood. POI is a common feature of postpubertal female individuals with CLPB deficiency. Patients with CLPB deficiency should be referred to pediatric gynecologists/endocrinologists for prompt POI diagnosis and hormone replacement therapy to minimize associated comorbidities.

Rare coding variants in DNA damage repair genes associated with timing of natural menopause.

The age of menopause is associated with fertility and disease risk, and its genetic control is of great interest. We use whole-exome sequences from 132,370 women in the UK Biobank to test for associations between rare damaging variants and age at natural menopause. Rare damaging variants in five genes are significantly associated with menopause: CHEK2 (p = 3.3 × 10-51), DCLRE1A (p = 8.4 × 10-13), and HELB (p = 5.7 × 10-7) with later menopause and TOP3A (p = 7.6 × 10-8) and CLPB (p = 8.1 × 10-7) with earlier menopause. Two additional genes are suggestive: RAD54L (p = 2.4 × 10-6) with later menopause and HROB (p = 2.9 × 10-6) with earlier menopause. In a follow-up analysis of repeated questionnaires in women who were initially premenopausal, CHEK2, TOP3A, and RAD54L genotypes are associated with subsequent menopause. Consistent with previous genome-wide association studies (GWASs), six of the seven genes are involved in the DNA damage repair pathway. Phenome-wide scans across 398,569 men and women revealed that in addition to known associations with cancers and blood cell counts, rare variants in CHEK2 are also associated with increased risk for uterine fibroids, polycystic ovary syndrome, and prostate hypertrophy; these associations are not shared with higher-penetrance breast cancer genes. Causal mediation analysis suggests that approximately 8% of the breast cancer risk conferred by CHEK2 pathogenic variants after menopause is mediated through delayed menopause.

AAA+ proteins: one motor, multiple ways to work.

Numerous ATPases associated with diverse cellular activities (AAA+) proteins form hexameric, ring-shaped complexes that function via ATPase-coupled translocation of substrates across the central channel. Cryo-electron microscopy of AAA+ proteins processing substrate has revealed non-symmetric, staircase-like hexameric structures that indicate a sequential clockwise/2-residue step translocation model for these motors. However, for many of the AAA+ proteins that share similar structural features, their translocation properties have not yet been experimentally determined. In the cases where translocation mechanisms have been determined, a two-residue translocation step-size has not been resolved. In this review, we explore Hsp104, ClpB, ClpA and ClpX as examples to review the experimental methods that have been used to examine, in solution, the translocation mechanisms employed by AAA+ motor proteins. We then ask whether AAA+ motors sharing similar structural features can have different translocation mechanisms. Finally, we discuss whether a single AAA+ motor can adopt multiple translocation mechanisms that are responsive to different challenges imposed by the substrate or the environment. We suggest that AAA+ motors adopt more than one translocation mechanism and are tuned to switch to the most energetically efficient mechanism when constraints are applied.

Backbone chemical shift assignment and dynamics of the N-terminal domain of ClpB from Francisella tularensis type VI secretion system.

The Hsp100 family member ClpB is a protein disaggregase which solubilizes and reactivates stress-induced protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. In the pathogenic bacterium Francisella tularensis, ClpB is involved in type VI secretion system (T6SS) disassembly through depolymerization of the IglA-IglB sheath. This leads to recycling and reassembly of T6SS components and this process is essential for the virulence of the bacterium. Here we report the backbone chemical shift assignments and 15N relaxation-based backbone dynamics of the N-terminal substrate-binding domain of ClpB (1-156).

Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX with Its Interacting Nucleoid Proteins, and of mtDNA.

Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP, cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are the main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brains. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with the nucleoid component POLDIP2, the mitochondrial poly(A) mRNA granule element LRPPRC, and tRNA processing factor GFM1 (in mouse, also GRSF1). Only in mouse did accumulated ClpX, GFM1, and GRSF1 appear in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely, OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids and show nucleoid enlargement in human as a key consequence.

Reconstruction of Three-Dimensional Conformations of Bacterial ClpB from High-Speed Atomic-Force-Microscopy Images.

ClpB belongs to the cellular disaggretase machinery involved in rescuing misfolded or aggregated proteins during heat or other cellular shocks. The function of this protein relies on the interconversion between different conformations in its native condition. A recent high-speed-atomic-force-microscopy (HS-AFM) experiment on ClpB from Thermus thermophilus shows four predominant conformational classes, namely, open, closed, spiral, and half-spiral. Analyses of AFM images provide only partial structural information regarding the molecular surface, and thus computational modeling of three-dimensional (3D) structures of these conformations should help interpret dynamical events related to ClpB functions. In this study, we reconstruct 3D models of ClpB from HS-AFM images in different conformational classes. We have applied our recently developed computational method based on a low-resolution representation of 3D structure using a Gaussian mixture model, combined with a Monte-Carlo sampling algorithm to optimize the agreement with target AFM images. After conformational sampling, we obtained models that reflect conformational variety embedded within the AFM images. From these reconstructed 3D models, we described, in terms of relative domain arrangement, the different types of ClpB oligomeric conformations observed by HS-AFM experiments. In particular, we highlighted the slippage of the monomeric components around the seam. This study demonstrates that such details of information, necessary for annotating the different conformational states involved in the ClpB function, can be obtained by combining HS-AFM images, even with limited resolution, and computational modeling.

Hsp100 Molecular Chaperone ClpB and Its Role in Virulence of Bacterial Pathogens.

This review focuses on the molecular chaperone ClpB that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and its biological function in selected bacterial pathogens, causing a variety of human infectious diseases, including zoonoses. It has been established that ClpB disaggregates and reactivates aggregated cellular proteins. It has been postulated that ClpB's protein disaggregation activity supports the survival of pathogenic bacteria under host-induced stresses (e.g., high temperature and oxidative stress), which allows them to rapidly adapt to the human host and establish infection. Interestingly, ClpB may also perform other functions in pathogenic bacteria, which are required for their virulence. Since ClpB is not found in human cells, this chaperone emerges as an attractive target for novel antimicrobial therapies in combating bacterial infections.

Bacterial RF3 senses chaperone function in co-translational folding.

Molecular chaperones assist with protein folding by interacting with nascent polypeptide chains (NCs) during translation. Whether the ribosome can sense chaperone defects and, in response, abort translation of misfolding NCs has not yet been explored. Here we used quantitative proteomics to investigate the ribosome-associated chaperone network in E. coli and the consequences of its dysfunction. Trigger factor and the DnaK (Hsp70) system are the major NC-binding chaperones. HtpG (Hsp90), GroEL, and ClpB contribute increasingly when DnaK is deficient. Surprisingly, misfolding because of defects in co-translational chaperone function or amino acid analog incorporation results in recruitment of the non-canonical release factor RF3. RF3 recognizes aberrant NCs and then moves to the peptidyltransferase site to cooperate with RF2 in mediating chain termination, facilitating clearance by degradation. This function of RF3 reduces the accumulation of misfolded proteins and is critical for proteostasis maintenance and cell survival under conditions of limited chaperone availability.

The Role of ClpB in Bacterial Stress Responses and Virulence.

Bacterial survival within a mammalian host is contingent upon sensing environmental perturbations and initiating an appropriate counter-response. To achieve this, sophisticated molecular machineries are used, where bacterial chaperone systems play key roles. The chaperones are a prerequisite for bacterial survival during normal physiological conditions as well as under stressful situations, e.g., infection or inflammation. Specific stress factors include, but are not limited to, high temperature, osmolarity, pH, reactive oxidative species, or bactericidal molecules. ClpB, a member of class 1 AAA+ proteins, is a key chaperone that via its disaggregase activity plays a crucial role for bacterial survival under various forms of stress, in particular heat shock. Recently, it has been reported that ClpB also regulates secretion of bacterial effector molecules related to type VI secretion systems. In this review, the roles of ClpB in stress responses and the mechanisms by which it promotes survival of pathogenic bacteria are discussed.

Structural basis for aggregate dissolution and refolding by the Mycobacterium tuberculosis ClpB-DnaK bi-chaperone system.

The M. tuberculosis (Mtb) ClpB is a protein disaggregase that helps to rejuvenate the bacterial cell. DnaK is a protein foldase that can function alone, but it can also bind to the ClpB hexamer to physically couple protein disaggregation with protein refolding, although the molecular mechanism is not well understood. Here, we report the cryo-EM analysis of the Mtb ClpB-DnaK bi-chaperone in the presence of ATPγS and a protein substrate. We observe three ClpB conformations in the presence of DnaK, identify a conserved TGIP loop linking the oligonucleotide/oligosaccharide-binding domain and the nucleotide-binding domain that is important for ClpB function, derive the interface between the regulatory middle domain of the ClpB and the DnaK nucleotide-binding domain, and find that DnaK binding stabilizes, but does not bend or tilt, the ClpB middle domain. We propose a model for the synergistic actions of aggregate dissolution and refolding by the Mtb ClpB-DnaK bi-chaperone system.

Resisting the Heat: Bacterial Disaggregases Rescue Cells From Devastating Protein Aggregation.

Bacteria as unicellular organisms are most directly exposed to changes in environmental growth conditions like temperature increase. Severe heat stress causes massive protein misfolding and aggregation resulting in loss of essential proteins. To ensure survival and rapid growth resume during recovery periods bacteria are equipped with cellular disaggregases, which solubilize and reactivate aggregated proteins. These disaggregases are members of the Hsp100/AAA+ protein family, utilizing the energy derived from ATP hydrolysis to extract misfolded proteins from aggregates via a threading activity. Here, we describe the two best characterized bacterial Hsp100/AAA+ disaggregases, ClpB and ClpG, and compare their mechanisms and regulatory modes. The widespread ClpB disaggregase requires cooperation with an Hsp70 partner chaperone, which targets ClpB to protein aggregates. Furthermore, Hsp70 activates ClpB by shifting positions of regulatory ClpB M-domains from a repressed to a derepressed state. ClpB activity remains tightly controlled during the disaggregation process and high ClpB activity states are likely restricted to initial substrate engagement. The recently identified ClpG (ClpK) disaggregase functions autonomously and its activity is primarily controlled by substrate interaction. ClpG provides enhanced heat resistance to selected bacteria including pathogens by acting as a more powerful disaggregase. This disaggregase expansion reflects an adaption of bacteria to extreme temperatures experienced during thermal based sterilization procedures applied in food industry and medicine. Genes encoding for ClpG are transmissible by horizontal transfer, allowing for rapid spreading of extreme bacterial heat resistance and posing a threat to modern food production.

A Review: Molecular Chaperone-mediated Folding, Unfolding and Disaggregation of Expressed Recombinant Proteins.

The advancements in biotechnology over time have led to an increase in the demand of pure, soluble and functionally active proteins. Recombinant protein production has thus been employed to obtain high expression of purified proteins in bulk. E. coli is considered as the most desirable host for recombinant protein production due to its inexpensive and fast cultivation, simple nutritional requirements and known genetics. Despite all these benefits, recombinant protein production often comes with drawbacks, such as, the most common being the formation of inclusion bodies due to improper protein folding. Consequently, this can lead to the loss of the structure-function relationship of a protein. Apart from various strategies, one major strategy to resolve this issue is the use of molecular chaperones that act as folding modulators for proteins. Molecular chaperones assist newly synthesized, aggregated or misfolded proteins to fold into their native conformations. Chaperones have been widely used to improve the expression of various proteins which are otherwise difficult to produce in E. coli. Here, we discuss the structure, function, and role of major E. coli molecular chaperones in recombinant technology such as trigger factor, GroEL, DnaK and ClpB.

Nonredundant functions of Mycobacterium tuberculosis chaperones promote survival under stress.

Bacterial chaperones ClpB and DnaK, homologs of the respective eukaryotic heat shock proteins Hsp104 and Hsp70, are essential in the reactivation of toxic protein aggregates that occur during translation or periods of stress. In the pathogen Mycobacterium tuberculosis (Mtb), the protective effect of chaperones extends to survival in the presence of host stresses, such as protein-damaging oxidants. However, we lack a full understanding of the interplay of Hsps and other stress response genes in mycobacteria. Here, we employ genome-wide transposon mutagenesis to identify the genes that support clpB function in Mtb. In addition to validating the role of ClpB in Mtb's response to oxidants, we show that HtpG, a homolog of Hsp90, plays a distinct role from ClpB in the proteotoxic stress response. While loss of neither clpB nor htpG is lethal to the cell, loss of both through genetic depletion or small molecule inhibition impairs recovery after exposure to host-like stresses, especially reactive nitrogen species. Moreover, defects in cells lacking clpB can be complemented by overexpression of other chaperones, demonstrating that Mtb's stress response network depends upon finely tuned chaperone expression levels. These results suggest that inhibition of multiple chaperones could work in concert with host immunity to disable Mtb.