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Summary DnaK is the bacterial homolog of Hsp70, an ATP-dependent chaperone that helps cofactor proteins to catalyze nascent protein folding and salvage misfolded proteins. In the pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), DnaK and its cofactors are proposed antimycobacterial targets, yet few small-molecule inhibitors or probes exist for these families of proteins. Here, we describe the repurposing of a drug called telaprevir that is able to allosterically inhibit the ATPase activity of DnaK and to prevent chaperone function by mimicking peptide substrates. In mycobacterial cells, telaprevir disrupts DnaK- and cofactor-mediated cellular proteostasis, resulting in enhanced efficacy of aminoglycoside antibiotics and reduced resistance to the frontline TB drug rifampin. Hence, this work contributes to a small but growing collection of protein chaperone inhibitors, and it demonstrates that these molecules disrupt bacterial mechanisms of survival in the presence of different antibiotic classes.

AMR is a global problem, especially for TB. Here, we show that mycobacterial chaperones support AMR in M. smegmatis, a nonpathogenic model of M. tuberculosis, the causative agent of TB., Chaperones aid in protein folding and maintenance of protein integrity. In doing so, they have the unique ability to directly stabilize resistance-conferring amino acid substitutions in drug targets and to counter the stress imparted by these substitutions, thus supporting heritable antimicrobial resistance (AMR). We asked whether chaperones support AMR in Mycobacterium smegmatis, a saprophytic model of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). We show that DnaK associates with many drug targets and that DnaK associates more with AMR-conferring mutant RNA polymerase (RNAP) than with wild-type RNAP. In addition, frequency-of-resistance (FOR) and fitness studies reveal that the DnaK system of chaperones supports AMR in antimicrobial targets in mycobacteria, including RNAP and the ribosome. These findings highlight chaperones as potential targets for drugs to overcome AMR in mycobacteria, including M. tuberculosis, as well as in other pathogens.

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.

SUMMARY 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., Graphical abstract, In brief Yin et al. use cryo-EM to analyze the structure of the Mycobacterium tuberculosis ClpB-DnaK bi-chaperone system. They find that the Mtb ClpB middle domain does not bend or tilt when interacting with DnaK. They therefore propose that the Mtb DnaK facilitates protein folding following protein disaggregation by ClpB.

INTRODUCTION Mycobacterium tuberculosis (Mtb) is the leading global cause of lethal infection in humans and accounts for the largest number of drug-resistant infections by a single bacterial pathogen. Resistance is particularly high against the most widely prescribed tuberculosis (TB) drug, isoniazid. Isoniazid blocks synthesis of mycolates, ultralong-chain fatty acids that provide structure to the waxy coat that surrounds Mtb cells and are incorporated into some of its virulence lipids. There is currently no known method to block the synthesis of both mycolates and nonmycolate-containing virulence lipids of Mtb at a single point of control. One such control point is phosphopantetheinyl transferase (PptT). PptT transfers 4′-phosphopantetheine (Ppt) from coenzyme A (CoA) to acyl carrier proteins (ACPs) that synthesize the lipids critical to Mtb structural integrity and virulence. RATIONALE TB drug discovery often begins with whole-cell, high-throughput screens that yield compounds that kill Mtb by unknown means. Selection of Mtb mutants resistant to these compounds can indicate candidate targets of the active compound, but experimental validation is required to confirm the functionally relevant target, which is often an enzyme. A suitable target must be essential in vivo, such that its inhibition precludes development of TB in animal models, but also “vulnerable,” meaning that a pharmacologically attainable level of inhibition should be lethal to Mtb within a patient. The inhibitor should act only on Mtb, and resistance should be rare. RESULTS Screening a chemical library revealed an amidino-urea compound called “8918” that kills Mtb, including drug-resistant clinical isolates. 8918 inhibits Mtb in mice and spares other bacteria, yeast, and mammalian cells. Rare Mtb mutants resistant to 8918 bore a point mutation in the PptT gene rv2794c, altering an amino acid residue overlying the Ppt-binding pocket of PptT. When Mtb carried the mutant allele as an extra copy of rv2794c, the Mtb was protected from 8918. 8918 inhibited recombinant PptT, albeit noncompetitively and incompletely. The impact of 8918 on the Mtb metabolome and lipids was consistent with inhibition of PptT in the intact cell. A crystal structure of the PptT-8918 complex at 1.8-Å resolution confirmed that 8918 binds within the Ppt binding pocket, adjacent to the phosphoadenosine phosphate portion of CoA. Intact CoA remained in the PptT-8918 complex, but the Ppt arm was displaced, decreasing but not abolishing PptT’s catalytic activity. Strains of Mtb producing reduced amounts of PptT became hypersensitive to 8918. It was puzzling that even partial inhibition of PptT killed Mtb. We observed that mutants with disruption of rv2795c, a gene encoding a hypothetical protein, were also highly resistant to 8918. Recombinant Rv2795c protein hydrolyzed Ppt from a mycolate-building holo-ACP that is a substrate for PptT. The action of this Ppt hydrolase (PptH) resembled that of nonhomologous enzymes called ACP hydrolases that remove Ppt from ACPs in vitro but whose physiological function is unknown. CONCLUSION We identified a small molecule that kills Mtb by inhibiting PptT, demonstrating that a key enzyme in CoA metabolism is a viable target for TB drug development. Even partial inhibition of PptT is toxic to Mtb, likely because PptH synergizes with the inhibitor by undoing the PptT reaction. PptT and PptH are co-regulated by translation from the same operon, and thus Mtb cannot respond to inhibition of PptT by making more PptT without also generating more PptH. The joint functioning of PptT and PptH suggests that Mtb closely regulates the activation of ACPs. The transcriptional co-regulation and constitutive function of both members of the PptT-PptH couple suggests that a posttranslational signal that impairs PptT more than PptH could allow Mtb to rapidly reverse a prior commitment to synthesis of its metabolically most costly lipids.

Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. To understand biotin biosynthesis in mycobacteria, we executed a genetic screen in Mycobacterium smegmatis for biotin auxotrophs and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. The biotin auxotrophy of the pyc::tn strain is due to failure to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of bioQ, the repressor of biotin biosynthesis, in the pyc::tn strain reverted biotin auxotrophy, as did reconstituting the last step of the pathway through heterologous expression of BioB and provision of its substrate DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Quantitation of the kinetics of depletion of biotinylated proteins after biotin withdrawal revealed that Pyc is the most rapidly depleted biotinylated protein and metabolomics revealed a broad metabolic shift in wild type cells upon biotin withdrawal which was blunted in cell lacking Pyc. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism.

Bacterial antimicrobial resistance is an escalating public health threat, yet the current antimicrobial pipeline remains alarmingly depleted, making the development of new antimicrobials an urgent need. Here, we identify a novel, potent, imidazoline antimicrobial compound, SKI-356313, with bactericidal activity against Mycobacterium tuberculosis and Gram-positive cocci, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). SKI-356313 is active in murine models of Streptococcus pneumoniae and MRSA infection and is potently bactericidal for both replicating and nonreplicating M. tuberculosis. Using a combination of genetics, whole genome sequencing, and a novel target ID approach using real time imaging of core macromolecular biosynthesis, we show that SKI-356313 inhibits DNA replication and displaces the replisome from the bacterial nucleoid. These results identify a new antimicrobial scaffold with a novel mechanism of action and potential therapeutic utility against nonreplicating M. tuberculosis and antibiotic resistant Gram-positive cocci.

RbpA and CarD are essential transcription regulators in mycobacteria. Mechanistic analyses of promoter open complex (RPo) formation establish that RbpA and CarD cooperatively stimulate formation of an intermediate (RP2) leading to RPo; formation of RP2 is likely a bottleneck step at the majority of mycobacterial promoters. Once RPo forms, CarD also disfavors its isomerization back to RP2. We determined a 2.76 Å-resolution crystal structure of a mycobacterial transcription initiation complex (TIC) with RbpA as well as a CarD/RbpA/TIC model. Both CarD and RbpA bind near the upstream edge of the −10 element where they likely facilitate DNA bending and impede transcription bubble collapse. In vivo studies demonstrate the essential role of RbpA, show the effects of RbpA truncations on transcription and cell physiology, and indicate additional functions for RbpA not evident in vitro. This work provides a framework to understand the control of mycobacterial transcription by RbpA and CarD. DOI: http://dx.doi.org/10.7554/eLife.22520.001

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 in Mycobacterium 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.

The requirement of peptidoglycan synthesis for growth complicates the analysis of interactions between proteins involved in this pathway. In particular, the latter steps that involve membrane-linked substrates have proven largely recalcitrant to in vivo analysis. Here, we have taken advantage of the peptidoglycan synthesis that occurs during sporulation in Bacillus subtilis to examine the interactions between SpoVE, a nonessential, sporulation-specific homolog of the well-conserved and essential SEDS (shape elongation, division, and sporulation) proteins, and SpoVD, a nonessential class B penicillin binding protein. We found that localization of SpoVD is dependent on SpoVE and that SpoVD protects SpoVE from in vivo proteolysis. Co-immunoprecipitations and fluorescence resonance energy transfer experiments indicated that SpoVE and SpoVD interact, and co-affinity purification in Escherichia coli demonstrated that this interaction is direct. Finally, we generated a functional protein consisting of an SpoVE-SpoVD fusion and found that a loss-of-function point mutation in either part of the fusion resulted in loss of function of the entire fusion that was not complemented by a wild-type protein. Thus, SpoVE has a direct and functional interaction with SpoVD, and this conclusion will facilitate understanding the essential function that SpoVE and related SEDS proteins, such as FtsW and RodA, play in bacterial growth and division.

Although peptidoglycan synthesis is one of the best-studied metabolic pathways in bacteria, the mechanism underlying the membrane translocation of lipid II, the undecaprenyl-disaccharide pentapeptide peptidoglycan precursor, remains mysterious. Recently, it was proposed that the essential Escherichia coli mviN gene encodes the lipid II flippase. Bacillus subtilis contains four proteins that are putatively homologous to MviN, including SpoVB, previously reported to be necessary for spore cortex peptidoglycan synthesis during sporulation. MviN complemented the sporulation defect of a Δ spoVB mutation, and SpoVB and another of the B. subtilis homologs, YtgP, complemented the growth defect of an E. coli strain depleted for MviN. Thus, these B. subtilis proteins are likely to be MviN homologs. However, B. subtilis strains lacking these four proteins have no defects in growth, indicating that they likely do not serve as lipid II flippases in this organism.

The Bacillus subtilis SpoVE integral membrane protein is essential for the heat resistance of spores, probably because of its involvement in spore peptidoglycan synthesis. We found that an SpoVE-yellow fluorescent protein (YFP) fusion protein becomes localized to the forespore during the earliest stages of engulfment, and this pattern is maintained throughout sporulation. SpoVE belongs to a well-conserved family of proteins that includes the FtsW and RodA proteins of B. subtilis . These proteins are involved in bacterial shape determination, although their function is not known. FtsW is necessary for the formation of the asymmetric septum in sporulation, and we found that an FtsW-YFP fusion localized to this structure prior to the initiation of engulfment in a nonoverlapping pattern with SpoVE-cyan fluorescent protein. Since FtsW and RodA are essential for normal growth, it has not been possible to identify loss-of-function mutations that would greatly facilitate analysis of their function. We took advantage of the fact that SpoVE is not required for growth to obtain point mutations in SpoVE that block the development of spore heat resistance but that allow normal protein expression and targeting to the forespore. These mutant proteins will be invaluable tools for future experiments aimed at elucidating the function of members of the SEDS (“shape, elongation, division, and sporulation”) family of proteins.

GHRH stimulates GH secretion in chickens as in mammals. However, nothing is known about the chicken GHRH receptor (GHRH-R). Here we report the cDNA sequence of chicken GHRH-R. Comparison of the cDNA sequence with the chicken genome localized the GHRH-R gene to chicken chromosome 2 and indicated that the chicken GHRH-R gene consists of 13 exons. Expression of all exons was confirmed by RT-PCR amplification of pituitary mRNA. The amino acid sequence predicted by the GHRH-R cDNA is homologous to that in other vertebrates and contains seven transmembrane domains and a conserved hormone-binding domain. The predicted size of the GHRH-R protein (48.9 kDa) was confirmed by binding of 125I-GHRH to chicken pituitary membranes and SDS-PAGE. GHRH-R mRNA was readily detected by RT-PCR in the pituitary but not in the hypothalamus, total brain, lung, adrenal, ovary, or pineal gland. Effects of corticosterone (CORT), GHRH, ghrelin, pituitary adenylate cyclase-activating peptide, somatostatin (SRIF), and TRH on GHRH-R and GH gene expression were determined in cultures of chicken anterior pituitary cells. GHRH-R and GH mRNA levels were determined by quantitative real-time RT-PCR. Whereas all treatments affected levels of GH mRNA, only CORT, GHRH, and SRIF significantly altered GHRH-R mRNA levels. GHRH-R gene expression was modestly increased by GHRH and suppressed by SRIF at 4 h, and CORT dramatically decreased levels of GHRH-R mRNA at 72 h. We conclude that adrenal glucocorticoids may substantially impact pituitary GH responses to GHRH in the chicken through modulation of GHRH-R gene expression.

pH-responsive transcription factors of the Rim101/PacC family govern virulence in many fungal pathogens. These family members control expression of target genes with diverse functions in growth, morphology and environmental adaptation, so the mechanistic relationship between Rim101/PacC and infection is unclear. We have focused on Rim101 from Candida albicans, which we find to be required for virulence in an oropharyngeal candidiasis model. Rim101 affects the yeast-hypha morphological transition, a major virulence requirement in disseminated infection models. However, virulence in the oropharyngeal candidiasis model is independent of the yeast-hypha transition because it is unaffected by an nrg1 mutation, which prevents formation of yeast cells. Here we have identified Rim101 target genes in an nrg1Delta/Delta mutant background and surveyed function using an overexpression-rescue approach. Increased expression of Rim101 target genes ALS3, CHT2, PGA7/RBT6, SKN1 or ZRT1 can partially restore pathogenic interaction of a rim101Delta/Delta mutant with oral epithelial cells. Four of these five genes govern cell wall structure. Our results indicate that Rim101-dependent cell wall alteration contributes to C. albicans pathogenic interactions with oral epithelial cells, independently of cell morphology.