A host-directed oxadiazole compound potentiates antituberculosis treatment via zinc poisoning in human macrophages and in a mouse model of infection.

Antituberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multiresistant strains of Mycobacterium tuberculosis (MTB) are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high-content imaging, we identified novel 1,2,4-oxadiazole-based compounds, which allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, MTB became even more sensitive to antituberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions.

An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death.

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases.

Modulation of bacterial membrane proteins activity by clustering into plasma membrane nanodomains.

Recent research has demonstrated specific protein clustering within membrane subdomains in bacteria, challenging the long-held belief that prokaryotes lack these subdomains. This mini review provides examples of bacterial membrane protein clustering, discussing the benefits of protein assembly in membranes and highlighting how clustering regulates protein activity.

MenT nucleotidyltransferase toxins extend tRNA acceptor stems and can be inhibited by asymmetrical antitoxin binding.

Mycobacterium tuberculosis, the bacterium responsible for human tuberculosis, has a genome encoding a remarkably high number of toxin-antitoxin systems of largely unknown function. We have recently shown that the M. tuberculosis genome encodes four of a widespread, MenAT family of nucleotidyltransferase toxin-antitoxin systems. In this study we characterize MenAT1, using tRNA sequencing to demonstrate MenT1 tRNA modification activity. MenT1 activity is blocked by MenA1, a short protein antitoxin unrelated to the MenA3 kinase. X-ray crystallographic analysis shows blockage of the conserved MenT fold by asymmetric binding of MenA1 across two MenT1 protomers, forming a heterotrimeric toxin-antitoxin complex. Finally, we also demonstrate tRNA modification by toxin MenT4, indicating conserved activity across the MenT family. Our study highlights variation in tRNA target preferences by MenT toxins, selective use of nucleotide substrates, and diverse modes of MenA antitoxin activity.

A Mycobacterium tuberculosis Effector Targets Mitochondrion, Controls Energy Metabolism, and Limits Cytochrome c Exit.

Host metabolism reprogramming is a key feature of Mycobacterium tuberculosis (Mtb) infection that enables the survival of this pathogen within phagocytic cells and modulates the immune response facilitating the spread of the tuberculosis disease. Here, we demonstrate that a previously uncharacterized secreted protein from Mtb, Rv1813c, manipulates the host metabolism by targeting mitochondria. When expressed in eukaryotic cells, the protein is delivered to the mitochondrial intermembrane space and promotes the enhancement of host ATP production by boosting the oxidative phosphorylation metabolic pathway. Furthermore, the release of cytochrome c from mitochondria, an early apoptotic event in response to short-term oxidative stress, is delayed in Rv1813c-expressing cells. This study reveals a novel class of mitochondria targeting effectors from Mtb that might participate in host cell metabolic reprogramming and apoptosis control during Mtb infections. IMPORTANCE In this article, using a combination of techniques (bioinformatics, structural biology, and cell biology), we identified and characterized a new class of effectors present only in intracellular mycobacteria. These proteins specifically target host cell mitochondria when ectopically expressed in cells. We showed that one member of this family (Rv1813c) affects mitochondria metabolism in a way that might twist the immune response. This effector also inhibits the cytochrome c exit from mitochondria, suggesting that it might alter normal host cell apoptotic capacities, one of the first defenses of immune cells against Mtb infection.

Maintenance of cell wall remodeling and vesicle production are connected in Mycobacterium tuberculosis.

Pathogenic and nonpathogenic mycobacteria secrete extracellular vesicles (EVs) under various conditions. EVs produced by Mycobacterium tuberculosis ( Mtb ) have raised significant interest for their potential in cell communication, nutrient acquisition, and immune evasion. However, the relevance of vesicle secretion during tuberculosis infection remains unknown due to the limited understanding of mycobacterial vesicle biogenesis. We have previously shown that a transposon mutant in the LCP-related gene virR ( virR mut ) manifested a strong attenuated phenotype during experimental macrophage and murine infections, concomitant to enhanced vesicle release. In this study, we aimed to understand the role of VirR in the vesicle production process in Mtb . We employ genetic, transcriptional, proteomics, ultrastructural and biochemical methods to investigate the underlying processes explaining the enhanced vesiculogenesis phenomenon observed in the virR mutant. Our results establish that VirR is critical to sustain proper cell permeability via regulation of cell envelope remodeling possibly through the interaction with similar cell envelope proteins, which control the link between peptidoglycan and arabinogalactan. These findings advance our understanding of mycobacterial extracellular vesicle biogenesis and suggest that these set of proteins could be attractive targets for therapeutic intervention.

Acid stress response in Escherichia coli: mechanism of regulation of gadA transcription by RcsB and GadE.

Escherichia coli can survive extreme acid stress for several hours. The most efficient acid resistance system is based on glutamate decarboxylation by the GadA and GadB decarboxylases and the import of glutamate via the GadC membrane protein. The expression of the corresponding genes is controlled by GadE, the central activator of glutamate-dependent acid resistance (GDAR). We have previously shown by genetic approaches that as well as GadE, the response regulator of the Rcs system, RcsB is absolutely required for control of gadA/BC transcription. In the presence of GadE, basal activity of RcsB stimulates the expression of gadA/BC, whereas activation of RcsB leads to general repression of the gad genes. We report here the results of various in vitro assays that show RcsB to regulate by direct binding to the gadA promoter region. Furthermore, activation of gadA transcription requires a GAD box and binding of an RcsB/GadE heterodimer. In addition, we have identified an RcsB box, which lies just upstream of the -10 element of gadA promoter and is involved in repression of this operon.

Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms.

The human pathogen Mycobacterium tuberculosis requires a P1B-ATPase metal exporter, CtpC (Rv3270), for resistance to zinc poisoning. Here, we show that zinc resistance also depends on a chaperone-like protein, PacL1 (Rv3269). PacL1 contains a transmembrane domain, a cytoplasmic region with glutamine/alanine repeats and a C-terminal metal-binding motif (MBM). PacL1 binds Zn2+, but the MBM is required only at high zinc concentrations. PacL1 co-localizes with CtpC in dynamic foci in the mycobacterial plasma membrane, and the two proteins form high molecular weight complexes. Foci formation does not require flotillin nor the PacL1 MBM. However, deletion of the PacL1 Glu/Ala repeats leads to loss of CtpC and sensitivity to zinc. Genes pacL1 and ctpC appear to be in the same operon, and homologous gene pairs are found in the genomes of other bacteria. Furthermore, PacL1 colocalizes and functions redundantly with other PacL orthologs in M. tuberculosis. Overall, our results indicate that PacL proteins may act as scaffolds that assemble P-ATPase-containing metal efflux platforms mediating bacterial resistance to metal poisoning.

Insights into the extracytoplasmic stress response of Xanthomonas campestris pv. campestris: role and regulation of {sigma}E-dependent activity.

Xanthomonas campestris pv. campestris is an epiphytic bacterium that can become a vascular pathogen responsible for black rot disease of crucifers. To adapt gene expression in response to ever-changing habitats, phytopathogenic bacteria have evolved signal transduction regulatory pathways, such as extracytoplasmic function (ECF) σ factors. The alternative sigma factor σ(E), encoded by rpoE, is crucial for envelope stress response and plays a role in the pathogenicity of many bacterial species. Here, we combine different approaches to investigate the role and mechanism of σ(E)-dependent activation in X. campestris pv. campestris. We show that the rpoE gene is organized as a single transcription unit with the anti-σ gene rseA and the protease gene mucD and that rpoE transcription is autoregulated. rseA and mucD transcription is also controlled by a highly conserved σ(E)-dependent promoter within the σ(E) gene sequence. The σ(E)-mediated stress response is required for stationary-phase survival, resistance to cadmium, and adaptation to membrane-perturbing stresses (elevated temperature and ethanol). Using microarray technology, we started to define the σ(E) regulon of X. campestris pv. campestris. These genes encode proteins belonging to different classes, including periplasmic or membrane proteins, biosynthetic enzymes, classical heat shock proteins, and the heat stress σ factor σ(H). The consensus sequence for the predicted σ(E)-regulated promoter elements is GGAACTN(15-17)GTCNNA. Determination of the rpoH transcription start site revealed that rpoH was directly regulated by σ(E) under both normal and heat stress conditions. Finally, σ(E) activity is regulated by the putative regulated intramembrane proteolysis (RIP) proteases RseP and DegS, as previously described in many other bacteria. However, our data suggest that RseP and DegS are not only dedicated to RseA cleavage and that the proteolytic cascade of RseA could involve other proteases.

SpxA1, a novel transcriptional regulator involved in X-state (competence) development in Streptococcus pneumoniae.

Streptococcus pneumoniae is a naturally transformable human pathogen. Genome and phylogenetic analyses uncovered two Spx-like global transcriptional regulators, SpxA1 and SpxA2, encoded by S. pneumoniae. spxA1 and spxA2 are not essential, but their simultaneous inactivation is lethal. SpxA1 represses transcription of the early competence operon comCDE and thereby negatively regulates the initiation of the X-state (competence). The molecular basis of this repression could be similar to that of SpxA of Bacillus subtilis, involving a specific interaction with the alpha subunit of RNA polymerase. S. pneumoniae lacks an SOS-like stress response and the X-state is proposed to be a general stress response mechanism in this species. In light of this, SpxA1-dependent repression could act to sense environmental or metabolic stresses and prevent launching of the X-state in the absence of stress.

Effects of access to preen gland secretions on mallard plumage.

Preen glands exist in almost every bird species and several non-exclusive functions have been proposed for this gland and the oils that it produces. One function generally admitted is that the oily secretions of the preen gland would provide a waterproofing layer when spread over feathers. Alternatively, several authors have proposed that plumage waterproofness is mostly due to the spatial micro-structure of feathers. The purpose of this study was to examine, by manipulating the access to the preen gland, the effect of the preen oil on the plumage waterproofness and condition. To explore this question, we carried out two independent experiments where we temporarily blocked access to the preen gland secretions with a removable mechanism in one group of captive mallards (Anas platyrhynchos), whilst a second group of birds had access to gland secretions. In a long-term experiment (3 months of treatment) and a short-term experiment (10 days), we measured plumage water retention and condition. After 3 months without access to preen glands, we found a significant decrease of plumage condition and an associated increase in plumage water retention. Moreover, we found a significant correlation between plumage condition and water retention ability. In contrast, after 10 days of treatment, no significant effect was found on plumage condition and water retention. Our study shows that preen oil acts to maintain plumage condition and suggests that feather microstructure is essential to maintain plumage waterproofness.

Multi-Stress Induction of the Mycobacterium tuberculosis MbcTA Bactericidal Toxin-Antitoxin System.

MbcTA is a type II toxin/antitoxin (TA) system of Mycobacterium tuberculosis. The MbcT toxin triggers mycobacterial cell death in vitro and in vivo through the phosphorolysis of the essential metabolite NAD+ and its bactericidal activity is neutralized by physical interaction with its cognate antitoxin MbcA. Therefore, the MbcTA system appears as a promising target for the development of novel therapies against tuberculosis, through the identification of compounds able to antagonize or destabilize the MbcA antitoxin. Here, the expression of the mbcAT operon and its regulation were investigated. A dual fluorescent reporter system was developed, based on an integrative mycobacterial plasmid that encodes a constitutively expressed reporter, serving as an internal standard for monitoring mycobacterial gene expression, and an additional reporter, dependent on the promoter under investigation. This system was used both in M. tuberculosis and in the fast growing model species Mycobacterium smegmatis to: (i) assess the autoregulation of mbcAT; (ii) perform a genetic dissection of the mbcA promoter/operator region; and (iii) explore the regulation of mbcAT transcription from the mbcA promoter (PmbcA) in a variety of stress conditions, including in vivo in mice and in macrophages.

An improved Xer-cise technology for the generation of multiple unmarked mutants in Mycobacteria.

Xer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild-type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.

A nucleotidyltransferase toxin inhibits growth of Mycobacterium tuberculosis through inactivation of tRNA acceptor stems.

Toxin-antitoxin systems are widespread stress-responsive elements, many of whose functions remain largely unknown. Here, we characterize the four DUF1814-family nucleotidyltransferase-like toxins (MenT1-4) encoded by the human pathogen Mycobacterium tuberculosis. Toxin MenT3 inhibited growth of M. tuberculosis when not antagonized by its cognate antitoxin, MenA3. We solved the structures of toxins MenT3 and MenT4 to 1.6 and 1.2 Å resolution, respectively, and identified the biochemical activity and target of MenT3. MenT3 blocked in vitro protein expression and prevented tRNA charging in vivo. MenT3 added pyrimidines (C or U) to the 3'-CCA acceptor stems of uncharged tRNAs and exhibited strong substrate specificity in vitro, preferentially targeting tRNASer from among the 45 M. tuberculosis tRNAs. Our study identifies a previously unknown mechanism that expands the range of enzymatic activities used by bacterial toxins, uncovering a new way to block protein synthesis and potentially treat tuberculosis and other infections.

Lysine represses transcription of the Escherichia coli dapB gene by preventing its activation by the ArgP activator.

The Escherichia coli dapB gene encodes one of the enzymes of the biosynthetic pathway leading to lysine and its immediate precursor, diaminopimelate. Expression of dapB is repressed by lysine, but no trans-acting regulator has been identified so far. Our analysis of the dapB regulatory region shows that sequences located in the -81/-118 interval upstream of the transcription start site are essential for full expression of dapB, as well as for lysine repression. Screening a genomic library for a gene that could alleviate lysine repression when present in multicopy led to the recovery of argP, a gene encoding an activating protein of the LysR-type family, known to use lysine as an effector. An argP null mutation strongly decreases dapB transcription that becomes insensitive to lysine. Purified His(6)-tagged ArgP protein binds with an apparent K(d) of 35 nM to the dapB promoter in a gel retardation assay, provided that sequences up to -103 are present. In the presence of L-lysine and L-arginine, the binding of ArgP to dapB is partly relieved. These results fit with a model in which ArgP contributes to enhanced transcription of dapB when lysine becomes limiting.

Osmotic regulation of transcription in Lactococcus lactis: ionic strength-dependent binding of the BusR repressor to the busA promoter.

The busA locus of Lactococcus lactis encodes a glycine betaine uptake system. At low osmolarity, the transcription of busA is repressed by the BusR protein, which is responsible for the osmotic inducibility of the busA promoter (busAp). In this work, we investigated the mechanism of the osmo-dependent repression by BusR. We found that BusR binding to the busA promoter is dependent on the ionic strength in vitro. Using a BusR derivative carrying a phosphorylation site and the Escherichia coli RNA polymerase holoenzyme, we showed that these proteins are able to form a stable ternary complex by both binding to the same busAp fragment. The association/dissociation of BusR to the RNA polymerase-busAp complex is strictly correlated to the surrounding ionic strength. Together, these results suggest that during growth at low osmolarity BusR represses transcription from busAp at a step further the recruitment of the RNA polymerase. At high osmolarity, an elevated cytoplasmic ionic strength would dissociate BusR from busAp, resulting in the osmotic induction of the busA operon.

Interactions between the 2.4 and 4.2 regions of sigmaS, the stress-specific sigma factor of Escherichia coli, and the -10 and -35 promoter elements.

The sigmas subunit of Escherichia coli RNA polymerase holoenzyme (EsigmaS) is a key factor of gene expression upon entry into stationary phase and in stressful conditions. The selectivity of promoter recognition by EsigmaS and the housekeeping Esigma70 is as yet not clearly understood. We used a genetic approach to investigate the interaction of sigmaS with its target promoters. Starting with down-promoter variants of a sigmaS promoter target, osmEp, altered in the -10 or -35 elements, we isolated mutant forms of sigmaS suppressing the promoter defects. The activity of these suppressors on variants of osmEp and ficp, another target of sigmaS, indicated that sigmaS is able to interact with the same key features within a promoter sequence as sigma70. Indeed, (i) sigmaS can recognize the -35 element of some but not all its target promoters, through interactions with its 4.2 region; and (ii) amino acids within the 2.4 region participate in the recognition of the -10 element. More specifically, residues Q152 and E155 contribute to the strong preference of sigmaS for a C in position -13 and residue R299 can interact with the -31 nucleotide in the -35 element of the target promoters.

Sigma(s)-dependent regulation of yehZYXW, which encodes a putative osmoprotectant ABC transporter of Escherichia coli.

The Escherichia coli yehZYXW operon encodes a putative osmoprotectant uptake system of the ABC transporter family. yehZ is identical to osmF, an osmotically inducible gene identified previously. Construction and analysis of a yehZ-lacZ transcriptional fusion demonstrated that yehZ is inducible not only by osmolarity, but also upon entry into stationary phase. The osmotic and growth-phase regulations operate at a unique promoter, yehZp, and are totally dependent on the stress specific sigma factor sigma(s). The yehZYXW encoded ABC transporter appears as an additional element of the global stress response controlled by sigma(s).