LysX2 is a Mycobacterium tuberculosis membrane protein with an extracytoplasmic MprF-like domain.

BACKGROUND: Aminoacyl-phosphatidylglycerol (aaPG) synthases are bacterial enzymes that usually catalyze transfer of aminoacyl residues to the plasma membrane phospholipid phosphatidylglycerol (PG). The result is introduction of positive charges onto the cytoplasmic membrane, yielding reduced affinity towards cationic antimicrobial peptides, and increased resistance to acidic environments. Therefore, these enzymes represent an important defense mechanism for many pathogens, including Staphylococcus aureus and Mycobacterium tuberculosis (Mtb), which are known to encode for lysyl-(Lys)-PG synthase MprF and LysX, respectively. Here, we used a combination of bioinformatic, genetic and bacteriological methods to characterize a protein encoded by the Mtb genome, Rv1619, carrying a domain with high similarity to MprF-like domains, suggesting that this protein could be a new aaPG synthase family member. However, unlike homologous domains of MprF and LysX that are positioned in the cytoplasm, we predicted that the MprF-like domain in LysX2 is in the extracytoplasmic region. RESULTS: Using genetic fusions to the Escherichia coli proteins PhoA and LacZ of LysX2, we confirmed this unique membrane topology, as well as LysX and MprF as benchmarks. Expression of lysX2 in Mycobacterium smegmatis increased cell resistance to human ß-defensin 2 and sodium nitrite, enhanced cell viability and delayed biofilm formation in acidic pH environment. Remarkably, MtLysX2 significantly reduced the negative charge on the bacterial surface upon exposure to an acidic environment. Additionally, we found LysX2 orthologues in major human pathogens and in rapid-growing mycobacteria frequently associated with human infections, but not in environmental and non-pathogenic mycobacteria. CONCLUSIONS: Overall, our data suggest that LysX2 is a prototype of a new class within the MprF-like protein family that likely enhances survival of the pathogenic species through its catalytic domain which is exposed to the extracytoplasmic side of the cell membrane and is required to decrease the negative charge on the bacterial surface through a yet uncharacterized mechanism.

Construction and Characterization of the Mycobacterium tuberculosis sigE fadD26 Unmarked Double Mutant as a Vaccine Candidate.

Despite the great increase in the understanding of the biology and pathogenesis of Mycobacterium tuberculosis achieved by the scientific community in recent decades, tuberculosis (TB) still represents one of the major threats to global human health. The only available vaccine (Mycobacterium bovis BCG) protects children from disseminated forms of TB but does not effectively protect adults from the respiratory form of the disease, making the development of new and more-efficacious vaccines against the pulmonary forms of TB a major goal for the improvement of global health. Among the different strategies being developed to reach this goal is the construction of attenuated strains more efficacious and safer than BCG. We recently showed that a sigE mutant of M. tuberculosis was more attenuated and more efficacious than BCG in a mouse model of infection. In this paper, we describe the construction and characterization of an M. tuberculosissigE fadD26 unmarked double mutant fulfilling the criteria of the Geneva Consensus for entering human clinical trials. The data presented suggest that this mutant is even more attenuated and slightly more efficacious than the previous sigE mutant in different mouse models of infection and is equivalent to BCG in a guinea pig model of infection.

The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of Mycobacterium tuberculosis.

The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H2O2 stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol-disulfide mechanism. We also found that Rv2466c uses a monothiol-disulfide exchange mechanism to reduce S-mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA-mycoredoxin cluster, we also characterized a homologous enzyme of Corynebacterium glutamicum (NCgl2339) and observed that it demycothiolates and reduces a mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free mycothiol and mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms.

The Alternative Sigma Factors SigE and SigB Are Involved in Tolerance and Persistence to Antitubercular Drugs.

The emergence and spread of drug-resistant Mycobacterium tuberculosis strains possibly threaten our ability to treat this disease in the future. Even though two new antitubercular drugs have recently been introduced, there is still the need to design new molecules whose mechanisms of action could reduce the length of treatment. We show that two alternative sigma factors of M. tuberculosis (SigE and SigB) have a major role in determining the level of basal resistance to several drugs and the amount of persisters surviving long-duration drug treatment. We also demonstrate that ethambutol, a bacteriostatic drug, is highly bactericidal for M. tuberculosis mutants missing either SigE or SigB. We suggest that molecules able to interfere with the activity of SigE or SigB not only could reduce M. tuberculosis virulence in vivo but also could boost the effect of other drugs by increasing the sensitivity of the organism and reducing the number of persisters able to escape killing.

Structure of the SigE regulatory network in Mycobacterium tuberculosis.

SigE is one of the main regulators of mycobacterial stress response and is characterized by a complex regulatory network based on two pathways, which have been partially characterized in conditions of surface stress. The first pathway is based on the induction of sigE transcription by the two-component system MprAB, while the second is based on the degradation of SigE anti-sigma factor RseA by ClpC1P2, a protease whose structural genes are induced by ClgR. We characterized the dynamics of the SigE network activation in conditions of surface stress and low pH in Mycobacterium tuberculosis. Using a series of mutants in which the main regulatory nodes of the network have been inactivated, we could explore their hierarchy, and we determined that MprAB had a key role in the network activation in both stress conditions through the induction of sigE. However, while in conditions of surface stress the absence of MprAB totally abrogated sigE induction, under low pH conditions it only resulted in a small delay of the induction of sigE. In this case, sigE induction was due to SigH, which acted as a MprAB backup system. The ClgR pathway, leading to the degradation of the SigE anti-sigma factor RseA, was shown to be essential for the activation of the SigE network only following surface stress, where it showed an equal hierarchy with the MprAB pathway.

SigE: A master regulator of Mycobacterium tuberculosis.

The Extracellular function (ECF) sigma factor SigE is one of the best characterized out of the 13 sigma factors encoded in the Mycobacterium tuberculosis chromosome. SigE is required for blocking phagosome maturation and full virulence in both mice and guinea pigs. Moreover, it is involved in the response to several environmental stresses as surface stress, oxidative stress, acidic pH, and phosphate starvation. Underscoring its importance in M. tuberculosis physiology, SigE is subjected to a very complex regulatory system: depending on the environmental conditions, its expression is regulated by three different sigma factors (SigA, SigE, and SigH) and a two-component system (MprAB). SigE is also regulated at the post-translational level by an anti-sigma factor (RseA) which is regulated by the intracellular redox potential and by proteolysis following phosphorylation from PknB upon surface stress. The set of genes under its direct control includes other regulators, as SigB, ClgR, and MprAB, and genes involved in surface remodeling and stabilization. Recently SigE has been shown to interact with PhoP to activate a subset of genes in conditions of acidic pH. The complex structure of its regulatory network has been suggested to result in a bistable switch leading to the development of heterogeneous bacterial populations. This hypothesis has been recently reinforced by the finding of its involvement in the development of persister cells able to survive to the killing activity of several drugs.

Role of the Extracytoplasmic Function Sigma Factor SigE in the Stringent Response of Mycobacterium tuberculosis.

Bacteria respond to nutrient starvation implementing the stringent response, a stress signaling system resulting in metabolic remodeling leading to decreased growth rate and energy requirements. A well-characterized model of stringent response in Mycobacterium tuberculosis is the one induced by growth in low phosphate. The extracytoplasmic function (ECF) sigma factor SigE was previously suggested as having a key role in the activation of stringent response. In this study, we challenge this hypothesis by analyzing the temporal dynamics of the transcriptional response of a sigE mutant and its wild-type parental strain to low phosphate using RNA sequencing. We found that both strains responded to low phosphate with a typical stringent response trait, including the downregulation of genes encoding ribosomal proteins and RNA polymerase. We also observed transcriptional changes that support the occurring of an energetics imbalance, compensated by a reduced activity of the electron transport chain, decreased export of protons, and a remodeling of central metabolism. The most striking difference between the two strains was the induction in the sigE mutant of several stress-related genes, in particular, the genes encoding the ECF sigma factor SigH and the transcriptional regulator WhiB6. Since both proteins respond to redox unbalances, their induction suggests that the sigE mutant is not able to maintain redox homeostasis in response to the energetics imbalance induced by low phosphate. In conclusion, our data suggest that SigE is not directly involved in initiating stringent response but in protecting the cell from stress consequent to the low phosphate exposure and activation of stringent response. IMPORTANCE Mycobacterium tuberculosis can enter a dormant state enabling it to establish latent infections and to become tolerant to antibacterial drugs. Dormant bacteria's physiology and the mechanism(s) used by bacteria to enter dormancy during infection are still unknown due to the lack of reliable animal models. However, several in vitro models, mimicking conditions encountered during infection, can reproduce different aspects of dormancy (growth arrest, metabolic slowdown, drug tolerance). The stringent response, a stress response program enabling bacteria to cope with nutrient starvation, is one of them. In this study, we provide evidence suggesting that the sigma factor SigE is not directly involved in the activation of stringent response as previously hypothesized, but it is important to help the bacteria to handle the metabolic stress related to the adaptation to low phosphate and activation of stringent response, thus giving an important contribution to our understanding of the mechanism behind stringent response development.

WhiB5, a transcriptional regulator that contributes to Mycobacterium tuberculosis virulence and reactivation.

The proteins belonging to the WhiB superfamily are small global transcriptional regulators typical of actinomycetes. In this paper, we characterize the role of WhiB5, a Mycobacterium tuberculosis protein belonging to this superfamily. A null mutant was constructed in M. tuberculosis H37Rv and was shown to be attenuated during both progressive and chronic mouse infections. Mice infected with the mutant had smaller bacillary burdens in the lungs but a larger inflammatory response, suggesting a role of WhiB5 in immunomodulation. Most interestingly, the whiB5 mutant was not able to resume growth after reactivation from chronic infection, suggesting that WhiB5 controls the expression of genes involved in this process. The mutant was also more sensitive than the wild-type parental strain to S-nitrosoglutathione (GSNO) and was less metabolically active following prolonged starvation, underscoring the importance of GSNO and starvation in development and maintenance of chronic infection. DNA microarray analysis identified 58 genes whose expression is influenced by WhiB5, including sigM, encoding an alternative sigma factor, and genes encoding the constituents of two type VII secretion systems, namely, ESX-2 and ESX-4.

Transcriptional regulation and drug resistance in Mycobacterium tuberculosis.

Bacterial drug resistance is one of the major challenges to present and future human health, as the continuous selection of multidrug resistant bacteria poses at serious risk the possibility to treat infectious diseases in the near future. One of the infection at higher risk to become incurable is tuberculosis, due to the few drugs available in the market against Mycobacterium tuberculosis. Drug resistance in this species is usually due to point mutations in the drug target or in proteins required to activate prodrugs. However, another interesting and underexplored aspect of bacterial physiology with important impact on drug susceptibility is represented by the changes in transcriptional regulation following drug exposure. The main regulators involved in this phenomenon in M. tuberculosis are the sigma factors, and regulators belonging to the WhiB, GntR, XRE, Mar and TetR families. Better understanding the impact of these regulators in survival to drug treatment might contribute to identify new drug targets and/or to design new strategies of intervention.

An integrated regulatory network including two positive feedback loops to modulate the activity of sigma(E) in mycobacteria.

sigma(E), one of the best characterized mycobacterial extracytoplasmic function sigma factors, is involved in virulence, surface stress response and modulation of the inflammatory response during infection. The regulation of its activity is very complex and involves transcriptional, translational and post-translational control. Post-translational regulation is controlled by RseA, an anti-sigma factor belonging to the zinc-associated anti-sigma factor family. In this issue of Molecular Microbiology, Barik et al. demonstrate that RseA is a redox-sensing protein that is able to bind sigma(E) only in reducing environment. Importantly, they describe a novel positive feedback loop responsible for sigma(E) release and activation following surface stress, due to ClpC1P2-dependent proteolytic degradation of RseA, depending on its phosphorylation by the eukaryotic-like Ser/Thr protein kinase PknB.

Xer site-specific recombination, an efficient tool to introduce unmarked deletions into mycobacteria.

Genetic manipulation of mycobacteria still represents a serious challenge due to the lack of tools and selection markers. In this report, we describe the development of an intrinsically unstable excisable cassette for introduction of unmarked mutations in both Mycobacterium smegmatis and Mycobacterium tuberculosis.

Tolerance and Persistence to Drugs: A Main Challenge in the Fight Against Mycobacterium tuberculosis.

The treatment of tuberculosis is extremely long. One of the reasons why Mycobacterium tuberculosis elimination from the organism takes so long is that in particular environmental conditions it can become tolerant to drugs and/or develop persisters able to survive killing even from very high drug concentrations. Tolerance develops in response to a harsh environment exposure encountered by bacteria during infection, mainly due to the action of the immune system, whereas persistence results from the presence of heterogeneous bacterial populations with different degrees of drug sensitivity, and can be induced by exposure to stress conditions. Here, we review the actual knowledge on the stress response mechanisms enacted by M. tuberculosis during infection, which leads to increased drug tolerance or development of a highly drug-resistant subpopulation.

Publisher Correction: Assessing the role of Rv1222 (RseA) as an anti-sigma factor of the Mycobacterium tuberculosis extracytoplasmic sigma factor SigE.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Assessing the role of Rv1222 (RseA) as an anti-sigma factor of the Mycobacterium tuberculosis extracytoplasmic sigma factor SigE.

σE is one of the 13 sigma factors encoded by the Mycobacterium tuberculosis chromosome, and its involvement in stress response and virulence has been extensively characterized. Several sigma factors are post-translationally regulated by proteins named anti-sigma factors, which prevent their binding to RNA polymerase. Rv1222 (RseA), whose gene lays immediately downstream sigE, has been proposed in the past as the σE-specific anti sigma factor. However, its role as anti-sigma factor was recently challenged and a new mechanism of action was hypothesized predicting RseA binding to RNA polymerase and DNA to slow down RNA transcription in a not specific way. In this manuscript, using specific M. tuberculosis mutants, we showed that by changing the levels of RseA expression, M. tuberculosis growth rate does not change (as hypothesized in case of non-specific decrease of RNA transcription) and has an impact only on the transcription level of genes whose transcriptional control is under σE, supporting a direct role of RseA as a specific anti-σE factor.

Improving the stability of the TetR/Pip-OFF mycobacterial repressible promoter system.

Tightly regulated gene expression systems are powerful tools to study essential genes and characterize potential drug targets. In a past work we reported the construction of a very stringent and versatile repressible promoter system for Mycobacterium tuberculosis based on two different repressors (TetR/Pip-OFF system). This system, causing the repression of the target gene in response to anhydrotetracycline (ATc), has been successfully used in several laboratories to characterize essential genes in different mycobacterial species both in vitro and in vivo. One of the limits of this system was its instability, leading to the selection of mutants in which the expression of the target gene was no longer repressible. In this paper we demonstrated that the instability was mainly due either to the loss of the integrative plasmid carrying the genes encoding the two repressors, or to the selection of a frameshift mutation in the gene encoding the repressors Pip. To solve these problems, we (i) constructed a new integrative vector in which the gene encoding the integrase was deleted to increase its stability, and (ii) developed a new integrative vector carrying the gene encoding Pip to introduce a second copy of this gene in the chromosome. The use of these new tools was shown to reduce drastically the selection of escape mutants.

Evidence of complex transcriptional, translational, and posttranslational regulation of the extracytoplasmic function sigma factor sigmaE in Mycobacterium tuberculosis.

The extracytoplasmic factor (ECF) sigma factor sigma(E) is one of the most studied sigma factors of Mycobacterium tuberculosis. It has been shown to be involved in virulence as well as in survival under conditions of high temperature, alkaline pH, and exposure to detergents and oxidative stress. Unlike many ECF sigma factors, sigma(E) does not directly regulate the transcription of its own gene. Two promoters have been identified upstream of the sigE gene; one is regulated by the two-component system MprAB, while the other has been shown to be sigma(H) dependent. In this paper, we further characterize the regulation of sigma(E) by identifying its anti-sigma factor and a previously unknown promoter. Finally, we show that sigE can be translated from three different translational start codons, depending on the promoter used. Taken together, our data demonstrate that sigma(E) not only is subjected to complex transcriptional regulation but is also controlled at the translational and posttranslational levels.

SigF controls carotenoid pigment production and affects transformation efficiency and hydrogen peroxide sensitivity in Mycobacterium smegmatis.

Carotenoids are complex lipids that are known for acting against photodynamic injury and free radicals. We demonstrate here that sigma(F) is required for carotenoid pigment production in Mycobacterium smegmatis. We further show that a sigF mutant exhibits a transformation efficiency 10(4)-fold higher than that of the parental strain, suggesting that sigma(F) regulates the production of components affecting cell wall permeability. In addition, a sigF mutant showed an increased sensitivity to hydrogen peroxide. An in silico search of the M. smegmatis genome identified a number of SigF consensus sites, including sites upstream of the carotenoid synthesis locus, which explains its SigF regulation.

The sigma factors of Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a remarkable pathogen capable of adapting and surviving in various harsh conditions. Correct gene expression regulation is essential for the success of this process. The reversible association of different sigma factors is a common mechanism for reprogramming bacterial RNA polymerase and modulating the transcription of numerous genes. Thirteen putative sigma factors are encoded in the M. tuberculosis genome, several being important for virulence. Here, we analyse the latest information available on mycobacterial sigma factors and discuss their roles in the physiology and virulence of M. tuberculosis.

Promoter mutagenesis for fine-tuning expression of essential genes in Mycobacterium tuberculosis.

A range of regulated gene expression systems has been developed for mycobacteria in the last few years to facilitate the study of essential genes, validate novel drug targets and evaluate their vulnerability. Among these, the TetR/Pip-OFF repressible promoter system was successfully used in several mycobacterial species both in vitro and in vivo. In the first version of the system, the repressible promoter was Pptr , a strong Pip-repressible promoter of Streptomyces pristinaespiralis, which might hamper effective downregulation of genes with a low basal expression level. Here, we report an enhanced system that allows more effective control of genes expressed at low level. To this end, we subjected Pptr to targeted mutagenesis and produced 16 different promoters with different strength. Three of them, weaker than the wild-type promoter, were selected and characterized showing that they can indeed improve the performances of TetR/Pip-OFF repressible system both in vitro and in vivo increasing its stringency. Finally, we used these promoters to construct a series of bacterial biosensors with different sensitivity to DprE1 inhibitors and developed a whole-cell screening assay to identify inhibitors of this enzyme.

Mapping and characterization of G-quadruplexes in Mycobacterium tuberculosis gene promoter regions.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), one of the top 10 causes of death worldwide in 2015. The recent emergence of strains resistant to all current drugs urges the development of compounds with new mechanisms of action. G-quadruplexes are nucleic acids secondary structures that may form in G-rich regions to epigenetically regulate cellular functions. Here we implemented a computational tool to scan the presence of putative G-quadruplex forming sequences in the genome of Mycobacterium tuberculosis and analyse their association to transcription start sites. We found that the most stable G-quadruplexes were in the promoter region of genes belonging to definite functional categories. Actual G-quadruplex folding of four selected sequences was assessed by biophysical and biomolecular techniques: all molecules formed stable G-quadruplexes, which were further stabilized by two G-quadruplex ligands. These compounds inhibited Mycobacterium tuberculosis growth with minimal inhibitory concentrations in the low micromolar range. These data support formation of Mycobacterium tuberculosis G-quadruplexes in vivo and their potential regulation of gene transcription, and prompt the use of G4 ligands to develop original antitubercular agents.