Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.
The enzyme pantothenate synthetase, PanC, is an attractive drug target in Mycobacterium tuberculosis. It is essential for the in vitro growth of M. tuberculosis and for survival of the bacteria in the mouse model of infection. PanC is absent from mammals. We developed an enzyme-based assay to identify inhibitors of PanC, optimized it for high-throughput screening, and tested a large and diverse library of compounds for activity. Two compounds belonging to the same chemical class of 3-biphenyl-4- cyanopyrrole-2-carboxylic acids had activity against the purified recombinant protein, and also inhibited growth of live M. tuberculosis in manner consistent with PanC inhibition. Thus we have identified a new class of PanC inhibitors with whole cell activity that can be further developed.
Identification of new drug targets is vital for the advancement of drug discovery against Mycobacterium tuberculosis, especially given the increase of resistance worldwide to first- and second-line drugs. Because traditional target-based screening has largely proven unsuccessful for antibiotic discovery, we have developed a scalable platform for target identification in M. tuberculosis that is based on whole-cell screening, coupled with whole-genome sequencing of resistant mutants and recombineering to confirm. The method yields targets paired with whole-cell active compounds, which can serve as novel scaffolds for drug development, molecular tools for validation, and/or as ligands for co-crystallization. It may also reveal other information about mechanisms of action, such as activation or efflux. Using this method, we identified resistance-linked genes for eight compounds with anti-tubercular activity. Four of the genes have previously been shown to be essential: AspS, aspartyl-tRNA synthetase, Pks13, a polyketide synthase involved in mycolic acid biosynthesis, MmpL3, a membrane transporter, and EccB3, a component of the ESX-3 type VII secretion system. AspS and Pks13 represent novel targets in protein translation and cell-wall biosynthesis. Both MmpL3 and EccB3 are involved in membrane transport. Pks13, AspS, and EccB3 represent novel candidates not targeted by existing TB drugs, and the availability of whole-cell active inhibitors greatly increases their potential for drug discovery.
Bacterial DNA gyrase is a validated target for antibacterial chemotherapy. It consists of two subunits, GyrA and GyrB, which form an A2B2 complex in the active enzyme. Sequence alignment of Mycobacterium tuberculosis GyrB with other bacterial GyrBs predicts the presence of 40 potential additional amino acids at the GyrB N-terminus. There are discrepancies between the M. tuberculosis GyrB sequences retrieved from different databases, including sequences annotated with or without the additional 40 amino acids. This has resulted in differences in the GyrB sequence numbering that has led to the reporting of previously known fluoroquinolone-resistance mutations as novel mutations.
We have expressed M. tuberculosis GyrB with and without the extra 40 amino acids in Escherichia coli and shown that both can be produced as soluble, active proteins. Supercoiling and other assays of the two proteins show no differences, suggesting that the additional 40 amino acids have no effect on the enzyme in vitro. RT-PCR analysis of M. tuberculosis mRNA shows that transcripts that could yield both the longer and shorter protein are present. However, promoter analysis showed that only the promoter elements leading to the shorter GyrB (lacking the additional 40 amino acids) had significant activity.
We conclude that the most probable translational start codon for M. tuberculosis GyrB is GTG (Val) which results in translation of a protein of 674 amino acids (74 kDa).
Gyrase; Topoisomerase; Mycobacterium tuberculosis
The ability of Mycobacterium tuberculosis (Mtb) to thrive in its phagosomal niche is critical for its establishment of a chronic infection. This requires that Mtb senses and responds to intraphagosomal signals such as pH. We hypothesized that Mtb would respond to additional intraphagosomal factors that correlate with maturation. Here, we demonstrate that [Cl−] and pH correlate inversely with phagosome maturation, and identify Cl− as a novel environmental cue for Mtb. Mtb responds to Cl− and pH synergistically, in part through the activity of the two-component regulator phoPR. Following identification of promoters responsive to Cl− and pH, we generated a reporter Mtb strain that detected immune-mediated changes in the phagosomal environment during infection in a mouse model. Our study establishes Cl− and pH as linked environmental cues for Mtb, and illustrates the utility of reporter bacterial strains for the study of Mtb-host interactions in vivo.
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a disease that remains a major global health problem. To ensure its long-term survival in the host, Mtb must be able to sense and respond to changes in its immediate environment, such as the pH differences that occur in the phagosome in which it lives. Knowledge of the external signals that Mtb recognizes during infection is critical for understanding the impact of the microenvironment on Mtb pathogenesis and persistence, and how Mtb interacts with its host cell. We show here that [Cl−] correlates inversely with pH as the phagosome matures, and identify [Cl−] as a novel cue that Mtb responds to, in synergism with pH. By constructing a Mtb strain that fluorescently reports on changes in [Cl−] and pH, we find using a mouse model of infection that environmental alterations in Mtb's phagosomal home are mediated at the local level by activities of the host immune system. Our study demonstrates how a pathogen can exploit linked environmental cues during infection, and shows the value of reporter bacterial strains for Mtb-host whole animal studies.
Tuberculosis is a serious global health problem caused by the bacterium Mycobacterium tuberculosis. There is an urgent need for discovery and development of new treatments, but this can only be accomplished through rapid and reproducible M. tuberculosis assays designed to identify potent inhibitors. We developed an automated 96-well assay utilizing a recombinant strain of M. tuberculosis expressing a far-red fluorescent reporter to determine the activity of novel compounds; this allowed us to measure growth by monitoring both optical density and fluorescence. We determined that optical density and fluorescence were correlated with cell number during logarithmic phase growth. Fluorescence was stably maintained without antibiotic selection over 5 days, during which time cells remained actively growing. We optimized parameters for the assay, with the final format being 5 days’ growth in 96-well plates in the presence of 2% w/v DMSO. We confirmed reproducibility using rifampicin and other antibiotics. The dual detection method allows for a reproducible calculation of the minimum inhibitory concentration (MIC), at the same time detecting artefacts such as fluorescence quenching or compound precipitation. We used our assay to confirm anti-tubercular activity and establish the structure activity relationship (SAR) around the imidazo[1,2-a]pyridine-3-carboxamides, a promising series of M. tuberculosis inhibitors.
Caseinolytic (Clp) proteases are widespread energy-dependent proteases; the functional ATP-dependent protease is comprised of multimers of proteolytic and regulatory subunits. Mycobacterium tuberculosis has two ClpP proteolytic subunits (ClpP1 and ClpP2), with both being essential for growth in vitro. ClpP1 and clpP2 are arranged in an apparent operon; we demonstrated that the two genes are co-expressed under normal growth conditions. We identified a single promoter region for the clpP1P2 operon; no promoter was detected upstream of clpP2 demonstrating that independent expression of clpP1 and clpP2 was highly unlikely. Promoter activity was not induced by heat shock or oxidative stress. We identified a regulatory region upstream of the promoter with a consensus sequence matching the ClgR regulator motif; we determined the limits of the region by mutagenesis and confirmed that positive regulation of the promoter occurs in M. tuberculosis. We developed a reporter system to monitor ClpP1 and ClpP2 enzymatic activities based on LacZ incorporating ssrAtag sequences. We showed that whilst both ClpP1 and ClpP2 degrade SsrA-tagged LacZ, ClpP2 (but not ClpP1) degrades untagged proteins. Our data suggest that the two proteolytic subunits display different substrate specificities and therefore have different, but overlapping roles in M. tuberculosis.
We investigated the effect of methionine sulfoximine (MetSox), a potent inhibitor of glutamine synthetase, on Mycobacterium tuberculosis. M. tuberculosis encodes four glutamine synthetases, of which MetSox targets the type I enzyme encoded by glnA1. Trancriptional profiling revealed that glutamate synthetase (gltB) and a type II glutamine synthetase (glnA3) were induced after exposure to MetSox. In addition, we observed a high rate (10−5) of spontaneous resistance to MetSox. All resistant strains had a single-nucleotide deletion in the 5′ region of glnA1, and Western analysis revealed that GlnA1 expression was increased in resistant as compared with sensitive strains. These data show that M. tuberculosis can respond to the effect of MetSox inhibition either by up-regulation of GlnA3 or by GlnA1. The high frequency of resistance suggests that MetSox and other compounds specifically targeting GlnA1 are not likely to become successful anti-mycobacterial agents.
Mycobacterium tuberculosis is a pathogen of major global importance. Validated drug targets are required in order to develop novel therapeutics for drug-resistant strains and to shorten therapy. The Clp protease complexes provide a means for quality control of cellular proteins; the proteolytic activity of ClpP in concert with the ATPase activity of the ClpX/ClpC subunits results in degradation of misfolded or damaged proteins. Thus, the Clp system plays a major role in basic metabolism, as well as in stress responses and pathogenic mechanisms. M. tuberculosis has two ClpP proteolytic subunits. Here we demonstrate that ClpP1 is essential for viability in this organism in culture, since the gene could only be deleted from the chromosome when a second functional copy was provided. Overexpression of clpP1 had no effect on growth in aerobic culture or viability under anaerobic conditions or during nutrient starvation. In contrast, clpP2 overexpression was toxic, suggesting different roles for the two homologs. We synthesized known activators of ClpP protease activity; these acyldepsipeptides (ADEPs) were active against M. tuberculosis. ADEP activity was enhanced by the addition of efflux pump inhibitors, demonstrating that ADEPs gain access to the cell but that export occurs. Taken together, the genetic and chemical validation of ClpP as a drug target leads to new avenues for drug discovery.
The current method for testing new drugs against tuberculosis in vivo is the enumeration of bacteria in organs by cfu assay. Owing to the slow growth rate of Mycobacterium tuberculosis (Mtb), these assays can take months to complete. Our aim was to develop a more efficient, fluorescence-based imaging assay to test new antibiotics in a mouse model using Mtb reporter strains.
A commercial IVIS Kinetic® system and a custom-built laser scanning system with fluorescence molecular tomography (FMT) capability were used to detect fluorescent Mtb in living mice and lungs ex vivo. The resulting images were analysed and the fluorescence was correlated with data from cfu assays.
We have shown that fluorescent Mtb can be visualized in the lungs of living mice at a detection limit of ∼8 × 107 cfu/lung, whilst in lungs ex vivo a detection limit of ∼2 × 105 cfu/lung was found. These numbers were comparable between the two imaging systems. Ex vivo lung fluorescence correlated to numbers of bacteria in tissue, and the effect of treatment of mice with the antibiotic moxifloxacin could be visualized and quantified after only 9 days through fluorescence measurements, and was confirmed by cfu assays.
We have developed a new and efficient method for anti-tuberculosis drug testing in vivo, based on fluorescent Mtb reporter strains. Using this method instead of, or together with, cfu assays will reduce the time required to assess the preclinical efficacy of new drugs in animal models and enhance the progress of these candidates into clinical trials against human tuberculosis.
antibiotics; drug testing; mycobacteria; optical imaging; TB
Thymidine biosynthesis is essential in all cells. Inhibitors of the enzymes involved in this pathway (e.g. methotrexate) are thus frequently used as cytostatics. Due to its pivotal role in mycobacterial thymidylate synthesis dUTPase, which hydrolyzes dUTP into the dTTP precursor dUMP, has been suggested as a target for new antitubercular agents. All mycobacterial genomes encode dUTPase with a mycobacteria-specific surface loop absent in the human dUTPase. Using Mycobacterium smegmatis as a fast growing model for Mycobacterium tuberculosis, we demonstrate that dUTPase knock-out results in lethality that can be reverted by complementation with wild-type dUTPase. Interestingly, a mutant dUTPase gene lacking the genus-specific loop was unable to complement the knock-out phenotype. We also show that deletion of the mycobacteria-specific loop has no major effect on dUTPase enzymatic properties in vitro and thus a yet to be identified loop-specific function seems to be essential within the bacterial cell context. In addition, here we demonstrated that Mycobacterium tuberculosis dUTPase is fully functional in Mycobacterium smegmatis as it rescues the lethal knock-out phenotype. Our results indicate the potential of dUTPase as a target for antitubercular drugs and identify a genus-specific surface loop on the enzyme as a selective target.
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a major global health threat. During infection, bacteria are believed to encounter adverse conditions such as iron depletion. Mycobacteria synthesize iron-sequestering mycobactins, which are essential for survival in the host, via the intermediate salicylate. Salicylate is a ubiquitous compound which is known to induce a mild antibiotic resistance phenotype. In M. tuberculosis salicylate highly induces the expression of Rv0560c, a putative methyltransferase. We identified and characterized the promoter and regulatory elements of Rv0560c. PRv0560c activity was highly inducible by salicylate in a dose-dependent manner. The induction kinetics of PRv0560c were slow, taking several days to reach maximal activity, which was sustained over several weeks. Promoter activity could also be induced by compounds structurally related to salicylate, such as aspirin or para-aminosalicylic acid, but not by benzoate, indicating that induction is specific to a structural motif. The −10 and −35 promoter elements were identified and residues involved in regulation of promoter activity were identified in close proximity to an inverted repeat spanning the −35 promoter element. We conclude that Rv0560c expression is controlled by a yet unknown repressor via a highly-inducible promoter.
We constructed recombinant strains of Mycobacterium tuberculosis in which expression of specific genes was downregulated to identify vulnerable drug targets. Growth phenotypes in macrophages and culture were used to rank targets: the dprE1, clpP1, and fadD32 operons were the best targets and glnA1, glnE, pknL, regX3, and senX3 were poor targets.
Two component regulatory systems are used widely by bacteria to coordinate changes in global gene expression profiles in response to environmental signals. The SenX3-RegX3 two component system of Mycobacterium tuberculosis has previously been shown to play a role in virulence and phosphate-responsive control of gene expression. We demonstrate that expression of SenX3-RegX3 is controlled in response to growth conditions, although the absolute changes are small. Global gene expression profiling of a RegX3 deletion strain and wild-type strain in different culture conditions (static, microaerobic, anaerobic), as well as in an over-expressing strain identified a number of genes with changed expression patterns. Among those were genes previously identified as differentially regulated in aerobic culture, including ald (encoding alanine dehydrogenase) cyd,encoding a subunit of the cytochrome D ubiquinol oxidase, and gltA1, encoding a citrate synthase. Promoter activity in the upstream regions of both cydB and gltA1 was altered in the RegX3 deletion strain. DNA-binding assays confirmed that RegX3 binds to the promoter regions of ald, cydB and gltA1 in a phosphorylation-dependent manner. Taken together these data suggest a direct role for the SenX-RegX3 system in modulating expression of aerobic respiration, in addition to its role during phosphate limitation.
Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in early infection and suggest novel diagnostic and therapeutic approaches.
The immune system of all jawed vertebrates harbors three distinct lymphocyte populations – αβ T cells, γδ T cells and B cells – yet only higher primates including humans possess so-called Vγ9/Vδ2 T cells, an enigmatic γδ T cell subset that uniformly responds to the majority of bacterial pathogens. For reasons that are not understood, this responsiveness is absent in all other animals although they too are constantly exposed to a plethora of potentially harmful micro-organisms. We here investigated how Vγ9/Vδ2 T cells respond to live microbes by mimicking physiological conditions in acute disease. Our experiments demonstrate that Vγ9/Vδ2 T cells recognize a small common molecule released when invading bacteria become ingested and killed by other white blood cells. The stimulation of Vγ9/Vδ2 T cells at the site of infection amplifies the inflammatory response and has important consequences for pathogen clearance and the development of microbe-specific immunity. However, if triggered at the wrong time or the wrong place, this rapid reaction toward bacteria may also lead to inflammation-related damage. These findings improve our insight into the complex cellular interactions in early infection, identify novel biomarkers of diagnostic and predictive value and highlight new avenues for therapeutic intervention.
The fatty acid synthase type II enzymatic complex of Mycobacterium tuberculosis (FAS-IIMt) catalyzes an essential metabolic pathway involved in the biosynthesis of major envelope lipids, mycolic acids. The partner proteins of this singular FAS-II system represent relevant targets for antituberculous drug design. Two heterodimers of the hydratase 2 protein family, HadAB and HadBC, were shown to be involved in the (3R)-hydroxyacyl-ACP dehydration (HAD) step of FAS-IIMt cycles. Recently, an additional member of this family, Rv0241c, was proposed to have the same function, based on the heterologous complementation of a HAD mutant of the yeast mitochondrial FAS-II system. In the present work, Rv0241c was able to complement a HAD mutant in the Escherichia coli model but not a dehydratase-isomerase deficient mutant. However, an enzymatic study of the purified protein demonstrated that Rv0241c possesses a broad chain length specificity for the substrate, unlike FAS-IIMt enzymes. Most importantly, Rv0241c exhibited a strict dependence on the coenzyme A (CoA) as opposed to AcpM, the natural acyl carrier protein bearing the chains elongated by FAS-IIMt. The deletion of Rv0241c showed that this gene is not essential to M. tuberculosis survival in vitro. The resulting mutant did not display any change in the mycolic acid profile. This demonstrates that Rv0241c is a trans-2-enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase that does not belong to FAS-IIMt. The relevance of a heterologous complementation strategy to identifying proteins of such a system is questioned.
The publication of the complete genome sequence for Mycobacterium tuberculosis H37Rv in 1998 has had a great impact on the research community. Nonetheless, it is suspected that genetic differences have arisen in stocks of H37Rv that are maintained in different laboratories. In order to assess the consistency of the genome sequences among H37Rv strains in use and the extent to which they have diverged from the original strain sequenced, we carried out whole-genome sequencing on six strains of H37Rv from different laboratories. Polymorphisms at 73 sites were observed, which were shared among the lab strains, though 72 of these were also shared with H37Ra and are likely to be due to sequencing errors in the original H37Rv reference sequence. An updated H37Rv genome sequence should be valuable to the tuberculosis research community as well as the broader microbial research community. In addition, several polymorphisms unique to individual strains and several shared polymorphisms were identified and shown to be consistent with the known provenance of these strains. Aside from nucleotide substitutions and insertion/deletions, multiple IS6110 transposition events were observed, supporting the theory that they play a significant role in plasticity of the M. tuberculosis genome. This genome-wide catalog of genetic differences can help explain any phenotypic differences that might be found, including a frameshift mutation in the mycocerosic acid synthase gene which causes two of the strains to be deficient in biosynthesis of the surface glycolipid phthiocerol dimycocerosate (PDIM). The resequencing of these six lab strains represents a fortuitous “in vitro evolution” experiment that demonstrates how the M. tuberculosis genome continues to evolve even in a controlled environment.
Mycobacterium tuberculosis synthesizes isoprenoids via the nonmevalonate or DOXP pathway. Previous work demonstrated that three enzymes in the pathway (Dxr, IspD, and IspF) are all required for growth in vitro. We demonstrate the essentiality of the key genes dxs1 and gcpE, confirming that the pathway is of central importance and that the second homolog of the synthase (dxs2) cannot compensate for the loss of dxs1. We looked at the effect of overexpression of Dxr, Dxs1, Dxs2, and GcpE on viability and on growth in M. tuberculosis. Overexpression of dxs1 or dxs2 was inhibitory to growth, whereas overexpression of dxr or gcpE was not. Toxicity is likely to be, at least partially, due to depletion of pyruvate from the cells. Overexpression of dxs1 or gcpE resulted in increased flux through the pathway, as measured by accumulation of the metabolite 4-hydroxy-3-methyl-but-2-enyl pyrophosphate. We identified the functional translational start site and promoter region for dxr and demonstrated that it is expressed as part of a polycistronic mRNA with gcpE and two other genes. Increased expression of this operon was seen in cells overexpressing Dxs1, indicating that transcriptional control is effected by the first enzyme of the pathway via an unknown regulator.
Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.
One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.
While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.
Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycobacterium tuberculosis expression of several of the far-red reporters was readily visualised by eye and three reporters (mCherry, tdTomato, and Turbo-635) fluoresced at a high intensity. Strains expressing mCherry showed no fitness defects in vitro or in macrophages. Treatment of cells with antibiotics demonstrated that mCherry could also be used as a reporter for cell death, since fluorescence decreased in the presence of a bactericidal compound, but remained stable in the presence of a bacteriostatic compound. mCherry was functional under hypoxic conditions; using mCherry we demonstrated that the PmtbB is expressed early in hypoxia and progressively down-regulated. mCherry and other far-red fluorescent proteins will have multiple uses in investigating the biology of mycobacteria, particularly under non-replicating, or low cell density conditions, as well as providing a novel means of detecting cell death rapidly.
Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome.
Mutants lacking either impA (Rv1604) or suhB (Rv2701c) were isolated in the absence of exogenous inositol, and no differences in levels of PIMs, LM, LAM or mycothiol were observed. Mutagenesis of cysQ (Rv2131c) was initially unsuccessful, but was possible when a porin-like gene of Mycobacterium smegmatis was expressed, and also by gene switching in the merodiploid strain. In contrast, we could only obtain mutations in impC (Rv3137) when a second functional copy was provided in trans, even when exogenous inositol was provided. Experiments to obtain a mutant in the presence of a second copy of impC containing an active-site mutation, in the presence of porin-like gene of M. smegmatis, or in the absence of inositol 1-phosphate synthase activity, were also unsuccessful. We showed that all four genes are expressed, although at different levels, and levels of inositol phosphatase activity did not fall significantly in any of the mutants obtained.
We have shown that neither impA, suhB nor cysQ is solely responsible for inositol synthesis. In contrast, we show that impC is essential for mycobacterial growth under the conditions we used, and suggest it may be required in the early stages of mycothiol synthesis.
Glutamine synthetase (GS) plays an important role in nitrogen assimilation. The major GS of Mycobacterium tuberculosis is GlnA1, a type I GS whose activity is controlled by posttranscriptional modification by GlnE. GlnE is an adenylyl transferase comprised of an adenylylating domain and a deadenylylating domain which modulate GS activity. We previously demonstrated that GlnE is essential in M. tuberculosis in normal growth medium. In this study, we further show that GlnE is required under multiple medium conditions, including in nitrogen-limited medium. We demonstrate that adenylylation is the critical activity for M. tuberculosis survival, since we were able to delete the deadenylylation domain with no apparent effect on growth or GS activity. Furthermore, we identified a critical aspartate residue in the proposed nucleotidyltransferase motif. Temperature-sensitive mutants of GlnE were generated and shown to have a defect in growth and GS activity in nitrogen-limited medium. Finally, we were able to generate a GlnE null mutant in the presence of l-methionine sulfoximine, a GS inhibitor, and glutamine supplementation. In the presence of these supplements, the null mutant was able to grow similarly to the wild type. Surprisingly, the GlnE mutant was able to survive and grow for extended periods in liquid medium, but not on solid medium, in the absence of GS inhibition. Thus, we have confirmed that the unusual requirement of M. tuberculosis for GlnE adenylylation activity is linked to the activity of GS in the cell.
Arabinan polymers are major components of the cell wall in Mycobacterium tuberculosis and are involved in maintaining its structure, as well as playing a role in host-pathogen interactions. In particular, lipoarabinomannan (LAM) has multiple immunomodulatory effects. In the nonpathogenic species Mycobacterium smegmatis, EmbC has been identified as a key arabinosyltransferase involved in the incorporation of arabinose into LAM, and an embC mutant is viable but lacks LAM. In contrast, we demonstrate here that in M. tuberculosis, embC is an essential gene under normal growth conditions, suggesting a more crucial role for LAM in the pathogenic mycobacteria. M. tuberculosis EmbC has an activity similar to that of M. smegmatis EmbC, since we were able to complement an embC mutant of M. smegmatis with embCMtb, confirming that it encodes a functional arabinosyltransferase. In addition, we observed that the size of LAM produced in M. smegmatis was dependent on the level of expression of embCMtb. Northern analysis revealed that embC is expressed as part of a polycistronic message encompassing embC and three upstream genes. The promoter region for this transcript was identified and found to be up-regulated in stationary phase but down-regulated during hypoxia-induced nonreplicating persistence. In conclusion, we have identified one of the key genes involved in LAM biosynthesis in M. tuberculosis and confirmed its essential role in this species.
Fosmidomycin is a phosphonic antibiotic which inhibits 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr), the first committed step of the non-mevalonate pathway of isoprenoid biosynthesis. In Mycobacterium tuberculosis Dxr is encoded by Rv2870c, and although the antibiotic has been shown to inhibit the recombinant enzyme , mycobacteria are intrinsically resistant to fosmidomycin at the whole cell level. Fosmidomycin is a hydrophilic molecule and in many bacteria its uptake is an active process involving a cAMP dependent glycerol-3-phosphate transporter (GlpT). The fact that there is no glpT homologue in the M. tuberculosis genome and the highly impervious nature of the hydrophobic mycobacterial cell wall suggests that resistance may be due to a lack of cellular penetration.
We demonstrated that dxr (Rv2780c) is an essential gene in M. tuberculosis, since we could not delete the chromosomal copy unless a second functional copy was provided on an integrating vector. This confirmed that the intracellular target of fosmidomycin was essential as well as sensitive. We looked at the uptake of fosmidomycin in two mycobacterial species, the slow-growing pathogenic M. tuberculosis and the fast-growing, saprophytic Mycobacterium smegmatis; both species were resistant to fosmidomycin to a high level. Fosmidomycin was not accumulated intra-cellularly in M. tuberculosis or M. smegmatis but remained in the extra-cellular medium. In contrast, fosmidomycin uptake was confirmed in the sensitive organism, Escherichia coli. We established that the lack of intra-cellular accumulation was not due to efflux, since efflux pump inhibitors had no effect on fosmidomycin resistance. Finally, we demonstrated that fosmidomycin was not modified by mycobacterial cells or by extracts but remained in a fully functional state.
Taken together, these data demonstrate that fosmidomycin resistance in M. tuberculosis and M. smegmatis results from a lack of penetration of the antibiotic to the site of the sensitive target.
Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway that forms phenylalanine and tyrosine in bacteria, fungi, plants, and apicomplexan parasites. Since this enzyme is absent from mammals, it represents a promising target for the development of new antimycobacterial drugs, which are needed to combat Mycobacterium tuberculosis, the causative agent of tuberculosis. Until recently, two putative open reading frames (ORFs), Rv0948c and Rv1885c, showing low sequence similarity to CMs have been described as “conserved hypothetical proteins” in the M. tuberculosis genome. However, we and others demonstrated that these ORFs are in fact monofunctional CMs of the AroQ structural class and that they are differentially localized in the mycobacterial cell. Since homologues to the M. tuberculosis enzymes are also present in Mycobacterium smegmatis, we cloned the coding sequences corresponding to ORFs MSMEG5513 and MSMEG2114 from the latter. The CM activities of both ORFs was determined, as well as their translational start sites. In addition, we analyzed the promoter activities of three M. tuberculosis loci related to phenylalanine and tyrosine biosynthesis under a variety of conditions using M. smegmatis as a surrogate host. Our results indicate that the aroQ (Rv0948c), *aroQ (Rv1885c), and fbpB (Rv1886c) genes from M. tuberculosis are constitutively expressed or subjected to minor regulation by aromatic amino acids levels, especially tryptophan.