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Microbial amino acid biosynthetic pathways are underexploited for the development of anti-bacterial agents. N-acetyl glutamate synthase (ArgA) catalyses the first committed step in L-arginine biosynthesis and is essential for M. tuberculosis growth. Here, we have purified and optimized assay conditions for the acetylation of l-glutamine by ArgA. Using the optimized conditions, high throughput screening was performed to identify ArgA inhibitors. We identified 2,5-Bis (2-chloro-4-guanidinophenyl) furan, a dicationic diaryl furan derivatives, as ArgA inhibitor, with a MIC 99 values of 1.56 μM against M. tuberculosis. The diaryl furan derivative displayed bactericidal killing against both M. bovis BCG and M. tuberculosis. Inhibition of ArgA by the lead compound resulted in transcriptional reprogramming and accumulation of reactive oxygen species. The lead compound and its derivatives showed micromolar binding with ArgA as observed in surface plasmon resonance and tryptophan quenching experiments. Computational and dynamic analysis revealed that these scaffolds share similar binding site residues with L-arginine, however, with slight variations in their interaction pattern. Partial restoration of growth upon supplementation of liquid cultures with either L-arginine or N-acetyl cysteine suggests a multi-target killing mechanism for the lead compound. Taken together, we have identified small molecule inhibitors against ArgA enzyme from M. tuberculosis., (Copyright © 2021. Published by Elsevier B.V.)

Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.

Peptidoglycan comprises repeating units of N-acetylmuramic acid, N-acetylglucosamine and short cross-linking peptides. After the conversion of UDP-N-acetylglucosamine (UNAG) to UDP-N-acetylmuramic acid (UNAM) by the MurA and MurB enzymes, an amino acid is added to UNAM by UDP-N-acetylmuramic acid L-alanine ligase (MurC). As peptidoglycan is an essential component of the bacterial cell wall, the enzymes involved in its biosynthesis represent promising targets for the development of novel antibacterial drugs. Here, the crystal structure of Mycobacterium bovis MurC (MbMurC) is reported, which exhibits a three-domain architecture for the binding of UNAM, ATP and an amino acid as substrates, with a nickel ion at the domain interface. The ATP-binding loop adopts a conformation that is not seen in other MurCs. In the UNAG-bound structure of MbMurC, the substrate mimic interacts with the UDP-binding domain of MbMurC, which does not invoke rearrangement of the three domains. Interestingly, the glycine-rich loop of the UDP-binding domain of MbMurC interacts through hydrogen bonds with the glucose moiety of the ligand, but not with the pyrophosphate moiety. These findings suggest that UNAG analogs might serve as potential candidates for neutralizing the catalytic activity of bacterial MurC.

An in situ screening assay for UDP-galactopyranose mutase (UGM, an essential enzyme of M. tuberculosis cell wall biosynthesis) has been developed to discover novel UGM inhibitors. The approach is based on the amide-forming reaction of an amino acid core with various cinnamic acids, followed by a direct fluorescence polarization assay to identify the best UGM binders without isolation and purification of the screened ligands. This assay allows us to perform one-pot high-throughput synthesis and screening of enzyme inhibitors in a 384-well plate format. UGM ligands were successfully identified by this technology and their inhibition levels were established from pure synthetic compounds in vitro and in a whole cell antibacterial assay. This study provides a blueprint for designing enamide structures as new UGM inhibitors and anti-mycobacterial agents.

The ability to perform genetic manipulation of mycobacteria is important for characterization of gene function. Homologous recombination-based protocols are frequently used for reverse genetics studies with mycobacteria. It is known that Mycobacteriumbovis BCG Russia, closely related to M. bovis BCG Moreau, is a natural recA deficient strain and is non-permissive to homologous recombination assays. In this work we show that M. bovis BCG Moreau is also deficient in homologous recombination, shown by a specialized transduction assay, but this phenotype can be reverted by complementation with heterologous recombinases, using a recombineering protocol. Sequence analysis of the genes known to be involved in homologous recombination annotated in the genome of BCG Moreau detected no differences compared to the genome of BCG Pasteur. Further studies are needed in order to determine the exact mechanism underlying this deficiency in BCG Moreau., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

CpG motifs in DNA sequences are recognized by Toll-like receptor 9 and activate immune cells. Bacterial genomic DNA (gDNA) has modified cytosine bases (5-methylcytosine [5 mC]) and modified adenine bases (6-methyladenine [6 mA]). 5 mC inhibits immune activation by CpG DNA; however, it is unclear whether 6 mA inhibits immune activation by CpG DNA. Mycobacterium bovis BCG (BCG) has three adenine methyltransferases (MTases) that act on specific target sequences. In this study, we examined whether the 6 mA at the target sites of adenine MTases affected the immunostimulatory activity of CpG DNA. Our results showed that only 6 mA located at the target sequence of mamA, an adenine MTase from BCG, enhanced interleukin (IL)-12p40 production from murine bone marrow-derived macrophages (BMDMs) stimulated with CpG DNA. Enhancement of IL-12p40 production in BMDMs was also observed when BMDMs were stimulated with CpG DNA ligated to oligodeoxynucleotides (ODNs) harboring 6 mA. Accordingly, we then evaluated whether gDNA from adenine MTase-deficient BCG was less efficient with regard to stimulation of BMDMs. Indeed, gDNA from a mamA-deficient BCG had less ability to activate BMDMs than that from wild-type BCG. We concluded from these results that adenine methylation on ODNs and bacterial gDNA may enhance immune activity induced by CpG DNA., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosis produces mycolic acids which are relevant for persistence, recalcitrance to antibiotics and defiance to host immunity. c-di-GMP is a second messenger involved in transition from planktonic cells to biofilms, whose levels are controlled by diguanylate cyclases (DGC) and phosphodiesterases (PDE). The transcriptional regulator dosR, is involved in response to low oxygen, a condition likely happening to a subset of cells within biofilms. Here, we found that in M. bovis BCG, expression of both BCG1416c and BCG1419c genes, which code for a DGC and a PDE, respectively, decreased in both stationary phase and during biofilm production. The kasA , kasB , and fas genes, which are involved in mycolic acid biosynthesis, were induced in biofilm cultures, as was dosR , therefore suggesting an inverse correlation in their expression compared with that of genes involved in c-di-GMP metabolism. The relative abundance within trehalose dimycolate (TDM) of α-mycolates decreased during biofilm maturation, with methoxy mycolates increasing over time, and keto species remaining practically stable. Moreover, addition of synthetic c-di-GMP to mid-log phase BCG cultures reduced methoxy mycolates, increased keto species and practically did not affect α-mycolates, showing a differential effect of c-di-GMP on keto- and methoxy-mycolic acid metabolism.

Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (Mtb), caused by amino acid substitutions in DNA gyrase, has been increasingly reported worldwide. WQ-3810 is a newly developed FQ that is highly active against FQ-resistant pathogens; however, its activity against Mtb has not been evaluated. Herein we examined the efficacy of WQ-3810 against Mtb through the use of recombinant Mtb DNA gyrases. In addition, in vitro antimycobacterial activity of WQ-3810 was evaluated against recombinant Mtb var. bovis Bacille Calmette-Guérin strains in which gyrase-coding genes were replaced with Mtb variants containing resistance-conferring mutations. WQ-3810 showed a higher inhibitory activity than levofloxacin against most recombinant DNA gyrases with FQ-resistance mutations. Furthermore, WQ-3810 showed inhibition even against a DNA gyrase variant harboring a G88C mutation which is thought to confer the highest resistance against FQs in clinical Mtb isolates. In contrast, the FQ susceptibility test showed that WQ-3810 had relatively weak mycobactericidal activity compared with moxifloxacin. However, the combination of WQ-3810 and ethambutol showed the greatest degree of synergistic activity against recombinant strains. Since FQs and ethambutol have been used in multi-drug therapy for tuberculosis, WQ-3810 might represent a new, potent anti-tuberculosis drug that can be effective even against FQ-resistant Mtb strains., Competing Interests: Declaration of competing interest None to declare., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) is a global health concern, and this situation has further worsened due to the emergence of drug-resistant strains and the failure of BCG vaccine to impart protection. There is an imperative need to develop highly sensitive, specific diagnostic tools, novel therapeutics, and vaccines for the eradication of TB. In the present study, a chemical screen of a pharmacologically active compound library was performed to identify antimycobacterial compounds. The phenotypic screen identified a few novel small-molecule inhibitors, including NU-6027, a known CDK-2 inhibitor. We demonstrate that NU-6027 inhibits Mycobacterium bovis BCG growth in vitro and also displayed cross-reactivity with Mycobacterium tuberculosis protein kinase D (PknD) and protein kinase G (PknG). Comparative structural and sequence analysis along with docking simulation suggest that the unique binding site stereochemistry of PknG and PknD accommodates NU-6027 more favorably than other M. tuberculosis Ser/Thr protein kinases. Further, we also show that NU-6027 treatment induces the expression of proapoptotic genes in macrophages. Finally, we demonstrate that NU-6027 inhibits M. tuberculosis growth in both macrophage and mouse tissues. Taken together, these results indicate that NU-6027 can be optimized further for the development of antimycobacterial agents., (Copyright © 2019 American Society for Microbiology.)

The Mycobacterium bovis Bacille Calmette Guérin (BCG) vaccine shows variable efficacy in protection against adult tuberculosis (TB). Earlier, we have described a BCG mutant vaccine with a transposon insertion in the gene coding for the secreted acid phosphatase SapM, which led to enhanced long-term survival of vaccinated mice challenged with TB infection. To facilitate development of this mutation as part of a future improved live attenuated TB vaccine, we have now characterized the genome and transcriptome of this sapM::Tn mutant versus parental BCG Pasteur. Furthermore, we show that the sapM::Tn mutant had an equal low pathogenicity as WT BCG upon intravenous administration to immunocompromised SCID mice, passing this important safety test. Subsequently, we investigated the clearance of this improved vaccine strain following vaccination and found a more effective innate immune control over the sapM::Tn vaccine bacteria as compared to WT BCG. This leads to a fast contraction of IFNγ producing Th1 and Tc1 cells after sapM::Tn BCG vaccination. These findings corroborate that a live attenuated vaccine that affords improved long-term survival upon TB infection can be obtained by a mutation that further attenuates BCG. These findings suggest that an analysis of the effectiveness of innate immune control of the vaccine bacteria could be instructive also for other live attenuated TB vaccines that are currently under development, and encourage further studies of SapM mutation as a strategy in developing a more protective live attenuated TB vaccine., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

M. tuberculosis (Mtb) , which causes tuberculosis disease, continues to be a major global health threat. Correct identification of valid drug targets is important for the development of novel therapeutics that would shorten the current 6-9 month treatment regimen and target resistant bacteria. Methionine aminopeptidases (MetAP), which remove the N-terminal methionine from newly synthesized proteins, are essential in all life forms (eukaryotes and prokaryotes). The MetAPs contribute to the cotranslational control of proteins as they determine their half life (N-terminal end rule) and facilitate further modifications such as acetylation and others. Mtb (and M. bovis ) possess two MetAP isoforms, MetAP1a and MetAP1c, encoded by the mapA and mapB genes, respectively. Conflicting evidence was reported in the literature on which of the two variants is essential. To resolve this question, we performed a targeted genetic deletion of each of these two genes. We show that a deletion mutant of mapA is viable with only a weak growth defect. In contrast, we provide two lines of genetic evidence that mapB is indispensable. Furthermore, construction of double-deletion mutants as well as the introduction of point mutations into mapB resulting in proteins with partial activity showed partial, but not full, redundancy between mapB and mapA . We propose that it is MetAP1c ( mapB ) that is essentially required for mycobacteria and discuss potential reasons for its vitality.

We developed a method involving recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) and recombinant Mycobacterium smegmatis to determine which mutations in Mycobacterium tuberculosis (Mtb) gyrBA are associated with fluoroquinolone (FQ) resistance. The minimal inhibitory concentration (MIC) for FQ for recombinant strains with wild-type Mtb gyrBA was equivalent to that for strains with intrinsic gyrBA. Among 27 gyrBA mutations, the fold-changes in FQ MIC for M. smegmatis and M. bovis BCG backgrounds were comparable and were in part equivalent to those previously reported for recombinant Mtb strains. Mutations at position 90 or 94 of gyrA conferred strong and synergistic FQ resistance, which may be associated with the clinical observation that isolates carrying these mutations are the most or second most frequent. Sitafloxacin hydrate had the lowest MIC among the FQs tested in this study, which is similar to findings from a previous in vivo animal study. Most gyrBA mutations detected in clinical Mtb isolates could confer FQ resistance, but several mutations reduced bacterial growth rates. Overall, recombinant M. smegmatis appears to be a beneficial surrogate system to evaluate FQ susceptibility of virulent mycobacteria.

Mycobacterium tuberculosis, the causative agent of tuberculosis, has surpassed HIV as the leading cause of death due to an infectious disease worldwide, being responsible for more than 1.5 million deaths in low-income countries. In response to a pandemic threat by drug resistant strains, the tuberculosis research community is searching for new chemical entities with novel mechanisms of action to avoid drug resistance and shorten treatment regimens using combinatorial chemotherapy. Herein, we have identified several novel chemical scaffolds, GSK97C (spiro-oxazolidin-2-one), GSK93A (2-amino-1,3-thiazole, GSK85A and GSK92A (enamides), which target M. tuberculosis aspartyl-tRNA synthetase (Mt-AspRS), an essential component of the protein synthesis machinery of tuberculosis, using a whole-cell target-based screening strategy against a genetically modified Mycobacterium bovis BCG strain. We also provide further evidence of protein inhibition and inhibitor profiling through a classical aminoacylation reaction and a tRNA-independent assay, respectively. Altogether, our results have identified a number of hit new molecules with novel mechanism of action for further development through medicinal chemistry as hits and leads.

Cyclic di-AMP (c-di-AMP) has been shown to play an important role in bacterial physiology and pathogen-host interactions. We previously reported that deletion of the sole c-di-AMP phosphodiesterase-encoding gene (cnpB) in Mycobacterium tuberculosis (Mtb) led to significant virulence attenuation. In this study, we found that ΔcnpB of M. bovisbacillus Calmette-Guerin (BCG) was unable to secrete c-di-AMP, which differs from Mtb ΔcnpB. We infected bone marrow-derived macrophages (BMDMs) with c-di-AMP-associated mutants generated from both Mtb and BCG. Our results showed that upon infection with Mtb ΔcnpB, BMDMs of wildtype mice secreted a large amount of interferon-β (IFN-β) post-infection similarly as we reported previously. In contrast, the response was less pronounced with BMDMs isolated from cGAS-/- mice and was nearly abolished with BMDMs prepared from STING-/- mice. Deletion of the region of difference 1 (RD1) locus in Mtb ΔcnpB did not alter the c-di-AMP secretion of ΔcnpB but eliminated the IFN-β production in the infected cells. In contrast, neither BCG ΔcnpB nor a recombinant BCG ΔcnpB with a pRD1 cosmid induced a type I interferon response. Interestingly, multiple studies have demonstrated that type I IFN enhances BCG's immunity. Thus, amending BCG based on our findings might improve BCG vaccination.

Mycobacterium tuberculosis is a major health threat, necessitating novel drug targets. Protein synthesis in bacteria uses initiator tRNA i charged with formylated methionine residue. Deletion of the formylase gene, tRNA fMet -formyl transferase (fmt), causes severe growth-retardation in E. coli and in S. pneumoniae, but not in P. aeruginosa or S. aureus. fmt was predicted to be essential in M. tuberculosis by transposon library analysis, but this was never formally tested in any mycobacteria. We performed a targeted deletion of fmt in M. smegmatis as well as Mtb-complex (M. bovis). In both cases, we created a mero-diploid strain, deleted the native gene by two-step allelic exchange or specialized-phage transduction, and then removed the complementing gene to create full deletion mutants. In M. smegmatis a full deletion strain could be easily created. In contrast, in M. bovis-BCG, a full deletion strain could only be created after incubation of 6 weeks, with a generation time ~2 times longer than for wt bacteria. Our results confirm the importance of this gene in pathogenic mycobacteria, but as the deletion mutant is viable, validity of fmt as a drug target remains unclear. Our results also refute the previous reports that fmt is essential in M. tuberculosis-complex.

Mycobacterium tuberculosis PtpA is a secreted effector protein that dephosphorylates several proteins in the host cell cytoplasm, such as p-JNK, p-p38, and p-VPS33B, leading to suppression of host innate immunity. Here we show that, in addition, PtpA enters the nucleus of host cells and regulates the expression of host genes, some of which are known to be involved in host innate immunity or in cell proliferation and migration (such as GADD45A). PtpA can bind directly to the promoter region of GADD45A in vitro. Both phosphatase activity and DNA-binding ability of PtpA are important in suppressing host innate immune responses. Furthermore, PtpA-expressing Mycobacterium bovis BCG promotes proliferation and migration of human lung adenoma A549 cells in vitro and in a mouse xenograft model. Further research is needed to test whether mycobacteria, via PtpA, might affect cell proliferation or migration in humans. Mycobacterium tuberculosis secretes a protein, PtpA, that dephosphorylates proteins in the host cell cytoplasm, weakening immune responses. Here, the authors show that PtpA also enters the nucleus, affects the expression of several host genes, and promotes proliferation and migration of a cancer cell line.

Signal transduction in bacteria is generally mediated via two-component systems. These systems depend on the transfer of a phosphate molecule from a donor to an acceptor by histidine kinases, thereby activating the acceptor to allow downstream signaling/activation. Several bacterial genomes, including the genome of M. tuberculosis, were shown to encode eukaryotic-like kinases. To better understand the function of these kinases and the regulatory networks within which they operate, identification of downstream targets is essential. We here present a straightforward approach for the identification of bacterial Ser/Thr-kinase substrates. This approach is based on the KESTREL (Kinase Tracking and Substrate Elucidation) procedure combined with reversed-phase chromatography and two-dimensional gel electrophoresis. Using this method, GarA was identified as one potential substrate for the mycobacterial Ser/Thr-protein kinase G (PknG). These results show that the modified KESTREL approach can be successfully employed for the identification of substrates for bacterial Ser/Thr-kinases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

As a eukaryotic-like Ser/Thr protein kinase, Mycobacterium tuberculosis virulent effector protein kinase G (PknG) mediates mycobacterial survival by regulating bacterial cell metabolic processes and preventing phagosome-lysosome fusion in host macrophages. Targeting PknG is an effective strategy for development of anti-tuberculosis (TB) drugs. In the study, we found that sclerotiorin, derived from marine fungi from the South China Sea, exhibited moderately strong inhibitory effects on recombinant PknG, with an IC 50 value of 76.5 μM, and acted as a non-competitive inhibitor. The dissociation constant (K D ) of sclerotiorin determined by MST was 11.4 μM, demonstrating a moderate binding strength between them. Sclerotiorin could substantially impair the mycobacterial survival in infected macrophages while the macrophage viability remained unaffected, though it did not inhibit the mycobacterial growth in culture. When sclerotiorin was used in combination with rifampicin, intracellular mycobacterial growth decreased as sclerotiorin concentration increased. Docking analysis suggested a binding mechanism of inhibition with performing interactions with the P-loop and catalytic loop of PknG. In summary, we reported that sclerotiorin had moderately strong PknG inhibitory activity, but no cytotoxicity, and it could substantially decrease the mycobacterial growth inside macrophages, suggesting that sclerotiorin has potential to supplement antibiotic therapy for TB., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This chapter provides a step-by-step protocol using activity-based protein profiling (ABPP) as a chemical-proteomic tool to survey the antibiotic properties of a small molecule. Here, we investigate the molecular mechanism behind the bactericidal activity of tetrahydrolipstatin (THL). ABPP relies on small molecule probes that target the active site of specific enzymes in complex proteomes. These probes in turn are equipped with a reporter tag that allows capturing, visualization, enrichment, identification, and quantification of its targets either in vitro or in situ. THL possesses bactericidal activities, but its precise spectrum of molecular targets is poorly characterized. Here, we used THL analogs functionalized to enable Huisgen-base cycloaddition, commonly known as "click chemistry," to identify target proteins after enrichment from mycobacterial cell lysates obtained from different physiological conditions.

High-throughput phenotypic screens have re-emerged as screening tools in antibiotic discovery. The advent of such technologies has rapidly accelerated the identification of 'hit' compounds. A pre-requisite to medicinal chemistry optimisation programmes required to improve the drug-like properties of a 'hit' molecule is identification of its mode of action. Herein, we have combined phenotypic screening with a biased target-specific screen. The inosine monophosphate dehydrogenase (IMPDH) protein GuaB2 has been identified as a drugable target in Mycobacterium tuberculosis, however previously identified compounds lack the desired characteristics necessary for further development into lead-like molecules. This study has identified 7 new chemical series from a high-throughput resistance-based phenotypic screen using Mycobacterium bovis BCG over-expressing GuaB2. Hit compounds were identified in a single shot high-throughput screen, validated by dose response and subjected to further biochemical analysis. The compounds were also assessed using molecular docking experiments, providing a platform for their further optimisation using medicinal chemistry. This work demonstrates the versatility and potential of GuaB2 as an anti-tubercular drug target.

Enhancement of the T cell-stimulating ability of Mycobacterium bovis BCG (BCG) is necessary to develop an effective tuberculosis vaccine. For this purpose, we introduced the PEST-HSP70-major membrane protein-II (MMPII)-PEST fusion gene into ureC-gene depleted recombinant (r) BCG to produce BCG-PEST. The PEST sequence is involved in the proteasomal processing of antigens. BCG-PEST secreted the PEST-HSP70-MMPII-PEST fusion protein and more efficiently activated human monocyte-derived dendritic cells (DCs) in terms of phenotypic changes and cytokine productions than an empty-vector-introduced BCG or HSP70-MMPII gene-introduced ureC gene-depleted BCG (BCG-DHTM). Autologous human naïve CD8 + T cells and naïve CD4 + T cells were effectively activated by BCG-PEST and produced IFN-γ in an antigen-specific manner through DCs. These T cell activations were closely associated with phagosomal maturation and intraproteasomal protein degradation in antigen-presenting cells. Furthermore, BCG-PEST produced long-lasting memory-type T cells in C57BL/6 mice more efficiently than control rBCGs. Moreover, a single subcutaneous injection of BCG-PEST more effectively reduced the multiplication of subsequent aerosol-challenged Mycobacterium tuberculosis of the standard H37Rv strain and clinically isolated Beijing strain in the lungs than control rBCGs. The vaccination effect of BCG-PEST lasted for at least 6months. These results indicate that BCG-PEST may be able to efficiently control the spread of tuberculosis in human., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unlabelled: Phthiocerol dimycocerosates (PDIM) are a group of cell surface-associated apolar lipids of Mycobacterium tuberculosis and closely related mycobacteria, such as Mycobacterium bovis and Mycobacterium leprae A characteristic methoxy group of these lipids is generated from the methylation of a hydroxyl group of the direct precursors, the phthiotriols. The precursors arise from the reduction of phthiodiolones, the keto intermediates, by a ketoreductase. The putative phthiodiolone ketoreductase (PKR) is encoded by Rv2951c in M. tuberculosis and BCG_2972c in M. bovis BCG, and these open reading frames (ORFs) encode identical amino acid sequences. We investigated the cofactor requirement of the BCG_2972c protein. A comparative analysis based on the crystallographic structures of similar enzymes identified structural elements for binding of coenzyme F420 and hydrophobic phthiodiolones in PKR. Coenzyme F420 is a deazaflavin coenzyme that serves several key functions in pathogenic and nonpathogenic mycobacteria. We found that an M. bovis BCG mutant lacking F420-dependent glucose-6-phosphate dehydrogenase (Fgd), which generates F420H2 (glucose-6-phosphate + F420 → 6-phosphogluconate + F420H2), was devoid of phthiocerols and accumulated phthiodiolones. When the mutant was provided with F420H2, a broken-cell slurry of the mutant converted accumulated phthiodiolones to phthiocerols; F420H2 was generated in situ from F420 and glucose-6-phosphate by the action of Fgd. Thus, the reaction mixture was competent in reducing phthiodiolones to phthiotriols (phthiodiolones + F420H2 → phthiotriols + F420), which were then methylated to phthiocerols. These results established the mycobacterial phthiodiolone ketoreductase as an F420H2-dependent enzyme (fPKR). A phylogenetic analysis of close homologs of fPKR revealed potential F420-dependent lipid-modifying enzymes in a broad range of mycobacteria., Importance: Mycobacterium tuberculosis is the causative agent of tuberculosis, and phthiocerol dimycocerosates (PDIM) protect this pathogen from the early innate immune response of an infected host. Thus, the PDIM synthesis system is a potential target for the development of effective treatments for tuberculosis. The current study shows that a PDIM synthesis enzyme is dependent on the coenzyme F420 F420 is universally present in mycobacteria and absent in humans. This finding expands the number of experimentally validated F420-dependent enzymes in M. tuberculosis to six, each of which helps the pathogen to evade killing by the host immune system, and one of which activates an antituberculosis drug, PA-824. This work also has relevance to leprosy, since similar waxy lipids are found in Mycobacterium leprae., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

A series of indole and pyridine based 1,3,4-oxadiazole derivatives 5a-t were synthesized and evaluated for their in vitro antitubercular activity against Mycobacterium tuberculosis H37Ra (MTB) and Mycobacterium bovis BCG both in active and dormant state. Compounds 5b, 5e, 5g and 5q exhibited very good antitubercular activity. All the newly synthesized compounds 5a-t were further evaluated for anti-proliferative activity against HeLa, A549 and PANC-1 cell lines using modified MTT assay and found to be noncytotoxic. On the basis of cytotoxicity and MIC values against Mycobacterium bovis BCG, selectivity index (SI) of most active compounds 5b, 5e, 5g and 5q was calculated (SI=GI50/MIC) in active and dormant state. Compounds 5b, 5e and 5g demonstrated SI values ⩾10 against all three cell lines and were found to safe for advance screening. Compounds 5a-t were further screened for their antibacterial activity against four bacteria strains to assess their selectivity towards MTB. In addition, the molecular docking studies revealed the binding modes of these compounds in active site of enoyl reductase (InhA), which in turn helped to establish a structural basis of inhibition of mycobacteria. The potency, low cytotoxicity and selectivity of these compounds make them valid lead compounds for further optimization., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: MDR Mycobacterium tuberculosis clinical strains that cause large outbreaks, particularly among HIV-negative patients, are likely to have undergone the most successful compensatory evolution. Hence, mutations secondary to the acquisition of drug resistance are worthy of consideration in these highly transmissible strains. Here, we assessed the role of a mutation within rpoB, rpoB V615M, secondary to the rifampicin resistance-conferring mutation rpoB S531L, which is associated with a major MDR tuberculosis outbreak strain that evolved in an HIV-negative context in northern Tunisia., Methods: Using BCG as a model organism, we engineered strains harbouring either the rpoB S531L mutation alone or the double mutation rpoB S531L, V615M. Individual and competitive in vitro growth assays were performed in order to assess the relative fitness of each BCG mutant., Results: The rpoB V615M mutation was found to be invariably associated with rpoB S531L. Structural analysis mapped rpoB V615M to the same bridge helix region as rpoB compensatory mutations previously described in Salmonella. Compared with the rpoB single-mutant BCG, the double mutant displayed improved growth characteristics and fitness rates equivalent to WT BCG. Strikingly, the rpoB double mutation conferred high-level resistance to rifampicin., Conclusions: Here, we demonstrated the fitness compensatory role of a mutation within rpoB, secondary to the rifampicin resistance mutation rpoB S531L, which is characteristic of an MDR M. tuberculosis major outbreak strain. The finding that this secondary mutation concomitantly increased the resistance level to rifampicin argues for its significant contribution to the successful transmission of the MDR-TB strain., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

We recently reported on our success to generate deletion mutants of the genes encoding glutamate dehydrogenase (GDH) and glutamine oxoglutarate aminotransferase (GOGAT) in M. bovis BCG, despite their in vitro essentiality in M. tuberculosis. We could use these mutants to delineate the roles of GDH and GOGAT in mycobacterial nitrogen metabolism by using M. bovis BCG as a model for M. tuberculosis specifically. Here, we extended our investigation towards the involvement of GDH and GOGAT in other aspects of M. bovis BCG physiology, including the use of glutamate as a carbon source and resistance to known phagosomal stresses, as well as in survival inside macrophages. We find that gdh is indispensable for the utilization of glutamate as a major carbon source, in low pH environments and when challenged with nitric oxide. On the other hand, the gltBD mutant had increased viability under low pH conditions and was unaffected by a challenge with nitric oxide. Strikingly, GDH was required to sustain M. bovis BCG during infection of both murine RAW 264.7 and bone-marrow derived and macrophages, while GOGAT was not. We conclude that the catabolism of glutamate in slow growing mycobacteria may be a crucial function during infection of macrophage cells and demonstrate a novel requirement for M. bovis BCG GDH in the protection against acidic and nitrosative stress. These results provide strong clues on the role of GDH in intracellular survival of M. tuberculosis, in which the essentiality of the gdh gene complicates knock out studies making the study of the role of this enzyme in pathogenesis difficult.

The study of genomic variability within various pathogenic and non-pathogenic strains of mycobacteria provides insight into their evolution and pathogenesis. The mycobacterial genome encodes seven cutinase-like proteins and each one of these exhibit distinct characteristics. We describe the presence of Cut5, a member of the cutinase family, in mycobacteria and the existence of a unique genomic arrangement in the cut5 gene of M. tuberculosis (Mtb) strains. A single nucleotide (T) insertion is observed in the cut5 gene, which is specific for Mtb strains. Using in silico analysis and RT-PCR, we demonstrate the transcription of Rv3724/cut5 as Rv3724a/cut5a and Rv3724b/cut5b in Mtb H37Rv and as full length cut5 in M. bovis. Cut5b protein of Mtb H37Rv (MtbCut5b) was found to be antigenically similar to its homologs in M. bovis and M. smegmatis, without any observed cross-reactivity with other Mtb cutinases. Also, the presence of Cut5b in Mtb and its homologs in M. bovis and M. smegmatis were confirmed by western blotting using antibodies raised against recombinant Cut5b. In Mtb H37Rv, Cut5b was found to be localized in the cell wall, cytosol and membrane fractions. We also report the vast prevalence of Cut5 homologs in pathogenic and non pathogenic species of mycobacteria. In silico analysis revealed that this protein has three possible organizations in mycobacteria. Also, a single nucleotide (T) insertion in Mtb strains and varied genomic arrangements within mycobacterial species make Rv3724/Cut5 a potential candidate that can be exploited as a biomarker in Mtb infection.

Modification of acyl carrier proteins (ACP) or domains by the covalent binding of a 4'-phosphopantetheine (4'-PP) moiety is a fundamental condition for activation of fatty acid synthases (FASes) and polyketide synthases (PKSes). Binding of 4'-PP is mediated by 4' phosphopantetheinyl transfersases (PPTases). Mycobacterium tuberculosis (Mtb) possesses two essential PPTases: acyl carrier protein synthase (Mtb AcpS), which activates the multidomain fatty acid synthase I (FAS I), and Mtb PptT, an Sfp-type broad spectrum PPTase that activates PKSes. To date, it has not been determined which of the two Mtb PPTases, AcpS or PptT, activates the meromycolate extension ACP, Mtb AcpM, en route to the production of mycolic acids, the main components of the mycobacterial cell wall. In this study, we tested the enzymatic activation of a highly purified Mtb apo-AcpM to Mtb holo-AcpM by either Mtb PptT or Mtb AcpS. By using SDS-PAGE band shift assay and mass spectrometry analysis, we found that Mtb PptT is the PPTase that activates Mtb AcpM. We measured the catalytic activity of Mtb PptT toward CoA, using an activation assay of a blue pigment synthase, BpsA (a nonribosomal peptide synthase, NRPS). BpsA activation by Mtb PptT was inhibited by Mtb apo-AcpM through competition for CoA, in accord with Mtb AcpM activation. A structural model of the putative interaction between Mtb PptT and Mtb AcpM suggests that both hydrophobic and electrostatic interactions stabilize this complex. To conclude, activation of Mtb AcpM by Mtb PptT reveals a potential target of the multistep mycolic acid biosynthesis.

PknJ (Rv2088) is a serine/threonine protein kinase of mycobacteria which is present in Mycobacterium tuberculosis (MTB), but its gene is absent in Mycobacterium smegmatis (MS); a fast grower and nonpathogenic species of mycobacteria. The heterologous expression of MTB-specific PknJ in MS altered the growth of recombinant mycobacteria highlighting one of the characteristics of this protein. This nature of the protein was further confirmed when Mycobacterium bovis BCG (BCG) containing antisense copy of pknJ resulted in the increased growth of BCG. The real-time RNA quantification analysis pointed out toward increased expression of this protein during infection of THP-1 macrophage cells which further emphasized that the protein is essential for the intracellular survival of mycobacteria. The differential in gel electrophoresis (DIGE) data followed by mass spectroscopy suggested that PknJ is involved in regulation of pyruvate kinase A (Rv1617). Since pyruvate kinase (PK) A is one of the key enzymes which controls glycolytic cycle in mycobacteria, we looked for its interaction with PknJ during extracellular and intracellular growth of mycobacteria. In order to identify the specific residue(s) involved in post-translational modification, the phospho-null mutants of PK were generated, and their substrate specificities in response to PknJ were assessed through kinase assay. The findings thus underlined that the PK activity is predominantly dependent on the threonine residue at the 94(th) position and further suggested that this site may be plausible in intracellular survival of mycobacteria upon phosphorylation with PknJ.

The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at (535)GAC>(535)AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å.

Objective: To clone, express, and characterize Mycobacterium tuberculosis (Mtb) ClpP2, and evaluated the potential usage of ClpP2 in clinical diagnosis of tuberculosis., Methods: Mtb ClpP2 was cloned into recombinant plasmid pET32a (+) and transformed into E. coli BL21 (DE3). SDS-PAGE and Western blot analysis were performed to detect the expression of the recombinant protein. The immunogenicity of Mtb ClpP2 was assessed with epitope prediction and antibody titer assay. Quantitative real-time PCR was performed to detect the influence of stress conditions on ClpP2 expression. ClpP2 antigen and antibody in patients with pulmonary diseases were detected by indirect ELISA. ROC curve was constructed to assess the diagnostic accuracy of Mtb ClpP2 for tuberculosis., Results: We had cloned and expressed recombinant Mtb ClpP2 in E. coli. Our results showed that Mtb ClpP2 had potent immunogenicity, and our own prepared polyclonal antibody could be used in detection and diagnostic tests. Results from Western blot showed that ClpP2 was mainly located in M. bovis BCG cytoplasm, and real-time PCR indicated that stress conditions could enhance the mRNA expression of ClpP2. Indirect ELISA suggested that, in tuberculosis patients, both the levels of ClpP2 antigen and antibody were increased, and the positive rates of ClpP2 were elevated. ROC curve had demonstrated satisfactory sensitivity and specificity of ClpP2-based diagnosis for tuberculosis., Conclusion: Our results suggest that Mtb ClpP2 antigens would be used as a biomarker in tuberculosis pathogenesis. These findings highlight the feasibility of the application of Mtb ClpP2 in the clinical diagnosis of tuberculosis.

Mycobacterium bovis is the etiological factor of bovine tuberculosis (BTB), posing a significant problem to domestic cattle. The bacterium is also zoonotic, affecting human health worldwide. Macrophage evasion of the bacterium involves mycobacterial molecules such as MB1684 (ornithine carbamoyltransferase). In this study, we confirmed a concentration-dependent decrease in proliferation of Ana-1 macrophages when treated with rMB1684 when compared with mycobacterium bovis purified protein derivative of tuberculosis (MbPPD) or phosphate buffer solution incubation groups. We examined the activation of nuclear factor-kappa B (NF-κB) upon exposure to MB1684, and its role in MB1684-induced upregulation of interferon (IFN)-γ and proinflammatory cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor-α) in Ana-1 macrophages. The levels of proinflammatory cytokines and IFN-γ were significantly high in MB1684-treated Ana-1 macrophages. The treatment led to an increase in NF-κB activation and a high expression of the just mentioned proinflammatory cytokines. NF-κB inhibition significantly abrogated MB1684-induced upregulation of proinflammatory cytokine mRNA expression, which suggests that MB1684-induced activation of NF-κB in turn stimulates gene expression of IFN-γ and proinflammatory cytokines in Ana-1 macrophages. The experiment was repeated in bone marrow macrophages, a more in-vivo-like model system, and similar results validated our conclusion. Further, we identified the possibility of the application of MB1684 antigen for the detection of BTB in cattle serum.

The flavin-dependent thymidylate synthase X (ThyX), rare in eukaryotes and completely absent in humans, is crucial in the metabolism of thymidine (a DNA precursor) in many microorganisms including several human pathogens. Conserved in mycobacteria, including Mycobacterium leprae, and Mycobacterium tuberculosis, it represents a prospective anti-mycobacterial therapeutic target. In a M. tuberculosis ThyX-enzyme inhibition assay, N-(3-(5-(2'-deoxyuridine-5'-phosphate))prop-2-ynyl)octanamide was reported to be the most potent and selective 5-substituted 2'-deoxyuridine monophosphate analogue. In this study, we masked the two charges at the phosphate moiety of this compound using our ProTide technology in order to increase its lipophilicity and then allow permeation through the complex mycobacterial cell wall. A series of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide phosphoroamidates were chemically synthesized and their biological activity as potential anti-tuberculars was evaluated. In addition to mycobacteria, several DNA viruses depend on ThyX for their DNA biosynthesis, thus these prodrugs were also screened for their antiviral properties., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Mycobacterium bovis bacillus Calmette-Guérin (BCG) is known to be only partially effective in inhibiting M. tuberculosis (MTB) multiplication in human. A new recombinant (r) urease-deficient BCG (BCG-dHCM) that secretes protein composed of heat shock protein (HSP)70, MTB-derived CysO and major membrane protein (MMP)-II was produced for the efficient production of interferon gamma (IFN-γ) which is an essential element for mycobacteriocidal action and inhibition of neutrophil accumulation in lungs., Methods: Human monocyte-derived dendritic cells (DC) and macrophages were differentiated from human monocytes, infected with BCG and autologous T cells-stimulating activity of different constructs of BCG was assessed. C57BL/6 mice were used to test the effectiveness of BCG for the production of T cells responsive to MTB-derived antigens (Ags)., Results: BCG-dHCM intracellularly secreted HSP70-CysO-MMP-II fusion protein, and activated DC by up-regulating Major Histcompatibility Complex (MHC), CD86 and CD83 molecules and enhanced various cytokines production from DC and macrophages. BCG-dHCM activated naïve T cells of both CD4 and CD8 subsets through DC, and memory type CD4+ T cells through macrophages in a manner dependent on MHC and CD86 molecules. These T cell activations were inhibited by the pre-treatment of Ag-presenting cells (APCs) with chloroquine. The single and primary BCG-dHCM-inoculation produced long lasting T cells responsive to in vitro secondarily stimulation with HSP70, CysO, MMP-II and H37Rv-derived cytosolic protein, and partially inhibited the replication of aerosol-challenged MTB., Conclusions: The results indicate that introduction of different type of immunogenic molecules into a urease-deficient rBCG is useful for providing polyclonal T cell activating ability to BCG and for production of T cells responsive to secondary stimulation.

3-Methyladenine DNA glycosylase recognizes and excises a wide range of damaged bases and thus plays a critical role in base excision repair. However, knowledge on the regulation of DNA glycosylase in prokaryotes and eukaryotes is limited. In this study, we successfully characterized a TetR family transcriptional factor from Mycobacterium bovis bacillus Calmette-Guerin (BCG), namely BCG0878c, which directly regulates the expression of 3-methyladenine DNA glycosylase (designated as MbAAG) and influences the base excision activity of this glycosylase at the post-translational level. Using electrophoretic mobility shift assay and DNase I footprinting experiments, we identified two conserved motifs within the upstream region of mbaag specifically recognized by BCG0878c. Significant down-regulation of mbaag was observed in BCG0878c-overexpressed M. bovis BCG strains. By contrast, about 12-fold up-regulation of mbaag expression was found in bcg0878c-deleted mutant M. bovis BCG strains. β-Galactosidase activity assays also confirmed these results. Thus, BCG0878c can function as a negative regulator of mbaag expression. In addition, the regulator was shown to physically interact with MbAAG to enhance the ability of the glycosylase to bind damaged DNA. Interaction between the two proteins was further found to facilitate AAG-catalyzed removal of hypoxanthine from DNA. These results indicate that a TetR family protein can dually regulate the function of 3-methyladenine DNA glycosylase in M. bovis BCG both at the transcriptional and post-translational levels. These findings enhance our understanding of the expression and regulation of AAG in mycobacteria.

Tetrahydrolipstatin (THL) is bactericidal but its precise target spectrum is poorly characterized. Here, we used a THL analog and activity-based protein profiling to identify target proteins after enrichment from whole cell lysates of Mycobacterium bovis Bacillus Calmette-Guérin cultured under replicating and non-replicating conditions. THL targets α/β-hydrolases, including many lipid esterases (LipD, G, H, I, M, N, O, V, W, and TesA). Target protein concentrations and total esterase activity correlated inversely with cellular triacylglycerol upon entry into and exit from non-replicating conditions. Cellular overexpression of lipH and tesA led to decreased THL susceptibility thus providing functional validation. Our results define the target spectrum of THL in a biological species with particularly diverse lipid metabolic pathways. We furthermore derive a conceptual approach that demonstrates the use of such THL probes for the characterization of substrate recognition by lipases and related enzymes.

For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8(+) T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8(+) T cells and perforin-producing effector CD8(+) T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.

The mechanistic basis for the resistance of Mycobacterium tuberculosis to para-aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursor para-aminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance in M. tuberculosis strain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates of M. tuberculosis and Mycobacterium bovis. From a panel of 85 multidrug-resistant M. tuberculosis clinical isolates, 5 were found to harbor mutations in the folC gene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics for M. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug.

Recent evidence suggests that the regulation of intracellular glutamate levels could play an important role in the ability of pathogenic slow-growing mycobacteria to grow in vivo. However, little is known about the in vitro requirement for the enzymes which catalyse glutamate production and degradation in the slow-growing mycobacteria, namely; glutamine oxoglutarate aminotransferase (GOGAT) and glutamate dehydrogenase (GDH), respectively. We report that allelic replacement of the Mycobacterium bovis BCG gltBD-operon encoding for the large (gltB) and small (gltD) subunits of GOGAT with a hygromycin resistance cassette resulted in glutamate auxotrophy and that deletion of the GDH encoding-gene (gdh) led to a marked growth deficiency in the presence of L-glutamate as a sole nitrogen source as well as reduction in growth when cultured in an excess of L-asparagine.

Background: Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid., Results: We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed., Conclusions: Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria.

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette-Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.

Objective: Mycobacterium tuberculosis can survive inside host cells for a long time. To elucidate the acid adaptation mechanism mediated by the asparagine (Asn) metabolic pathway, the activity of asparaginase (AnsA) and growth of mycobacteria were characterized., Methods: The nonpathogenic M. bovis BCG was used as a model strain to characterize AnsA function. The recombinant AnsA was further expressed in E. coli, purified by Co2+ charged resin, and analyzed by enzymatic method. M. bovis BCG mutant (DansA) was constructed by homologous recombination. The growth and ammonia generation by M. bovis BCG wild type and mutant were evaluated in acidic medium., Results: Ammonia was detected in the presence of AnsA and Asn. Deletion of ansA significantly delayed the growth of M. bovis BCG for 10 days in acidic medium. Asn was used by M. bovis BCG to produce ammonia, increasing pH., Conclusion: The acid adaptation of M. bovis BCG was mediated by ammonia, the metabolic product of asparagine. This provides new clue to uncover the survival mechanism of M. tuberculosis inside the acidic niche environment of host macrophage.

The cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, contains lipids with unusual structures. These lipids play a key role in both virulence and resistance to the various hostile environments encountered by the bacteria during infection. They are synthesized by complex enzymatic systems, including type-I polyketide synthases and type-I and -II fatty acid synthases, which require a post-translational modification to become active. This modification consists of the covalent attachment of the 4'-phosphopantetheine moiety of Coenzyme A catalyzed by phosphopantetheinyl transferases (PPTases). PptT, one of the two PPTases produced by mycobacteria, is involved in post-translational modification of various type-I polyketide synthases required for the formation of both mycolic acids and lipid virulence factors in mycobacteria. Here we identify PptT as a new target for anti-tuberculosis drugs; we address all the critical issues of target validation to demonstrate that PptT can be used to search for new drugs. We confirm that PptT is essential for the growth of M. bovis BCG in vitro and show that it is required for persistence of M. bovis BCG in both infected macrophages and immunodeficient mice. We generated a conditional expression mutant of M. tuberculosis, in which the expression of the pptT gene is tightly regulated by tetracycline derivatives. We used this construct to demonstrate that PptT is required for the replication and survival of the tubercle bacillus during the acute and chronic phases of infection in mice. Finally, we developed a robust and miniaturized assay based on scintillation proximity assay technology to search for inhibitors of PPTases, and especially of PptT, by high-throughput screening. Our various findings indicate that PptT meets the key criteria for being a therapeutic target for the treatment of mycobacterial infections.

Pathogenic mycobacteria survive within macrophages by residing in phagosomes, which they prevent from maturing and fusing with lysosomes. Although several bacterial components were seen to modulate phagosome processing, the molecular regulatory mechanisms taking part in this process remain elusive. We investigated whether the phagosome maturation block (PMB) could be modulated by signaling through Ser/Thr phosphorylation. Here, we demonstrated that mycolic acid cyclopropane synthase PcaA, but not MmaA2, was phosphorylated by mycobacterial Ser/Thr kinases at Thr-168 and Thr-183 both in vitro and in mycobacteria. Phosphorylation of PcaA was associated with a significant decrease in the methyltransferase activity, in agreement with the strategic structural localization of these two phosphoacceptors. Using a BCG ΔpcaA mutant, we showed that PcaA was required for intracellular survival and prevention of phagosome maturation in human monocyte-derived macrophages. The physiological relevance of PcaA phosphorylation was further assessed by generating PcaA phosphoablative (T168A/T183A) or phosphomimetic (T168D/T183D) mutants. In contrast to the wild-type and phosphoablative pcaA alleles, introduction of the phosphomimetic pcaA allele in the ΔpcaA mutant failed to restore the parental mycolic acid profile and cording morphotype. Importantly, the PcaA phosphomimetic strain, as the ΔpcaA mutant, exhibited reduced survival in human macrophages and was unable to prevent phagosome maturation. Our results add new insight into the importance of mycolic acid cyclopropane rings in the PMB and provide the first evidence of a Ser/Thr kinase-dependent mechanism for modulating mycolic acid composition and PMB.

Background: Intrinsic multidrug resistance of the Mycobacterium avium-intracellulare complex presents a serious problem in the treatment of the diseases caused by these bacteria. Recently, it was shown that deletion of a polyketide synthase, Pks12, in an M. avium laboratory strain decreases this intrinsic resistance., Methods: We investigated Pks12 expression and its enzymatic activity in 9 clinical isolates of M. intracellulare, and compared their drug susceptibilities to 4 drugs. Also, we made pks12-disrupted M. bovis bacillus Calmette-Guérin (BCG) mutant and its complemented strain. Using these BCG and M. intracellulare strains, we observed intracellular accumulation of ethidium bromide (EtBr)., Results: We found positive correlations between Pks12 and drug resistance for all of the antibiotics tested. The drug susceptible M. intracellulare strain showed higher EtBr accumulation. Consistent with this, EtBr was much more accumulated in pks12-disrupted BCG than wild-type or the complemented strains., Conclusions: Collectively, these results suggest that Pks12 controls the multidrug resistance in part through intracellular drug accumulation., (Copyright © 2012 S. Karger AG, Basel.)

A new vaccination strategy is urgently needed for improved control of the global tuberculosis (TB) epidemic. Using a mouse aerosol Mycobacterium tuberculosis challenge model, we investigated the protective efficacy of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG (ΔmmaA4BCG) formulated in dimethyl dioctadecyl ammonium bromide (DDA) - D(+) trehalose 6,6 dibenenate (TDB) (DDA/TDB) adjuvant. In previous studies, deletion of the mmaA4 gene was shown to reduce the suppression of IL-12 production often seen after mycobacterial infections. While the non-adjuvanted ΔmmaA4BCG strain did not protect mice substantially better than conventional BCG against a tuberculous challenge in four protection experiments, the protective responses induced by the ΔmmaA4BCG vaccine formulated in DDA/TDB adjuvant was consistently increased relative to nonadjuvanted BCG controls. Furthermore, the ΔmmaA4BCG-DDA/TDB vaccine induced significantly higher frequencies of multifunctional (MFT) CD4 T cells expressing both IFNγ and TNFα (double positive) or IFNγ, TNFα and IL-2 (triple positive) than CD4 T cells derived from mice vaccinated with BCG. These MFT cells were characterized by having higher IFNγ and TNFα median fluorescence intensity (MFI) values than monofunctional CD4 T cells. Interestingly, both BCG/adjuvant and ΔmmaA4BCG/adjuvant formulations induced significantly higher frequencies of CD4 T cells expressing TNFα and IL-2 than nonadjuvanted BCG or ΔmmaA4BCG vaccines indicating that BCG/adjuvant mixtures may be more effective at inducing central memory T cells. Importantly, when either conventional BCG or the mutant were formulated in adjuvant and administered to SCID mice or immunocompromised mice depleted of IFNγ, significantly lower vaccine-derived mycobacterial CFU were detected relative to immunodeficient mice injected with non-adjuvanted BCG. Overall, these data suggest that immunization with the ΔmmaA4BCG/adjuvant formulation may be an effective, safe, and relatively inexpensive alternative to vaccination with conventional BCG.

NADH pyrophosphatase (NudC) catalyses the hydrolysis of NAD(H) to AMP and NMN(H) [nicotinamide mononucleotide (reduced form)]. NudC multiple sequence alignment reveals that homologues from most Mycobacterium tuberculosis isolates, but not other mycobacterial species, have a polymorphism at the highly conserved residue 237. To elucidate the functional significance of this polymorphism, comparative analyses were performed using representative NudC isoforms from M. tuberculosis H37Rv (NudC(Rv)) and M. bovis BCG (NudC(BCG)). Biochemical analysis showed that the P237Q polymorphism prevents dimer formation, and results in a loss of enzymatic activity. Importantly, NudC(BCG) was found to degrade the active forms of isoniazid (INH), INH-NAD and ethionamide (ETH), ETH-NAD. Consequently, overexpression of NudC(BCG) in Mycobacterium smegmatis mc(2)155 and M. bovis BCG resulted in a high level of resistance to both INH and ETH. Further genetic studies showed that deletion of the nudC gene in M. smegmatis mc(2)155 and M. bovis BCG resulted in increased susceptibility to INH and ETH. Moreover, inactivation of NudC in both strains caused a defect in drug tolerance phenotype for both drugs in exposure assays. Taken together, these data suggest that mycobacterial NudC plays an important role in the inactivation of INH and ETH., (© 2011 Blackwell Publishing Ltd.)

Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using ¹³C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with ¹³C labeled glycerol or sodium bicarbonate. Through measurements of the ¹³C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate--oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO₂ into biomass. As the human host is abundant in CO₂ this finding requires further investigation in vivo as CO₂ fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using ¹³C-MFA.

An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1-bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB~DbjA>DhlA~DhaA~DbeA~DmbA>DatA~DmbC~DrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.

Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c(+) MHC-II(int) CD40(int) dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination., (Copyright © 2011 EMBO Molecular Medicine.)

Pathogenic strains of mycobacteria produce copious amounts of glutamine synthetase (GS) in the culture medium. The enzyme activity is linked to synthesis of poly-α-l-glutamine (PLG) in the cell walls. This study describes a glnA-1 mutant of Mycobacterium bovis that produces reduced levels of GS. The mutant was able to grow in enriched 7H9 medium without glutamine supplementation. The glnA-1 strain contained no detectable PLG in the cell walls and showed marked sensitivity to different chemical and physical stresses such as lysozyme, SDS and sonication. The sensitivity of the mutant to two antitubercular drugs, rifampicin and d-cycloserine, was also increased. The glnA-1 strain infected THP-1 cells with reduced efficiency and was also attenuated for growth in macrophages. A Mycobacterium smegmatis strain containing the M. bovis glnA-1 gene survived longer in THP-1 cells than the wild-type strain and also produced cell wall-associated PLG. The M. bovis mutant was not able to replicate in the organs of BALB/c mice and was cleared within 4-6 weeks of infection. Disruption of the glnA-1 gene adversely affected biofilm formation on polystyrene surfaces. The results of this study demonstrate that the absence of glnA-1 not only attenuates the pathogen but also affects cell surface properties by altering the cell wall chemistry of the organism via the synthesis of PLG; this may be a target for drug development.