EmbR2, a structural homologue of EmbR, inhibits the Mycobacterium tuberculosis kinase/substrate pair PknH/EmbR

International audienceEmbR is a transcriptional regulator that is phosphorylated by the cognate mycobacterial STPK (serine/threonine protein kinase) PknH. Recent studies demonstrated that PknH-dependent phosphorylation of EmbR enhances its DNA-binding activity and activates the transcription of the embCAB genes encoding arabinosyltransferases, which participate in arabinan biosynthesis. In the present study, we identified a genomic region of 4425 bp, which is present in Mycobacterium tuberculosis CDC1551, but absent from M. tuberculosis H37Rv, comprising the MT3428 gene, which is homologous with embR. Homology modelling of the MT3428 gene product illustrated its close relationship (56% identity) to EmbR, and it was hence termed EmbR2. In marked contrast with EmbR, EmbR2 was not phosphorylated by PknH, although it is a substrate of other M. tuberculosis kinases, including PknE and PknF. Tryptophan fluorescence emission of EmbR2 was monitored in the presence of three different PknH-derived phosphopeptides and demonstrated that EmbR2 binds to at least two of the threonine sites known to undergo autophosphorylation in PknH. We observed that the capacity of EmbR2 to interact physically with PknH without being phosphorylated was a result of EmbR2-mediated inhibition of kinase activity: incubation of PknH with increasing concentrations of EmbR2 led to a dose-response inhibition of the autokinase activity, similarly to O6-cyclohexylmethylguanine, a known inhibitor of eukaryotic cyclin-dependent kinases. Moreover, EmbR2 inhibited PknH-dependent phosphorylation of EmbR in a dose-dependent manner. Together, these results suggest that EmbR2 is a regulator of PknH activation, thus directly participating in the control of the PknH/EmbR pair and potentially in mycobacterial physiology/virulence of M. tuberculosis CDC1551

A truncated lipoglycan from mycobacteria with altered immunological properties

Maintenance of cell-wall integrity in Mycobacterium tuberculosis is essential and is the target of several antitubercular drugs. For example, ethambutol targets arabinogalactan and lipoarabinomannan (LAM) biosynthesis through the inhibition of several arabinofuranosyltransferases. Apart from their role in cell-wall integrity, mycobacterial LAMs also exhibit important immunomodulatory activities. Here we report the isolation and detailed structural characterization of a unique LAM molecule derived from Mycobacterium smegmatis deficient in the arabinofuranosyltransferase AftC (AftC-LAM). This mutant LAM expresses a severely truncated arabinan domain completely devoid of 3,5-Araf–branching residues, revealing an intrinsic involvement of AftC in the biosynthesis of LAM. Furthermore, we found that ethambutol efficiently inhibits biosynthesis of the AftC-LAM arabinan core, unambiguously demonstrating the involvement of the arabinofuranosyltransferase EmbC in early stages of LAM-arabinan biosynthesis. Finally, we demonstrate that AftC-LAM exhibits an enhanced proinflammatory activity, which is due to its ability to activate Toll-like receptor 2 (TLR2). Overall, our efforts further describe the mechanism of action of an important antitubercular drug, ethambutol, and demonstrate a role for specific arabinofuranosyltransferases in LAM biosynthesis. In addition, the availability of sufficient amounts of chemically defined wild-type and isogenic truncated LAMs paves the way for further investigations of the structure–function relationship of TLR2 activation by mycobacterial lipoglycans

Biosynthesis of mycobacterial arabinogalactan: identification of a novel (13)arabinofuranosyltransferase

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis and is the target of several anti-tubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. A bioinformatics approach identified putative integral membrane proteins, MSMEG2785 in Mycobacterium smegmatis, Rv2673 in Mycobacterium tuberculosis and NCgl1822 in Corynebacterium glutamicum, with 10 predicted transmembrane domains and a glycosyltransferase motif (DDX), features that are common to the GT-C superfamily of glycosyltransferases. Deletion of M. smegmatis MSMEG2785 resulted in altered growth and glycosyl linkage analysis revealed the absence of AG (13)-linked arabinofuranosyl (Araf) residues. Complementation of the M. smegmatis deletion mutant was fully restored to a wild type phenotype by MSMEG2785 and Rv2673, and as a result, we have now termed this previously uncharacterized open reading frame, arabinofuranosyltransferase C (aftC). Enzyme assays using the sugar donor -D-arabinofuranosyl-1-monophosphoryldecaprenol (DPA) and a newly synthesized linear (15)-linked Ara5 neoglycolipid acceptor together with chemical identification of products formed, clearly identified AftC as a branching (13) arabinofuranosyltransferase. This newly discovered glycosyltransferase sheds further light on the complexities of Mycobacterium cell wall biosynthesis, such as in M. tuberculosis and related species and represents a potential new drug target

Elucidation of a protein-protein interaction network involved in <i>Corynebacterium glutamicum</i> cell wall biosynthesis as determined by bacterial two-hybrid analysis

Mycobacterium species have a highly complex and unique cell wall that consists of a large macromolecular structure termed the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex. This complex is essential for growth, survival and virulence of the human pathogen Mycobacterium tuberculosis, and is the target of several anti-tubercular drugs. The closely related species Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes and proteins involved in mAGP synthesis. This study examines the construction of a protein-protein interaction network for the major cell wall component arabinogalactan in C. glutamicum based on the use of a bacterial two-hybrid system. We have identified twenty-four putative homotypic and heterotypic protein interactions in vivo. Our results demonstrate an association between glycosyltransferases, GlfT1 and AftB, and interaction between the sub-units of decaprenylphosphoribose epimerase, DprE1 and DprE2. These analyses have also shown that AftB interacts with AftA, which catalyzes the addition of the first three arabinose units onto the galactan chain. Both AftA and AftB associate with other arabinofuranosyltransferases, including Emb and AftC, that elongate and branch the arabinan domain. Moreover, a number of proteins involved in arabinogalactan biosynthesis were shown to form dimers or multimers. These findings provide a useful recourse for understanding the biosynthesis and function of the mycobacterial cell wall, as well as providing new therapeutic targets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10719-014-9549-3) contains supplementary material, which is available to authorized users

Synthesis of α-glucan in mycobacteria involves a hetero-octameric complex of trehalose synthase TreS and Maltokinase Pep2

Recent evidence established that the cell envelope of Mycobacterium tuberculosis, the bacillus causing tuberculosis (TB), is coated by an α-glucan-containing capsule that has been implicated in persistence in a mouse infection model. As one of three known metabolic routes to α-glucan in mycobacteria, the cytoplasmic GlgE-pathway converts trehalose to α(1 → 4),α(1 → 6)-linked glucan in 4 steps. Whether individual reaction steps, catalyzed by trehalose synthase TreS, maltokinase Pep2, and glycosyltransferases GlgE and GlgB, occur independently or in a coordinated fashion is not known. Here, we report the crystal structure of M. tuberculosis TreS, and show by small-angle X-ray scattering and analytical ultracentrifugation that TreS forms tetramers in solution. Together with Pep2, TreS forms a hetero-octameric complex, and we demonstrate that complex formation markedly accelerates maltokinase activity of Pep2. Thus, complex formation may act as part of a regulatory mechanism of the GlgE pathway, which overall must avoid accumulation of toxic pathway intermediates, such as maltose-1-phosphate, and optimize the use of scarce nutrients

Direct liquid extraction surface analysis mass spectrometry of cell wall lipids from mycobacteria: Salt additives for decreased spectral complexity

RationaleLipids are important mycobacterium cell wall constituents; changes are linked with drug resistance. Liquid extraction surface analysis (LESA) enables direct sampling in a highly sensitive manner. Here we describe protocols for the analysis of lipids from bacterial colonies. Lipids form various adducts, complicating spectra. Salt additives were investigated to circumvent this problem.MethodsChloroform:methanol mixtures were studied for lipid extraction and analysis by LESA‐MS. The inclusion of (ESI‐compatible) acetate salts of sodium, potassium or lithium in the extraction solvent was investigated.ResultsWe report the detection of bacterial cell wall lipids from mycobacterial species using LESA for the first time. Sampling protocols were optimised for the use of volatile extraction solvents. The inclusion of acetate salt additives in the sampling solvent significantly reduces spectral complexity in comparison with no additives being used.ConclusionsLESA offers a sensitive technique for bacterial lipid phenotyping. The inclusion of an acetate salt in the sampling solvent drives adduct formation towards a specific adduct type and thus significantly reduces spectral complexity

Inhibition of Mycobacterium tuberculosis InhA: Design, synthesis and evaluation of new di-triclosan derivatives

Multi-drug resistant tuberculosis (MDR-TB) represents a growing problem for global healthcare systems. In addition to 1.3 million deaths in 2018, the World Health Organisation reported 484,000 new cases of MDR-TB. Isoniazid is a key anti-TB drug that inhibits InhA, a crucial enzyme in the cell wall biosynthesis pathway and identical in Mycobacterium tuberculosis and M. bovis. Isoniazid is a pro-drug which requires activation by the enzyme KatG, mutations in KatG prevent activation and confer INH-resistance. ‘Direct inhibitors’ of InhA are attractive as they would circumvent the main clinically observed resistance mechanisms. A library of new 1,5-triazoles, designed to mimic the structures of both triclosan molecules uniquely bound to InhA have been synthesised. The inhibitory activity of these compounds was evaluated using isolated enzyme assays with 2 (5-chloro-2-(4-(5-(((4-(4-chloro-2-hydroxyphenoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) exhibiting an IC50 of 5.6 µM. Whole-cell evaluation was also performed, with 11 (5-chloro-2-(4-(5-(((4-(cyclopropylmethoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) showing the greatest potency, with an MIC99 of 12.9 µM against M. bovis

Utilisation of the Prestwick Chemical Library to identify drugs that inhibit the growth of mycobacteria.

Tuberculosis (TB) is an infectious bacterial disease that kills approximately 1.3 million people every year. Despite global efforts to reduce both the incidence and mortality associated with TB, the emergence of drug resistant strains has slowed any progress made towards combating the spread of this deadly disease. The current TB drug regimen is inadequate, takes months to complete and poses significant challenges when administering to patients suffering from drug resistant TB. New treatments that are faster, simpler and more affordable are urgently required. Arguably, a good strategy to discover new drugs is to start with an old drug. Here, we have screened a library of 1200 FDA approved drugs from the Prestwick Chemical library using a GFP microplate assay. Drugs were screened against GFP expressing strains of Mycobacterium smegmatis and Mycobacterium bovis BCG as surrogates for Mycobacterium tuberculosis, the causative agent of TB in humans. We identified several classes of drugs that displayed antimycobacterial activity against both M. smegmatis and BCG, however each organism also displayed some selectivity towards certain drug classes. Variant analysis of whole genomes sequenced for resistant mutants raised to florfenicol, vanoxerine and pentamidine highlight new pathways that could be exploited in drug repurposing programmes

Identification and structural characterisation of a partially arabinosylated lipoarabinomannan variant isolated from a Corynebacterium glutamicum ubiAmutant

Arabinan polysaccharide side-chains are present in both Mycobacterium tuberculosis and Corynebacterium glutamicum in the heteropolysaccharide arabinogalactan (AG), and in M. tuberculosis in the lipoglycan, lipoarabinomannan (LAM). Herein, we show by quantitative sugar and glycosyl linkage analysis that C. glutamicum possesses a much smaller LAM version, Cg-LAM, characterised by single t-Araf residues linked to th

HIV Drugs Inhibit Transfer of Plasmids Carrying Extended-Spectrum β-Lactamase and Carbapenemase Genes

More and more bacterial infections are becoming resistant to antibiotics. This has made treatment of many infections very difficult. One of the reasons this is such a large problem is that bacteria are able to share their genetic material with other bacteria, and these shared genes often include resistance to a variety of antibiotics, including some of our drugs of last resort. We are addressing this problem by using a fluorescence-based system to search for drugs that will stop bacteria from sharing resistance genes. We uncovered a new role for two drugs used to treat HIV and show that they are able to prevent the sharing of two different types of resistance genes in two unique bacterial strains. This work lays the foundation for future work to reduce the prevalence of resistant infections.Antimicrobial-resistant (AMR) infections pose a serious risk to human and animal health. A major factor contributing to this global crisis is the sharing of resistance genes between different bacteria via plasmids. The WHO lists Enterobacteriaceae, such as Escherichia coli and Klebsiella pneumoniae, producing extended-spectrum β-lactamases (ESBL) and carbapenemases as “critical” priorities for new drug development. These resistance genes are most often shared via plasmid transfer. However, finding methods to prevent resistance gene sharing has been hampered by the lack of screening systems for medium-/high-throughput approaches. Here, we have used an ESBL-producing plasmid, pCT, and a carbapenemase-producing plasmid, pKpQIL, in two different Gram-negative bacteria, E. coli and K. pneumoniae. Using these critical resistance-pathogen combinations, we developed an assay using fluorescent proteins, flow cytometry, and confocal microscopy to assess plasmid transmission inhibition within bacterial populations in a medium-throughput manner. Three compounds with some reports of antiplasmid properties were tested; chlorpromazine reduced transmission of both plasmids and linoleic acid reduced transmission of pCT. We screened the Prestwick library of over 1,200 FDA-approved drugs/compounds. From this, we found two nucleoside analogue drugs used to treat HIV, abacavir and azidothymidine (AZT), which reduced plasmid transmission (AZT, e.g., at 0.25 μg/ml reduced pCT transmission in E. coli by 83.3% and pKpQIL transmission in K. pneumoniae by 80.8% compared to untreated controls). Plasmid transmission was reduced by concentrations of the drugs which are below peak serum concentrations and are achievable in the gastrointestinal tract. These drugs could be used to decolonize humans, animals, or the environment from AMR plasmids