Lynn G. Dover

Current Status and Research Strategies in Tuberculosis Drug Development

In this Miniperspective we broadly cover the recent developments and strategies that are being adopted globally in the battle to urgently discover and develop new drugs to treat tuberculosis (TB). It highlights the impact and outcomes of recent nongovernment organizations (NGO) and academic research consortia that have made a significant impact in recent years in the field, providing the reader with an insight into the overall status of TB drug discovery and development while illustrating the current strategies and directions that are currently being adopted to find new drugs to control the disease.

Thiacetazone, an Antitubercular Drug that Inhibits Cyclopropanation of Cell Wall Mycolic Acids in Mycobacteria

Background Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets. Methodology/Principle Findings We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the α- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs. Conclusions/Significance This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

Lipoteichoic acid biosynthesis: two steps forwards, one step sideways?

Lipoteichoic acids (LTAs) are membrane-anchored molecules in the cell envelopes of Gram-positive bacteria. Until recently, they were considered to be restricted to the Firmicutes, which include important pathogens such as Staphylococcus aureus and Streptococcus pneumoniae. Polyanionic LTAs have fundamentally important roles in divalent cation retention within the Gram-positive cell envelope and thereby influence bacterial cell division. Thus, LTA biosynthesis provides an attractive target for the development of novel antimicrobial interventions. Recent studies, notably two investigations of S. aureus and another of Bacillus subtilis, have greatly improved our understanding of the genetic basis of LTA biosynthesis. In addition, reports have revealed that at least some members of the Actinobacteria (another phylum of Gram-positive bacteria) produce LTAs, rather than the lipoglycans previously assumed to be typical of this taxon. The availability of whole bacterial genome sequences has enabled us to perform comparative analyses to shed light on the distribution of putative LTA biosynthetic genes among bacteria. Here, we discuss the results of these genomic analyses, together with the current literature, and propose that LTA biosynthesis in Actinobacteria might be fundamentally different to that in most Firmicutes.

The rhodococcal cell envelope: composition, organisation and biosynthesis

The cell envelopes of rhodococci and their closest relatives are domi- natedbythepresence oflargebranchedchainfattyacids,themycolicacids.Herewe review the structural features underlying the incorporation of the mycolic acids into the rhodococcal cell envelope, notably their covalent anchoring to the peptidogly- can-arabinogalactan complex and their organisation into an outer lipid permeability barrier. Rhodococcal cell envelopes also accommodate diverse non-covalently associated components such as channel-forming porin proteins, free lipids,

New drugs and vaccines for drug-resistant Mycobacterium tuberculosis infections

Tuberculosis remains the most common cause of death due to a single infective organism. Despite the availability of a vaccine and chemotherapeutic options, the global disease burden remains relatively unaffected. The ability of the mycobacterial etiological agents to adopt a semidormant, phenotypically drug-resistant state requires that chemotherapy is both complex and lengthy. The emergence of drug resistance has raised the possibility of virtually untreatable tuberculosis. Furthermore, the currently used bacillus Calmette–Guerin vaccine has had mixed success in protecting susceptible populations. Given this backdrop, the need for novel anti-infectives and more effective vaccines is clearly evident. Recent progress, described herein, has seen the development and entry into clinical trials of several new drugs and vaccine candidates.

Characterization of Mycobacterium tuberculosis diaminopimelic acid epimerase: paired cysteine residues are crucial for racemization

Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K(M) value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis.

Conformational Dynamics, Ligand Binding and Effects of Mutations in NirE an S-Adenosyl-L-Methionine Dependent Methyltransferase

Heme d1, a vital tetrapyrrol involved in the denitrification processes is synthesized from its precursor molecule precorrin-2 in a chemical reaction catalysed by an S-adenosyl-L-methionine (SAM) dependent Methyltransferase (NirE). The NirE enzyme catalyses the transfer of a methyl group from the SAM to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. An important insight into the structure-activity relationships of NirE has been revealed by elucidating its crystal structure, but there is still no understanding about how conformational flexibility influences structure, cofactor and substrate binding by the enzyme as well as the structural effects of mutations of residues involved in binding and catalysis. In order to provide this missing but very important information we performed a comprehensive atomistic molecular dynamics study which revealed that i) the binding of the substrate contributes to the stabilization of the structure of the full complex; ii) conformational changes influence the orientation of the pyrrole rings in the substrate, iii) more open conformation of enzyme active site to accommodate the substrate as an outcome of conformational motions; and iv) the mutations of binding and active site residues lead to sensitive structural changes which influence binding and catalysis.

Structural characterisation of the virulence-associated protein VapG from the horse pathogen Rhodococcus equi.

Highlights • The 3-dimensional structure of a Rhodococcus equi virulence protein was determined.• VapG comprises a closed beta barrel domain preceded by a natively disordered region.• The structures of VapB, VapD and VapG are closely superimposable.• The VAP structures lack recognisable ligand or protein binding sites.• Phagosome-induced conformational changes may be required for virulence.

Synthesis of novel Iron(III) chelators based on triaza macrocycle backbone and 1-hydroxy-2(H)-pyridin-2-one coordinating groups and their evaluation as antimicrobial agents

Several novel chelators based on 1-hydroxy-2(1H)-pyridinone coordinating groups decorating a triaza macrocyclic backbone scaffold were synthesised as potential powerful Fe(3+) chelators capable of competing with bacterial siderophores. In particular, a novel chloromethyl derivative of 1-hydroxy-2(1H)-pyridinone exploiting a novel protective group for this family of coordinating groups was developed. These are the first examples of hexadentate chelators based on 1-hydroxy-2(1H)-pyridinone to be shown to have a biostatic activity against a range of pathogenic bacteria. Their efficacy as biostatic agents was assessed revealing that minor variations in the structure of the chelator can affect efficacy profoundly. The minimal inhibitory concentrations of our best tested novel chelators approach or are comparable to those for 1,4,7-tris(3-hydroxy-6-methyl-2-pyridylmethyl)-1,4,7-triazacyclononane, the best Fe(3+) chelator known to date. The retarding effect these chelators have on microbial growth suggests that they could have a potential application as a co-active alongside antibiotics in the fight against infections.

Comment on Tocheva et al. [ldquo]Sporulation, bacterial cell envelopes and the origin of life[rdquo]

In their recent Opinion article (Sporulation, bacterial cell envelopes and the origin of life. Nat. Rev. Microbiol. 14, 535–542 (2016))1, Tocheva and colleagues build on their elegant electron cryotomography studies to derive an intriguing hypothesis that sporulation in a primordial monoderm bacterium (having a single membrane) could have resulted in the evolution of sporulating diderm bacteria (having a double membrane) and thus all extant bacteria. The evolution of outer membranes as a consequence of the topography of sporulation is a plausible hypothesis; however, it should be highlighted that Tocheva et al. consider a relatively limited range of the known and candidate phyla in their model. Thus, it will be of interest to see how their hypothesis stands up when tested against expanded phylo genetic analyses; for example, including the many new phyla that were identified in recent large-scale genomic analyses, which include novel monoderm phyla and possibly phyla with novel cell envelope architectures2,3. Although the hypothesis of Tocheva et al.1 may help to explain the evolution of most types of outer membrane, we question their suggestions that all diderm bacteria have arisen from an ancestral sporulating diderm and that outer membranes have probably only evolved once. As briefly acknowledged by Tocheva et al., the uniqueness of the mycolic acid-based outer membranes (MOMs) of members of the suborder Corynebacterineae challenges these assumptions. The ‘mycolata’ are significant because they belong to the predominantly monoderm phylum Actinobacteria and contain several major pathogens, including Mycobacterium tuberculosis. Several lines of evidence support the uniqueness of the MOM. First, mycolic acids are structurally distinct, being formed from condensed fatty acids4. Second, in contrast to the outer membranes of all other diderm bacteria, mycolic acids are the only outer-membrane lipids that are described as being covalently anchored to the underlying arabinogalactan–peptidoglycan4,5, which results in a unique architecture that is unlikely to have arisen from remodelling of typical outer membranes, as suggested by Tocheva et al.1 Third, all other characterized outer membranes contain BamA family proteins, which have a central role in β-barrel protein insertion, along with several specialized protein secretion systems5,6. It is notable that neither BamA proteins nor type I–VI secretion systems have been identified in any mycolata5–7. Although bioinformatic evidence suggests that proteins with high β-sheet content are found in the MOM, relatively few MOM proteins have been characterized in detail, and orthology with β-barrels in other membranes cannot be assumed. Notably, the structures of MOM porins differ from the canonical β-barrels of porins in other diderm taxa8. The homology of the carboxyl terminus of mycobacterial Rv0899 with proteobacterial outer-membrane protein A (OmpA) proteins (highlighted by Tocheva et al.1), seems to reflect its function as a periplasmic peptidoglycanbinding domain, but its overall topology and location remain unclear9. Cumulatively, these observations argue against a common evolutionary history for the MOM of Corynebacterineae and the outer membranes in other diderm bacteria. Thus, we argue, in contrast to the conclusions of Tocheva et al.1, that structurally distinct outer membranes probably evolved at least twice.