Two-Way Regulation of MmpL3 Expression Identifies and Validates Inhibitors of MmpL3 Function in Mycobacterium tuberculosis.

MmpL3, an essential mycolate transporter in the inner membrane of Mycobacterium tuberculosis (Mtb), has been identified as a target of multiple, chemically diverse antitubercular drugs. However, several of these molecules seem to have secondary targets and inhibit bacterial growth by more than one mechanism. Here, we describe a cell-based assay that utilizes two-way regulation of MmpL3 expression to readily identify MmpL3-specific inhibitors. We successfully used this assay to identify a novel guanidine-based MmpL3 inhibitor from a library of 220 compounds that inhibit growth of Mtb by largely unknown mechanisms. We furthermore identified inhibitors of cytochrome bc1-aa3 oxidase as one class of off-target hits in whole-cell screens for MmpL3 inhibitors and report a novel sulfanylacetamide as a potential QcrB inhibitor.

Benzothiazinones mediate killing of Corynebacterineae by blocking decaprenyl phosphate recycling involved in cell wall biosynthesis.

Benzothiazinones (BTZs) are a new class of sulfur containing heterocyclic compounds that target DprE1, an oxidoreductase involved in the epimerization of decaprenyl-phosphoribose (DPR) to decaprenyl-phosphoarabinose (DPA) in the Corynebacterineae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis. As a result, BTZ inhibition leads to inhibition of cell wall arabinan biosynthesis. Previous studies have demonstrated the essentiality of dprE1. In contrast, Cg-UbiA a ribosyltransferase, which catalyzes the first step of DPR biosynthesis prior to DprE1, when genetically disrupted, produced a viable mutant, suggesting that although BTZ biochemically targets DprE1, killing also occurs through chemical synthetic lethality, presumably through the lack of decaprenyl phosphate recycling. To test this hypothesis, a derivative of BTZ, BTZ043, was examined in detail against C. glutamicum and C. glutamicum::ubiA. The wild type strain was sensitive to BTZ043; however, C. glutamicum::ubiA was found to be resistant, despite possessing a functional DprE1. When the gene encoding C. glutamicum Z-decaprenyl-diphosphate synthase (NCgl2203) was overexpressed in wild type C. glutamicum, resistance to BTZ043 was further increased. This data demonstrates that in the presence of BTZ, the bacilli accumulate DPR and fail to recycle decaprenyl phosphate, which results in the depletion of decaprenyl phosphate and ultimately leads to cell death.

Genetics of Mycobacterial Arabinogalactan and Lipoarabinomannan Assembly.

The cell wall of Mycobacterium tuberculosis is unique in that it differs significantly from those of both Gram-negative and Gram-positive bacteria. The thick, carbohydrate- and lipid-rich cell wall with distinct lipoglycans enables mycobacteria to survive under hostile conditions such as shortage of nutrients and antimicrobial exposure. The key features of this highly complex cell wall are the mycolyl-arabinogalactan-peptidoglycan (mAGP)-based and phosphatidyl-myo-inositol-based macromolecular structures, with the latter possessing potent immunomodulatory properties. These structures are crucial for the growth, viability, and virulence of M. tuberculosis and therefore are often the targets of effective chemotherapeutic agents against tuberculosis. Over the past decade, sophisticated genomic and molecular tools have advanced our understanding of the primary structure and biosynthesis of these macromolecules. The availability of the full genome sequences of various mycobacterial species, including M. tuberculosis, Mycobacterium marinum, and Mycobacterium bovis BCG, have greatly facilitated the identification of large numbers of drug targets and antigens specific to tuberculosis. Techniques to manipulate mycobacteria have also improved extensively; the conditional expression-specialized transduction essentiality test (CESTET) is currently used to determine the essentiality of individual genes. Finally, various biosynthetic assays using either purified proteins or synthetic cell wall acceptors have been developed to study enzyme function. This article focuses on the recent advances in determining the structural details and biosynthesis of arabinogalactan, lipoarabinomannan, and related glycoconjugates.

IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria.

BACKGROUND: The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown. RESULTS: Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An ipsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Further studies revealed the absence of inositol-derived lipids in the cell wall and a complete loss of mycothiol biosynthesis. The phenotype of the C. glutamicum ipsA deletion mutant was complemented to different extend by homologs from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. Myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro. CONCLUSIONS: This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development.

Arabinogalactan and lipoarabinomannan biosynthesis: structure, biogenesis and their potential as drug targets.

Mycobacterium tuberculosis, the etiological agent of TB, remains the leading cause of mortality from a single infectious organism. The persistence of this human pathogen is associated with its distinctive lipid-rich cell wall structure that is highly impermeable to hydrophilic chemical drugs. This highly complex and unique structure is crucial for the growth, viability and virulence of M. tuberculosis, thus representing an attractive target for vaccine and drug development. It contains a large macromolecular structure known as the mycolyl-arabinogalactan-peptidoglycan complex, as well as phosphatidyl-myo-inositol derived glycolipids with potent immunomodulatory activity, notably lipomannan and lipoarabinomannan. These cell wall components are often the targets of effective chemotherapeutic agents against TB, such as ethambutol. This review focuses on the structural details and biosynthetic pathways of both arabinogalactan and lipoarabinomannan, as well as the effects of potent drugs on these important (lipo)polysaccharides.