Reinterpreting the Mechanism of Inhibition of <i>Mycobacterium tuberculosis</i> d‑Alanine:d‑Alanine Ligase by d‑Cycloserine

d-Cycloserine is a second-line drug approved for use in the treatment of patients infected with <i>Mycobacterium tuberculosis</i>, the etiologic agent of tuberculosis. The unique mechanism of action of d-cycloserine, compared with those of other clinically employed antimycobacterial agents, represents an untapped and exploitable resource for future rational drug design programs. Here, we show that d-cycloserine is a slow-onset inhibitor of MtDdl and that this behavior is specific to the <i>M. tuberculosis</i> enzyme orthologue. Furthermore, evidence is presented that indicates d-cycloserine binds exclusively to the C-terminal d-alanine binding site, even in the absence of bound d-alanine at the N-terminal binding site. Together, these results led us to propose a new model of d-alanine:d-alanine ligase inhibition by d-cycloserine and suggest new opportunities for rational drug design against an essential, clinically validated mycobacterial target

Correction to Reinterpreting the Mechanism of Inhibition of <i>Mycobacterium tuberculosis</i> d‑Alanine:d‑Alanine Ligase by d‑Cycloserine

Correction to Reinterpreting the Mechanism of Inhibition of <i>Mycobacterium tuberculosis</i> d‑Alanine:d‑Alanine Ligase by d‑Cycloserin

Nitazoxanide Disrupts Membrane Potential and Intrabacterial pH Homeostasis of <i>Mycobacterium tuberculosis</i>

Nitazoxanide (Alinia), a nitro-thiazolyl antiparasitic drug, kills diverse microorganisms by unknown mechanisms. Here we identified two actions of nitazoxanide against <i>Mycobacterium tuberculosis</i> (Mtb): disruption of Mtb’s membrane potential and pH homeostasis. Both actions were shared by a structurally related antimycobacterial compound, niclosamide. Reactive nitrogen intermediates were reported to synergize with nitazoxanide and its deacetylated derivative tizoxanide in killing Mtb. Herein, however, we could not attribute this to increased uptake of nitazoxanide or tizoxanide as monitored by targeted metabolomics, nor to increased impact of nitazoxanide on Mtb’s membrane potential or intrabacterial pH. Thus, further mechanisms of action of nitazoxanide or tizoxanide may await discovery. The multiple mechanisms of action may contribute to Mtb’s ultralow frequency of resistance against nitazoxanide

The Mechanism of Acetyl Transfer Catalyzed by <i>Mycobacterium tuberculosis</i> GlmU

The biosynthetic pathway of peptidoglycan is essential for <i>Mycobacterium tuberculosis</i>. We report here the acetyltransferase substrate specificity and catalytic mechanism of the bifunctional <i>N</i>-acetyltransferase/uridylyltransferase from <i>M. tuberculosis</i> (GlmU). This enzyme is responsible for the final two steps of the synthesis of UDP-<i>N</i>-acetylglucosamine, which is an essential precursor of peptidoglycan, from glucosamine 1-phosphate, acetyl-coenzyme A, and uridine 5′-triphosphate. GlmU utilizes ternary complex formation to transfer an acetyl from acetyl-coenzyme A to glucosamine 1-phosphate to form <i>N</i>-acetylglucosamine 1-phosphate. Steady-state kinetic studies and equilibrium binding experiments indicate that GlmU follows a steady-state ordered kinetic mechanism, with acetyl-coenzyme A binding first, which triggers a conformational change in GlmU, followed by glucosamine 1-phosphate binding. Coenzyme A is the last product to dissociate. Chemistry is partially rate-limiting as indicated by pH–rate studies and solvent kinetic isotope effects. A novel crystal structure of a mimic of the Michaelis complex, with glucose 1-phosphate and acetyl-coenzyme A, helps us to propose the residues involved in deprotonation of glucosamine 1-phosphate and the loop movement that likely generates the active site required for glucosamine 1-phosphate to bind. Together, these results pave the way for the rational discovery of improved inhibitors against <i>M. tuberculosis</i> GlmU, some of which might become candidates for antibiotic discovery programs

TPL-2 Regulates Macrophage Lipid Metabolism and M2 Differentiation to Control T_H2-Mediated Immunopathology

<div><p>Persistent T_H2 cytokine responses following chronic helminth infections can often lead to the development of tissue pathology and fibrotic scarring. Despite a good understanding of the cellular mechanisms involved in fibrogenesis, there are very few therapeutic options available, highlighting a significant medical need and gap in our understanding of the molecular mechanisms of T_H2-mediated immunopathology. In this study, we found that the Map3 kinase, TPL-2 (<i>Map3k8</i>; Cot) regulated T_H2-mediated intestinal, hepatic and pulmonary immunopathology following <i>Schistosoma mansoni</i> infection or <i>S</i>. <i>mansoni</i> egg injection. Elevated inflammation, T_H2 cell responses and exacerbated fibrosis in <i>Map3k8</i>^–/–mice was observed in mice with myeloid cell-specific (LysM) deletion of <i>Map3k8</i>, but not CD4 cell-specific deletion of <i>Map3k8</i>, indicating that TPL-2 regulated myeloid cell function to limit T_H2-mediated immunopathology. Transcriptional and metabolic assays of <i>Map3k8</i>^–/–M2 macrophages identified that TPL-2 was required for lipolysis, M2 macrophage activation and the expression of a variety of genes involved in immuno-regulatory and pro-fibrotic pathways. Taken together this study identified that TPL-2 regulated T_H2-mediated inflammation by supporting lipolysis and M2 macrophage activation, preventing T_H2 cell expansion and downstream immunopathology and fibrosis.</p></div

T cell-intrinsic <i>Map3k8</i> does not contribute to exacerbated inflammation and pathology following <i>S</i>. <i>mansoni</i> infection.

<p><i>Cd4</i>^<i>Cre</i><i>Map3k8</i>^<i>+/+</i> and <i>Cd4</i>^<i>Cre</i><i>Map3k8</i>^<i>fl/fl</i> mice were infected percutenously with 50 <i>S</i>. <i>mansoni</i> cercariae and analysed at 8 weeks post-infection. A–C) Perfused tissue was fixed and embedded in paraffin before sectioning and staining with Masson’s trichrome. B) Granuloma size was determined from 10–20 individual granulomas per sample measured using Image J. D) Expression of <i>Col3</i> and <i>Col6</i> was determined from RNA extracted from liver or small intestinal tissue. Data is expressed relative to HPRT and shown as a fold-change relative to uninfected mice. E) Mesenteric lymph node cells were re-stimulated with anti-CD3 for 3 days. Cytokines were measured in supernatants, by ELISA. F) Naive T cells (CD4⁺CD44⁻CD25⁻CD62L⁺) were FACS purified from WT and <i>Map3k8</i>^{<i>–/–</i>}mice and cultured under T_H1 and T_H2 conditions. Frequencies of CD44⁺IFNγ⁺ and CD44⁺IL-4⁺ cells were determined by intracellular FACS analysis on day 7. All experiments are representative of 2–3 independent experiments with 5–10 mice/genotype. * p< 0.05 as assessed by two-tailed Mann-Whitney test.</p

TPL-2 is required for M2 activation of Macrophages, <i>in vivo</i>.

<p>WT C57BL/6 mice were lethally irradiated (900rad) and reconstituted with 50% CD45.1+ WT bone marrow and 50% CD45.2+ <i>Map3k8</i>^–/–bone marrow and left for 6–8 weeks, prior to infection with 50 <i>S</i>. <i>mansoni</i> cercariae. A) After 8 weeks of infection, mice were sacrificed and CD3⁻CD19⁻CD11b⁺F4/80⁺ Macrophages were FACS-sorted. B-C) Expression of <i>Arg1</i>, <i>Relma</i>, <i>Chi3l3</i>, <i>Col1a1</i>, <i>Col3a1</i> and <i>Ctgf</i> was determined from RNA extracted from purified macrophages. Data is expressed relative to HPRT and presented as a fold-change relative in genotype-controlled naïve bone marrow derived macrophages. Experiments are representative of 2 independent experiments with 5 mice/genotype. * p< 0.05 as assessed by two-tailed Mann-Whitney test.</p

TPL-2 regulates pro-fibrotic and immuno-regulatory pathways in M2 macrophages.

<p>Bone marrow-derived macrophages (BMDM) were stimulated with IL-4 and IL-13 for 24 hours. Cells were harvested, RNA extracted and genome-wide transcriptional expression was determined by microarray analysis using 3 biological replicates. A) Heat map of differentially regulated genes in un-stimulated and IL-4+IL-13 stimulated cells. B) Ingenuity pathways analysis of transcriptional profiles of differentially regulated genes. C-F) Venn diagram and bar graphs of TPL-2 dependent (1), common (2) and TPL-2-regulated genes (3). G and H) Ratio of Ratios graph (top) and bar graphs (H) showing increased pro-fibrotic (red) and decreased Immunoregulatory (blue) genes in <i>Map3k8</i>^{<i>–/–</i>}macrophages, relative to WT macrophages (x-axis) and un-stimulated macrophages (y-axis).</p

<i>Map3k8</i>^–/–mice develop increased hepatic and intestinal inflammation and fibrosis following <i>S</i>. <i>mansoni</i> infection.

<p>WT and <i>Map3k8</i>^{<i>–/–</i>}mice were infected percutenously with 50 <i>S</i>. <i>mansoni</i> cercariae and analysed at 8 weeks post-infection. A & C) Perfused tissue was fixed and embedded in paraffin before sectioning and staining with Masson’s trichrome. B) Granuloma size was determined from 10–20 individual granulomas per sample measured using Image J. Scale bars are 1000μm (top), 200μm (middle) and 100μm (bottom). D) Intestinal pathology score, as described in methods. E) Expression of <i>Col3</i> and <i>Col6</i> was determined from RNA extracted from liver or small intestinal tissue. Data is expressed relative to HPRT. F) Hydroxyproline was quantified in liver tissue from naïve and infected animals. G) Frequency of T_REG (CD4⁺CD25⁺<i>Foxp3</i>^<i>RFP</i>+) and T_H2 (CD4⁺CD44⁺<i>Il4</i>^<i>GFP</i>+) cells in the spleen, mesenteric lymph nodes (MLN) and liver were determined by FACS. All experiments are representative of 2–3 independent experiments with 5–10 mice/genotype. * p< 0.05 as assessed by two-tailed Mann-Whitney test.</p

Myeloid cell (<i>LysM</i>⁺) expression of Map3k8 regulates T_H2-mediated immunopathology and fibrosis following <i>S</i>. <i>mansoni</i> infection.

<p><i>LysM</i>^<i>Cre</i><i>Map3k8</i>^<i>+/+</i> and <i>LysM</i>^<i>Cre</i><i>Map3k8</i>^<i>fl/fl</i> mice were infected percutenously with 50 <i>S</i>. <i>mansoni</i> cercariae and analysed at 8 weeks post-infection. A–B) Perfused tissue was fixed and embedded in paraffin before sectioning and staining with Masson’s trichrome. Scale bar is 200μm. C) Granuloma size was determined from 10–20 individual granulomas per sample measured using Image J. D) Intestinal pathology score, as described in methods. E) Expression of <i>Col3</i> and <i>Col6</i> was determined from RNA extracted from liver. Data is expressed relative to HPRT and presented as a fold-change relative in infected WT mice. F) Hydroxyproline was quantified in liver tissue from naïve and infected animals. G) Mesenteric lymph node cells were re-stimulated with anti-CD3 for 3 days. Cytokines were measured in supernatants, by ELISA. All experiments are representative of 2–3 independent experiments with 5–10 mice/genotype. * p< 0.05 as assessed by two-tailed Mann-Whitney test.</p