Proximity-dependent biotin identification links cholesterol catabolism with branched-chain amino acid degradation in Mycobacterium smegmatis.

Cholesterol is a crucial component in Mycobacterium tuberculosis virulence as it is required for phagocytosis of mycobacteria by macrophages. In addition, the tubercle bacilli can grow using cholesterol as the sole carbon source. Thus, cholesterol catabolism represents a valuable target for the development of new antitubercular drugs.

Interconnection of the mycobacterial heparin-binding hemagglutinin with cholesterol degradation and heme/iron pathways identified by proximity-dependent biotin identification in Mycobacterium smegmatis.

Deciphering protein-protein interactions is a critical step in the identification and the understanding of biological mechanisms deployed by pathogenic bacteria. The development of in vivo technologies to characterize these interactions is still in its infancy, especially for bacteria whose subcellular organization is particularly complex, such as mycobacteria. In this work, we used the proximity-dependent biotin identification (BioID) to define the mycobacterial heparin-binding hemagglutinin (HbhA) interactome in the saprophytic bacterium Mycobacterium smegmatis.

Protein scaffold involving MSMEG_1285 maintains cell wall organization and mediates penicillin sensitivity in mycobacteria.

Protein-protein interactions are key in mycobacterial physiology, notably during the biosynthesis of the very peculiar mycobacterial cell wall. In this paper, we demonstrate that MSMEG_1285 interacts with PonA1, a bifunctional penicillin-binding protein involved in peptidoglycan biosynthesis. Deletion of MSMEG_1285 enhances Mycobacterium smegmatis resistance to penicillin antibiotics, a phenotype that is exacerbated by the additional deletion of hbhA.

In Vivo Methods to Study Protein-Protein Interactions as Key Players in Virulence.

Studies on protein-protein interactions (PPI) can be helpful for the annotation of unknown protein functions and for the understanding of cellular processes, such as specific virulence mechanisms developed by bacterial pathogens. In that context, several methods have been extensively used in recent years for the characterization of PPI to further decipher tuberculosis (TB) pathogenesis. This review aims at compiling the most striking results based on in vivo methods (yeast and bacterial two-hybrid systems, protein complementation assays) for the specific study of PPI in mycobacteria.

Rv0613c/MSMEG_1285 Interacts with HBHA and Mediates Its Proper Cell-Surface Exposure in Mycobacteria.

Heparin-binding haemagglutinin (HBHA) is a surface-exposed virulence factor of and is involved in the binding of mycobacteria to non-phagocytic cells, allowing for extra-pulmonary dissemination of the bacilli. Despite its surface exposure, HBHA is not produced as a pre-protein containing a typical cleavable N-terminal signal peptide and is thus likely secreted by a Sec-independent, as of yet unknown mechanism. Here, we used the bacterial adenylate cyclase two-hybrid system to identify the proteins encoded by and as being able to interact with HBHA.

ArfGAP1 restricts entry by controlling the actin cytoskeleton.

The interaction of (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F-actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP-ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (MR).

STAT3 Represses Nitric Oxide Synthesis in Human Macrophages upon Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis is a successful intracellular pathogen. Numerous host innate immune responses signaling pathways are induced upon mycobacterium invasion, however their impact on M. tuberculosis replication is not fully understood.

LppM impact on the colonization of macrophages by Mycobacterium tuberculosis.

Mycobacterium tuberculosis produces several bacterial effectors impacting the colonization of phagocytes. Here, we report that the putative lipoprotein LppM hinders phagocytosis by macrophages in a toll-like receptor 2-dependent manner. Moreover, recombinant LppM is able to functionally complement the phenotype of the mutant, when exogenously added during macrophage infection.

Synthetic variants of mycolactone bind and activate Wiskott-Aldrich syndrome proteins.

Mycolactone is a complex macrolide toxin produced by Mycobacterium ulcerans, the causative agent of skin lesions called Buruli ulcers. Mycolactone-mediated activation of neural (N) Wiskott-Aldrich syndrome proteins (WASP) induces defects in cell adhesion underpinning cytotoxicity and disease pathogenesis. We describe the chemical synthesis of 23 novel mycolactone analogues that differ in structure and modular assembly of the lactone core with its northern and southern polyketide side chains.

Phosphorylation of KasB regulates virulence and acid-fastness in Mycobacterium tuberculosis.

Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment.

A microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs.

Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis.

We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis by the previously uncharacterized mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser55 site in Pks13 conferred TP resistance in M. tuberculosis.

Mycolactone activation of Wiskott-Aldrich syndrome proteins underpins Buruli ulcer formation.

Mycolactone is a diffusible lipid secreted by the human pathogen Mycobacterium ulcerans, which induces the formation of open skin lesions referred to as Buruli ulcers. Here, we show that mycolactone operates by hijacking the Wiskott-Aldrich syndrome protein (WASP) family of actin-nucleating factors. By disrupting WASP autoinhibition, mycolactone leads to uncontrolled activation of ARP2/3-mediated assembly of actin in the cytoplasm.

The Mycobacterium tuberculosis FAS-II dehydratases and methyltransferases define the specificity of the mycolic acid elongation complexes.

Background: The human pathogen Mycobacterium tuberculosis (Mtb) has the originality of possessing a multifunctional mega-enzyme FAS-I (Fatty Acid Synthase-I), together with a multi-protein FAS-II system, to carry out the biosynthesis of common and of specific long chain fatty acids: the mycolic acids (MA). MA are the main constituents of the external mycomembrane that represents a tight permeability barrier involved in the pathogenicity of Mtb. The MA biosynthesis pathway is essential and contains targets for efficient antibiotics.

Mycolactone diffuses into the peripheral blood of Buruli ulcer patients--implications for diagnosis and disease monitoring.

Background: Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established.

Methodology/principal Finding: Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast.

Expression of OmpATb is dependent on small membrane proteins in Mycobacterium bovis BCG.

Small membrane proteins emerge as a novel class of regulatory molecules in bacteria. Experiments carried out in Mycobacterium bovis BCG indicate that the ompATb gene (Rv0899), encoding a major outer membrane protein, is organized in operon with Rv0900 and Rv0901, encoding two small proteins with a predicted transmembrane domain. Fractioning experiment confirmed the association of Rv0901 with the membrane fraction.

Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis.

The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation.

Phosphorylation of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein reductase MabA regulates mycolic acid biosynthesis.

Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential beta-ketoacyl-acyl carrier protein reductase, MabA.

The Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III activity is inhibited by phosphorylation on a single threonine residue.

Mycolic acids are hallmark features of the Mycobacterium tuberculosis cell wall. They are synthesized by the condensation of two fatty acids, a C56-64-meromycolyl chain and a C24-26-fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid synthases (FAS-I and FAS-II).

The Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitment of Rv2175c.

Although Mycobacterium tuberculosis (M. tb) comprises 11 serine/threonine protein kinases, the mechanisms of regulation of these kinases and the nature of their endogenous substrates remain largely unknown. Herein, we characterized the M.

The biosynthesis of mycolic acids in Mycobacterium tuberculosis relies on multiple specialized elongation complexes interconnected by specific protein-protein interactions.

Tuberculosis kills about two million people every year and remains one of the leading causes of mortality worldwide. As a result of the increasing antibiotic resistance of Mycobacterium tuberculosis (Mtb) strains, there is an urgent need for new antitubercular drugs. Several efficient antibiotics, including isoniazid, specifically target the fatty acid synthase-II (FAS-II) complex of mycolic acid biosynthesis.

Protein-protein interactions within the Fatty Acid Synthase-II system of Mycobacterium tuberculosis are essential for mycobacterial viability.

Despite the existence of efficient chemotherapy, tuberculosis remains a leading cause of mortality worldwide. New drugs are urgently needed to reduce the potential impact of the emergence of multidrug-resistant strains of the causative agent Mycobacterium tuberculosis (Mtb). The front-line antibiotic isoniazid (INH), and several other drugs, target the biosynthesis of mycolic acids and especially the Fatty Acid Synthase-II (FAS-II) elongation system.