Fragment-based discovery of a new class of inhibitors targeting mycobacterial tRNA modification.

Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs

Stepwise pathogenic evolution of Mycobacterium abscessus.

Although almost all mycobacterial species are saprophytic environmental organisms, a few, such as Mycobacterium tuberculosis, have evolved to cause transmissible human infection. By analyzing the recent emergence and spread of the environmental organism M. abscessus through the global cystic fibrosis population, we have defined key, generalizable steps involved in the pathogenic evolution of mycobacteria. We show that epigenetic modifiers, acquired through horizontal gene transfer, cause saltational increases in the pathogenic potential of specific environmental clones. Allopatric parallel evolution during chronic lung infection then promotes rapid increases in virulence through mutations in a discrete gene network; these mutations enhance growth within macrophages but impair fomite survival. As a consequence, we observe constrained pathogenic evolution while person-to-person transmission remains indirect, but postulate accelerated pathogenic adaptation once direct transmission is possible, as observed for M. tuberculosis Our findings indicate how key interventions, such as early treatment and cross-infection control, might restrict the spread of existing mycobacterial pathogens and prevent new, emergent ones

Euro Surveill

In September 2023, a severe outbreak of type B botulism with fifteen cases was linked to consumption of canned sardines at a restaurant in Bordeaux, France, during the Rugby World Cup. The cases were from seven countries. One death was recorded. Outbreak investigation using credit card data, rapid communication between health authorities of the affected countries and broad media communication allowed identification of cases and exposed persons and prevented further severe outcomes

Décryptage de la voie de biosynthèse des polyphléates de tréhalose, une famille de glycolipides de surface largement distribuée chez les mycobactéries non tuberculeuses

Les bactéries du genre Mycobacterium possèdent une enveloppe très caractéristique composée d'une membrane plasmique, une paroi et une capsule. Le compartiment pariétal est le plus remarquable, notamment grâce à la présence de lipides non liés de façon covalente à la structure : les lipides extractibles. Ubiquitaires ou spécifiques à une espèce, ces derniers sont retrouvés en surface, où ils peuvent jouer un rôle clé dans les interactions hôte-pathogène. La synthèse et le transport de ces lipides nécessite l'intervention d'une enzyme modulaire appelée polykétide synthase (Pks), d'une enzyme activant le substrat de la Pks, d'une autre transférant le produit de la Pks sur une molécule acceptrice et enfin d'un transporteur de la famille RND (résistance, nodulation, division cellulaire). Un locus regroupant les gènes codant pour des protéines de biosynthèse et de transport de lipides extractibles a été retrouvé sur le chromosome de M. smegmatis entre les gènes MSMEG_0406 et MSMEG_0413. Nous avons alors entrepris de caractériser le rôle de chacun de ces gènes. Notre stratégie a consisté à inactiver ces gènes par recombinaison homologue puis à étudier la nature et la localisation des dérivés lipidiques de l'enveloppe des mutants. Suite à la caractérisation structurale des dérivés lipidiques affectés par ces mutations, nous avons montré que ce locus est lié aux polyphléates de tréhalose (PPT), auparavant décrits chez M. phlei. Le composé mature consiste en un tréhalose estérifié par sept acides gras polyinsaturés appelés acides phléiques. L'étude des génomes mycobactériens a montré que ces gènes sont conservés par un large panel de mycobactéries non tuberculeuses, en particulier M. avium et M. abscessus, deux pathogènes opportunistes. L'analyse du contenu lipidique de ces deux espèces a mis en évidence un composé possédant des propriétés similaires aux PPT. Par ailleurs, l'inactivation chez M. abscessus de l'orthologue du gène codant la Pks abolit la production de ces composés, confirmant ainsi le rôle de la Pks. Par analogie avec les voies de biosynthèse des glycolipides produits par M. tuberculosis, agent de la tuberculose, nous avons pu établir un modèle de la voie de biosynthèse des PPT chez les mycobactéries.The unique mycobacterial envelope can be divided into three entities: a plasma membrane, a cell wall and a capsule. The parietal compartment is remarkable for its lipid content notably the extractible lipids, not covalently bound to the structure. Either ubiquitous or species-specific, these surface lipids can play key roles in host-pathogen interactions. The synthesis and transport of many extractible lipids require at least a modular enzyme called polyketide synthase (Pks), a protein allowing the activation of the Pks substrate, another transferring the Pks product onto an acceptor molecule and a RND transporter (resistance-nodulation-division). A locus containing synthesis and transport coding genes was found on the M. smegmatis chromosome between MSMEG_0406 and MSMEG_0413. We then undertook to characterize the role of each gene composing this locus. Our strategy was to disrupt these genes by homologous recombination and then to analyze the lipid content and localization of the mutant strain envelope. Structural characterization of the impacted lipids showed a link between this locus and trehalose polyphleates (PPT) synthesis. The mature compound is a trehalose acylated by seven polyunsaturated fatty acids called phleic acid and previously described in M. phlei. Analysis of mycobacterial genomes showed that these genes are conserved in several non tuberculous mycobacteria, including the opportunistic pathogens M. avium and M. abscessus. Analysis of the lipid content of both species showed a compound with the same properties than PPT. Moreover, inactivation of the M. abscessus pks orthologue abolished its production, thus confirming the Pks implication in the PPT synthesis. By analogy with M. tuberculosis glycolipid biosynthesis, we could propose a model of the PPT biosynthetic pathway in mycobacteria

Deciphering the biosynthetic pathway of trehalose polyphleates, a family of surface-exposed glycolipids broadly distributed in nontuberculous mycobacteria

Les bactéries du genre Mycobacterium possèdent une enveloppe très caractéristique composée d'une membrane plasmique, une paroi et une capsule. Le compartiment pariétal est le plus remarquable, notamment grâce à la présence de lipides non liés de façon covalente à la structure : les lipides extractibles. Ubiquitaires ou spécifiques à une espèce, ces derniers sont retrouvés en surface, où ils peuvent jouer un rôle clé dans les interactions hôte-pathogène. La synthèse et le transport de ces lipides nécessite l'intervention d'une enzyme modulaire appelée polykétide synthase (Pks), d'une enzyme activant le substrat de la Pks, d'une autre transférant le produit de la Pks sur une molécule acceptrice et enfin d'un transporteur de la famille RND (résistance, nodulation, division cellulaire). Un locus regroupant les gènes codant pour des protéines de biosynthèse et de transport de lipides extractibles a été retrouvé sur le chromosome de M. smegmatis entre les gènes MSMEG_0406 et MSMEG_0413. Nous avons alors entrepris de caractériser le rôle de chacun de ces gènes. Notre stratégie a consisté à inactiver ces gènes par recombinaison homologue puis à étudier la nature et la localisation des dérivés lipidiques de l'enveloppe des mutants. Suite à la caractérisation structurale des dérivés lipidiques affectés par ces mutations, nous avons montré que ce locus est lié aux polyphléates de tréhalose (PPT), auparavant décrits chez M. phlei. Le composé mature consiste en un tréhalose estérifié par sept acides gras polyinsaturés appelés acides phléiques. L'étude des génomes mycobactériens a montré que ces gènes sont conservés par un large panel de mycobactéries non tuberculeuses, en particulier M. avium et M. abscessus, deux pathogènes opportunistes. L'analyse du contenu lipidique de ces deux espèces a mis en évidence un composé possédant des propriétés similaires aux PPT. Par ailleurs, l'inactivation chez M. abscessus de l'orthologue du gène codant la Pks abolit la production de ces composés, confirmant ainsi le rôle de la Pks. Par analogie avec les voies de biosynthèse des glycolipides produits par M. tuberculosis, agent de la tuberculose, nous avons pu établir un modèle de la voie de biosynthèse des PPT chez les mycobactéries.The unique mycobacterial envelope can be divided into three entities: a plasma membrane, a cell wall and a capsule. The parietal compartment is remarkable for its lipid content notably the extractible lipids, not covalently bound to the structure. Either ubiquitous or species-specific, these surface lipids can play key roles in host-pathogen interactions. The synthesis and transport of many extractible lipids require at least a modular enzyme called polyketide synthase (Pks), a protein allowing the activation of the Pks substrate, another transferring the Pks product onto an acceptor molecule and a RND transporter (resistance-nodulation-division). A locus containing synthesis and transport coding genes was found on the M. smegmatis chromosome between MSMEG_0406 and MSMEG_0413. We then undertook to characterize the role of each gene composing this locus. Our strategy was to disrupt these genes by homologous recombination and then to analyze the lipid content and localization of the mutant strain envelope. Structural characterization of the impacted lipids showed a link between this locus and trehalose polyphleates (PPT) synthesis. The mature compound is a trehalose acylated by seven polyunsaturated fatty acids called phleic acid and previously described in M. phlei. Analysis of mycobacterial genomes showed that these genes are conserved in several non tuberculous mycobacteria, including the opportunistic pathogens M. avium and M. abscessus. Analysis of the lipid content of both species showed a compound with the same properties than PPT. Moreover, inactivation of the M. abscessus pks orthologue abolished its production, thus confirming the Pks implication in the PPT synthesis. By analogy with M. tuberculosis glycolipid biosynthesis, we could propose a model of the PPT biosynthetic pathway in mycobacteria

Trehalose Polyphleates Are Produced by a Glycolipid Biosynthetic Pathway Conserved across Phylogenetically Distant Mycobacteria

International audienc

4′-Phosphopantetheinyl Transferase PptT, a New Drug Target Required for Mycobacterium tuberculosis Growth and Persistence In Vivo

International audienceThe cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, contains lipids with unusual structures. These lipids play a key role in both virulence and resistance to the various hostile environments encountered by the bacteria during infection. They are synthesized by complex enzymatic systems, including type-I polyketide synthases and type-I and -II fatty acid synthases, which require a post-translational modification to become active. This modification consists of the covalent attachment of the 4'-phosphopantetheine moiety of Coenzyme A catalyzed by phosphopantetheinyl transferases (PPTases). PptT, one of the two PPTases produced by mycobacteria, is involved in post-translational modification of various type-I polyketide synthases required for the formation of both mycolic acids and lipid virulence factors in mycobacteria. Here we identify PptT as a new target for anti-tuberculosis drugs; we address all the critical issues of target validation to demonstrate that PptT can be used to search for new drugs. We confirm that PptT is essential for the growth of M. bovis BCG in vitro and show that it is required for persistence of M. bovis BCG in both infected macrophages and immunodeficient mice. We generated a conditional expression mutant of M. tuberculosis, in which the expression of the pptT gene is tightly regulated by tetracycline derivatives. We used this construct to demonstrate that PptT is required for the replication and survival of the tubercle bacillus during the acute and chronic phases of infection in mice. Finally, we developed a robust and miniaturized assay based on scintillation proximity assay technology to search for inhibitors of PPTases, and especially of PptT, by high-throughput screening. Our various findings indicate that PptT meets the key criteria for being a therapeutic target for the treatment of mycobacterial infections

Cross-talk between Staphylococcus aureus leukocidins-intoxicated macrophages and lung epithelial cells triggers chemokine secretion in an inflammasome-dependent manner.

International audienceStaphylococcus aureus is a major pathogen responsible for both nosocomial and community-acquired infections. Central to its virulence is its ability to secrete haemolysins, pore-forming toxins and cytolytic peptides. The large number of membrane-damaging toxins and peptides produced during S. aureus infections has hindered a precise understanding of their specific roles in diseases. Here, we used comprehensive libraries of recombinant toxins and synthetic cytolytic peptides, of S. aureus mutants and clinical strains to investigate the role of these virulence factors in targeting human macrophages and triggering IL-1β release. We found that the Panton Valentine leukocidin (PVL) is the major trigger of IL-1β release and inflammasome activation in primary human macrophages. The cytolytic peptides, δ-haemolysin and PSMα3; the pore-forming toxins, γ-haemolysin and LukDE; and β-haemolysin synergize with PVL to amplify IL-1β release, indicating that these factors cooperate with PVL to trigger inflammation. PVL(+) S. aureus causes necrotizing pneumonia in children and young adults. The severity of this disease is due to the massive recruitment of neutrophils that cause lung damage. Importantly, we demonstrate that PVL triggers IL-1β release in human alveolar macrophages. Furthermore, IL-1β released by PVL-intoxicated macrophages stimulates the secretion of the neutrophil attracting chemokines, IL-8 and monocyte chemotactic protein-1, by lung epithelial cells. Finally, we show that PVL-induced IL-8/monocyte chemotactic protein-1 release is abolished by the inclusion of IL-1 receptor antagonist (IL-1Ra) in a mixed culture of lung epithelial cells and macrophages. Together, our results identify PVL as the predominant S. aureus secreted factor for triggering inflammasome activation in human macrophages and demonstrate how PVL-intoxicated macrophages orchestrate inflammation in the lung. Finally, our work suggests that anakinra, a synthetic IL-1Ra, may be an effective therapeutic agent to reduce the massive neutrophils infiltration observed during necrotizing pneumonia and decrease the resulting host-mediated lung injury

<i>In vitro</i> activity of PptT.

<p><b>A.</b> Diagrammatic representation of the domain organization of PKS13 and of ACP and ACPb domains. ACP: Acyl Carrier protein, KS: ketosynthase, AT: acyltransferase, TE: thioesterase. <b>B.</b> ACP activation with CoA and acetyl-CoA. The <i>apo</i>-ACP module was incubated with (+) or without (−) PptT in the presence of either CoA (left panel) or acetyl-CoA (right panel). <i>apo</i>- (a) and <i>holo</i>-ACP (h) forms were separated on urea polyacrylamide gels and stained with Coomassie blue (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003097#s4" target="_blank">materials and methods</a>). M: PageRuler prestained protein ladder plus (Fermentas). <b>C.</b> ACP activation with CoA analogs. PptT or Sfp were incubated with the <i>apo</i>-ACP domain in the presence of either fluorescent CoA analogs (CoA488 or CoA547) or CoA-biotin. Fluorescent <i>holo</i>-ACP forms (h) were resolved by SDS-PAGE and visualized by fluorescence scanning using a Typhoon scanner (GE Healthcare) (upper panel). Biotin-labeled ACP was detected by spotting 5 µl of the reaction mix onto the nitrocellulose membrane and incubation with streptavidin peroxidase followed by enhanced chemiluminescence detection (lower panel). The dashed-line circle shows the drop zone for the PptT reaction.</p

Effect of PptT depletion on the persistence of <i>M. tuberculosis</i> H37Rv in BALB/c mice.

<p>Mice were infected with strain PMM168 and treated with 0.1 mg/ml doxycycline from 1 day post-infection. Thirty-five days post-infection mice were split in two groups: group 1 continued to receive doxycycline and doxycycline treatment was withdrawn from group 2. Numbers of CFU present in lungs (upper panel) and spleen (lower panel) of treated (closed circles) and of untreated (open circles) mice were determined 1 (D1), 35 (D35), 63 (D63), 91 (D91), 120 (D120) and 160 (D160) days post-infection (experiments 1 and 2, black and blue circles) or 1, 35 and 160 days post-infection (experiment 3, green circles). Each circle represents CFU obtained from one mouse. The dashed line corresponds to the detection limit. When counts in infected mice were below the detection limit, the number of CFU scored was 50 CFU per organ (1.7 log).</p