a-microscopic-phenotypic-assay-for-quantification-intracellular

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 ...

Université de Lille

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. This biosynthetic pathway is essential and specific for mycobacteria and still represents a valuable system for the search of new anti-tuberculous agents. Several data, in the literature, suggest the existence of protein-protein interactions within the FAS-II system. These interactions themselves might serve as targets for a new generation of drugs directed against Mtb. By using an extensive in vivo yeast two-hybrid approach and in vitro co-immunoprecipitation, we have demonstrated the existence of both homotypic and heterotypic interactions between the known components of FAS-II. The condensing enzymes KasA, KasB and mtFabH interact with each other and with the reductases MabA and InhA. Furthermore, we have designed and constructed point mutations of the FAS-II reductase MabA, able to disrupt its homotypic interactions and perturb the interaction pattern of this protein within FAS-II. Finally, we showed by a transdominant genetic approach that these mutants are dominant negative in both non-pathogenic and pathogenic mycobacteria. These data allowed us to draw a dynamic model of the organization of FAS-II. They also represent an important step towards the design of a new generation of anti-tuberculous agents, as being inhibitors of essential protein-protein interactions.

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

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. We have previously shown that there are protein-protein interactions between the components of FAS-II that are essential for mycobacterial survival. We have now looked at the potential partners of FAS-II, mtFabD, the methyltransferases MmaAs, and Pks13. A combination of yeast two-hybrid and co-immunoprecipitation experiments showed that mtFabD interacts with each beta-ketoacyl-synthase (KasA, KasB and mtFabH) and with the core of FAS-II (InhA and MabA). The methyltransferases have a greater affinity for KasA and KasB than for mtFabH, suggesting that modifications on the meromycolic chains may occur during their elongation. Finally, Pks13, which catalyzes the final Claisen condensation of mycolic acids, interacts specifically with KasB. These data allowed us to determine the architecture of the multiple specialized FAS-II complexes, giving us insights into the organization of the complete mycolic acids biosynthesis. Our studies suggest a new and crucial interaction (KasB-Pks13) as a putative target for peptidomimetic antibiotics.

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

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. tb kinase PknL by demonstrating that it expresses autophosphorylation activity and phosphorylates Rv2175c. On-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, five phosphorylated threonine residues were identified in PknL. Among them, we showed that the activation loop phosphorylated residues Thr173 and Thr175 were essential for the autophosphorylation activity of PknL. Phosphorylation of the activation loop Thr173 residue is also required for optimal PknL-mediated phosphorylation of Rv2175c. Together, our results indicate that phosphorylation of the PknL activation loop Thr residues not only controls PknL kinase activity but is also required for recruitment and phosphorylation of its substrate. Rv2175c was found to be phosphorylated when overexpressed and purified from Mycobacterium smegmatis as 2-DE indicated the presence of different phosphorylated isoforms. Given the presence of the dcw gene cluster in the close vicinity of the pknL/Rv2175c locus, and its conservation in all mycobacterial species, we propose that PknL/Rv2175c may represent a functional pair in the regulation of mycobacterial cell division and cell envelope biosynthesis.

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.

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 beta-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP. Because mtFabH represents a potential regulatory key point of the mycolic acid pathway, we investigated the hypothesis that phosphorylation of mtFabH controls its activity. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism of regulation in prokaryotes. We demonstrate here that mtFabH was efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknF and PknA, as well as in vivo in mycobacteria. Analysis of the phosphoamino acid content indicated that mtFabH was phosphorylated exclusively on threonine residues. Mass spectrometry analyses using liquid chromatography-electrospray ionization/tandem mass spectrometry identified Thr45 as the unique phosphoacceptor. This was further supported by complete loss of PknF- or PknA-dependent phosphorylation of a mtFabH mutant. Mapping Thr45 on the crystal structure of mtFabH illustrates that this residue is located at the entrance of the substrate channel, suggesting that the phosphate group may alter accessibility of the substrate and thus affect mtFabH enzymatic activity. A T45D mutant of mtFabH, designed to mimic constitutive phosphorylation, exhibited markedly decreased transacylation, malonyl-AcpM decarboxylation, and condensing activities compared with the wild-type protein or the T45A mutant. Together, these findings not only represent the first demonstration of phosphorylation of a beta-ketoacyl-ACP synthase III enzyme but also indicate that phosphorylation of mtFabH inhibits its enzymatic activity, which may have important consequences in regulating mycolic acid biosynthesis.

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

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. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr(191) being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr(191) alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through Ser/Thr protein kinase-dependent phosphorylation of a type II fatty acid synthase enzyme.

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

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. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA_T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.

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

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. To investigate the role of Rv0900 and Rv0901 in M. bovis BCG, we have constructed a strain deleted for the whole operon as well as complemented strains carrying a deletion of Rv0900 or a frameshift mutation in either Rv0900 or Rv0901. Importantly, mutations in Rv0900 and/or Rv0901 strongly altered OmpATb expression, demonstrating that Rv0900 and Rv0901 play a regulatory role, which appears to occur at a post-transcriptional level.

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

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.

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

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. We have demonstrated previously that proteins of FAS-II interact specifically to form specialized and interconnected complexes. This finding suggested that the organization of FAS-II resemble to the architecture of multifunctional mega-enzyme like the mammalian mFAS-I, which is devoted to the fatty acid biosynthesis.

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

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. In epithelial cells, mycolactone-induced stimulation of ARP2/3 concentrated in the perinuclear region, resulting in defective cell adhesion and directional migration. In vivo injection of mycolactone into mouse ears consistently altered the junctional organization and stratification of keratinocytes, leading to epidermal thinning, followed by rupture. This degradation process was efficiently suppressed by coadministration of the N-WASP inhibitor wiskostatin. These results elucidate the molecular basis of mycolactone activity and provide a mechanism for Buruli ulcer pathogenesis. Our findings should allow for the rationale design of competitive inhibitors of mycolactone binding to N-WASP, with anti-Buruli ulcer therapeutic potential.

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

Mycolactone est un lipide diffusible s&eacute;cr&eacute;t&eacute; par le pathog&egrave;ne humain Mycobacterium ulcerans, qui induit la formation d&#39;ouvrir des l&eacute;sions cutan&eacute;es appel&eacute;es ulc&egrave;res de Buruli. Ici, nous montrons que mycolactone op&egrave;re en d&eacute;tournant la famille de prot&eacute;ine (WASP) de Wiskott - Aldrich, syndrome de facteurs de nucl&eacute;ation de l&#39;actine. En perturbant la gu&ecirc;pe auto-inhibition, mycolactone m&egrave;ne &agrave; activation incontr&ocirc;l&eacute;e de ARP2/3-m&eacute;di&eacute;e par l&#39;Assembl&eacute;e de l&#39;actine dans le cytoplasme. Dans les cellules &eacute;pith&eacute;liales, mycolactone induite par la stimulation de ARP2/3 concentr&eacute;s dans la r&eacute;gion p&eacute;rinucl&eacute;aire, aboutissant &agrave; des cellules d&eacute;fectueuses adh&eacute;rence et directionnelle de migration. L&#39;injection in vivo de la mycolactone dans les oreilles de souris constamment modifi&eacute; l&#39;organisation jonctionnelle et stratification des k&eacute;ratinocytes, conduisant &agrave; un amincissement &eacute;pidermique, suivie de la rupture. Ce processus de d&eacute;gradation a &eacute;t&eacute; efficacement supprim&eacute; par l&#39;administration concomitante de la N-WASP inhibiteur wiskostatin. Ces r&eacute;sultats &eacute;lucider les bases mol&eacute;culaires de l&#39;activit&eacute; de la mycolactone et fournissent un m&eacute;canisme pour la pathog&eacute;nie de l&#39;ulc&egrave;re de Buruli. Nos conclusions devraient permettre la conception de la raison d&#39;&ecirc;tre des inhibiteurs comp&eacute;titifs de la liaison de la mycolactone &agrave; N-WASP, avec le potentiel th&eacute;rapeutique de l&#39;anti-Buruli ulc&egrave;re.

Mycolactone activation des prot&eacute;ines de Wiskott - Aldrich, syndrome sous-tend la formation de l&#39;ulc&egrave;re de Buruli.

Mycolactone &egrave; una lipide diffusibile secernute dall&#39;agente patogeno umano Mycobacterium ulcerans, che induce la formazione di aprire le lesioni cutanee denominate ulcere di Buruli. Qui, ci mostra che mycolactone opera dal dirottamento della famiglia di proteine (WASP) Wiskott - Aldrich-Syndroms fattori actina-nucleare. Interrompendo WASP autoinhibition, mycolactone conduce all&#39;attivazione incontrollata dell&#39;Assemblea ARP2/3-mediata di actina nel citoplasma. Nelle cellule epiteliali, stimolazione indotta da mycolactone di ARP2/3 concentrato nella regione perinucleare, con conseguente migrazione di adesione e direzionale delle cellule difettose. Iniezione in vivo di mycolactone nelle orecchie del mouse costantemente alterato l&#39;organizzazione giunzionale e stratificazione dei cheratinociti, portando ad un assottigliamento dell&#39;epidermide, seguita dalla rottura. Questo processo di degradazione in modo efficiente fu soppressa dal coadministration del N-WASP inibitore della wiskostatin. Questi risultati delucidare le basi molecolari dell&#39;attivit&agrave; mycolactone e forniscono un meccanismo per la patogenesi di ulcera di Buruli. I nostri risultati dovrebbero consentire la progettazione logica di inibitori competitivi dell&#39;associazione mycolactone a N-WASP, con potenziale terapeutico ulcera di Buruli anti.

Mycolactone attivazione delle proteine di Wiskott - Aldrich-Syndroms sottende la formazione di ulcere di Buruli.

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. Overexpression of wild-type Pks13 resulted in TP resistance, and overexpression of the Pks13(F79S) mutant conferred high resistance. In vitro, TP inhibited fatty acyl-AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type M. tuberculosis, but it did so to a much lesser extent in TP-resistant M. tuberculosis. TP treatment was bactericidal and equivalent to treatment with the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment resulted in sterilizing activity. Computational docking identified a possible TP-binding groove within the Pks13 acyl carrier protein domain. This study confirms that M. tuberculosis Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway.

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

Nous rapportons une nouvelle classe de compos&eacute;s de thioph&egrave;ne (TP) qui tuent Mycobacterium tuberculosis par le m&eacute;canisme non d&eacute;finie auparavant d&#39;inhibition de la Pks13. Une mutation de F79S pr&egrave;s du site catalytique de Ser55 en Pks13 conf&egrave;re r&eacute;sistance TP chez M. tuberculosis. Surexpression de Pks13 sauvage a donn&eacute; lieu &agrave; la r&eacute;sistance de TP, et la surexpression du mutant Pks13(F79S) conf&eacute;r&eacute; haute r&eacute;sistance. In vitro, TP inhibe gras acyl-AMP chargement dans Pks13. TP inhib&eacute;e mycoliques biosynth&egrave;se de l&#39;acide chez M. tuberculosis de type sauvage, mais elle l&#39;a fait dans une bien moindre mesure chez M. TP-resistant tuberculosis. TP traitement &eacute;tait bact&eacute;ricide et &eacute;quivalent &agrave; un traitement par l&#39;isoniazide de premi&egrave;re intention, mais c&#39;&eacute;tait moins susceptible de permettre une r&eacute;sistance &eacute;mergente. Isoniazide combin&eacute;e et le traitement de la TP a entra&icirc;n&eacute; dans l&#39;activit&eacute; de st&eacute;rilisation. Calcul d&#39;accueil identifi&eacute; une rainure de TP-liaison possible au sein du domaine de prot&eacute;ine de transporteur d&#39;acyl Pks13. Cette &eacute;tude confirme que M. tuberculosis Pks13 est n&eacute;cessaire pour la biosynth&egrave;se de l&#39;acide mycolique, il valide comme cible th&eacute;rapeutiques et montre le potentiel th&eacute;rapeutique d&#39;inhiber simultan&eacute;ment plusieurs cibles dans la m&ecirc;me voie de biosynth&egrave;se.

Article	Year
Protein-protein interactions within the Fatty Acid Synthase-II system of Mycobacterium tuberculosis are essential for mycobacterial viability. Molecular microbiology\| PubMed ID: 15554959	2004
The biosynthesis of mycolic acids in Mycobacterium tuberculosis relies on multiple specialized elongation complexes interconnected by specific protein-protein interactions. Journal of molecular biology\| PubMed ID: 16213523	2005
The Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitmen... Proteomics\| PubMed ID: 18175374	2008
The Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III activity is inhibited by phosphorylation on a single threonine residue. The Journal of biological chemistry\| PubMed ID: 19074144	2009
Phosphorylation of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein reductase MabA regulates mycolic acid biosynthesis. The Journal of biological chemistry\| PubMed ID: 20178986	2010
Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis. Molecular microbiology\| PubMed ID: 21143326	2010
Expression of OmpATb is dependent on small membrane proteins in Mycobacterium bovis BCG. Tuberculosis (Edinburgh, Scotland)\| PubMed ID: 21802366	2011
Mycolactone diffuses into the peripheral blood of Buruli ulcer patients--implications for diagnosis and disease monitoring. PLoS neglected tropical diseases\| PubMed ID: 21811642	2011
The Mycobacterium tuberculosis FAS-II dehydratases and methyltransferases define the specificity of the mycolic acid elongation complexes. PloS one\| PubMed ID: 22216317	2011
Mycolactone activation of Wiskott-Aldrich syndrome proteins underpins Buruli ulcer formation. The Journal of clinical investigation\| PubMed ID: 23549080	2013
Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis. Nature chemical biology\| PubMed ID: 23770708	2013

Romain Veyron-Churlet

Biography

JoVE Journal Publications

Other Publications