Arthur_michel

Ecole Doctorale COMPLEXITE DU VIVANT – Fiche Projet CONCOURS

Nom et prénom du directeur de thèse (et si besoin du co-directeur) : ARTHUR Michel

Coordonnées Tel : 01 44 27 54 55
e-mail : michel.arthur@crc.jussieu.fr
Nom et prénom du responsable de l’équipe : ARTHUR Michel
Nombre de chercheurs et enseignants-chercheurs statutaires de l’équipe titulaires d’une HDR : 2 Nom et prénom du responsable du laboratoire : Wolf-Herman FRIDMAN Intitulé du laboratoire et N° d’unité : Structures bactérienne impliquées dans les modulation de la résistance aux
antibiotiques (LRMA), Equipe 12, Centre de Recherche des Cordeliers (UMR S 872)
Titre du projet de thèse : Role of five L,D-transpeptidase paralogues in peptidoglycan synthesis and maturation
in Mycobacterium tuberculosis

Résumé du projet de thèse
Despite intensive efforts to improve tuberculosis (TB) care and control, the global burden of TB is falling only
slowly and drug resistance is increasing. According to the WHO report 2012, there were 8.7 million new cases
and 1.4 million deaths in 2011 (1). In recent years, TB treatment has become more complicated due to the
emergence of multidrug-resistant (MDR) TB, i.e. TB due to strains resistant to isoniazid and rifampin, the two
main antituberculous drugs. In 2011, the WHO estimated that there were 310,000 new cases of infections due to
MDR-TB. Unfortunately, the extensive use of second-line drugs has led to the emergence of extensively drug
resistant M. tuberculosis (XDR-TB) that shows a very poor prognosis with mortality rates in the order of 26 %
(2-4), due to the lack of an efficient therapy. These alarming data highlight the urgent need for new
antituberculosis drugs since no novel antituberculosis antibiotic, except fluoroquinolones and bedaquiline, has
been introduced in the past 45 years.
Beta-lactams have not been considered for the treatment of TB since M. tuberculosis naturally produces an extended spectrum class A β-lactamase (BlaC) (5). However, very recent data have opened new avenues toward
the potential use of β-lactams in the treatment of tuberculosis, especially against MDR- and XDR-TB. First,
BlaC was found to be irreversibly inactivated by clavulanic acid (5). In combination with this β-lactamase
inhibitor, one member of the carbapenem family, meropenem, was uniformly active against a panel of XDR
strains (MIC of meropenem < 1 mg/l in the presence of 2.5 mg/l of clavulanate) (6). L,D-transpeptidases are
likely to be the target of carbapenems since these enzymes are responsible for formation of 80% of the cross-
links and are irreversibly inactivated by these drugs (7). Moreover, one of the five L,D-transpeptidases of M.
tuberculosis (LdtMt2) is essential for virulence in a mouse model of acute infection (8). Inhibition of L,D-
transpeptidases by carbapenems may also have potential applications for the treatment of susceptible TB since
the meropenem-clavulanate combination is active against non-replicative forms (“dormant” forms) of M.
tuberculosis (6), that are difficult to eradicate even with isoniazid and rifampin. The adaptive response of M.
tuberculosis during the transition from aerobic growth to stationary phase results in the activation of several
genes that are likely to play an essential role in the long-term survival of the bacteria and are therefore encoding
potential targets for the development of sterilizing drugs. Among these genes, Rv0116c, the gene encoding L,D-
transpeptidase LdtMt1, is up-regulated 17 fold (9). Thus, LdtMt1 could be an essential target accounting for the
bactericidal activity of the meropenem–clavulanate combination against dormant forms of M. tuberculosis in
vitro.
In this context, the specific aims of the thesis can be outlined as follows:
1. Identification of the transpeptidases essential for the growth and persistence of M. tuberculosis. We have
identified a total of five L,D-transpeptidase paralogs that are present and conserved in the genome of all strains of
M. tuberculosis available in sequence databases. Selection of meropenem resistant-mutants and genome
sequencing will be used to identify essential transpeptidases. In order to analyze the regulation of the L,D-
transpeptidases in response to environmental conditions, we will (i) raise rat polyclonal antibodies specific of
each of the five proteins for Western blot analyses; (ii) use RT-PCR to monitor the level of expression of each of
the corresponding genes; (iii) design an MS-based assay to detect transpeptidases covalently labeled by a
carbapenem containing a biotinyl group (synthesized by one of our collaborators). These assays will be applied
to the detection of the five L,D-transpeptidases in the exponential and stationary phases of growth and in the
nutrient starvation and oxygen depletion dormancy models.
2. Characterization of the adaptation of peptidoglycan structure to the growth and persistence of M.
Ecole Doctorale COMPLEXITE DU VIVANT – Fiche Projet CONCOURS

tuberculosis. Changes in peptidoglycan structure will be investigated during the transition from exponential
growth to the non-replicative state in in vitro models of dormancy by a combination of conventional liquid
chromatography-mass spectrometry analysis (LC-MS) and solid-state NMR as developed in a previous ANR
program. By these approaches, snapshots of peptidoglycan structure during the cell cycle will be obtained.
“Pulse” labeling techniques will also be developed to determine whether differences identified in peptidoglycan
structure results from changes in the mode of synthesis of the polymer or from its maturation, i.e. changes
affecting the activity of peptidoglycan polymerases (glycosyltransferases and transpeptidases) or the activity of
enzymes that remodel the peptidoglycan network after its polymerization. For this approach, “pulse” isotope
labeling methods will be developed based on combination of growth media containing “light” (12C and 14N) and
“heavy” (13C and 15N) nutriments. By using these techniques, it will be feasible to determine how M. tuberculosis
adapts its peptidoglycan structure to generate the highly resistant forms that survive for extended time periods in
a “dormant” persistent state.
The thesis will contribute to the development β-lactams useful for the treatment of XDR-TB, which is one of our
main objectives in collaboration with two teams, specialized in organic synthesis and structural biology.
1. Anonymous. WHO, 2012, Global TB control report 2012. in World Health Organization Report
2. Gandhi, N. R., Moll, A., Sturm, A. W., Pawinski, R., Govender, T., Lalloo, U., Zeller, K., Andrews, J., and
Friedland, G. (2006) Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with
tuberculosis and HIV in a rural area of South Africa. The Lancet 368, 1575-1580
3. Kliiman, K., and Altraja, A. (2009) Predictors of poor treatment outcome in multi- and extensively drug- resistant pulmonary TB. European Respiratory Journal 33, 1085-1094
4. Veziris, N., Martin, C., Brossier, F., Bonnaud, F., Denis, F., and Aubry, A. (2007) Treatment failure in a case of extensively drug-resistant tuberculosis associated with selection of a GyrB mutant causing fluoroquinolone
resistance. European Journal of Clinical Microbiology &amp; Infectious Diseases 26, 423-425
5. Hugonnet, J. E., and Blanchard, J. S. (2007) Irreversible inhibition of the Mycobacterium tuberculosis beta- lactamase by clavulanate. Biochemistry 46, 11998-12004
6. Hugonnet, J. E., Tremblay, L. W., Boshoff, H. I., Barry, C. E. r., and Blanchard, J. S. (2009) Meropenem- clavulanate is effective against extensively drug-resistant Mycobacterium tuberculosis. Science 323, 1215-
1218
7. Lavollay, M., Arthur, M., Fourgeaud, M., Dubost, L., Marie, A., Veziris, N., Blanot, D., Gutmann, L., and Mainardi, J. L. (2008) The peptidoglycan of stationary-phase Mycobacterium tuberculosis predominantly
contains cross-links generated by L,D-transpeptidation. J Bacteriol 190, 4360-4366
8. Gupta, R., Lavollay, M., Mainardi, J. L., Arthur, M., Bishai, W. R., and Lamichhane, G. (2010) The Mycobacterium tuberculosis protein Ldt(Mt2) is a nonclassical transpeptidase required for virulence and
resistance to amoxicillin. Nat Med 16, 466-469
9. Betts, J. C., Lukey, P. T., Robb, L. C., McAdam, R. A., and Duncan, K. (2002) Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol
Microbiol 43, 717-731
Thèses actuellement en cours dans l’équipe
Trois publications récentes du directeur de thèse
Fonvielle, M**, M. Chemama,** M. Lecerf, R. Villet, P. Busca, A. Bouhss, M. Ethève-Quelquejeu,* and
M. Arthur* Co first** and corresponding* authors. Decoding the logic of the tRNA regiospecificity of
non-ribosomal FemXWv aminoacyl transferase. Angew. Chem. Int. Ed. Engl. 2010. 49:5115-5119.
Triboulet, S., M. Arthur, J. L. Mainardi, C. Veckerle, V. Dubée, A. Nguekam-Moumi, L. Gutmann, L. B.
Rice, and J. E. Hugonnet. Inactivation kinetics of a new target of beta-lactam antibiotics. J. Biol. Chem.
2011. 286:22777-22784.
Lecoq, L., C. Bougault, J.E. Hugonnet, C. Veckerlé, O. Pesseya, M. Arthur*, and JP. Simorre*.
Corresponding authors*. 2012. Dynamics induced by β-lactam antibiotics in the active site of Bacillus
subtilis L,D-transpeptidase. Structure. 20:850-861.
Ecole Doctorale COMPLEXITE DU VIVANT – Fiche Projet CONCOURS

Docteurs encadrés par le directeur de thèse ayant soutenu après septembre 2006 et publications relatives à leur sujet de thèse.
Mettre en gras le nom du directeur de thèse et celui du docteur.
Publications :
Mainardi JL, Hugonnet JE, Rusconi F, Fourgeaud M, Dubost L, Moumi AN, Delfosse V, Mayer C, Gutmann L, Rice
LB, Arthur M. Unexpected inhibition of peptidoglycan LD-transpeptidase from Enterococcus faecium by the beta-
lactam imipenem. J Biol Chem. 2007 Oct 19;282(42):30414-22.
Hugonnet JE, Blanchard JS. Irreversible inhibition of the Mycobacterium tuberculosis beta-lactamase by clavulanate.
Tremblay LW, Hugonnet JE, Blanchard JS. Structure of the covalent adduct formed between Mycobacterium
tuberculosis beta-lactamase and clavulanate. Biochemistry. 2008 13:5312-6.
Hugonnet JE, Tremblay LW, Boshoff HI, Barry CE 3rd, Blanchard JS. Meropenem-clavulanate is effective against
extensively drug-resistant Mycobacterium tuberculosis. Science. 2009 323:1215-8. Hugonnet JE. Efficient combination of clavulanate and beta-lactam antibiotics against extensively drug-resistant M.
tuberculosis. Med Sci (Paris). 2009 25:661-3. Sacco E*, Hugonnet JE*, Josseaume N, Cremniter J, Dubost L, Marie A, Patin D, Blanot D, Rice LB, Mainardi JL,
Arthur M. *equal contribution. Activation of the L,D-transpeptidation peptidoglycan cross-linking pathway by a
metallo-D,D-carboxypeptidase in Enterococcus faecium. Mol Microbiol. 2010 Feb;75(4):874-85.
Triboulet, S., M. Arthur, J. L. Mainardi, C. Veckerle, V. Dubée, A. Nguekam-Moumi, L. Gutmann, L. B. Rice, and J.
E. Hugonnet. Inactivation kinetics of a new target of beta-lactam antibiotics. J. Biol. Chem. 2011. 286:22777-
22784.
Publications :
Villet R., M. Fonvielle., P. Busca, M. Chemama, A.P. Maillard, J.E. Hugonnet, L. Dubost, A. Marie, N. Josseaume,
S. Mesnage, C. Mayer, J.M. Valéry, M. Ethève-Quelquejeu, and M. Arthur. Idiosyncratic features in tRNAs
participating in bacterial cell wall synthesis. Nucl. Acids Res. 2007. 35:6870-6883.
Chemama M., M. Fonvielle, R. Villet, M. Arthur, J.M. Valéry, and M. Etheve-Quelquejeu. Stable analogues of
aminoacyl-tRNA for inhibition of an essential step of bacterial cell wall synthesis. J. Am. Chem. Soc. 2007. 129:12642-12643. Fonvielle M., M. Chemama, R., Villet, M. Lecerf, M., A. Bouhss, J.M. Valéry, M. Ethève-Quelquejeu, and M.
Arthur. Aminoacyl-tRNA recognition by the FemXWv transferase for bacterial cell wall synthesis. Nucl. Acids
Res. 2009. 37:1589-1601.
Fonvielle, M**, M. Chemama,** M. Lecerf, R. Villet, P. Busca, A. Bouhss, M. Ethève-Quelquejeu,* and M. Arthur*
Co first** and corresponding* authors. Decoding the logic of the tRNA regiospecificity of non-ribosomal FemXWv aminoacyl transferase. Angew. Chem. Int. Ed. Engl. 2010. 49:5115-5119. IF : 12.73 Mainardi, J.L., R. Villet, T.D. Bugg C. Mayer, and M. Arthur Evolution of peptidoglycan synthesis under the
selective pressure of antibiotics in Gram-positive bacteria. FEMS Microbiol. Rev. 2008. 32:386-408.

Source: http://www.ed515.upmc.fr/doc/fiches_projet2013/ARTHUR_Michel.pdf