Université Paris Descartes

Faculté de Médecine
Paris Descartes (site Necker)

Unité de Pathogénie
des Infections Systémiques
UMR 570

Head: Prof. Xavier Nassif

156, rue de Vaugirard
75730 Paris Cedex 15
FRANCE
The mycobacterial genus encompasses saprophytic, environmental opportunistic pathogenic and pathogenic species that that are responsible of human threats such as leprosy, tuberculosis and Buruli ulcer, all being diseases of developing country. The mycobacterial cell envelope is a multilaminar structure that has been shown to protect the cell from the stress encountered in the environment but also to play an important role against the bactericidal activity of the immune cells. Mycobacterial species secrete in the medium but also onto their cell surface a very complex array of small hydrophobic molecules, some of which have been shown to play a role in the pathology. Some of these small molecules such as the trehalose mycolates and the phosphatidylinositolmannoside are ubiquitous glycolipids that are found in the entire Corynebacterinae suborder within the Actinobacteria division, while others such as PGL (phenolglycolipids) or GPL (glycopeptidolipid) are species specific. Currently, the mechanism of how these glycolipids are addressed to the cell surface is not understood.

We have shown, by using a transposon library of M. smegmatis and a simple dye assay, that 6 previously uncharacterised genes are involved in GPL biosynthesis. All of these genes are clustered in a single genomic region of approximately 60 kb, called extended mps locus. We demonstrated that a small integral membrane protein of 272 amino acids named Gap (gap: GPL addressing protein) is specifically required for the transport of the GPLs to the cell surface. This protein is predicted to contain six transmembrane segments and possesses homologs across the mycobacterial genus, thus delineating a new protein family. In addition, we have shown that Gtf3, which is part of the extended mps locus, is a glycosyltransferase responsible for the synthesis of the triglycosylated GPLs. Recently, we identified Lsr2 as a negative regulator of the glycopeptidolipid synthesis and characterized it, in collaboration with Jun Liu (Univ. Toronto), as a new DNA-bridging protein, which might explain its regulatory activity.


Left pannel, upper pannel. Wild-type and gpl- strains of M. smegmatis. Lower pannel., left, sliding motility of M. smegmatis, right, mass-spectrometry analysis of M. smegmatis. GPLs, Right pannel, structure of the GPLs of M. smegmatis Artwork by C. Deshayes & D. Kocincova.
Our project is aimed at characterising the genes that are necessary for the biosynthesis and export of the small metabolites that constitute the mycobacterial cell envelope. In some cases, an association between the production of these compounds and the virulence has been proved. Indeed, some of these metabolites strongly interfere with the immune system. The modularity of the small molecules metabolism is by far not yet unravelled, and one might guess that the combination of genetic manipulation of these pathways with high through put screening of in vitro biological activity may well identify new drugs for the future




Bianca Audrain (Master student, U. Paris Diderot).
Daniel Euphrasie (Technician, U. Paris Descartes).
Rachid Nessar (PhD student, U. Paris Diderot (MRT)).
Halima Medjahed (PhD student, U. Paris Diderot (VLM)).
Anil Kumar Singh (Post-doc, (AXA funds)).
Jean-Marc Reyrat (PI, DR2 Inserm).

PAST MEMBERS

Berit Sonden, Post-doc, 2001-2003.
Stephane Calmat, Master Student, 2003.
Marcin Golec, MD, 2004.
Lamya Rhayat, Master Student, 2005.
Andra Waagmeester, PhD student, 2005.
Nina Semiramoth, Master Student, 2005.
Fanny Beneyt, Master Student, 2006.
Dana Kocincova, PhD Student, 2002-2006.
Shaghayegh Rouzbeh, Master Student, 2007.
Caroline Deshayes PhD student, 2004-2007.
Angela Cordone Post doc, 2007-2008.
Neike Fernandez Master student, 2008.





Deshayes, C., Kocincova, D., Etienne, G., and Reyrat, J. M. 2008. Glycopeptidolipids: a complex pathway for small pleiotropic molecules. In The Mycobacterial Cell Envelope (J. M. Reyrat and M. Daffe, Eds), ch 21, 345-366. (ASM press). (book chapter).

Stocker, N. G., Sander, P. and Reyrat, J. M. 2005. Gene Replacement Systems. In Tuberculosis (S. T. Cole et al., Eds), ch 12, 183-190. (ASM press). (book chapter).

Reyrat J.-M. and Kahn D. 2001. Mycobacterium smegmatis : an absurd model for tuberculosis? Trends in Microbiology, 9: 472-473. (comments).

Reyrat J.-M., Pelicic V., Gicquel B. and Rappuoli R. 1998. Counter-selectable markers: untapped tools for bacterial genetics and pathogenesis. Infect. Immun., 66: 4011-4017. (review).

Pelicic V., Reyrat J.-M. and Gicquel B. 1998. Genetic advances for studying Mycobacterium tuberculosis pathogenicity. Mol. Microbiol., 28: 413-420. (review).




The most significant are indicated in deep purple

Medjahed H and Reyrat JM. 2009. Construction of Mycobacterium abscessus defined glycopeptidolipid mutants: comparison of genetic tools. Appl Environ Microbiol., 75: 1331-1338.

Kocincova D, Winter N, Euphrasie D, Daffe M, Reyrat JM and Etienne G. 2009. The cell surface-exposed glycopeptidolipids confer a selective advantage to the smooth variants of Mycobacterium smegmatis in vitro.. FEMS Microbiol Lett., 290: 39-44.

Gallien S, Perrodou E, Carapito C, Deshayes C, Reyrat JM, Van Dorsselaer A, Poch O, Schaeffer C and Lecompte O. 2009. Ortho-proteogenomics: multiple proteomes investigation through orthology and a new MS-based protocol. Genome Res., 19: 128-135.

Malaga W, Constant P, Euphrasie D, Cataldi A, Daffe M, Reyrat JM and Guilhot C. 2008. Deciphering the genetic bases of the structural diversity of phenolic glycolipids in strains of the Mycobacterium tuberculosis complex. J Biol Chem., 283: 15177-15184.

 Provvedi R, Kocincova D, Dona V, Euphrasie D, Daffe M, Etienne G, Manganelli R and Reyrat JM. 2008. SigF controls carotenoid pigment production and affects transformation efficiency and hydrogen peroxide sensitivity in Mycobacterium smegmatis. J. Bacteriol.,190: 7859-7863

 Kocincova, D., Singh, A.K., Beretti, J. L., Ren, H., Euphrasie, D., Liu, J., Daffe, M., Etienne, G. and Reyrat J. M. 2008. Spontaneous transposition of IS1096 or ISMsm3 leads to glycopeptidolipid overproduction and affects surface properties in M. smegmatis. Tuberculosis,, 88: 390-398.

Chen J. M, Ren H, Shaw J. E, Wang Y. J, Li M, Leung A. S, Tran V, Berbenetz N. M, Kocincova D, Yip C. M, Reyrat J. M and Liu J. 2008. Lsr2 of Mycobacterium tuberculosis is a DNA-bridging protein N. A. R.,, 36: 2123-2135.

Biet F, Bay S, Thibault V. C, Euphrasie D, Grayon M, Ganneau C, Lanotte P, Daffe M, Gokhale R, Etienne G and Reyrat J. M.. 2008. Lipopentapeptide induces a strong host humoral response and distinguishes Mycobacterium avium subsp. paratuberculosis from M. avium subsp. avium Vaccine,, 26: 257-268.

Ripoll F, Deshayes C, Pasek S, Laval F, Beretti J. L, Biet F, Risler J. L, Daffe M, Etienne G, Gaillard J. L and Reyrat J. M. 2007. Genomics of glycopeptidolipid biosynthesis in Mycobacterium abscessus and M. chelonae. BMC Genomics, 8: 114.

Deshayes, C., Perrodou E., Gallien S., Euphrasie, D., Schaeffer C., Van-Dorsselaer A., Poch O., Lecompte O. and Reyrat J. M. 2007. Interrupted coding sequences in Mycobacterium smegmatis: Authentic Mutations or sequencing errors ? Gen. Biol., 7: 8 (2): R20.

 Vultos T. D, Mederle I., Abadie V., Pimentel M., Moniz-Pereira J., Gicquel B., Reyrat J. M., and Winter N. 2006. Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria. BMC Mol Biol., 7: 47.

Perrodou E, Deshayes C, Muller J, Schaeffer C, Van Dorsselaer A, Ripp R, Poch O, Reyrat JM, Lecompte O. 2006. ICDS database: interrupted CoDing sequences in prokaryotic genomes. N. A. R., 34: D338-43.

Deshayes, C., Laval, F., Montrozier, H., Daffe, M., Etienne, G., and Reyrat, J. M.. 2005. A glycosyltransferase involved in the biosynthesis of triglycosylated glycopeptidolipids in Mycobacterium smegmatis: impact on surface properties. J. Bacteriol., 87: 7283-7291.

Sonden, B., Kocincova, D., Deshayes, C., Euphrasie, D., Rhayat, L., Laval, F., Frehel, C., Daffe, M., Etienne, G., and Reyrat, J. M. 2005. Gap, a mycobacterial specific integral membrane protein, is required for glycolipid transport to the cell surface. Mol. Microbiol., 58: 426-440.

L. Grode, J.Hess, P.Seiler, V. Brinkmann, S. Baumann, P. Mann, D. Smith, G. J. Bancroft, J-M.Reyrat, D. van Soolingen, B. Raupach and S. H. E. Kaufmann. 2005. Efficacious Vaccination against Tuberculosis by Mycobacterium bovis Bacille Calmette-Guerin Mutants Secreting Listeriolysin. J. Clin. Invest., 115: 2472-2479.

Waagmeester, A., Thompson, J., and Reyrat, J. M. 2005. Comparative genomic analysis of the sigma factors content between Mycobacterium smegmatis and Mycobacterium tuberculosis. Trends in Microbiol. 13: 505-509.

Rhodes, M. W., Kator, H., McNabb, A., Deshayes, C., Reyrat, J. M., Brown-Elliott, B. A., Wallace Jr, R., Trott, K. A., Parker, J. M., Lifland, B., Osterhout, G., Kaattari, I., Reece, K., Volgelbein, W., Ottinger, C. A. 2005. Mycobacterium pseudoshottsii sp. nov., a slowly growing chromogenic species isolated from Chesapeake Bay striped bass (Morone saxatilis). Int. J. Sys. Evol. Microbiol55: 1139-1147.

Wagner, D., Maser, J., Moric, I., Boechat, N., Vogt, S., Gicquel., B., Lai, B., Reyrat, J. M., and Bermudez, L. 2005. Changes of the phagosomal element concentrations by Mycobacterium tuberculosis Mramp. Microbiology, 151: 323-332.

Khalid, D., Deghmane, A. E., Reyrat, J. M., Talal, A., and Hmama, Z. 2004. Mycobacterium bovis BCG urease attenuates MHC class II trafficking to the macrophage cell surface. Inf. Immun., 72 :4200-4209.

Kocincova, D., Sonden, B., de Mendonça-Lima L., Gicquel B., and Reyrat J.-M. 2004. The Erp protein is anchored at the surface by a carboxy-terminal hydrophobic domain and is important for cell-wall structure in M. smegmatis. FEMS microbiol. letters,. 231: 191-196.

Kocincova, D., Sonden, B., Bordat, Y., Pivert, E., de Mendonça-Lima L., Gicquel B., and Reyrat J.-M. 2004. The hydrophobic domain of the mycobacterial Erp protein is not essential for the virulence of M. tuberculosis. Inf. Immun., 72 : 2379-2382..

Recchi, C., Sclavi, B., Rauzier, J., Gicquel, B., and Reyrat J.-M. 2003. Mycobacterium tuberculosis Rv1395 is a class III transcriptional regulator of the AraC family involved in cytochrome P450 regulation. J. Biol. Chem., 278 : 33763-33773.
de Mendonça-Lima L., Bordat, Y., Pivert, E., Recchi, C., Neyrolles, O., Maitournam, A., Gicquel., B., and Reyrat, J. M. 2003. The allele encoding the mycobacterial Erp protein affects lung disease in mice. Cell. Microbiol., 5 : 65-73.

Boechat N., Lagier, B., Petit, S., Bordat, Y., Rauzier, J., Hance, A. J., Gicquel, B. and Reyrat J.-M. 2002. Disruption of the gene homologous to mamalian Nramp in Mycobacterium tuberculosis does not affect virulence in Balb/c mice. Infect. Immun., 70: 4124-4131.

Recchi C., Rauzier J., Gicquel B. and Reyrat J.-M. 2002. Signal-sequence-independant secretion of the staphylococcal nuclease in Mycobacterium smegmatis. Microbiol, 148: 529-535.

de Mendonça-Lima L., Picardeau M., Raynaud C., Rauzier J., Goguet de la Salmonière Y.-O., Barker L., Bigi F., Cataldi A., Gicquel B. and Reyrat J.-M. 2001. Erp, a surface-exposed protein familly specific to mycobacteria. Microbiol., 147: 2315-2320..

Pelicic V., Jackson M., Reyrat J.-M., Jacobs W.-R., Gicquel B. and Guilhot C. 1997. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc. Natl Acad. Sci., USA., 94: 10955-10960.

Quinting B., Reyrat J.-M., Monnaie D., Amicosante G., Pelicic V., Gicquel B., Frère J.-M. and Galeni M. 1997. Contribution of ß-lactamase production to the resistance of mycobacteria to ß-lactam antibiotic. FEBS Lett., 406: 275-278.

Pelicic V., Reyrat J.-M. and Gicquel B. 1996. Positive selection of allelic exchange events in slow-growing mycobacteria. FEMS Microbiol. Lett., 144: 161-166.

Reyrat J.-M., Lopez-Ramirez G., Ofredo C., Gicquel B. and Winter N. 1996. Urease activity does not contribute dramatically to persistence of M. bovis BCG. Infect. Immun., 64: 3934-3936.

Pelicic V., Reyrat J.-M. and Gicquel B. 1996. Generation of unmarked directed mutations in mycobacteria, using sucrose counterselectable suicide vectors. Mol. Microbiol., 20: 919-925.
Pelicic V., Reyrat J.-M. and Gicquel B. 1996. Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria. J. Bacteriol., 178: 1197-1199.

Reyrat J.-M., Berthet F.-X. and Gicquel B. 1995. The ureABC locus of M. tuberculosis and its utilization for the demonstration of allelic echange in M. bovis BCG. Proc. Natl. Acad. Sci., USA, 92: 8768-8772.