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Molecular mechanisms of microbial adaptation

Researchers
Hubert BECKER, MdC, Paris VI
Ursula LIEBL, DR, CNRS
Roxane LESTINI, Asst Prof, Ecole Polytechnique
Hannu MYLLYKALLIO, DR, CNRS
Stephane SKOULOUBRIS, MdC, Paris –Sud XI

Technicians & engineers
Isabelle LAMARRE-JOUENNE
Caroline L'HERMITTE STEAD

Thesis students
Yoann COLLIEN
Oscar CASTANON

For inquiries on the different research projects and possibilities for joining us, please contact us by email (Nous contacter)

Research topics
Our principal research goal is to understand how microorganisms adapt to different metabolic changes, crucial for their survival. Our studies concern sensing and signaling of different gas conditions, the availability of DNA building blocks and the reciprocal interactions of DNA replication and repair, responsible for guaranteeing genomic integrity at extreme conditions.

Ongoing projects
Our research interests include three major topics:

 

 

Project 1: Mechanism and targeting of the alternative thymidylate synthase ThyX

Much recent work has been devoted to molecular biological and biochemical studies, with the goal to unravel the reaction mechanism and inhibition of alternative thymidylate synthase ThyX, discovered in the laboratory. ThyX proteins are essential for de novo synthesis of the letter “T” (thymidylate) in DNA in many prokaryotes. Our work has shed new light on the evolution of prokaryotic genomes, since the low catalytic activity of ThyX limits DNA replication and consequently is found in prokaryotic species with small genomes, whereas organisms with more complex genomes use thymidylate synthase ThyA, the enzyme present also in humans. As ThyX is present in many human pathogens such as Helicobacter pylori or Mycobacterium tuberculosis, but absent in human cells, this opens the way to the development of novel antimicrobial strategies by identifying new inhibitors of this enzyme. In collaboration with Team 2, a number of complementary techniques have been applied to research on ThyX. In particular, we have observed an unusually high flexibility of the active site of ThyX proteins and have proposed how complex substrate interactions modulate oxygen reactivity of ThyX proteins. We are also analyzing folate metabolic and r elated pathways in the human pathogenic bacterium H. pylori.
We have obtained proof–of-concept using animal models of infection for the anti-microbial activity of 1,4- naphthoquinones that we patented as efficient and specific inhibitors of the alternative thymidylate synthase X (Figure 1). Our primary goal is to render our inhibitor series for eventual preclinical development through collaborative efforts. This will require synthesis of new lead derivatives to optimize anti-microbial activity of already identified compounds.

Figure 1: Identification and anti-microbial activity of H. pylori ThyX inhibitors

Project 1 - Recent publications:
  1. Laptenok SP, Bouzhir-Sima L, Lambry JC, Myllykallio H, Liebl U, Vos MH (2013) Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX. Proceedings of the National Academy of Sciences of the United States of America 110(22):8924-8929
  2. Basta T, Boum Y, Briffotaux J, Becker HF, Lamarre-Jouenne I, Lambry JC, Skouloubris S, Liebl U, Graille M, van Tilbeurgh H, Myllykallio H (2012) Mechanistic and structural basis for inhibition of thymidylate synthase ThyX. Open biology 2: 120
  3. Escartin F, Skouloubris S, Liebl U, Myllykallio H (2008) Flavin-dependent thymidylate synthase X limits chromosomal DNA replication. Proceedings of the National Academy of Sciences of the United States of America 105: 9948-9952
  4. Esra-Onen F, Boum Y, Jacquement C, Spanedda MV, Jaber N, Scherman D, Myllykallio H, Herscovici J (2008) Design, synthesis and evaluation of potent thymidylate synthase X inhibitors. Bioorganic & medicinal chemistry letters 18: 3628-3631
  5. International patents PCT N°PCT/EP201110555 and BET10P0152 on antimicrobial activity of ThyX inhibitors (INSERM/CNRS)

 

Project 2 – DNA replication and repair in Archaea

How Archaea, the third domain of Life, replicate their DNA and maintain their genomes intact under deleterious environmental conditions is also investigated by our team. A novel family of specific DNA endonucleases, dubbed NucS, has been identified and its action mechanism deciphered. We have established Haloferax volcanii as a unique model for studying archaeal DNA replication and repair in living cells using fluorescence microscopy (Figure 2). Moreover, a project on understanding the physiological function of a dimeric RNA ligase found in many archaeal species is underway. High throughput sequencing approaches will allow future studies of the chromosomal location of archaeal DNA replication and repair complexes.

Figure 2: Intracellular dynamics of a GFP-tagged Hef protein participating in replication restart in living archaeal cells. Aphidicolin (APD) treatment results in replication arrest and increase in the number of fluorescence foci reflecting Hef recruitment onto chromatin (≈ 17 500 cells analyzed).
 

Project 2 - Recent publications:

 

  1. Lestini R, Laptenok SP, Kuhn J, Hink MA, Schanne-Klein M-C, Liebl U, Myllykallio H (2013) Intracellular dynamics of archaeal FANCM homologue Hef in response to halted DNA replication. Nucleic Acid Research. Dec 1;41(22):10358-70.
  2. Creze C, Ligabue A, Laurent S, Lestini R, Laptenok SP, Khun J, Vos MH, Czjzek M, Myllykallio H, Flament D (2012) Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels. The Journal of biological chemistry 287: 15648-15660.
  3. Creze C, Lestini R, Kuhn J, Ligabue A, Becker HF, Czjzek M, Flament D, Myllykallio H (2011) Structure and function of a novel endonuclease acting on branched DNA substrates. Biochemical Society transactions 39: 145-149.
  4. Ren B, Kuhn J, Meslet-Cladiere L, Briffotaux J, Norais C, Lavigne R, Flament D, Ladenstein R, Myllykallio H (2009) Structure and function of a novel endonuclease acting on branched DNA substrates. The EMBO journal 28: 2479-2489

 

Project 3-Bacterial heme sensor proteins

Our third research avenue concerns bacterial heme-based gas sensor proteins (in close collaboration with M. Vos and J.-Christophe Lambry, team 2). These proteins are essential components of signal transduction chains and the binding of gas effector molecules induces structural changes that eventually lead to adaptation of the activity of other proteins and specific changes in gene expression (Figure 3). We aim at understanding the mechanism of signaling initiation and propagation, as well as the dynamic events that lead to protein function. Many sensors have been investigated, most recently two mycobacterial sensor proteins, where complementary response mechanisms to hypoxia signals were found, important for transition between the virulent and persistent state of M. tuberculosis.

Figure 3: Dormancy regulator DosT from Mycobacterium tuberculosis: different ligation states and heme pocket of 02 complex.
 

Project 3 – Recent publications
  1. Liebl U, Lambry JC, Vos MH. Primary processes in heme-based sensor proteins. Biochim Biophys Acta. 2013 Sep;1834(9):1684-92.
  2. Vos MH, Bouzhir-Sima L, Lambry JC, Luo H, Eaton-Rye JJ, Ioanoviciu A, Ortiz de Montellano PR, Liebl U. Ultrafast ligand dynamics in the heme-based GAF sensor domains of the histidine kinases DosS and DosT from Mycobacterium tuberculosis. Biochemistry. 2012 Jan 10;51(1):159-66.
  3. Lechauve C, Bouzhir-Sima L, Yamashita T, Marden MC, Vos MH, Liebl U, Kiger L. Ultrafast heme-residue bond formation in six-coordinate heme proteins: implications for functional ligand exchange. J Biol Chem. 2009 Dec 25;284(52):36146-59.
  4. Vos MH, Battistoni A, Lechauve C, Marden MC, Kiger L, Desbois A, Pilet E, de Rosny E, Liebl U. Ultrafast heme-residue bond formation in six-coordinate heme proteins: implications for functional ligand exchange. Biochemistry. 2008 May 27;47(21):5718-23.

Technical approaches

In silico methods and phylogenomics, microbiology and cellular biology, molecular biology, protein biochemistry and enzymology, inhibitory screening, rapid mixing spectroscopic techniques; steady-state and time-resolved spectroscopy (absorption, fluorescence including FRET), life cell imaging, high throughput sequencing (DNA, RNA).

Collaborations 2008-2013:

B. Golinelli, Collège de France, Paris (ANR PCV publications 2010, 2011)
D. Flament, IFREMER (ANR Blanc, publications 2009, 2011, 2012)
D. Lechardeur, A. Gruss, INRA UMR 1319, Jouy-en-Josas (publication 2012)
G. Marverti, U. Modena, Italie (publication 2012)
H. de Reuse, I. Pasteur (ANR Emergence, manuscript in preparation)
H. van Tilbeurgh, M. Graille, U. Paris Sud, Orsay (ANR EMERGENGE, publication 2012)
J. Eaton-Rye, H. Luo, U. Otago, New Zealand (F-NZ Lottery Health Grant; publication 2011)
J. Herscovici, D. Scherman, CNRS UMR 8151 - Inserm U1022 (publication 2008)
L. Kiger, C. Lechauve, M. C. Marden, INSERM U779, Le Kremlin-Bicêtre (publications 2008, 2009)
M.A. Hink, EuroBioimaging/U. Amsterdam, The Netherlands (publication 2013)
P. R. Ortiz de Montellano, A. Ioanoviciu, UCSF, USA (publication 2011)
R. Ladenstein, Karolinska Institute, Sweden (publication 2009)
R. Wade Heidelberg Institute for Theoretical Studies and U. Heidelberg, Germany (review 2010)
S. Krumova, Bulgarian Academy of Sciences, IBBME, Sofia, Bulgaria (RILA 2013-2014)
Y. Boum, Epicentre Research Station, Mbara, Uganda (collaboration, MTA signed, publication 2012)

Former members including current affiliation

Véronique BALLAND (post-doctoral fellow, 2003-2004), now MdC Paris VII
Tamara BASTA (post-doctoral fellow 2008-2010), now MdC Paris XI
Yap BOUM (PhD student 2006-2008), now research director, Epicentre, Uganda
Julien BRIFFOTAUX (post-doctoral fellow 2011-1012),
Frédéric ESCARTIN (PhD student 2004-2008), post-doc INRA
Sébastien GRAZIANI (PhD student 2002-2005), now researcher at DGA
Klara HOLA (Marie Curie fellow, 2004)
Magda KOSMOPOULOU (Marie Curie fellow, 2006)
Damien LEDUC (PhD student 2001-2004), Professor of Biology, Agrégé en Biochimie Génie Biologique
Alessio LIGABUE (visiting student, post-doctoral fellow 2008-2010), now research associate at U. Washinton, Seattle
Gérard LIPOWSKI (PhD student 1999-2003), now managing director at Quintesensia, Tokyo, Japan
Laurence MESLET-CLADIERE (post-doctoral fellow, 2003-2006), now MdC U. Bretagne
Cédric NORAIS (PhD student 2003-2007), now MdC E. polytechnique
Stéphane SKOULOUBRIS (INSERM Jeune Chercheur 2003-2006), now MdC Paris XI.
Andreea SODOLESCU (TODEA) (PhD student 2006-2009), now Medical Science Liaison Officer, Chugai Pharma France
John ULMER (post-doctoral fellow, 2007-2009), post-doc Paris VII.