Bacterial Pathogeny and Anti-Infectious Strategies

The projects

 1- Mechanisms allowing Brucella persistence under conditions of oxygen depletion

 Classical Brucella species pathogenic for humans have the capacity to persist, resulting in chronic infections of specific target organs such as liver and spleen, often going with the formation of granulomas or abscesses.  In general, the chronic stage of infection is very difficult to treat. Therefore, brucellae are, together with M. tuberculosis, among the most suited model pathogens for the study of chronic bacterial infections. The sites of persistence are characterized by hypoxic or anoxic conditions, requiring specific adaptation of the pathogen. Having shown in vitro and in vivo the central role of the two-component system RegB/RegA in this adaptation, we have evidenced in B. suis the role of RegA as a master controller of persistence. Recently performed transcriptome analysis comparing wild-type and ΔregA mutant strains in our in vitro model of persistence based on a progressive depletion in oxygen has primed our study of the metabolic pathways linked to energy production under these conditions. The RegA-controlled gene aceA encodes isocitrate lyase, which is essential for the replication and persistence of B. suis during in vivo infection, as observed for M. tuberculosis. In addition, the enzyme of B. suis favors persistence in vitro in the presence of fatty acids. In the host, B. suis might therefore be able to use fatty acids as source of energy. Therefore, ongoing studies are focused on the regulation and the role of genes of interest such as aceA in the degradation of fatty acids potentially used by this bacterium during adaptation to the specific conditions of infection. The phenotype of the corresponding mutants will be analyzed in vitro in our model of persistence and/or under direct anoxia, in cellulo by infection of hypoxic macrophages, and in vivo. The biological functions of these genes will be characterized by enzymatic tests. In addition, potential RegA-binding sites identified in silico will be validated by electrophoretic mobility shift assays (EMSA) with previously purified, recombinant RegA. The characterization of these genes and of their mode of RegA-dependent regulation will allow a better comprehension of the mechanisms of adaptation brought into play during hypoxic persistence in a Brucella species classically encountered in the cases of human contamination.

In collaboration with a team of clinicians, these approaches could be applied to homologous systems involved in chronic infections caused by the opportunistic pathogen Pseudomonas aeruginosa.





2- Physiology and pathogenic potential of new Brucella species

The recently described atypical species of Brucella include, besides B. microti and B. inopinata, strains isolated from African frogs, thereby challenging the dogma that Brucella sp. is associated exclusively to mammal hosts. As their pathogenic potential in humans is yet mostly unknown, the team is following up these emerging species to characterize their atypical capacity of adaptation to the environment and rapid growth, and to identify possible virulence factors. Adaptation to stresses such as low pH encountered in the environment, in food and in the host (gastric juice and early intracellular vacuole), and to the intracellular environment of the macrophage is part of our ongoing studies, using global "RNA Seq" high-throughput transcriptome analysis. We expect to identify the corresponding bacterial factors and to elucidate the mechanisms at the origin of the observed increased growth rates of B. microti and of its resistance to acid pH in vitro and in cellulo. In orally acquired pathogens such as Escherichia coli and Shigella flexneri, but also in B. microti, the GAD (glutamate decarboxlase) system is one of the major systems involved in resistance to extreme acid pH. The team pursues its work on this topic in this model organism: functional characteristics of GAD are compared between atypical Brucella strains, and the roles of the other genes located in the same operon as gadBC are presently studied. We have also recently identified and characterized a second system of acid resistance, AR2_Q, active in B. microti and the other atypical species of Brucella, which is glutaminase (GlsA)-dependent and whose activity appears to be coupled to that of the GAD system. Last not least, we are working on the identification of regulatory factors of the GAD system and we envisage to apply EMSA using B. microti proteins and DNA sequences located upstream of the gad locus. This project is achieved in close collaboration with the group of Prof. D. De Biase (Institut Pasteur, Univ. La Sapienza, Rome). 


 







Post-Docs, Ph.D.s, Trainees

We welcome candidates trained in microbiology, molecular genetics or biochemistry, but also senior microbiologists (with CNRS, INSERM, or University positions), interested in the team projects.

Enregistrer

2018

Pennacchietti E, D’Alonzo C, Freddi L, Occhialini A, De Biase D.
The glutaminase-dependent acid resistance system: qualitative and quantitative assays and analysis of its distribution in enteric bacteria. Front Microbiol (in press).


2017

1. Abdou E, Jiménez de Bagüés MP, Martínez-Abadía I, Ouahrani-Bettache S, Pantesco V, Occhialini A, Al Dahouk S, Köhler S, Jubier-Maurin V.
RegA plays a key role in oxygen-dependent establishment of persistence and in isocitrate lyase activity, a critical determinant of in vivo Brucella suis pathogenicity.
Front Cell Infect Microbiol, 2017, 7: 186.

2. Al Dahouk S, Köhler S, Occhialini A, Jiménez de Bagüés MP, Hammerl JA, Eisenberg T, Vergnaud J, Cloeckaert A, Zygmunt MS, Whatmore AM, Melzer F, Drees KP, Foster JT, Wattam AR, Scholz HC.
Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts.
Sci Rep (Sci Rep UK), 2017, 7: 44420.

3. Freddi L, Damiano MA, Chaloin L, Pennacchietti E, Al Dahouk S, Köhler S, De Biase D, Occhialini A.The glutaminase-dependent system confers extreme acid resistance to new species and atypical strains of Brucella.
Front Microbiol. 2017, 8: 2236.


2015

1. Damiano MA, Bastianelli D, Al Dahouk S, Köhler S, Cloeckaert A, De Biase D, Occhialini A.
Glutamate decarboxylase-dependent acid resistance in Brucella spp.: Distribution and contribution to fitness under extreme acid conditions.
Appl Environ Microbiol, 2015, 81: 578-586.

2. Grassini G, Pennachietti E, Cappadocio F, Occhialini A, De Biase D.
Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system.
FEBS Open Bio, 2015, 5: 209-218.

3. Ombouma J, Vullo D, Köhler S, Supuran CT, Winum JY.
N-glycosyl-N-hydroxysulfamides as potent inhibitors of Brucella suis carbonic anhydrases.
J Enzyme Inhib Med Chem, 2015, 30:1010-1012.

4. Riafrecha L, Vullo D, Ouahrani-Bettache S, Köhler S, Dumy P,  Winum JY,   Supuran CT, Colinas PA.
Inhibition of b-carbonic anhydrases from Brucella suis with C-cinnamoyl glycosides incorporating the phenol moiety.
J Enzyme Inhib Med Chem, 2015, 30:1017-1020.

2014

1. D’ambrosio K, Lopez M, Dathan NA, Ouahrani-Bettache S, Köhler S, Ascione G, Monti SM, Winum J-Y, De Simone G.
Structural basis for the rational design of new anti-Brucella agents: the crystal structure of the C366S mutant of L-histidinol dehydrogenase from Brucella suis.
Biochimie, 2014, 97: 114-120.

2. Turtaut F, Lopez M, Ouahrani-Bettache S, Köhler S, Winum JY.
Oxo- and thiooxo-imidazo(1,5-c)pyrimidine molecule library: beyond their interest in inhibition of Brucella suis histidinol dehydrogenase, a powerful protection tool in the synthesis of histidine analogues.
Bioorg Med Chem Lett, 2014, 24: 5008-5010.

2013

1. Abdou E, Deredjian A, Jiménez de Bagüés MP, Köhler S, Jubier-Maurin V.
RegA, the transcriptional regulator of the two-component system RegB/RegA of Brucella suis, is a controller of both oxidative respiration and denitrification, required for chronic infection in mice.
Infect Immun, 2013, 81: 2053-2061.

2. Al Dahouk S, Jubier-Maurin V, Neubauer H, Köhler S.
Quantitative analysis of the Brucella suis proteome reveals metabolic adaptation to long-term nutrient starvation.
BMC Microbiology, 2013, 13:199.

3. Hanna N, Ouahrani-Bettache S, Drake KL, Adams LG, Köhler S, Occhialini A.
Global Rsh-dependent transcription profile of Brucella suis during stringent response unravels adaptation to nutrient starvation and cross-talk with other stress responses.
BMC Genomics, 2013, 14: 459.

2012

1. Maresca A, Scozzafava A, Köhler S, Winum JY, Supuran C.
Inhibition of beta-carbonic anhydrases from the bacterial pathogen Brucella suis with inorganic anions.
J Inorgan Biochem, 2012, 110: 36-39.

 2. Occhialini A, Jiménez de Bagüés MP, Saadeh B, Bastianelli D, Hanna N, de Biase D, Köhler S.
The glutamic acid decarboxylase system of the new species Brucella microti contributes to its acid resistance and to oral infection of mice. 
J Infect Dis, 2012, 206: 1424-1432.

2011

1. Abdo MR, Joseph P, Mortier J, Turtaut F, Montero JL, Masereel B, Köhler S, Winum JY.
Anti-virulence strategy against Brucella suis: synthesis, biological evaluation and molecular modeling of selective histidinol dehydrogenase inhibitors.
Org Biomol Chem, 2011, 9: 3681-3690.
Sélectionné comme "HOT article" par le Board Editorial.

2. Hanna N, Jiménez de Bagüés MP, Ouahrani-Bettache S, El yakhlifi Z, Köhler S, Occhialini A.
The virB operon is essential for lethality of Brucella microti in the Balb/c murine model of infection.
J Infect Dis, 2011, 203: 1129-1135.

3. Joseph P, Ouahrani-Bettache S, Montero JL, Nishimori I, Minakuchi T, Vullo D, Scozzafava A, Winum JY, Köhler S, Supuran CT.
A new beta-carbonic anhydrase from Brucella suis, its cloning, characterization and inhibition with sulfonamides and sulfamates, leading to impaired pathogen growth.
Bioorg Med Chem, 2011, 19: 1172-1178.

4. Turtaut F, Ouahrani-Bettache S, Montero JL, Köhler S, Winum JY.
Synthesis and biological evaluation of a new class of anti-Brucella compounds targeting histidinol dehydrogenase: a-O-arylketones and a-S-arylketones derived from histidine.
Med Chem Commun, 2011, 2: 995-1000.

2010

1. Jiménez de Bagüés MP, Ouahrani-Bettache S, Quintana JF, Mitjana O, Hanna N, Bessoles S, Sanchez F, Scholz HC, Lafont V, Köhler S, Occhialini A.
The new species Brucella microti replicates in macrophages and causes death in murine models of infection.
J Infect Dis, 2010, 202: 3-10.
A fait l'objet d'un commentaire éditorial dans le même numéro:
Morris JG, Southwick FS. Brucella, voles, and emerging pathogens. J Infect Dis, 2010, 202: 1-2.

2. Joseph P, Turtaut F, Ouahrani-Bettache S, Montero JL, Nishimori I, Minakuchi T, Vullo D, Scozzafava A, Köhler S, Winum JY, Supuran C.
Cloning, characterization and inhibition studies of a  b-carbonic anhydrase from Brucella suis.
J Med Chem, 2010, 53: 2277-2285.

3. Vullo D, Nishimori I, Scozzafava A, Köhler S, Winum JY, Supuran C.
Inhibition studies of a b-carbonic anhydrase from Brucella suis with a series of water soluble glycosyl sulfanilamides.
Bioorg Med Chem Lett, 2010, 20: 2178-2182.

Review articles

2017

Köhler S, Ouahrani-Bettache S, Winum J-Y.
Brucella suis carbonic anhydrases and their inhibitors: Towards alternative antibiotics?
J Enzyme Inhib Med Chem 32: 683-687.

2012

Lopez M, Köhler S, Winum J-Y.
Zinc metalloenzymes as new targets against the bacterial pathogen Brucella.
J Inorgan Biochem 111: 138-145.

2010

Winum J-Y, Köhler S, Supuran C.T.
Brucella carbonic anhydrase: New targets for designing anti-infective agents.
Curr Pharmaceut Design 16: 3310-3316.

 

Book chapters

2016

1. Köhler S, Dessolin J, Winum J-Y.
Inhibitors of histidinol dehydrogenase.
Top. Med. Chem. 22: 35-46 (chapter 7), Springer International Publishing.

2. Köhler S, Hanna N, Ouahrani-Bettache S, Drake KL, Adams LG, Occhialini A.
Global Rsh-dependent transcription profile of Brucella suis during stringent response unravels adaptation to nutrient starvation and cross-talk with other stress responses.
In Stress and Environmental Control of Gene Expression in Bacteria; F. J. de Bruijn (editor); Chapter 7.2., p. 489-499. Wiley-Blackwell Publishers.

2012

Baron C, Winum JY, Köhler S. Novel targets for antibacterial agents in Brucella sp.  In Brucella: Molecular Microbiology and Genomics; I. Lopez-Goni and D. O'Callaghan (editors); Chapter 12, p. 225-241. ISBN: 978-1-904455-93-6. Caister Academic Press.

Other publications

2011


Brèves du CNRS
Article suite à la publication d'Abdo et al., Org. Biomol. Chem. , 2011 (voir ci-dessus). Disponible sur le site suivant:

http://www.cnrs.fr/insb/recherche/parutions/articles2011/s-kohler.htm


Patents

2010

Extension internationale brevet N° 2 938 258:
"Histidinol dehydrogenase inhibitors and their use as drugs". PCT WO 2010/055254 A1.
J-Y. Winum, J-L. Montero, S. Köhler.


Brevet Français, 14 mai 2010, N° 2 938 258 (publication définitive):
"Inhibiteurs de l’histidinol déshydrogénase et leur utilisation en tant que médicament".

Local :

  • L. CHALOIN, IRIM – UMR 9004, Montpellier.
  • J.-Y. WINUM, IBMM - UMR 5247, Montpellier.
  • V. PANTESCO, IRMB - INSERM, CHRU, Montpellier.

National :

  • A. CLOECKAERT, UMR 1282 INRA, Tours.
  • J. DESSOLIN, CBMN - UMR 5248, Bordeaux.

International :

  • S. AL DAHOUK, BfR Berlin, Germany.
  • D. DE BIASE, Univ. La Sapienza / Institut Pasteur, Rome, Italy.
  • M.-P. JIMENEZ DE BAGUES, CITA Zaragoza, Spain.
  • P. MATTARELLI, Univ. of Bologna, Italy.
  • I. MORIYON, Univ. of Pamplona, Spain.
  • C. SUPURAN, Univ. of Florence, Italy

Team members


 







Stephan Köhler
- Senior scientist DR2 INSERM, HDR Bacteriology.









Véronique Jubier-Maurin
- Senior scientist CR1 CNRS, HDR Bacteriology.










Alessandra Occhialini-Cantet
- Senior scientist MCF Université Montpellier, HDR Bacteriology.









Safia Ouahrani-Bettache,
IEHC  Engineer INSERM.







Jorge de la Garza
- PhD student CBS2 Montpellier. Financial support : Infectiopôle Sud/Conacyt, Mexico.

 







Alumni

Postdoctoral scientists

2014 à 2016 : Maria Alessandra DAMIANO, Infectiopôle Sud fellowship

Molecular mechanisms of acid stress resistance in Brucella.  

Scientific supervisor : A. Occhialini

2012 : Abdelkarim MAHDHI, Infectiopôle Sud fellowship

Mechanisms of rapid growth and acid resistance of new Brucella species.

Scientific supervisor: A. Occhialini

Present situation : University teacher in Tunisia

2010 à 2013: Marie LOPEZ, ANR grant

The histidinol dehydrogenase: A novel therapeutic target against the etiological agents of brucellosis and tuberculosis.

Scientific supervisors: S. Köhler – J.-Y. Winum

Present position : Senior scientist CR CNRS, IBMM UMR5247 Montpellier

 

Ph.D. students

Since 2016 : Jorge DE LA GARZA, CBS2 doctoral school, Univ. of Montpellier

Pathogenic potential and adaptative capacities of Brucella spp. under acid stress conditions and in the phagocytic host cells.

Thesis directors: S. Köhler - A. Occhialini

2014 to 2017 : Luca FREDDI, CBS2 doctoral school, Univ. of Montpellier

Mechanisms of extreme acid  resistance in new and atypical Brucella strains

Thesis director: A. Occhialini

Present position :  Senior scientist, Anses, Maisons-Alfort


2010 to 2013 : Bashir SAADEH, CBS2 doctoral school, Univ. of Montpellier

Characterization and search for virulence-related factors in “Classical” and “New” Brucella species.

Thesis directors: D. O'Callaghan - A. Occhialini

 
2009 to 2013 : Elias ABDOU, CBS2 doctoral school, Univ. Montpellier 2

RegBA-dependent regulation in the establishment of persistence in Brucella suis.

Thesis director: V. Jubier-Maurin

Present position : University Lecturer, Beirut, Lebanon

2008 à 2011 : Nabil HANNA, CBS2 doctoral school, Univ. Montpellier 2

Characterization of the stringent response regulon in Brucella suis and study of the virulence factors of Brucella microti participating in the atypical infection phenotype.

Thesis directors: S. Köhler - A. Occhialini

Present position : Assistant, University of Geneva, Switzerland.


2008 to 2011 : François TURTAUT, Chemical Sciences doctoral school, Univ.
Montpellier 2

Design, synthesis and biological evaluation of inhibitors of histidinol dehydrogenase from intracellular bacteria.

Thesis directors: S. Köhler – J.Y. Winum

 

Visiting Ph.D. students

2014 : Federica ARMAS, Doctoral school Univ. Rome 1, Rome, Italy

Fellowship of the Italian Society of Veterinary Laboratory Diagnostics

Identification of differentially expressed genes in Brucella microti

and Brucella suis during their growth in TS broth.

Thesis director: C. Marianelli, Superior Health Institute, Rome.

2012 : Daniela BASTIANELLI, Thesis Institut Pasteur, Rome, Italy

Galilei Program France-Italy, Hubert Curien Partnership.

Implication of the Gad system in acid stress resistance of the new species

Brucella microti.

Thesis director: D. de Biase, Univ. La Sapienza, Rome.

2011 and 2012 : Maria-Alessandra DAMIANO, Animal Biology doctoral school, Univ. Palermo

Financial support : Fellowship of the Italian Ministry of Research.

Further evidences of antimicrobial activities in marine invertebrates.    

Thesis director: N. Parrinello, Univ Palermo.

 

Master 2 students

2016 : Donia ZMANDER, M2, IMHE, Univ. of Montpellier

Identification of the DNA binding sites of the transcriptional regulator RegA of Brucella suis.

Scientific supervisor: V. Jubier-Maurin

2014 : Guillaume GERVAIS, M2 Biology and Health, Univ. Montpellier 2

Identification and role of the GAD system in acid stress resistance of anaerobic bacteria of the human buccal flora.

Scientific supervisor: A. Occhialini

2010: Zoubida EL YAKIFI, M2 Biology and Health, Univ. Montpellier 2

Role of the GAD system in acid pH resistance of B. microti.

Scientific supervisor: A. Occhialini


2009 : Elias BOU SAMRA, M2 Biology and Health, Univ. Montpellier 2

Role of the transcriptional regulator RegA from the two-component system RegB/RegA in the adaptation of Brucella suis to oxygen deficiency.

Scientific supervisor: V. Jubier-Maurin


Engineer's Degree, Ecole Nationale Supérieure de Chimie of Montpellier

2011 : François HALLOY

 

Licence pro Analytical and Experimental Biology, IUT, Univ. Montpellier

2013 : Emilie BIANCHINI

 

DUT GB, option Biological and Biochemical Analyses, Univ. Montpellier

2018 : Alice TRAUSCH

2017 : Julien DU PASQUIER

2016 : Margot BICHON

2015 : Julien AUGIER

2012 : Fanny ALLAYAUD

2011 : Manon KORRICHI

2010 : Elena HAUSER

 

Brevet de Technicien Supérieur (Technician Diploma), Lycée A. Camus, Nîmes

2016 : Angélique PERRET, BTS Biotechnologie

 

Brevet de Technicien Supérieur (Technician Diploma), Lycée J. Mermoz, Montpellier

2017 : Chloé MONTOYA, BTS Bio-analyses and Controls

2013 and 2014 : Damien SERY, BTS Biotechnology

2013 : Laura MANEZ, BTS Bio-analyses and Controls

2012 and 2013 : Jade SALVI-LEYRAL, BTS Biotechnology

2011 et 2012 : Emilie BIANCHINI, BTS Bio-analyses and Controls

 

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Team leader

Stephan Köhler

Research Director INSERM, HDR Bacteriology

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AT A GLANCE

Model organisms

Brucella

Biological process studied

Brucella virulence factors and mechanisms of host cell adaptation


Techniques used

  • Microbiology
  • Genetics
  • Transcriptomics
  • Models of infection and persistance
  • Molecular Biology
  • Biochemistry

Medical applications

New targets for anti-bacterial agentsEnregistrerEnregistrer

Financial support

   
 


 
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IRIM
Institut de Recherche en Infectiologie de Montpellier
UMR 9004 - CNRS / UM
1919 route de Mende - 34293 Montpellier cedex 5
FRANCE

 

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