In the tug of war between microbes and their hosts, the manipulation of eukaryotic signalling pathways by bacterial effector proteins plays a crucial role in virulence. Our research focuses on Coxiella burnetii and Brucella, two intravacuolar bacterial pathogens that evolved specific strategies to evade host cell recognition and develop stealthy, chronic infections.
Towards a systems biology approach to host/pathogen interactions
In recent years, the effective identification of bacterial virulence determinants and the study of their diverse mechanisms of action represented a real technical challenge for which the available laboratory techniques are often not suited. Technological advances towards laboratory automation have encouraged the development of high-throughput and high-content screens (HTS and HCS respectively) that revolutionised our approach to scientific questions. Our team has been designing new functional assays for the large-scale identification and characterisation of host/pathogen interactions. Since then, we have set up multi-parametric phenotypic screening approaches aiming at a comprehensive study of the cell biology of intracellular bacterial pathogens.
Cell biology of infection
The development of an intracellular replicative niche is a fundamental step in the pathogenesis of intravacuolar bacterial pathogens and involves reprogramming multiple host membrane trafficking pathways. If on the one hand, Brucella diverts the tight-fitting BCVs (Brucella-containing vacuoles) from the endocytic maturation pathway to escape lysosomal degradation, Coxiella subverts membrane traffic to provide membranes and nutrients for the development of a large, lysosomal-like CCV (Coxiella-containing vacuole). The radical differences between these bacterial replicative compartments make Coxiella and Brucella excellent models to investigate how bacteria differentially manipulate host cell functions. Following our recent advances on the role of autophagy and lipid metabolism in the development of CCVs, we explore how Rab GTPases and lipid metabolism differentially contribute to the development of these two radically different replicative compartments. Subversion of host cell functions relies on the translocation of bacterial effector proteins by specialised secretion systems. Genome analysis using state-of-the-art bioinformatics tools effectively predicts genes encoding candidate T4SS substrates in C. burnetii and Brucella spp. Combined with transcriptomics analysis on both bacteria in the course of infection, these data will allow us to shed light on the arsenal used by these microbes to manipulate host cell functions to protect infected cells from apoptosis, prevent inflammasome activation and suppress the innate immune response. The role of candidate Coxiella and Brucella virulence determinants will be assessed using the techniques already available in our teams and taking advantage of transposon mutant libraries and/or targeted mutants.
Advances in Next-Gen sequencing techniques allow nowadays to trace the transcriptional landscape of bacterial infections. On the microbial side, this allows pinpointing virulence genes involved in the adaptation of bacteria to the host environment and the manipulation of host cell functions. On the host side, transcriptional profiling highlights specific signalling pathways activated upon infection and, using specific bacterial mutants, how these are suppressed or enhanced during infections. Using dual RNA-Seq, we investigate candidate virulence determinants in Coxiella and Brucella, and we use gene set enrichment analysis to characterise the host cell response to these microbes. The characteristic profiles of Coxiella- and Brucella-infected cells will be compared to identify shared signalling pathways that might be used as targets for the development of antimicrobials. In recent years, Next-Gen Seq also highlighted an emerging role of the non-coding genome in the regulation of eukaryotic and prokaryotic functions and how these can play an important role in host/pathogen interactions. Accordingly, several intergenic mutations in Coxiella result in severe intracellular growth defects.
Infections represent a stress signal for both the host cell and the pathogen. On the one hand, eukaryotic cells respond to pathogens invasion and intracellular replication by triggering many response pathways, some of which are pathogen-specific, which collectively aim at clearing the infection. On the microbial side, upon host cell invasion, bacteria are exposed to a radically different environment as compared to the extracellular milieu. Coxiella and Brucella represent an excellent model to investigate the stress response associated with infections in this context. Both are stealth pathogens, capable of repressing cellular stress response using translocated effector proteins. Moreover, pH variations are sensed by both pathogens to trigger a transcriptional response. Coxiella seeks a hostile environment for optimal replication, whereas Brucella actively escapes lysosomal compartments by modifying the composition of its replicative niche. We have recently observed that Coxiella infections elicit a stress response in infected cells which, similarly to abiotic stress, may lead to the expression of long non-coding RNAs that serves as platforms to transiently sequesters proteins in the nucleus. Also, we observed that bacterial effector proteins might interfere with this mechanism.
Matteo Bonazzi DR2 CNRS, HDR
Stephan Köhler DR2 Inserm, HDR
Eric Martinez CRCN CNRS, HDR
Veronique Maurin CRHC CNRS, HDR
Alessandra Occhialini MCF, UM, HDR
Franck Cantet IEHC, CNRS
Safia Bettache IEHC, Inserm
Arthur Bienvenu PhD student, UM
Former lab members
Mélanie Burette, PhD student, UM
Karine Lambou, MCF, UM
Amelie Machura, Licence Pro BAE
Angelique Perret, Master 1
Fernande Siadous, PhD Student, UM
Lucien Platon, Master 1
Julie Allombert, Post-Doc
Betty Cotteux, Licence Pro BAE
Sarah Scussel, Master 1
And so it began: the team in 2011
- Modulation of innate immune signaling by a Coxiella burnetii eukaryotic-like effector protein. Burette M, Allombert J, Lambou K, Maarifi G, Nisole S, Di Russo Case E, Blanchet FP, Hassen-Khodja C, Cabantous S, Samuel J, Martinez E, Bonazzi M. PNAS. 2020 Jun 16;117(24):13708-13718. doi: 10.1073/pnas.1914892117. Epub 2020 Jun 1. PMID: 32482853
- The secreted protein kinase CstK from Coxiella burnetii influences vacuole development and interacts with the GTPase-activating host protein TBC1D5. Martinez E, Huc-Brandt S, Brelle S, Allombert J, Cantet F, Gannoun-Zaki L, Burette M, Martin M, Letourneur F, Bonazzi M, Molle V. J Biol Chem. 2020 Apr 17. pii: jbc.RA119.010112. doi: 10.1074/jbc.RA119.010112. [Epub ahead of print] PMID:32303638
- From neglected to dissected: How technological advances are leading the way to the study of Coxiella burnetii pathogenesis. Burette M, Bonazzi M. Cell Microbiol. 2020 Apr;22(4):e13180. doi:10.1111/cmi.13180. Review. PMID:32185905
- Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence. Siadous FA, Cantet F, Van Schaik E, Burette M, Allombert J, Lakhani A, Bonaventure B, Goujon C, Samuel J, Bonazzi M, Martinez E. Autophagy. 2020 Mar 1:1-17. doi: 10.1080/15548627.2020.1728098. [Epub ahead of print] PMID:32116095
- A CsrA-Binding, trans-Acting sRNA of Coxiella burnetii Is Necessary for Optimal Intracellular Growth and Vacuole Formation during Early Infection of Host Cells. Wachter S, Bonazzi M, Shifflett K, Moses AS, Raghavan R, Minnick MF. J Bacteriol. 2019 Oct 21;201(22). pii: e00524-19. doi: 10.1128/JB.00524-19. Print 2019 Nov 15. PMID: 31451541
- Lethality of Brucella microti in a murine modele of infection depends on the wbkE gene involved in O-polysaccharide synthesis. Ouahrani-Bettache S, Jiménez de Bagüés MP, Jorge De La Garza, Freddi L, Juan P. Buezo, Sebastien Lyonnais, Al Dahouk, de Biase D, Köhler S, Occhialini A. Virulence, 2019, 10: 268-278.
- Tiny architects: biogenesis of intracellular replicative niches by bacterial pathogens. Martinez E, Siadous FA, Bonazzi M. FEMS Microbiol Rev. 2018 Mar 27. doi: 10.1093/femsre/fuy013.
- Multiple Substrate Usage of Coxiella burnetii to Feed a Bipartite Metabolic Network Häuslein I, Cantet F, Reschke S, Chen F, Bonazzi M, Eisenreich W. Front Cell Infect Microbiol. 2017 Jun 29;7:285. doi: 10.3389/fcimb.2017.00285. eCollection 2017.
- Horizontally Acquired Biosynthesis Genes Boost Coxiella burnetii's Physiology Moses AS, Millar JA, Bonazzi M, Beare PA, Raghavan R. Front Cell Infect Microbiol. 2017 May 10;7:174. doi: 10.3389/fcimb.2017.00174. eCollection 2017.
- The SCID Mouse Model for Identifying Virulence Determinants in Coxiella burnetii Erin J. van Schaik, Elizabeth D. Case, Eric Martinez, Matteo Bonazzi and James E. Samuel.
Front Cell Infect Microbiol. 2017 Feb 3;7:25. doi: 10.3389/fcimb.2017.00025. eCollection 2017.
Beginning to Understand the Role of the Type IV Secretion System Effector Proteins in Coxiella burnetii Pathogenesis. Lührmann A, Newton HJ, Bonazzi M. Curr Top Microbiol Immunol. 2017;413:243-268. doi: 10.1007/978-3-319-75241-9_10.
- 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. Abdou E, Jiménez de Bagüés MP, Martínez-Abadía I, Ouahrani-Bettache S, Pantesco V, Occhialini A, Al Dahouk S, Köhler S, Jubier-Maurin V. Front Cell Infect Microbiol, 2017, 7: 186.
- Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts. 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. Sci Rep (Sci Rep UK), 2017, 7: 44420.
- The glutaminase-dependent system confers extreme acid resistance to new species and atypical strains of Brucella. Freddi L, Damiano MA, Chaloin L, Pennacchietti E, Al Dahouk S, Köhler S, De Biase D, Occhialini A. Front Microbiol, 2017, 8: 2236.
- Brucella suis carbonic anhydrases and their inhibitors: Towards alternative antibiotics? Köhler S, Ouahrani-Bettache S, Winum J-Y. J Enzyme Inhib Med Chem 32: 683-687.
- Right on Q: genetics begin to unravel Coxiella burnetii host cell interactions Charles L Larson, Eric Martinez, Paul A Beare, Brendan Jeffrey, Robert A Heinzen & Matteo Bonazzi. Future Microbiol. 2016 Jul;11:919-39. doi: 10.2217/fmb-2016-0044
- Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development Martinez E, Allombert J, Cantet F, Lakhani A, Yandrapalli N, Neyret A, Norville IH, Favard C, Muriaux D, Bonazzi M. PNAS. 2016 Jun 7;113(23):E3260-9. doi: 10.1073/pnas.1522811113.
- Inhibitors of histidinol dehydrogenase. Köhler S, Dessolin J, Winum J-Y. Top. Med. Chem. 22: 35-46 (chapter 7), Springer International Publishing.
- Global Rsh-dependent transcription profile of Brucella suis during stringent response unravels adaptation to nutrient starvation and cross-talk with other stress responses. Köhler S, Hanna N, Ouahrani-Bettache S, Drake KL, Adams LG, Occhialini A. Stress and Environmental Control of Gene Expression in Bacteria; F. J. de Bruijn (editor); Chapter 7.2., p. 489-499. Wiley-Blackwell Publishers.
- Generation and multi-phenotypic high-content screening of Coxiella burnetii transposon mutants. Martinez E, Cantet F, Bonazzi M. J Vis Exp. 2015 May 13;(99):e52851. doi: 10.3791/52851.
- The Recent Evolution of a Maternally-Inherited Endosymbiont of Ticks Led to the Emergence of the Q Fever Pathogen, Coxiella burnetii. Duron O, Noël V, McCoy KD, Bonazzi M, Sidi-Boumedine K, Morel O, Vavre F, Zenner L, Jourdain E, Durand P, Arnathau C, Renaud F, Trape JF, Biguezoton AS, Cremaschi J, Dietrich M, Léger E, Appelgren A, Dupraz M, Gómez-Díaz E, Diatta G, Dayo GK, Adakal H, Zoungrana S, Vial L, Chevillon C. PLoS Pathog. 2015 May 15;11(5):e1004892. doi: 10.1371/journal.ppat.1004892. eCollection 2015 May.
- Glutamate decarboxylase-dependent acid resistance in Brucella spp.: Distribution and contribution to fitness under extreme acid conditions. Damiano MA, Bastianelli D, Al Dahouk S, Köhler S, Cloeckaert A, De Biase D, Occhialini A. Appl Environ Microbiol, 2015, 81: 578-586.
- Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system. Grassini G, Pennachietti E, Cappadocio F, Occhialini A, De Biase D. FEBS Open Bio, 2015, 5: 209-218.
- N-glycosyl-N-hydroxysulfamides as potent inhibitors of Brucella suis carbonic anhydrases. Ombouma J, Vullo D, Köhler S, Supuran CT, Winum JY. J Enzyme Inhib Med Chem, 2015, 30:1010-1012.
- Inhibition of b-carbonic anhydrases from Brucella suis with C-cinnamoyl glycosides incorporating the phenol moiety. Riafrecha L, Vullo D, Ouahrani-Bettache S, Köhler S, Dumy P, Winum JY, Supuran CT, Colinas PA. J Enzyme Inhib Med Chem, 2015, 30:1017-1020.
- Galleria mellonella as an alternative model of Coxiella burnetii infection. Norville IH, Hartley MG, Martinez E, Cantet F, Bonazzi M, Atkins TP. Microbiology. 2014 Jun;160(Pt 6):1175-81. doi: 10.1099/mic.0.077230-0. Epub 2014 Mar 27.
- Identification of OmpA, a Coxiella burnetii protein involved in host cell invasion, by multi-phenotypic high-content screening.
Martinez E, Cantet F, Fava L, Norville I, Bonazzi M. PLoS Pathog. 2014 Mar 20;10(3):e1004013. doi: 10.1371/journal.ppat.1004013. eCollection 2014 Mar.
- 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. D’ambrosio K, Lopez M, Dathan NA, Ouahrani-Bettache S, Köhler S, Ascione G, Monti SM, Winum J-Y, De Simone G. Biochimie, 2014, 97: 114-120.
- 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. Turtaut F, Lopez M, Ouahrani-Bettache S, Köhler S, Winum JY. Bioorg Med Chem Lett, 2014, 24: 5008-5010.
- 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. Abdou E, Deredjian A, Jiménez de Bagüés MP, Köhler S, Jubier-Maurin V. Infect Immun, 2013, 81: 2053-2061.
- Quantitative analysis of the Brucella suis proteome reveals metabolic adaptation to long-term nutrient starvation. Al Dahouk S, Jubier-Maurin V, Neubauer H, Köhler S. BMC Microbiology, 2013, 13:199.
- Global Rsh-dependent transcription profile of Brucella suis during stringent response unravels adaptation to nutrient starvation and cross-talk with other stress responses. Hanna N, Ouahrani-Bettache S, Drake KL, Adams LG, Köhler S, Occhialini A. BMC Genomics, 2013, 14: 459.
- Inhibition of beta-carbonic anhydrases from the bacterial pathogen Brucella suis with inorganic anions. Maresca A, Scozzafava A, Köhler S, Winum JY, Supuran C. J Inorgan Biochem, 2012, 110: 36-39.
- The glutamic acid decarboxylase system of the new species Brucella microti contributes to its acid resistance and to oral infection of mice. Occhialini A, Jiménez de Bagüés MP, Saadeh B, Bastianelli D, Hanna N, de Biase D, Köhler S. J Infect Dis, 2012, 206: 1424-1432.
- Zinc metalloenzymes as new targets against the bacterial pathogen Brucella. Lopez M, Köhler S, Winum J-Y. J Inorgan Biochem 111: 138-145.
- Novel targets for antibacterial agents in Brucella sp. Baron C, Winum JY, Köhler S. 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.
- Anti-virulence strategy against Brucella suis: synthesis, biological evaluation and molecular modeling of selective histidinol dehydrogenase inhibitors. Abdo MR, Joseph P, Mortier J, Turtaut F, Montero JL, Masereel B, Köhler S, Winum JY. Org Biomol Chem, 2011, 9: 3681-3690. Selected as "HOT article" par le Board Editorial.
- The virB operon is essential for lethality of Brucella microti in the Balb/c murine model of infection. Hanna N, Jiménez de Bagüés MP, Ouahrani-Bettache S, El yakhlifi Z, Köhler S, Occhialini A. J Infect Dis, 2011, 203: 1129-1135.
- A new beta-carbonic anhydrase from Brucella suis, its cloning, characterization and inhibition with sulfonamides and sulfamates, leading to impaired pathogen growth. Joseph P, Ouahrani-Bettache S, Montero JL, Nishimori I, Minakuchi T, Vullo D, Scozzafava A, Winum JY, Köhler S, Supuran CT. Bioorg Med Chem, 2011, 19: 1172-1178.
- 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. Turtaut F, Ouahrani-Bettache S, Montero JL, Köhler S, Winum JY. Med Chem Commun, 2011, 2: 995-1000.
- The new species Brucella microti replicates in macrophages and causes death in murine models of infection. 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. J Infect Dis, 2010, 202: 3-10. Commentary article in the same issue:Morris JG, Southwick FS. Brucella, voles, and emerging pathogens.
- Cloning, characterization and inhibition studies of a b-carbonic anhydrase from Brucella suis. Joseph P, Turtaut F, Ouahrani-Bettache S, Montero JL, Nishimori I, Minakuchi T, Vullo D, Scozzafava A, Köhler S, Winum JY, Supuran C. J Med Chem, 2010, 53: 2277-2285.
- Inhibition studies of a b-carbonic anhydrase from Brucella suis with a series of water soluble glycosyl sulfanilamides. Vullo D, Nishimori I, Scozzafava A, Köhler S, Winum JY, Supuran C. Bioorg Med Chem Lett, 2010, 20: 2178-2182.
- Brucella carbonic anhydrase: New targets for designing anti-infective agents. Winum J-Y, Köhler S, Supuran C.T. Curr Pharmaceut Design 16: 3310-3316.
International Extention Patent 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.
French Patent, 14 mai 2010, N° 2 938 258 (final publication):
"Inhibiteurs de l’histidinol déshydrogénase et leur utilisation en tant que médicament".
- D. Muriaux & C. Favard, IRIM, Montpellier. Role of lipids in Coxiella vacuole biogenesis.
- V. Molle, DIMNP, Montpellier. Ser/Thr kinases in Coxiella infections.
- Olivier Duron, MIVEGEC, Montpellier. Coxiella and Ticks
- D. Meyer, CIRAD, Guadeloupe. Bioinformatics tools & virulence factors
- I. Norville, DSTL, Porton Down (UK). Development of in vivo models for Coxiella infections.
- E. Van Schaik, Texas A&M, Austin (TX). Development of in vivo models for Coxiella infections.
- R. Raghavan, Portland State University, Portland (OR). Horizontal gene transfer.
- W. Eisenreich, Technical University of Munich, Munich (GER). Coxiella metabolism.
- Anja Lührmann, University of Erlangen, (GER). Characterisation of Coxiella virulence factors
- Sacha AlDahouk, BfR (GER). Identification and characterisation of bacterial sRNAs
Matteo BonazziDR2 CNRS, HDR
En savoir +
AT A GLANCE
Model OrganismCoxiella burnetii
Biological ProcessHost/pathogen Interactions
- Multi-parametric Phenotypic Screening
- Systems Biology
- Identification of potential targets for the development of anti-infectious molecules