Biologie cellulaire des infections bactériennes



Towards a systems biology approach to host/pathogen interactions

Infection by bacterial pathogens are characterised by the subversion of host cell functions, which is orchestrated by an array of bacterial proteins referred to as virulence factors. The diverse strategies elaborated by bacterial pathogens to take control of the infected cell are globally defined as host/pathogen interactions. Cellular microbiology fostered remarkable advances towards the understanding of bacterial pathogenesis, by stressing the importance of studying pathogens in relation to their host. However, the effective identification of bacterial virulence determinants and the study of their diverse mechanisms of action represents a real technical challenge for which the available laboratory techniques are often not suited. Technological advances towards laboratory automation has encouraged the development of high-throughput and high-content screens (HTS and HCS respectively) that promise to revolutionise our approach to scientific questions. Our team has been created in 2011, with the aim of 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.

Coxiella burnetii

Coxiella burnetii is a Gram-negative, obligate intracellular bacterium responsible for severe outbreaks of the worldwide zoonosis Q fever. Infections spread from animals to humans through the contamination of hay and dust, mostly in farms, where exposed subjects may be infected by the inhalation of contaminated particles. Despite the relatively low incidence of Q fever cases, C. burnetii is an extremely infectious agent and is classified as a class B biothreat. The primary target of C. burnetii are alveolar macrophages, however this microbe is capable of invading and replicating in non-phagocytic cells as well. Upon internalisation, bacteria remain confined within a tight-fitting vacuole which follows the endocytic pathway from early endosomes to lysosomes. Vacuolar acidification triggers the activation of the Dot/Icm, T4BSS, thus allowing the translocation of effector proteins to the host cell cytoplasm. Bioinformatics analysis predicted ~200 effector proteins, half of which have been already validated. Given the relatively recent development of genetic tools to investigate C. burnetii infections, the role of most effector protein remain under-investigated. It is clear however that some of these effectors contribute to the biogenesis of the Coxiella Containing Vacuole (CCV), whereas others regulate signalling pathways involved in cytoskeleton remodelling and apoptosis.

In our team we use C. burnetii as a model system to develop high-content screening approaches aiming at the large-scale, unbiased identification of bacterial and host factors involved in infections.

Phenotypic screens of C. burnetii infections.

To tackle C. burnetii infections by phenotypic screens, we first generated a GFP-tagged library of transposon mutants, which was initially screened using a phenotypic screening approached based on 3 parameters: internalisation and replication within host cells and cytotoxicity. This allowed us to 1) investigate the role of Dot/Icm core proteins in vacuole biogenesis; 2) validate the role of a large number of effector proteins and identify new candidates and 3) identify the first C. burnetii invasin OmpA, which allows bacteria to invade non-phagocytic cells.

We have recently implemented this screening approach by raising the number of analysed parameters to more than 20, thus allowing to gain an unparalleled resolution in the characterisation of phenotypes deriving from transposon insertions. This is being applied to a targeted siRNA screen against host proteins involved in membrane traffic as well as a small molecule screen using inhibitors of eukaryotic protein kinases. Transcriptome analysis of both bacteria and infected cells is being integrated to our screening approaches to better characterise the role of coding and non-coding regions of the bacterial genome and the host response to C. burnetii interactions.


 

Vacuole biogenesis

Our screening approaches allowed us to identify a number of effector proteins involved in the biogenesis of the CCV. We have reported that the Dot/Icm protein IcmS and the effector protein CvpB are involved in the homotypic fusion between CCVs and their function has a clear relevance in virulence. We are thus pursuing the characterisation of the host and bacterial factors involved in the biogenesis of CCVs exploiting our phenotypic screening data, in combination with quantitative proteomic data of the CCV proteome. In parallel, following our observation that CvpB manipulates phosphoinositides (PIs) metabolism for vacuole biogenesis, we are aiming at defining the role of PIs in CCV biogenesis.

 




 

 











Membres de l’équipe








Matteo Bonazzi

DR2 CNRS, HDR.



Eric Martinez 
CR CNRS.



Karine Lambou
Maitre de Conférence, UM.



Franck Cantet 
Ingénieur d'étude, CNRS.



Fernande Siadous 
Doctorante, UM.


Melanie Burette






Mélanie Burette
 
Doctorante, UM.


Ils ont travaillé avec nous


Julie Allombert









Julie Allombert

Post-Doctorante

                                                                    
Sarah Scussel
      







Sarah Scussel

Master1











Betty Cotteux
Licence Pro BAE
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2017

2016

2015

2014

Locales

  • D. Muriaux & C. Favard, IRIM, Montpellier. Role of phosphoinositides in Coxiella vacuole biogenesis.
  • V. Molle, DIMNP, Montpellier. Ser/Thr kinases in Coxiella infections.

Nationales

  • D. Meyer, CIRAD, Guadeloupe. Bioinformatics tools & virulence factors

Internationales

  • 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.

 

Responsable

Matteo Bonazzi

Matteo Bonazzi

DR2 CNRS, HDR
En savoir +

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En bref

Organisme(s) modèle(s)

Coxiella burnetii

Processus biologique étudié

Interactions hôte/pathogène

Techniques utilisées

  • Criblage phénotypique multi-paramétrique
  • Biologie des systèmes
  • Protéomique

Applications médicales

  • Identification de cibles potentielles pour le développement des molécules anti-infectieuses
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Financements

                                     






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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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