Research
Published on December 14, 2018 | Updated on December 14, 2018

LEGCOXINET Project

Exploring the functional, ecological and evolutionary relationships between Legionella and Coxiella T4SS effectors and the host autophagy machinery

Co-leaders: Patricia Doublet (CIRI) & Fabrice Vavre (LBBE)

Autophagy is a universal innate immunity mechanism involving several autophagyrelated (ATG) gene-encoded proteins. To avoid their degradation, numerous intracellular pathogens have evolved molecular strategies to avoid autophagy, or even to hijack this process to their own benefit. Legionella pneumophila (Lp), the etiological agent of the severe pneumonia legionellosis and Coxiella burnetii (Cb), which is responsible of the zoonosis Q fever, are paradigms of highly adapted “intravacuolar” pathogens that acquired abilities to evade endocytic degradation and replicate within host cells. Despite subtle differences, a common trait of Lp and Cb infectious cycles is the biogenesis of a vacuole permissive to their intracellular replication concomitantly with the subversion of the host autophagy process. Lp and Cb also share a common molecular tool for virulence, i.e. a Type 4 Secretion System (T4SS) that is essential for host cell subversion and intracellular replication. Thus, Lp and Cb actually hijack host autophagy to their benefit, most likely by delivering T4SS effectors into the host cytosol. Yet, the interplay between autophagy and Lp or Cb has not been formally determined and the nature of the virulence factors responsible for autophagy modulation remains unknown.

LEGCOXiNET gathers complementary skills in infectiology, eco-immunology and bioinformatics to (1) gain further knowledge on the molecular mechanisms by which Lp and Cb manipulate host autophagy to successfully infect mammals, and to (2) lay foundations for understanding the evolutionary scenarii that could allow the emergence of common strategies to escape host defenses, in particular autophagy, for successful dissemination in environment and during infection. First, the common T4SS effectors in Lp and Cb will be identified by a deep bioinformatic and phylogenomic search of T4SS effectors orthologs in 6 Lp and 11 Cb strains from various geographical origins by using robust approach relying on orthology pattern search in gene family tree. Common T4SS effectors will also be assessed in the
context of symbiosis or pathogenesis of environmental vectors or reservoirs by screening amoeba-isolated Lp strains as well as tick-isolated Cb and Cb-like strains that will be isolated and fully sequenced.

To gain insight on the molecular mechanisms by which Lp and Cb modulate autophagy, the T4SS effectors of Lp conserved in Cb will be assayed pairwise for their putative interaction with a matrix of 35 autophagy-associated proteins in a yeast twohybrid array. An in silico interactome will be also inferred by using conserved interactions prediction, and it will be used to confirm and extend the experimental interactome. The resulting T4SS effector/autophagy protein interaction network will be modelled and visualized using graph theory algorithms. The T4SS effectors found to interact with ATG proteins will be assayed for their potency to modulate the autophagy process. Reciprocally, involvement of the targeted autophagic proteins in this process will be evaluated using siRNA shutting down their respective expression. Finally, the ability of Lp proteins to modulate autophagy will be investigated in mammalian cells and amoebas by using genetically-deleted Lp strains for the considered virulence factors. Conservation of the strategy for autophagy subversion in Cb will be challenged by trans-complementation of Lp mutant phenotypes by Cb genesencoding vectors. Finally, the presence and the polymorphism of genes encoding Legionella/Coxiella T4SS effectors that hijack autophagy will be investigated in a panel of environmental Lp, Cb and Cb-like samples. RNA microarrays will allow verifying that the investigated effectors are expressed in environmental strains isolated in task 1.

Together, these data will provide new molecular model and knowledge about the emergence of common strategies for successful evasion of host defenses and adaptation to diverse environmental conditions.

Co-leaders : Patricia Doublet, team "Pathogenèse des Légionnelles" (CIRI) & Fabrice Vavre, team "Génétique et évolution des interactions hôtes-parasites" (LBBE).
Collaborative teamsGuy Perrière, team "Bioinformatique, Phylogénie et Génomique Évolutive" (LBBE),  Mathias Faure, team "Autophagie, infections, immunité" (CIRI), Lionel Zenner, team "Génétique et évolution des interactions hôte-parasite" (LBBE) and Elsa Jourdain, team "Epidémiologie Animale" (INRA).
Project duration :
3 years
Financing :
PhD fellowship & consumable money
PhD Fellow :
Virginie Lelogeais