Transport des acides aminés et virulence nutritionnelle de Francisella tularensis

Abstract : Francisella tularensis is the causative agent of tularemia. This facultative intracellular pathogen can infect a broad variety of mammalian cells. To multiply actively in the cytoplasm of host cells, the bacteria must be able to capture in this environment all necessary nutrients, including amino acids, that constitute its principal carbon and nitrogen sources. Intracellular pathogens have established a set of mechanisms aimed at: i) hijacking the metabolic pathways of the host, to increase the pool of nutrients available, and ii) expressing transporters dedicated to the optimal transport of these nutrients. These mechanisms are grouped under the term "nutritional virulence." In Legionella pneumophila, several members of a sub-family of secondary active transporters (designated Pht for phagosomal transporter), responsible for the uptake of amino acids in the intracellular phase of the bacteria, have been characterized. In this work, we have established the role of two Pht members (designated AnsP and IleP) in amino acid transport and virulence of Francisella. The AnsP protein is an asparagine transporter. Deletion of the gene encoding the transporter has no effect on the growth of Francisella in synthetic medium, but lead to a drastic decrease in intracellular multiplication in all cell types tested, and significant attenuation of virulence in mouse. The loss of virulence of the ansP mutant was reversible in vivo and in vitro by addition of excess asparagine. F. tularensis, which is prototrophic for asparagine in minimum medium, becomes auxotrophic under its intracellular growth and requires transport of the amino acid via AnsP. This change of asparagine requirement between an environmental and an intracellular lifestyle illustrates the nutritional adaptation achieved by bacteria during infection. The deletion of the second transporter, IleP, causes a growth defect in minimum medium, reversible by adding threonine, a metabolic precursor of isoleucine in F. tularensis subsp. novicida. Transport asays with radiolabelled isoleucine have highlighted the role of IleP protein in isoleucine uptake. Deletion of the ileP gene caused a severe defect in intracellular multiplication, combined with a slight delay exit the phagosome and a significant attenuation of virulence in mice. Notably, the biosynthesis pathway of isoleucine via the threonine is interrupted in holarctica and tularensis subspecies. The growth of these subspecies, more virulent than novicida, in the infected cell becomes strictly dependent on the transport of isoleucine by IleP. This example is a perfect illustration of the specialization of pathogenic strains to nutritional condition of their host. In conclusion, the work presented in this thesis has demonstrated the participation of two amino acid transporters to the nutritional virulence of the Francisella.
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Gaël Gesbert. Transport des acides aminés et virulence nutritionnelle de Francisella tularensis. Microbiologie et Parasitologie. Université René Descartes - Paris V, 2014. Français. ⟨NNT : 2014PA05T023⟩. ⟨tel-01071293⟩

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