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Métabolisme des acides aminés dans l’échappement de Francisella tularensis du phagosome des macrophages infectés

Abstract : Francisella tularensis, the etiologic agent of the zoonotic disease tularemia, is a facultative intracellular bacterium which can infect multiple cell types with specific tropism for macrophages. This bacterium is responsible for severe infections in numerous animal species and in humans. Of note, F. tularensis subsp. tularensis has been classified as a type A bioterrorism agent because of its high infectivity and very low infectious dose. Genome sequence analyses and genome-scale genetic studies have revealed the importance of genes involved in metabolic functions throughout the bacterial intracellular cycle. Among these genes, several amino acid transporter where found to belong to the amino-acid-polyamine organocation subfamily (APC), prompting us to address the role of these transporters in bacterial virulence. We first focused on the APC transporter encoded by gene FTN_0571 in F. tularensis subsp. novicida and named GadC. We showed that GadC was a genuine glutamate importer, necessary for Francisella intracellular multiplication and virulence. gadC inactivation completely blocked bacterial phagosomal escape. Remarkably, multiplication of a gadC mutant was restored in gp91phox-/- macrophages that are unable to generate reactive oxygen species. Altogether, our study revealed that glutamate uptake was critical in bacterial oxidative stress resistance in the phagosomal compartment and highlighted possible links between glutamate utilization and the tricarboxylic acid (TCA) cycle. These results prompted us to address the role of a second APC transporter sharing 33 % amino acid identity with GadC and named ArgP. argP inactivation severely delayed bacterial phagosomal escape, thus impairing intracellular multiplication and virulence. We demonstrated that ArgP was a high affinity arginine transporter, suggesting that impaired phagosomal escape might be directly linked to an arginine import defect. argP inactivation in the F. tularensis subsp. holarctica Live vaccine strain also leads to a severe intracellular multiplication defect, consistent with a conserved role among all F. tularensis subspecies. Arginine is an essential amino acid for F. tularensis. To understand the importance of this amino acid during the phagosomal phase of the Francisella intracellular life cycle, a proteomic analysis of the bacteria, in conditions of arginine limitation, was carried out. This analysis revealed that arginine limitation affected in the argP mutant the expression of a series of proteins and in particular of all the ribosomal proteins. One may imagine that intracellular bacteria could also sense nutrient limitations in the phagosome as a subcellular localization signal. Altogether, these studies constitute the first demonstration of the importance of amino acid acquisition during F. tularensis phagosomal escape.
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Submitted on : Friday, October 10, 2014 - 1:52:24 PM
Last modification on : Thursday, April 9, 2020 - 11:55:41 AM
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  • HAL Id : tel-01073776, version 1


Elodie Ramond. Métabolisme des acides aminés dans l’échappement de Francisella tularensis du phagosome des macrophages infectés. Biologie cellulaire. Université René Descartes - Paris V, 2014. Français. ⟨NNT : 2014PA05T026⟩. ⟨tel-01073776⟩



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