Importance of Host Cell Arginine Uptake in Francisella Phagosomal Escape and Ribosomal Protein Amounts*[S

Elodie Ramond,Gael Gesbert,Ida Chiara Guerrera,Cerina Chhuon,Marion Dupuis,Mélanie Rigard,Thomas Henry,Monique Barel,Alain Charbit

doi:10.1074/mcp.m114.044552

Abstract

Upon entry into mammalian host cells, the pathogenic bacterium Francisella must import host cell arginine to multiply actively in the host cytoplasm. We identified and functionally characterized an arginine transporter (hereafter designated ArgP) whose inactivation considerably delayed bacterial phagosomal escape and intracellular multiplication. Intramacrophagic growth of the ΔargP mutant was fully restored upon supplementation of the growth medium with excess arginine, in both F. tularensis subsp. novicida and F. tularensis subsp. holarctica LVS, demonstrating the importance of arginine acquisition in these two subspecies. High-resolution mass spectrometry revealed that arginine limitation reduced the amount of most of the ribosomal proteins in the ΔargP mutant. In response to stresses such as nutritional limitation, repression of ribosomal protein synthesis has been observed in all kingdoms of life. Arginine availability may thus contribute to the sensing of the intracellular stage of the pathogen and to trigger phagosomal egress. All MS data have been deposited in the ProteomeXchange database with identifier PXD001584 (http://proteomecentral.proteomexchange.org/dataset/PXD001584).

Highlights

Francisella tularensis is a Gram-negative coccobacillus responsible for the zoonotic disease tularemia
We recently demonstrated that Francisella possessed amino acid transporters that were required to fulfill some of its metabolic requirements during the different stages of its intracellular progression [11,12,13]
We show that ArgPmediated arginine uptake is crucial for efficient phagosomal escape, highlighting for the first time the importance of essential amino acids during early stage infection

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains, Media, and Chemicals—F. tularensis subsp. novicida (F. novicida) strain U112, its ⌬FPI derivative, F. tularensis subsp. holartica strain LVS, its ⌬iglC derivative, and all the mutant strains constructed in this work, were grown as described in supplementary Materials and Methods. Bacterial Strains, Media, and Chemicals—F. tularensis subsp. Novicida (F. novicida) strain U112, its ⌬FPI derivative, F. tularensis subsp. Holartica strain LVS, its ⌬iglC derivative, and all the mutant strains constructed in this work, were grown as described in supplementary Materials and Methods. Plasmids, and primers used in this study are listed in supplemental Table S2. Details of the construction and characterization of mutant and complemented strains; macrophage preparation and infections, are described in supplementary Materials and Methods. Quantitative (q)RT-PCR (real-time PCR) was performed with gene-specific primers (supplemental Table S2), using an ABI PRISM 7700 and SYBR green PCR master mix (Applied Biosystems, Foster city, CA), are described in supplementary Materials and Methods. Confocal and electron microscopy complete descriptions, real time cell death, and phagosome permeabilization assays, are described in supplementary Materials and Methods

Stress Survival Assays

RESULTS

Gene product

DISCUSSION