The Reduced Genome of the Francisella tularensis Live Vaccine Strain (LVS) Encodes Two Iron Acquisition Systems Essential for Optimal Growth and Virulence

Natalie Marie Pérez,Girija Ramakrishnan,Alain Charbit

doi:10.1371/journal.pone.0093558

Natalie Marie Pérez, Girija Ramakrishnan + Show 1 more

Open Access

https://doi.org/10.1371/journal.pone.0093558

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Journal: PLoS ONE	Publication Date: Apr 2, 2014
Citations: 76	License type: CC BY 4.0

Affiliation: University of Virginia

Abstract

Bacterial pathogens require multiple iron-specific acquisition systems for survival within the iron-limiting environment of the host. Francisella tularensis is a virulent intracellular pathogen that can replicate in multiple cell-types. To study the interrelationship of iron acquisition capability and virulence potential of this organism, we generated single and double deletion mutants within the ferrous iron (feo) and ferric-siderophore (fsl) uptake systems of the live vaccine strain (LVS). The Feo system was disrupted by a partial deletion of the feoB gene (ΔfeoB′), which led to a growth defect on iron-limited modified Muller Hinton agar plates. 55Fe uptake assays verified that the ΔfeoB′ mutant had lost the capacity for ferrous iron uptake but was still competent for 55Fe-siderophore-mediated ferric iron acquisition. Neither the ΔfeoB′ nor the siderophore-deficient ΔfslA mutant was defective for replication within J774A.1 murine macrophage-like cells, thus demonstrating the ability of LVS to survive using either ferrous or ferric sources of intracellular iron. A LVS ΔfslA ΔfeoB′ mutant defective for both ferrous iron uptake and siderophore production was isolated in the presence of exogenous F. tularensis siderophore. In contrast to the single deletion mutants, the ΔfslA ΔfeoB′ mutant was unable to replicate within J774A.1 cells and was attenuated in virulence following intraperitoneal infection of C57BL/6 mice. These studies demonstrate that the siderophore and feoB-mediated ferrous uptake systems are the only significant iron acquisition systems in LVS and that they operate independently. While one system can compensate for loss of the other, both are required for optimal growth and virulence.

Highlights

Iron is an essential element for most organisms, and plays a critical role in enzymatic catalysis, electron transfer, and amino acid and DNA synthesis
To maintain the live vaccine strain (LVS) DfslA DfeoB9 mutant, F. tularensis siderophore-active culture supernatant was obtained from LVS cultures grown in iron limiting che-Chamberlin’s defined media (CDM) liquid and 100 mL of this filter sterilized (0.22 mm) supernatant was topically added to MHA+ agar plates
This study comparing single and double deletion mutants in siderophore biosynthesis and ferrous iron uptake has established that the F. tularensis strain LVS possesses only two mechanisms for iron acquisition: the fsl-locus encoded ferric-siderophore and feoBmediated ferrous iron acquisition systems. 55Fe uptake assays clearly demonstrated that the DfeoB9 mutant is completely deficient in ferrous iron uptake, revealing the Feo system as the sole transporter of ferrous iron across the inner membrane of F. tularensis

Summary

Introduction

Iron is an essential element for most organisms, and plays a critical role in enzymatic catalysis, electron transfer, and amino acid and DNA synthesis. Iron is largely sequestered and bound by host proteins such as heme, ferritin, and transferrin, but is available in small amounts within the labile iron pool (LIP) of the cytoplasm [2,3,4]. In this iron-limiting intracellular environment, bacterial pathogens acquire iron through the use of specific acquisition systems that are characteristically regulated by the ferric uptake regulator, Fur [5,6]. While specific iron acquisition mechanisms are not necessarily indicators of pathogenicity, the ability to acquire iron within the host can be a determinant of bacterial virulence potential [7]

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