Mechanisms of heme utilization by Francisella tularensis.

Helena Lindgren,Lena Lindgren,Igor Golovliov,Anders Sjöstedt,Alain Charbit

doi:10.1371/journal.pone.0119143

Abstract

Francisella tularensis is a highly virulent facultative intracellular pathogen causing the severe disease tularemia in mammals. As for other bacteria, iron is essential for its growth but very few mechanisms for iron acquisition have been identified. Here, we analyzed if and how F. tularensis can utilize heme, a major source of iron in vivo. This is by no means obvious since the bacterium lacks components of traditional heme-uptake systems. We show that SCHU S4, the prototypic strain of subspecies tularensis, grew in vitro with heme as the sole iron source. By screening a SCHU S4 transposon insertion library, 16 genes were identified as important to efficiently utilize heme, two of which were required to avoid heme toxicity. None of the identified genes appeared to encode components of a potential heme-uptake apparatus. Analysis of SCHU S4 deletion mutants revealed that each of the components FeoB, the siderophore system, and FupA, contributed to the heme-dependent growth. In the case of the former two systems, iron acquisition was impaired, whereas the absence of FupA did not affect iron uptake but led to abnormally high binding of iron to macromolecules. Overall, the present study demonstrates that heme supports growth of F. tularensis and that the requirements for the utilization are highly complex and to some extent novel.

Highlights

Francisella tularensis is a highly virulent facultative intracellular pathogen causing the severe disease tularemia in mammals [1]
150 μM of heme was required for SCHU S4 to grow as efficiently as in CCDM supplemented with 26 μM FeSO4, containing equimolar amount of Fe as heme (Fig. 1)
The amounts of iron incorporated by SCHU S4 growing at stationary phase in C-Chamberlain’s defined medium (CDM) supplemented with increasing concentrations of heme or FeSO4 was measured by the ferrozine assay

Summary

Introduction

Francisella tularensis is a highly virulent facultative intracellular pathogen causing the severe disease tularemia in mammals [1]. Two of which are of clinical importance; the highly virulent subspecies tularensis (type A), which causes disease with high mortality if untreated, and the less aggressive subspecies holarctica (type B), which despite its lower virulence, may cause serious illness in humans. Regardless of subspecies, it is highly contagious with an infectious dose of less than 10 bacteria and in the mouse model it reaches high bacterial numbers within a few days of infection [1]. In view of its high pathogenicity and ability to rapidly cause lethal infection and the low free iron concentration in vivo (~ 10-18 M), F. tularensis must possess mechanisms that despite its inability of effective iron utilization in vitro, must allow effective iron utilization in vivo.

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Conclusion