Pyrophosphate-Mediated Iron Acquisition from Transferrin in Neisseria meningitidis Does Not Require TonB Activity Francis Biville*, Christophe Bre ´ zillon, Dario Giorgini, Muhamed-Kheir Taha Unite ´ des Infections Bacte ´riennes invasives, De ´partement Infection et Epide ´miologie, Institut Pasteur, Paris, France Abstract The ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Neisseria meningitidis. Outside the cells, iron is bound to transferrin in serum, or to lactoferrin in mucosal secretions. Meningococci can extract iron from iron-loaded human transferrin by the TbpA/TbpB outer membrane complex. Moreover, N. meningitidis expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin. Iron transport through the outer membrane requires energy provided by the ExbB-ExbD-TonB complex. After transportation through the outer membrane, iron is bound by periplasmic protein FbpA and is addressed to the FbpBC inner membrane transporter. Iron-complexing compounds like citrate and pyrophosphate have been shown to support meningococcal growth ex vivo. The use of iron pyrophosphate as an iron source by N. meningitidis was previously described, but has not been investigated. Pyrophosphate was shown to participate in iron transfer from transferrin to ferritin. In this report, we investigated the use of ferric pyrophosphate as an iron source by N. meningitidis both ex vivo and in a mouse model. We showed that pyrophosphate was able to sustain N. meningitidis growth when desferal was used as an iron chelator. Addition of a pyrophosphate analogue to bacterial suspension at millimolar concentrations supported N. meningitidis survival in the mouse model. Finally, we show that pyrophosphate enabled TonB-independent ex vivo use of iron-loaded human or bovine transferrin as an iron source by N. meningitidis. Our data suggest that, in addition to acquiring iron through sophisticated systems, N. meningitidis is able to use simple strategies to acquire iron from a wide range of sources so as to sustain bacterial survival. Citation: Biville F, Bre ´zillon C, Giorgini D, Taha M-K (2014) Pyrophosphate-Mediated Iron Acquisition from Transferrin in Neisseria meningitidis Does Not Require TonB Activity. PLoS ONE 9(10): e107612. doi:10.1371/journal.pone.0107612 Editor: Eric Cascales, Centre National de la Recherche Scientifique, Aix-Marseille Universite ´, France Received May 12, 2014; Accepted August 14, 2014; Published October 7, 2014 Copyright: ß 2014 Biville et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: This work was supported by Pasteur Institute and the Institut de veille sanitaire. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: fbiville@pasteur.fr Introduction Neisseria meningitidis (Nm) is found exclusively in humans, and although it is frequently present in the nasopharynx of asymp- tomatic carriers, it may be the causative agent of life-threatening invasive infections such as septicemia and meningitis [1]. Ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Nm [2]. In mammals, iron sequestration is the main form of nutritional immunity [3], [4]. Obtaining iron required for bacterial growth is a challenge, since 99.9% of total body iron is sequestered inside the cells [5]. Outside the cells, iron is bound to transferrin in the serum or to lactoferrin in mucosal secretions [2]. Another iron source in mammals is heme, mainly contained in hemoproteins like hemoglobin. When freed after erythrocyte lysis, most hemoglobin is bound by haptoglobin. Hemoglobin degradation allows the release of heme that is sequestered by hemopexin to prevent its toxicity [5]. Bacterial acquisition of iron in mammals requires the activity of transport systems allowing uptake of iron and/or heme bound to proteins. In Nm, the HmbR [6] and HpuAB outer membrane transport systems [7] allow the bacteria to use heme- loaded proteins as a heme source. HmbR and HpuAB systems differ according to their substrate specificity. HmbR can obtain heme from hemoglobin with better efficiency for human hemoglobin [6]. In contrast, HpuAB not does not exhibit specificity toward the human forms of its two substrates, characterized as hemoglobin and haptoglobin-hemoglobin com- plexes [8]. Nm strains express HmbR, HpuAB or both systems [9]. Most invasive strains express HmbR alone or both heme uptake systems, as reported in isolates of the hyperinvasive genotype ST- 11 [9]. Strains expressing only the HpuAB heme transport system were mostly described as carriage strains [9]. The periplasmic heme binding protein and the inner membrane heme transporter are not yet identified. Inside the cytoplasm, heme is degraded by HemO, a bacterial heme oxygenase, thus allowing the release of iron [10]. The main source of iron in blood is iron-loaded transferrin. Iron is extracted from iron-loaded human transferrin by the TbpA/ TbpB outer membrane complex [11]. Also, Nm expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin [12]. After transportation through the outer membrane, iron is bound by the periplasmic protein FbpA and directed to the FbpBC inner membrane PLOS ONE | www.plosone.org 1 October 2014 | Volume 9 | Issue 10 | e107612