Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

et al. (2014) Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp.. PLoS ONE 9(8): e105805. doi:10.1371/journal.pone.0105805 Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp. Mirelle Garcia Silva-Baila o 0 Elisa Fla via Luiz Cardoso Baila o 0 Beatrix Elisabeth Lechner 0 Gregory M. Gauthier 0 Herbert Lindner 0 Alexandre Melo Baila o 0 Hubertus Haas 0 Ce lia Maria de Almeida Soares 0 Roy Martin RoopII, East Carolina University School of Medicine, United States of America 0 1 Laborato rio de Biologia Molecular, Instituto de Ciencias Biolo gicas, Universidade Federal de Goia s , Goiania, Goia s, Brazil , 2 Programa de Po s-Graduac a o em Patologia Molecular, Universidade de Bras lia, Bras lia, Brazil, 3 Unidade Universita ria de Ipora , Universidade Estadual de Goia s , Ipora , Goia s, Brazil , 4 Division of Molecular Biology/ Biocenter, Innsbruck Medical University , Innsbruck , Austria , 5 Division of Clinical Biochemistry/Biocenter, Innsbruck Medical University , Innsbruck , Austria , 6 Department of Medicine, Section of Infectious Diseases, University of Wisconsin , Madison, Wisconsin , United States of America Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity. - Funding: Work at Universidade Federal de Goias was supported by grants from Financiadora de Estudos e Projetos (FINEP), Conselho Nacional de Desenvolvimento Cientfico e Tecnolo gico (CNPq), Fundacao de Amparo a` Pesquisa do Estado de Goias (FAPEG), Coordenacao de Pessoal do Ensino Superior (CAPES) and Pronex. Work at Innsbruck Medical University was supported by the Austrian Science Foundation (FWF P21643-B11 to HH). MGSB was supported by a fellowship from CAPES. 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. The requirement of iron for growth and proliferation is a feature of virtually all organisms, with the exception of a few bacteria [1,2]. The biological significance of iron lies on its ability to cycle between two oxidation states: the reduced ferrous (Fe2+) and oxidized ferric (Fe3+). The capacity to accept and donate electrons gives iron a redox versatility to function as a cofactor for various cellular enzymes involved in several essential biological processes including respiration, the tricarboxylic acid cycle, synthesis of amino acids, deoxyribonucleotides, lipids and sterols as well as oxidative stress detoxification [1]. Although essential, iron can also be toxic in high concentrations since Fe2+ has the potential to generate cell damaging reactive oxygen species (ROS) via the Fenton/Haber Weiss reaction [3,4]. Thereby, cellular iron homeostasis depends on the precise regulation of iron acquisition, utilization and storage. Under aerobic conditions, iron is oxidized and Fe3+ is essentially insoluble in water at neutral pH [5]. Beyond the environmental low iron availability, pathogenic microorganisms are also confronted by iron scarcity during interaction with the host. In mammalian hosts, the assimilated iron is bound to proteins, such as hemoglobin, transferrin, ferritin and lactoferrin [6]. Following infection, iron concentrations in extracellular fluid and plasma decrease [7]. Macrophages play an important role in the iron withholding. These defense cells limit the release of iron obtained from damaged and senescent erythrocytes and, under the influence of cytokines, inhibit multiplication of phagocytosed microorganisms by moving iron from the phagosome to cytoplasmic ferritin [8,9]. Since both host and pathogen require iron for metabolism, the control over access to this nutrient can dictate the fate of an infection. Microorganisms, including fungi, have evolved high affinity uptake strategies for iron acquisition in order to overcome the low bioavailability of this ion in aqueous environments (concentration of free Fe3+ approximately 10218 M at pH 7) and within mammalian hosts (concentration of free iron in serum in the order of 10224 M) [10]. One of these strategies consists of the synthesis and secretion of siderophores, defined as low molecular weight organic chelators with high affinity for Fe3+ [11]. Such molecules are produced under iron limiting conditions and make insoluble Fe3+ available for consumption [11]. The high affinity for iron allows siderophores to compete with host proteins transferrin and lactoferrin. Indeed, the pathogen Aspergillus fumigatus overcomes the iron limitation of serum by secreting siderophores which remove iron from serum transferrin [12,13]. With the exception of carboxylates produced by zygomycetes [14], virtually all fungal siderophores are hydroxamates, derived from the non proteinogenic amino acid ornithine [15] (Figure S1). In the proposed biosynthetic pathway for fungal hydroxamates, ornithine-N5-monooxygenase (SidA) catalyzes N5-hydroxylation of ornithine [13,15,16]. The hydroxamate group is formed next by N5-acylation of N5-hydroxyornithine catalyzed by N5-transacylases [15]. In A. fumigatus two transacylases, which add different acyl groups to hydroxyornithine, were identified thus far: SidF [17], which adds anhydromevalonyl-CoA, and (...truncated)