The crystal structure of Klebsiella pneumoniae FeoA reveals a site for protein-protein interactions

In order to establish infection, pathogenic bacteria must obtain essential nutrients such as iron. Under acidic and/or anaerobic conditions, most bacteria utilize the Feo system in order to acquire ferrous iron (Fe²⁺) from their host environment. The mechanism of this process, including its regulation, remains poorly understood. In this work, we have determined the crystal structure of FeoA from the nosocomial agent Klebsiella pneumoniae (KpFeoA). Our structure reveals an SH3-like domain that mediates interactions between neighboring polypeptides via hydrophobic intercalations into a Leu-rich surface ridge. Using docking of a small peptide corresponding to a postulated FeoB partner binding site, we demonstrate that KpFeoA can assume both “open” and “closed” conformations, controlled by binding at this Leu-rich ridge. We propose a model in which a “C-shaped” clamp along the FeoA surface mediates interactions with its partner protein, FeoB. These findings are the first to demonstrate atomic-level details of FeoA-based protein-protein interactions and provide a framework for testing FeoA-FeoB interactions, which could be exploited for future antibiotic developments.     

Roles of the Yfe and Feo transporters of Yersinia pestis in iron uptake and intracellular growth

In Yersinia pestis, the Yfe and Feo systems likely function to transport ferrous iron. Both FeoA and FeoB are essential for iron acquisition activity while FeoC is not. Mutations in yfe and feo had an additive effect on microaerophilic growth under iron-chelating conditions. Y. pestis cells lacking the Ybt siderophore-dependent system, the Yfe or the Feo system grow normally in J774A.1 cells. However, a double yfeAB feoB mutant was no longer able to grow in this murine macrophage cell line. This growth defect likely resulted from iron and not manganese deprivation since a yfeAB mntH mutant grew normally in J774A.1 cells. These results suggest that the Yfe and Feo systems are somewhat redundant ferrous iron transporters capable of iron acquisition during intracellular growth of the plague bacterium.     

Genetics and environmental regulation of Shigella iron transport systems

Shigella spp. have transport systems for both ferric and ferrous iron. The iron can be taken up as free iron or complexed to a variety of carriers. All Shigella species have both the Feo and Sit systems for acquisition of ferrous iron, and all have at least one siderophore-mediated system for transport of ferric iron. Several of the transport systems, including Sit, Iuc/IutA (aerobactin synthesis and transport), Fec (ferric di-citrate uptake), and Shu (heme transport) are encoded within pathogenicity islands. The presence and the genomic locations of these islands vary considerably among the Shigella species, and even between isolates of the same species. The expression of the iron transport systems is influenced by the concentration of iron and by environmental conditions including the level of oxygen. ArcA and FNR regulate iron transport gene expression as a function of oxygen tension, with the sit and iuc promoters being highly expressed in aerobic conditions, while the feo ferrous iron transporter promoter is most active under anaerobic conditions. The effects of oxygen are also seen in infection of cultured cells by Shigella flexneri; the Sit and Iuc systems support plaque formation under aerobic conditions, whereas Feo allows plaque formation anaerobically.     

Iron acquisition in Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron and must obtain this element in the human host as well as in its varied environmental niches. It has multiple systems for iron acquisition, including the TonB-dependent transport of heme, the endogenous siderophore vibriobactin and several siderophores that are produced by other microorganisms. There is also a Feo system for the transport of ferrous iron and an ABC transporter, Fbp, which transports ferric iron. There appears to be at least one additional high affinity iron transport system that has not yet been identified. In iron replete conditions, iron acquisition genes are repressed by Fur. Fur also represses the synthesis of a small, regulatory RNA, RyhB, which negatively regulates genes for iron-containing proteins involved in the tricarboxylic acid cycle and respiration as well as genes for motility and chemotaxis. The redundancy in iron transport systems has made it more difficult to determine the role of individual systems in vivo and in vitro, but it may reflect the overall importance of iron in the growth and survival of V. cholerae.     

Bacterial iron homeostasis

Iron is essential to virtually all organisms, but poses problems of toxicity and poor solubility. Bacteria have evolved various mechanisms to counter the problems imposed by their iron dependence, allowing them to achieve effective iron homeostasis under a range of iron regimes. Highly efficient iron acquisition systems are used to scavenge iron from the environment under iron-restricted conditions. In many cases, this involves the secretion and internalisation of extracellular ferric chelators called siderophores. Ferrous iron can also be directly imported by the G protein-like transporter, FeoB. For pathogens, host-iron complexes (transferrin, lactoferrin, haem, haemoglobin) are directly used as iron sources. Bacterial iron storage proteins (ferritin, bacterioferritin) provide intracellular iron reserves for use when external supplies are restricted, and iron detoxification proteins (Dps) are employed to protect the chromosome from iron-induced free radical damage. There is evidence that bacteria control their iron requirements in response to iron availability by down-regulating the expression of iron proteins during iron-restricted growth. And finally, the expression of the iron homeostatic machinery is subject to iron-dependent global control ensuring that iron acquisition, storage and consumption are geared to iron availability and that intracellular levels of free iron do not reach toxic levels.     

Iron and Pathogenesis of Shigella: Iron Acquisition in the Intracellular Environment

Shigella species are able to grow in a variety of environments, including intracellularly in host epithelial cells. Shigella have a number of different iron transport systems that contribute to their ability to grow in these diverse environments. Siderophore iron uptake systems, heme transporters, and ferric and ferrous iron transport systems are present in these bacteria, and the genes encoding some of these systems appear to have spread among the Shigella species by horizontal transmission. Iron is not only essential for growth of Shigella but also plays an important role in regulation of metabolic processes and virulence determinants in Shigella. This regulation is mediated by the repressor protein Fur and the small RNA RyhB.     

A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe²⁺ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe²⁺ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein–protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen–deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein–protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA–NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes.     

革兰氏阴性菌亚铁离子转运系统的组成及作用机制

几乎所有细菌的生长都离不开铁元素。在有氧的环境中,三价铁离子几乎无法被细菌直接利用。但是在宿主胃肠道中,铁元素主要以可溶性的亚铁离子形式存在,它们可通过革兰氏阴性菌外膜直接进入胞周质,在周质通过亚铁离子转运系统,将铁离子转运至胞浆供细菌利用。绝大多数阴性菌主要是通过Feo转运系统利用亚铁离子,大肠杆菌的Feo转运系统由feoA、feoB和feoC3个基因组成。除Feo转运系统外,还发现Yfe转运系统、Efe转运系统、Sit转运系统等。本文重点介绍革兰氏阴性菌Feo转运系统的组成及作用机制,以期为进一步研究细菌亚铁离子的转运机制提供参考。