Starvation helps transition to abundance - a ferrosome story

Abstract

Iron is an essential nutrient for bacterial pathogenesis. In their study Skaar and colleagues (Pi. et al. 2023) discovered and determined the detailed structure of ferrosomes within Clostridioides difficile, the iron storage organelles that form under iron limited conditions in anticipation for future iron overload.

Iron is an essential metal necessary for fundamental cellular processes [1]. Low iron availability impairs metabolism and excess iron is toxic, hence organisms have evolved iron homeostasis mechanisms. Due to low bio-availability of iron found in nature and occasional high iron abundance in disease states, iron import and export, along with homeostasis mechanisms are tightly regulated [1–4].

Iron regulation occurs via the ferric uptake regulator Fur in bacteria [1,3]. When there is excess iron in the cytoplasm, iron forms a complex with Fur, that represses genes responsible for iron uptake. Under iron limited conditions, the Fur mediated repression is lifted [1–3,5]. Iron acquisition contributes to pathogenesis of the bacterium Clostridioides difficile, a Gram-positive, human intestinal pathogen that is a major public health threat [5,6]. It encodes the putative hydroxamate (fhu) and catecholate (fpi) ABC transporter systems to import iron-bound siderophores that are Fur regulated (Fig. 1) [3,5]. While siderophore biosynthesis genes have been identified in some Clade 3 C. difficile genomes, most strains of C. difficile do not encode these genes, suggesting that C. difficile likely uses siderophores produced by other gut commensals [3,5]. In the anaerobic gut environment, C. difficile directly imports soluble ferrous iron using ferrous iron permeases known as Feo transporters (Fig. 1) [3].

Fig. 1. Ferric uptake regulator and ferrosome formation regulate iron homeostasis in Clostridioides difficile.

Clostridioides difficile directly imports soluble ferrous iron via ferrous iron permeases known as Feo transporters and also expresses putative hydroxamate (fhu) and catecholate (fpi) ABC transporters to import ferric iron-bound xenosiderophores. To maintain iron homeostasis, it expresses fezXAB genes that encode ferrosome organelles that can store iron. Pi. et al. 2023 discovered and elucidates this ferrosome ultrastructure and function in C. difficile. The iron uptake and ferrosome genes are regulated by ferric uptake regulator (Fur). (a) Under iron rich condition, iron forms a complex with Fur, that inhibits the expression of these genes. (b) Under iron poor condition, the complex of iron and Fur breaks down and these genes are upregulated. (c) When there is a transition from iron starvation to iron overload, there is a sudden influx of iron through the transporters, which are still open. The excess iron is toxic. In this situation, the ferrosome that were expressed under iron limited condition can store iron in membrane bound organelles. This prevents immediate iron toxicity that can impair proliferation, while the Fur system acts in the background to downregulate the genes, thereby maintaining iron homeostasis. Created with BioRender.com.

In their study, Pi et al. has discovered an iron homeostasis mechanism in C. difficile and revealed the detailed nanoscopic molecular structure (ultrastructure) of membrane bound iron granules called ferrosomes using advanced visualization techniques [4]. The authors identify three genes in C. difficile: fezB, fezA and fezX, that are upregulated under iron limited condition, are Fur regulated, and share sequence homology to ferrosome forming genes previously discovered in three Gram-negative anaerobes [7] (Fig. 1). Generation of a C. difficile ΔfezB mutant revealed no growth defects both in iron excess and limited conditions in comparison to wildtype C. difficile. This result demonstrates that FezB is not responsible for iron import or export, likely because both ABC and Feo transporters are still intact and are expressed in a Fur dependent manner in the ΔfezB mutant. The ΔfezB mutant exhibits a moderate extended lag phase in the presence of both iron-chelator dipyridyl and H₂O₂, suggesting the importance of ferrosome in oxidative stress protection under iron limiting condition. Intriguingly the ΔfezB mutant also displays an extended lag phase and reduced intracellular iron levels when transitioning from iron deficiency to abundance, demonstrating the importance of FezB in iron homeostasis and storage. During the transition, wildtype C. difficile can internalize the sudden influx of iron in ferrosomes, preventing iron toxicity that can impair growth, which is dysfunctional in ΔfezB mutant. However, the Fur system is still active in the background and downregulates the expression of ABC and Feo transporters (Fig. 1c). This can explain the less dramatic growth defect in ΔfezB mutant. The authors postulate that Fez system is induced upon iron starvation in anticipation of iron overload and functions as a defense mechanism against transient iron abundance while simultaneously storing iron for use during low iron supply.

Next, the authors investigated the mechanistic and biophysical properties of ferrosomes using an impressive array of imaging techniques. Scanning transmission electron microscopy (STEM) was used to visualize, at nanometer range, electron dense granules within C. difficile. To understand ferrosome composition, the authors use energy dispersive X-ray spectroscopy (EDS) which is a technique used to determine chemical composition of a sample and confirmed that ferrosomes within C. difficile cells are similarly composed of iron, phosphorus, and oxygen [8]. EDS of isolated ferrosome also confirmed this composition. Ferrosomes were localized in the cytosol using electron tomography, a technique of three-dimensional reconstruction of subcellular objects using STEM images. Ultrathin sections of cells were then processed and subjected to STEM-EDS imaging. STEM imaging re-confirmed ferrosome localization in the cytoplasm and EDS analysis re-confirmed ferrosome composition. Liquid chromatography tandem mass spectrometry and targeted proteomics confirmed the ferrosome was comprised of FezB and FezA proteins. FezX was not detected in the ferrosome, though this observation could be a technical, rather than biological, limitation of identifying the small FezX protein by mass spectrometry as the entire fezXAB operon is required for the heterologous expression of ferrosome in Bacillus subtilis that do not form ferrosome naturally. Next, cryo-electron tomography (cryo-ET) was performed to understand the ultrastructure of ferrosome. In this technique a biological sample is flash frozen or vitrified in liquid ethane, then imaged using a transmission electron microscope under cryogenic condition (below −150°C). 3D reconstruction of cryo-ET images revealed ferrosomes are membrane bound and are found in proximity to cell membrane. To better visualize ferrosome membrane structures in situ, ultrathin sections of vitrified cells were prepared using cryofocused ion beam scanning electron microscopy (cryo-FIB-SEM) and imaged with cryo-ET. It confirmed that ferrosomes are membrane-bound and frequently localized adjacent to the cell membrane. Combined, these techniques revealed the ultrastructure and composition of ferrosome.

Experiments in wild-type and calprotectin deficient (S100a9^−/−) mice revealed that ferrosome formation is an important determinant in C. difficile host colonization only in hosts that can restrict iron access, and there is a positive association between bacterial ferrosome and host calprotectin expression, which contributes to nutritional immunity by limiting iron availability [4,9].

These findings open new research avenues, such as understanding how iron uptake and ferrosome formation are linked, which can be studied by following bacterial growth kinetics and ferrosome formation in Feo and ABC transporter mutants. Future research should also focus on answering questions on ferrosome biogenesis, how iron is internalized into ferrosomes, how ferrosomes release iron, and whether there is an association between ferrosome membrane and the cell membrane. Answers to these essential questions will provide insight into the fate of stored iron. Iron acquisition is important for toxin production by C. difficile [10]. Therefore, investigating how the ferrosome effects toxin production and whether ferrosomes provide a competitive advantage for C. difficile over gut commensals will provide important insight into C. difficile pathogenesis. Overall, the study by Pi et al. [4] significantly advances our understanding of the ultrastructure of bacterial cells and the potential role of ferrosome in circumventing nutritional immunity.