Bacterial Weapon Turned Iron Thief: Yersinia Secretes a Ferrousophore to Starve the Starver

Iron sits at the heart of one of the most ancient arms races in biology. Every pathogenic bacterium needs it, and every vertebrate host works hard to deny it. A new study published in Stress Biology by Qingyun Dai, Hongxin Guan, Jianan Huang and colleagues at Northwest A&F University and Fujian Normal University has now revealed a strikingly unexpected strategy in this contest: the intestinal pathogen Yersinia pseudotuberculosis uses its type VI secretion system, a molecular spear gun better known for attacking rival bacteria and host cells, to fire out a tiny protein that grabs ferrous iron from the environment and hauls it back into the cell. The finding adds a previously unrecognized chapter to the biology of nutritional immunity, the host strategy of starving microbes of essential metals.

The target of this bacterial maneuver is calprotectin, one of the most abundant antimicrobial proteins in the human body. Composed of the calcium-binding subunits S100A8 and S100A9, calprotectin makes up roughly forty percent of the cytoplasmic protein in neutrophils and is dumped rapidly into infected tissues. Scientists have long known that it sequesters manganese and zinc, but more recent work showed that it also binds ferrous iron, Fe(II), with high affinity. What remained unclear was whether this iron-withholding function actually matters during infection, and whether pathogens have evolved dedicated countermeasures. The new study answers both questions with unusually complete evidence, spanning structural biology, biophysics, genetics and mouse models.

The story began with a simple observation about stress. When the researchers deleted clpV1, a gene encoding the ATPase motor essential for the operation of Y. pseudotuberculosis’s type VI secretion system 1, or T6SS1, the mutant bacteria became markedly more sensitive to hydrogen peroxide and to acidic conditions at pH 5.0. Complementing the mutation restored normal resistance, and growth curves under non-stress conditions showed no baseline fitness defect, indicating that the secretion system itself was doing something specific to help the bacteria weather oxidative and acidic assault. Both forms of stress are highly relevant inside a host, where neutrophils bombard invaders with reactive oxygen species and the stomach presents a formidable acid barrier.

To find out what T6SS1 was secreting, the team systematically scanned the gene cluster for small hypothetical proteins, prioritizing uncharacterized open reading frames at the cluster terminus, a region known to harbor lineage-specific accessory genes. That search yielded YPK_0411, a diminutive 93-residue protein that structural modeling suggested might resemble iron-sulfur cluster-binding proteins. Secretion assays using a VSVG tag detected the protein in culture supernatants of wild-type bacteria but not of the clpV1 mutant, confirming that its export depends on the secretion machinery. The researchers named the protein SfeP, for T6SS-secreted ferrous iron-binding effector protein.

The biophysical characterization of SfeP is where the study becomes technically compelling. Using isothermal titration calorimetry under carefully controlled, oxidation-minimizing conditions, with ferrous iron freshly prepared in deoxygenated buffer supplemented with ascorbate and verified by ferrozine assays, the team showed that SfeP binds Fe(II) with a dissociation constant of about 1.013 nanomolar, an extraordinarily tight interaction. Just as telling was the specificity: SfeP ignored magnesium, manganese, zinc, copper and nickel, and it preferred Fe(II) over Fe(III). Inductively coupled plasma mass spectrometry then showed that deleting sfeP specifically depleted intracellular iron while leaving magnesium, zinc and manganese pools untouched, exactly the signature expected of a dedicated iron acquisition factor.

Functionally, the sfeP mutant grew normally in rich medium but faltered when iron was chelated away with EDDHA and EDDA, and the defect could be rescued either by restoring the gene or by flooding the medium with ferrous iron. The mutant was also hypersensitive to peroxide and acid, and, crucially, supplemental Fe(II) protected wild-type and complemented strains under those stresses but offered the mutant far less benefit, consistent with a broken uptake pipeline. A clever solvent-partitioning assay using chloroform affinity suggested that the mutant carried a reduced intracellular pool of electron-donating species, again pointing to diminished ferrous iron. Together these experiments establish that SfeP is not merely an iron-binding curiosity but a working component of an iron import pathway.

To understand how SfeP actually moves iron across the bacterial outer membrane, the team solved its crystal structure at 1.76 angstrom resolution. The protein folds into a compact four-helix bundle, an architecture reminiscent of copper storage proteins such as Csp3 and Csp1, but without their cysteine-rich metal-binding motifs, and without the ferroxidase centers of ferritins or the dimeric iron-coordination interfaces of encapsulin cargo proteins. No bound iron was visible in the electron density, likely because ferrous iron oxidizes readily and associates transiently, so the structure serves as a framework while the calorimetry establishes the binding chemistry. The authors lay out a careful roadmap for future anaerobic co-crystallization and metal-substitution experiments to pin down the binding site itself.

The delivery partner turned out to be OmpF, a well-known outer membrane porin. A GST pull-down screen with SfeP-coated beads specifically retained a 40-kilodalton protein from bacterial lysates that mass spectrometry identified as OmpF, which shares 57 percent sequence identity with the ferrous uptake porin of Escherichia coli. Deleting ompF lowered both total and ferrous intracellular iron, and the interaction was confirmed by in vivo and in vitro pull-downs and far-western blotting with purified proteins. Molecular docking suggests SfeP binds near the pore region of OmpF, supporting a model in which the secreted effector captures extracellular Fe(II) and escorts protein-complexed iron through the porin into the periplasm. The authors propose the term ferrousophore for this class of secreted ferrous iron-binding protein, drawing an analogy to the HasA hemophore system that delivers heme to its outer membrane receptor.

The in vivo consequences are dramatic. When mice were infected orally with the sfeP mutant, every animal survived to 21 days, whereas fewer than ten percent of mice infected with wild-type bacteria lived that long. Histopathology revealed severe intestinal damage in wild-type infections, including mucosal abscission and epithelial disruption, that was absent in mutant infections, and the mutant could barely be recovered from the stomach, small intestine, cecum, spleen or feces. Even neutralizing stomach acid with bicarbonate only partially rescued the mutant, showing that SfeP contributes to virulence well beyond acid tolerance, supporting gut colonization and systemic spread. The mutant also formed weaker biofilms on living nematodes and abiotic surfaces, produced less extracellular polysaccharide, showed altered surface hydrophobicity and swam more actively, painting a picture of a factor that reshapes multiple lifestyle traits through iron homeostasis.

Perhaps the most elegant experiment addresses calprotectin directly. In calprotectin-deficient S100a9 knockout mice, the severe colonization defect of the sfeP mutant was largely reversed, with high bacterial burdens recovered from intestinal tissues, demonstrating that calprotectin-mediated iron withholding is a genuine force shaping infection outcomes. Conversely, the knockout mice proved more susceptible to wild-type Yersinia, confirming the protective role of the protein for the host. Because the rescue was incomplete, SfeP evidently also promotes fitness through calprotectin-independent mechanisms. The broader significance is twofold: it validates ferrous iron sequestration as a real pillar of nutritional immunity, and it expands the known repertoire of the type VI secretion system, already implicated in manganese, zinc and copper scavenging, into the ferrous realm. Given that SfeP homologs are distributed across many bacterial taxa and that OmpF is widespread, the ferrousophore strategy may be far more common than anyone suspected, and disrupting the calprotectin-iron axis or the SfeP-OmpF partnership could offer a new anti-virulence target that avoids the selective pressures of conventional antibiotics.

Subject of Research: T6SS-mediated ferrous iron acquisition by Yersinia pseudotuberculosis to evade calprotectin-mediated nutritional immunity

Article Title: Yersinia pseudotuberculosis secretes an Fe (II)-binding effector to evade calprotectin-mediated nutritional immunity

Article References: Dai, Q., Guan, H., Huang, J., Hou, J., Zhang, M., Wang, Y., Zhang, P., Xu, L., Gu, H., Wang, Y., Ouyang, S., & Shen, X. (2026). Yersinia pseudotuberculosis secretes an Fe (II)-binding effector to evade calprotectin-mediated nutritional immunity. Stress Biology, 6(1), Article 29. https://doi.org/10.1007/s44154-026-00304-6

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00304-6

Keywords: Yersinia pseudotuberculosis, type VI secretion system, SfeP, ferrous iron, calprotectin, nutritional immunity, OmpF, ferrousophore, oxidative stress, acid stress, virulence, iron homeostasis