Exploring Iron Withholding by the Innate Immune Protein Human Calprotectin

Abstract

Calprotectin (CP) is a versatile player in the metal-withholding innate immune response, a process termed "nutritional immunity." CP is a heterooligomer of the polypeptides S100A8 and S100A9 and houses two transition-metal-binding sites at its S100A8/S100A9 heterodimer interface. During infection, CP is released from host cells and sequesters "bioavailable" transition metal ions in the extracellular space, thereby preventing microbial acquisition of these essential nutrients. For many years, the role of CP in nutritional immunity was interpreted in the contexts of Mn(II) and Zn(II) limitation, but recent work has broadened our understanding of its contributions to this process. We uncovered that CP provides a form of nutritional immunity that has previously received little attention: the battle between host and microbe for ferrous iron (Fe(II)). In this Account, we present our current understanding of Fe(II) coordination by CP and its role in Fe(II) withholding as well as considerations for future discovery. Nutritional immunity was first described in the context of host-microbe competition for ferric iron (Fe(III)). The battle for Fe(II) has received comparably little attention because the abundance of Fe(II) at infection sites and the importance of Fe(II) acquisition for microbial pathogenesis were recognized only recently. Several years ago, we discovered that human CP sequesters Fe(II) at its His₆ site with subpicomolar affinity and thus hypothesized that it provides a means for Fe(II) limitation by the host during microbial infection. Fe(II) coordination by CP is unprecedented in biology because of its novel hexahistidine coordination sphere and its high-affinity binding, which surpasses that of other known Fe(II)-binding proteins. CP is also capable of shifting the Fe redox equilibrium by stabilizing Fe(II) in aerobic solution and can thereby sequester Fe in both reducing and nonreducing environments. These coordination chemistry studies allowed us to hypothesize that CP provides a means for Fe(II) limitation by the host during microbial infection. While investigating this putative Fe(II)-sequestering function, we discovered that CP withholds Fe from diverse bacterial pathogens. Recent studies by our lab and others of the bacterial pathogens Pseudomonas aeruginosa and Acinetobacter baumannii have shown that, by preventing sufficient Fe acquisition, CP induces Fe starvation responses in these organisms. As a result, CP affects bacterial virulence and metabolism. We also elucidated a complex interplay between CP and secondary metabolites produced by P. aeruginosa during the competition for Fe. Our work provides a foundation for understanding how CP affects Fe homeostasis during microbial infection. We believe that understanding how bacterial physiology is altered when challenged with Fe(II) withholding by CP will likely reveal crucial determinants of bacterial survival within the host.

Figure 1.

Crystal structure of Ni(II)-, Ca(II), and Na(I)-bound CP-Ser (PDB 5WIF). CP-Ser is composed of the S100A8(C42S) and S100A9(C3S) subunits. This variant has been employed for many metal-binding and microbiology studies. A heterodimer unit is taken from the structure of the heterotetramer. S100A8 is shown in green; S100A9 is shown in blue; Ni(II)-binding residues are shown in orange; Ni(II) is shown in teal; Ca(II) is shown in yellow; Na(I) is shown in purple. The N- and C-termini of S100A8 and S100A9 are labeled. The His₃Asp site is shown expanded on the left of the dimer, and the His₆ site is shown expanded on the right of the dimer.

Figure 2.

Fe(II) binding by CP.^, (A) Depletion of Fe from Tris:TSB medium [62:38 20 mM Tris, 100 mM NaCl, pH 7.5:Tryptic soy broth (TSB) medium] supplemented with ~3 mM β-mercaptoethanol and ≈2 mM Ca(II) by CP-Ser. The mean and SDM are reported (n = 5). (B) The 4.2 K/53 mT Mössbauer spectrum for ⁵⁷Fe(II)-bound CP-Ser prepared with excess Ca(II) and 0.83 equiv of ⁵⁷Fe(II) sulfate per CP heterodimer is shown as black vertical parts. The simulation of this spectrum as a single quadrupole doublet with an isomer shift (δ) of 1.20 mm/s and a quadrupole splitting parameter (ΔE_Q) of 1.78 mm/s is shown as the blue line. The Mössbauer spectrum of ⁵⁷Fe(II) sulfate in 50 mM Tris, pH 7.5 is shown as the red line. (C) The 5 K, 7 T NIR MCD spectra for the titration of CP-Ser with Fe(II) in the presence of excess Ca(II). Panel C was reproduced with permission from ref. . Copyright 2017 the Royal Society of Chemistry.

Figure 3.

CP sequesters Fe(II) under aerobic conditions and shifts Fe redox equilibrium to favor Fe(II). (A) Depletion of Fe from Tris:TSB medium (62:38 20 mM Tris, 100 mM NaCl, pH 7.5:TSB medium) supplemented with 2 mM Ca(II) by 10.5 μM CP-Ser in the absence or presence of ~3 mM β-mercaptoethanol (BME). The mean and SDM are reported (n = 3). (B & C) Fe(III) citrate (10 mM) was incubated with 10.5 μM (B) CP-Ser or (C) ΔΔ variant in the presence of 2 mM Ca(II) and the Fe redox speciation was monitored by the ferrozine assay (75 mM HEPES, 100 mM NaCl, pH 7.0 at 30 °C, 150 rpm). The mean and SDM are reported (n = 6). Panels B and C were reproduced with permission from ref. . Copyright 2017 the Royal Society of Chemistry.

Figure 4.

Analysis of cell-associated Fe levels shows that CP inhibits Fe uptake by several bacterial pathogens during aerobic culture. Bacteria (P. aeruginosa PA14, E. coli UTI89, S. Typhimurium ATCC 14028, K. pneumoniae ATCC 13883, A. baumannii ATCC 17978, and S. aureus USA300 JE2) were grown in Tris:TSB or LB medium in the absence or presence of 10 μM CP-Ser (in Tris:TSB) or 20 μM CP-Ser (in LB) at 37°C for 8 h. Cell-associated Fe corresponds to the concentration of Fe in an OD₆₀₀ = 10 cell suspension (n = 5, *P < 0.05; **P < 0.01). Reproduced with permission from ref. . Copyright 2019 the American Society for Biochemistry and Molecular Biology

Figure 5.

CP induces Fe starvation responses in P. aeruginosa. CP inhibits Fe uptake, and apo-Fur derepresses the transcription of prrF and pvdS. Subsequently produced PrrF sRNAs repress antR translation, and PvdS promotes pyoverdine biosynthesis. As a result, antR translation is inhibited by CP, and pyoverdine production is promoted by CP.

Figure 6.

Effect of siderophores and phenazines on P. aeruginosa Fe homeostasis and Fe(II) sequestration by CP.