Transition Metal Sequestration by the Host-Defense Protein Calprotectin.
ABSTRACT: In response to microbial infection, the human host deploys metal-sequestering host-defense proteins, which reduce nutrient availability and thereby inhibit microbial growth and virulence. Calprotectin (CP) is an abundant antimicrobial protein released from neutrophils and epithelial cells at sites of infection. CP sequesters divalent first-row transition metal ions to limit the availability of essential metal nutrients in the extracellular space. While functional and clinical studies of CP have been pursued for decades, advances in our understanding of its biological coordination chemistry, which is central to its role in the host-microbe interaction, have been made in more recent years. In this review, we focus on the coordination chemistry of CP and highlight studies of its metal-binding properties and contributions to the metal-withholding innate immune response. Taken together, these recent studies inform our current model of how CP participates in metal homeostasis and immunity, and they provide a foundation for further investigations of a remarkable metal-chelating protein at the host-microbe interface and beyond.
Project description: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.
Project description:The human innate immune protein calprotectin (CP, S100A8/S100A9 oligomer, calgranulin A/calgranulin B oligomer, MRP-8/MRP-14 oligomer) chelates a number of first-row transition metals, including Mn(II), Fe(II), and Zn(II), and can withhold these essential nutrients from microbes. Here we elucidate the Ni(II) coordination chemistry of human CP. We present a 2.6-Å crystal structure of Ni(II)- and Ca(II)-bound CP, which reveals that CP binds Ni(II) ions at both its transition-metal-binding sites: the His3Asp motif (site 1) and the His6 motif (site 2). Further biochemical studies establish that coordination of Ni(II) at the hexahistidine site is thermodynamically preferred over Zn(II). We also demonstrate that CP can sequester Ni(II) from two human pathogens, Staphylococcus aureus and Klebsiella pneumoniae, that utilize this metal nutrient during infection, and inhibit the activity of the Ni(II)-dependent enzyme urease in bacterial cultures. In total, our findings expand the biological coordination chemistry of Ni(II)-chelating proteins in nature and provide a foundation for evaluating putative roles of CP in Ni(II) homeostasis at the host-microbe interface and beyond.
Project description:Human calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is an abundant host-defense protein that is involved in the metal-withholding innate immune response. CP coordinates a variety of divalent first-row transition metal ions, which is implicated in its antimicrobial function, and its ability to sequester nutrient Zn(II) ions from microbial pathogens has been recognized for over two decades. CP has two distinct transition-metal-binding sites formed at the S100A8/S100A9 dimer interface, including a histidine-rich site composed of S100A8 residues His17 and His27 and S100A9 residues His91 and His95. In this study, we report that CP binds Zn(II) at this site using a hexahistidine motif, completed by His103 and His105 of the S100A9 C-terminal tail and previously identified as the high-affinity Mn(II) and Fe(II) coordination site. Zn(II) binding at this unique site shields the S100A9 C-terminal tail from proteolytic degradation by proteinase K. X-ray absorption spectroscopy and Zn(II) competition titrations support the formation of a Zn(II)-His6 motif. Microbial growth studies indicate that the hexahistidine motif is important for preventing microbial Zn(II) acquisition from CP by the probiotic Lactobacillus plantarum and the opportunistic human pathogen Candida albicans. The Zn(II)-His6 site of CP expands the known biological coordination chemistry of Zn(II) and provides new insight into how the human innate immune system starves microbes of essential metal nutrients.
Project description:Human calprotectin (CP) is a metal-chelating antimicrobial protein of the innate immune response. The current working model states that CP sequesters manganese and zinc from pathogens. We report the discovery that CP chelates iron and deprives bacteria of this essential nutrient. Elemental analysis of CP-treated growth medium establishes that CP reduces the concentrations of manganese, iron and zinc. Microbial growth studies reveal that iron depletion by CP contributes to the growth inhibition of bacterial pathogens. Biochemical investigations demonstrate that CP coordinates Fe(II) at an unusual hexahistidine motif, and the Mössbauer spectrum of (57)Fe(II)-bound CP is consistent with coordination of high-spin Fe(II) at this site (? = 1.20 mm/s, ?EQ = 1.78 mm/s). In the presence of Ca(II), CP turns on its iron-sequestering function and exhibits subpicomolar affinity for Fe(II). Our findings expand the biological coordination chemistry of iron and support a previously unappreciated role for CP in mammalian iron homeostasis.
Project description:Calprotectin (CP) is an abundant host-defense protein that contributes to the metal-withholding innate immune response by sequestering nutrient metal ions from microbial pathogens in the extracellular space. Over the past decade, murine models of infectious disease have advanced understanding of the physiological functions of CP and its ability to compete with microbes for essential metal nutrients. Despite this extensive work, murine CP (mCP) has not been biochemically evaluated, and structural and biophysical understanding of CP is currently limited to the human orthologue. We present the reconstitution, purification, and characterization of mCP as well as the cysteine-null variant mCP-Ser. Apo mCP is a mS100A8/mS100A9 heterodimer, and Ca(II) binding causes two heterodimers to self-associate and form a heterotetramer. Initial metal-depletion studies demonstrate that mCP depletes multiple first-row transition metal ions, including Mn, Fe, Ni, Cu, and Zn, from complex microbial growth medium, indicating that mCP binds multiple nutrient metals with high affinity. Moreover, antibacterial activity assays show that mCP inhibits the growth of a variety of bacterial species. The metal-depletion and antibacterial activity studies also provide evidence that Ca(II) ions enhance these functional properties of mCP. This contribution provides the groundwork for understanding the similarities and differences between the human and murine orthologues of CP and for further elucidation of its biological coordination chemistry.
Project description:Calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/14 oligomer, calgranulins A and B) is a protein component of the innate immune system that contributes to the metal-withholding response by sequestering bioavailable transition metal ions at sites of infection. Human CP employs Ca(II) ions to modulate its quaternary structure, transition metal binding properties, and antimicrobial activity. In this work, we report the discovery that Ca(II)-induced self-association of human CP to afford heterotetramers protects the protein scaffold from degradation by host serine proteases. We present the design and characterization of two new human CP-Ser variants, S100A8(C42S)(I60E)/S100A9(C3S) and S100A8(C42S)(I60K)/S100A9(C3S), that exhibit defective tetramerization properties. Analytical size exclusion chromatography and analytical ultracentrifugation reveal that both variants, hereafter I60E and I60K, persist as heterodimers in the presence of Ca(II) only, and form heterotetramers in the presence of Mn(II) only and both Ca(II) and Mn(II). Coordination to Fe(II) also causes I60E and I60K to form heterotetramers in both the absence and presence of Ca(II). The Ca(II)-bound I60E and I60K heterodimers are readily degraded by trypsin, chymotrypsin and human neutrophil elastase, whereas the Ca(II)-bound CP-Ser heterotetramers and the Ca(II)- and Mn(II)-bound I60E and I60K heterotetramers are resistant to degradation by these host proteases. The staphylococcal extracellular protease GluC cuts the S100A8 subunit of CP-Ser at the C-terminal end of residue 89 to afford a ?SKHE variant. The GluC cleavage site is in close proximity to the His3Asp metal-binding site, which coordinates Zn(II) with high affinity, and Zn(II) chelation protects the S100A8 subunit from GluC cleavage. Taken together, these results provide new insight into how Ca(II) ions and transition metals modulate the chemistry and biology of CP, and indicate that coordination to divalent cations transforms human CP into a protease-resistant form and enables innate immune function in the hostile conditions of an infection site.
Project description:Transition metals are essential nutrients for all organisms and important players in the host-microbe interaction. During bacterial infection, a tug-of-war between the host and microbe for nutrient metals occurs: the host innate immune system responds to the pathogen by reducing metal availability and the pathogen tries to outmaneuver this response. The outcome of this competition, which involves metal-sequestering host-defense proteins and microbial metal acquisition machinery, is an important determinant for whether infection occurs. One strategy bacterial pathogens employ to overcome metal restriction involves hijacking abundant host metalloproteins. The obligate human pathogens Neisseria meningitidis and N. gonorrhoeae express TonB-dependent transport systems that capture human metalloproteins, extract the bound metal ions, and deliver these nutrients into the bacterial cell. This review highlights structural and mechanistic investigations that provide insights into how Neisseria acquire iron from the Fe(III)-transport protein transferrin (TF), the Fe(III)-chelating host-defense protein lactoferrin (LF), and the oxygen-transport protein hemoglobin (Hb), and obtain zinc from the metal-sequestering antimicrobial protein calprotectin (CP).
Project description:We report that the metal-sequestering human host-defense protein calprotectin (CP, S100A8/S100A9 oligomer) affects the redox speciation of iron (Fe) in bacterial growth media and buffered aqueous solution. Under aerobic conditions and in the absence of an exogenous reducing agent, CP-Ser (S100A8(C42S)/S100A9(C3S) oligomer) depletes Fe from three different bacterial growth media preparations over a 48 h timeframe (T = 30 °C). The presence of the reducing agent ?-mercaptoethanol accelerates this process and allows CP-Ser to deplete Fe over a ?1 h timeframe. Fe-depletion assays performed with metal-binding-site variants of CP-Ser show that the hexahistidine (His6) site, which coordinates Fe(ii) with high affinity, is required for Fe depletion. An analysis of Fe redox speciation in buffer containing Fe(iii) citrate performed under aerobic conditions demonstrates that CP-Ser causes a time-dependent increase in the [Fe(ii)]/[Fe(iii)] ratio. Taken together, these results indicate that the hexahistidine site of CP stabilizes Fe(ii) and thereby shifts the redox equilibrium of Fe to the reduced ferrous state under aerobic conditions. We also report that the presence of bacterial metabolites affects the Fe-depleting activity of CP-Ser. Supplementation of bacterial growth media with an Fe(iii)-scavenging siderophore (enterobactin, staphyloferrin B, or desferrioxamine B) attenuates the Fe-depleting activity of CP-Ser. This result indicates that formation of Fe(iii)-siderophore complexes blocks CP-mediated reduction of Fe(iii) and hence the ability of CP to coordinate Fe(ii). In contrast, the presence of pyocyanin (PYO), a redox-cycling phenazine produced by Pseudomonas aeruginosa that reduces Fe(iii) to Fe(ii), accelerates Fe depletion by CP-Ser under aerobic conditions. These findings indicate that the presence of microbial metabolites that contribute to metal homeostasis at the host/pathogen interface can affect the metal-sequestering function of CP.
Project description:To combat infections, the mammalian host limits availability of essential transition metals such as iron (Fe), zinc (Zn), and manganese (Mn) in a strategy termed “nutritional immunity”. The innate immune protein calprotectin (CP) contributes to nutritional immunity by sequestering these metals to exert antimicrobial activity against a broad range of microbial pathogens. One such pathogen is Pseudomonas aeruginosa, which causes opportunistic infections in vulnerable populations including individuals with cystic fibrosis. CP was previously shown to withhold Fe(II) and Zn(II) from P. aeruginosa and induce Fe- and Zn-starvation responses in this pathogen. In this work, we performed quantitative, label-free proteomics to further elucidate how CP impacts metal homeostasis pathways in P. aeruginosa. We report that CP induces an incomplete Fe-starvation response, as many Fe-containing proteins that are repressed by Fe limitation are not affected by CP treatment. The Zn-starvation response elicited by CP seems to be more complete than the Fe-starvation response and includes increases in Zn transporters and Zn-independent proteins. CP also induces the expression of membrane-modifying enzymes, and metal-depletion studies indicate this response results from the sequestration of multiple metals. Moreover, the increased expression of membrane-modifying enzymes upon CP treatment correlates with increased tolorance to polymyxin B. Thus, response of P. aeruginosa to CP treatment includes both single and multi-metal starvation responses and includes many factors related to virulence potential, broadening our understanding of this pathogen’s interaction with the host.
Project description:Manganese is an essential metal ion that bacterial pathogens need to acquire from the vertebrate host during infection. In the mammalian nutritional immunity strategy to combat bacterial infection, the host restricts bacterial access to Mn(II) by sequestering this metal nutrient using the protein calprotectin (CP). The role of murine calprotectin (mCP) in Mn(II) sequestration has been demonstrated in vivo, but the molecular basis of this function has not been evaluated. Herein, biochemical assays and electron paramagnetic resonance (EPR) spectroscopy are employed to characterize the Mn(II) binding properties of mCP. We report that mCP has one high-affinity Mn(II) binding site. This site is a His6 site composed of His17 and His27 of mS100A8 and His92, His97, His105, and His107 of mS100A9. Similar to the human ortholog (hCP), Ca(II) binding to the EF-hand domains of mCP enhances the Mn(II) affinity of the protein; however, this effect requires ?10-fold more Ca(II) than was previously observed for hCP. Mn(II) coordination to the His6 site also promotes self-association of two mCP heterodimers to form a heterotetramer. Low-temperature X-band EPR spectroscopy revealed a nearly octahedral Mn(II) coordination sphere for the Mn(II)-His6 site characterized by the zero-field splitting parameters D = 525 MHz and E/D = 0.3. Further electron-nuclear double resonance studies with globally 15N-labeled mCP provided hyperfine couplings from the coordinating ?-nitrogen atoms of the His ligands (aiso = 4.3 MHz) as well as the distal ?-nitrogen atoms (aiso = 0.25 MHz). Mn(II) competition assays between mCP and two bacterial Mn(II) solute-binding proteins, staphylococcal MntC and streptococcal PsaA, showed that mCP outcompetes both proteins for Mn(II) under conditions of excess Ca(II). In total, this work provides the first coordination chemistry study of mCP and reveals striking similarities in the Mn(II) coordination sphere as well as notable differences in the Ca(II) sensitivity and oligomerization behavior between hCP and mCP.