Zinc sequestration by human calprotectin facilitates manganese binding to the bacterial solute-binding proteins PsaA and MntC.

Zinc is an essential transition metal nutrient for bacterial survival and growth but may become toxic when present at elevated levels. The Gram-positive bacterial pathogen Streptococcus pneumoniae is sensitive to zinc poisoning, which results in growth inhibition and lower resistance to oxidative stress. Streptococcus pneumoniae has a relatively high manganese requirement, and zinc toxicity in this pathogen has been attributed to the coordination of Zn(II) at the Mn(II) site of the solute-binding protein (SBP) PsaA, which prevents Mn(II) uptake by the PsaABC transport system. In this work, we investigate the Zn(II)-binding properties of pneumococcal PsaA and staphylococcal MntC, a related SBP expressed by another Gram-positive bacterial pathogen, Staphylococcus aureus, which contributes to Mn(II) uptake. X-ray absorption spectroscopic studies demonstrate that both SBPs harbor Zn(II) sites best described as five-coordinate, and metal-binding studies in solution show that both SBPs bind Zn(II) reversibly with sub-nanomolar affinities. Moreover, both SBPs exhibit a strong thermodynamic preference for Zn(II) ions, which readily displace bound Mn(II) ions from these proteins. We also evaluate the Zn(II) competition between these SBPs and the human S100 protein calprotectin (CP, S100A8/S100A9 oligomer), an abundant host-defense protein that is involved in the metal-withholding innate immune response. CP can sequester Zn(II) from PsaA and MntC, which facilitates Mn(II) binding to the SBPs. These results demonstrate that CP can inhibit Zn(II) poisoning of the SBPs and provide molecular insight into how S100 proteins may inadvertently benefit bacterial pathogens rather than the host.

The copper-linked Escherichia coli AZY operon: Structure, metal binding, and a possible physiological role in copper delivery.

The Escherichia coli yobA-yebZ-yebY (AZY) operon encodes the proteins YobA, YebZ, and YebY. YobA and YebZ are homologs of the CopC periplasmic copper-binding protein and the CopD putative copper importer, respectively, whereas YebY belongs to the uncharacterized Domain of Unknown Function 2511 family. Despite numerous studies of E. coli copper homeostasis and the existence of the AZY operon in a range of bacteria, the operon's proteins and their functional roles have not been explored. In this study, we present the first biochemical and functional studies of the AZY proteins. Biochemical characterization and structural modeling indicate that YobA binds a single Cu2+ ion with high affinity. Bioinformatics analysis shows that YebY is widespread and encoded either in AZY operons or in other genetic contexts unrelated to copper homeostasis. We also determined the 1.8 Å resolution crystal structure of E. coli YebY, which closely resembles that of the lantibiotic self-resistance protein MlbQ. Two strictly conserved cysteine residues form a disulfide bond, consistent with the observed periplasmic localization of YebY. Upon treatment with reductants, YebY binds Cu+ and Cu2+ with low affinity, as demonstrated by metal-binding analysis and tryptophan fluorescence. Finally, genetic manipulations show that the AZY operon is not involved in copper tolerance or antioxidant defense. Instead, YebY and YobA are required for the activity of the copper-related NADH dehydrogenase II. These results are consistent with a potential role of the AZY operon in copper delivery to membrane proteins.

High-Field EPR Spectroscopic Characterization of Mn(II) Bound to the Bacterial Solute-Binding Proteins MntC and PsaA.

During infection, the bacterial pathogens Staphylococcus aureus and Streptococcus pneumoniae employ ATP-binding cassette (ABC) transporters to acquire Mn(II), an essential nutrient, from the host environment. Staphylococcal MntABC and streptococcal PsaABC attract the attention of the biophysical and bacterial pathogenesis communities because of their established importance during infection. Previous biophysical examination of Mn(II)-MntC and Mn(II)-PsaA using continuous-wave (≈9 GHz) electron paramagnetic resonance (EPR) spectroscopy revealed broad, difficult-to-interpret spectra (Hadley et al. J. Am. Chem. Soc. 2018, 140, 110-113). Herein, we employ high-frequency (>90 GHz), high-field (>3 T) EPR spectroscopy to investigate the Mn(II)-binding sites of these proteins and determine the spin Hamiltonian parameters. Our analyses demonstrate that the zero-field splitting (ZFS) is large for Mn(II)-MntC and Mn(II)-PsaA at +2.72 and +2.87 GHz, respectively. The measured 55Mn hyperfine coupling values for Mn(II)-MntC and Mn(II)-PsaA of 241 and 236 MHz, respectively, demonstrate a more covalent interaction between Mn(II) and the protein compared to Mn(II) in aqueous solution (≈265 MHz). These studies indicate that MntC and PsaA bind Mn(II) in a similar coordination geometry. Comparison of the ZFS values determined herein with those ascertained for other Mn(II) proteins suggests that the Mn(II)-MntC and Mn(II)-PsaA coordination spheres are not five-coordinate in solution.

Murine Calprotectin Coordinates Mn(II) at a Hexahistidine Site with Ca(II)-Dependent Affinity.

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 ε-nitrogen atoms of the His ligands (aiso = 4.3 MHz) as well as the distal δ-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.

Preparation and Iron Redox Speciation Study of the Fe(II)-Binding Antimicrobial Protein Calprotectin.

Calprotectin (CP, S100A8/S100A9 heterooligomer) is an abundant metal-sequestering host-defense protein expressed by neutrophils, other white blood cells, and epithelial cells. The apoprotein is a S100A8/S100A9 heterodimer that contains two sites for transition metal binding at the S100A8/S100A9 interface: a His3Asp motif (site 1) and a His6 motif (site 2). In this chapter, we provide a step-by-step protocol for the overexpression and purification of the human and murine orthologues of CP that affords each apo heterodimer in high yield and purity. In these procedures, the S100A8 and S100A9 subunits are overexpressed in Escherichia coli BL21(DE3), and each apo heterodimer is obtained following cell lysis, folding, column chromatography, and dialysis against Chelex resin to reduce metal contamination. Recent studies demonstrated that human CP coordinates Fe(II) and that the protein affects the redox speciation of Fe in solution. An Fe redox speciation assay employing ferrozine is described that demonstrates the ability of both the human and murine orthologues of CP to shift the redox speciation of Fe from the ferric to the ferrous oxidation state over time.

Initial Biochemical and Functional Evaluation of Murine Calprotectin Reveals Ca(II)-Dependence and Its Ability to Chelate Multiple Nutrient Transition Metal Ions.

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.

Biochemical and Spectroscopic Observation of Mn(II) Sequestration from Bacterial Mn(II) Transport Machinery by Calprotectin.

Human calprotectin (CP, S100A8/S100A9 oligomer) is a metal-sequestering host-defense protein that prevents bacterial acquisition of Mn(II). In this work, we investigate Mn(II) competition between CP and two solute-binding proteins that Staphylococcus aureus and Streptococcus pneumoniae, Gram-positive bacterial pathogens of significant clinical concern, use to obtain Mn(II) when infecting a host. Biochemical and electron paramagnetic resonance (EPR) spectroscopic analyses demonstrate that CP outcompetes staphylococcal MntC and streptococcal PsaA for Mn(II). This behavior requires the presence of excess Ca(II) ions, which enhance the Mn(II) affinity of CP. This report presents new spectroscopic evaluation of two Mn(II) proteins important for bacterial pathogenesis, direct observation of Mn(II) sequestration from bacterial Mn(II) acquisition proteins by CP, and molecular insight into the extracellular battle for metal nutrients that occurs during infection.

Transition metals at the host-pathogen interface: how Neisseria exploit human metalloproteins for acquiring iron and zinc.

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).