Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase

Carbapenems, 'last-resort' β-lactam antibiotics, are inactivated by zinc-dependent metallo-β-lactamases (MBLs). The host innate immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. We show that metal sequestration is detrimental for the accumulation of MBLs in the bacterial periplasm, because those enzymes are readily degraded in their nonmetallated form. However, the New Delhi metallo-β-lactamase (NDM-1) can persist under conditions of metal depletion. NDM-1 is a lipidated protein that anchors to the outer membrane of Gram-negative bacteria. Membrane anchoring contributes to the unusual stability of NDM-1 and favors secretion of this enzyme in outer-membrane vesicles (OMVs). OMVs containing NDM-1 can protect nearby populations of bacteria from otherwise lethal antibiotic levels, and OMVs from clinical pathogens expressing NDM-1 can carry this MBL and the bla[subscript NDM] gene. We show that protein export into OMVs can be targeted, providing possibilities of new antibacterial therapeutic strategies.Kinship Foundation. Searle Scholars ProgramMassachusetts Institute of Technology. Department of Chemistr

Metal sequestration of the host-defense protein human calprotectin

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.Transition metals are essential nutrients for all organisms. Microbial pathogens must acquire first-row transition metals such as manganese (Mn), iron (Fe), and zinc (Zn) to colonize the mammalian host and to cause disease. As part of the first line of defense against pathogenic infection, the host innate immune response deploys a number of metal-chelating host-defense factors that restrict the access of nutrient metals from these microbes. One of these host-defense factors is the abundant neutrophil protein calprotectin (CP, Si 00A8/S 1 00A9, MRP-8/MRP- 14 oligomer, calgranulins A and B). This calcium (Ca)-binding EF-hand domain protein exhibits two transition-metal-binding sites at the Si 00A8/S 1 00A9 heterodimer interface, and Ca(II) coordination enhances the transition-metal-binding affinities of CP. At the start of this dissertation research, the accepted model stated that CP exhibits antimicrobial activity by withholding bioavailable Mn(II) and Zn(II). In our work, we investigate the coordination chemistry and antimicrobial activity of human CP. We discovered that CP also has the capacity to scavenge Fe(II) from microbes by chelating this metal ion with high affinity at its unusual hexahistidine (His 6) coordination motif, and we show that this site influences the redox speciation of iron in solution. Subsequent studies establish that the His 6 site also contributes to nickel (Ni) withholding, and we evaluate the structural basis for the Ca(II)-modulated Ni(II)- binding properties of CP. Finally, we demonstrate that the functionally versatile His6 motif of CP also contributes to Zn(II) sequestration. In total, our investigations into the bio-inorganic chemistry of CP provide molecular-level insights into host-mediated metal sequestration.by Toshiki G. Nakashige.Ph. D

Nickel Sequestration by the Host-Defense Protein Human Calprotectin

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

Contributions of the S100A9 C‑Terminal Tail to High-Affinity Mn(II) Chelation by the Host-Defense Protein Human Calprotectin

Human calprotectin (CP) is an antimicrobial protein that coordinates Mn­(II) with high affinity in a Ca­(II)-dependent manner at an unusual histidine-rich site (site 2) formed at the S100A8/S100A9 dimer interface. We present a 16-member CP mutant family where mutations in the S100A9 C-terminal tail (residues 96–114) are employed to evaluate the contributions of this region, which houses three histidines and four acidic residues, to Mn­(II) coordination at site 2. The results from analytical size-exclusion chromatography, Mn­(II) competition titrations, and electron paramagnetic resonance spectroscopy establish that the C-terminal tail is essential for high-affinity Mn­(II) coordination by CP in solution. The studies indicate that His103 and His105 (HXH motif) of the tail complete the Mn­(II) coordination sphere in solution, affording an unprecedented biological His₆ site. These solution studies are in agreement with a Mn­(II)-CP crystal structure reported recently (Damo, S. M.; et al. <i>Proc. Natl. Acad. Sci. U.S.A. </i> <b>2013</b>, <i>110</i>, 3841). Remarkably high-affinity Mn­(II) binding is retained when either H103 or H105 are mutated to Ala, when the HXH motif is shifted from positions 103–105 to 104–106, and when the human tail is substituted by the C-terminal tail of murine S100A9. Nevertheless, antibacterial activity assays employing human CP mutants reveal that the native disposition of His residues is important for conferring growth inhibition against <i>Escherichia coli</i> and <i>Staphylococcus aureus</i>. Within the S100 family, the S100A8/S100A9 heterooligomer is essential for providing high-affinity Mn­(II) binding; the S100A7, S100A9­(C3S), S100A12, and S100B homodimers do not exhibit such Mn­(II)-binding capacity

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

Functional Multigenomic Screening of Human-Associated Bacteria for NF-κB-Inducing Bioactive Effectors

Human-associated bacteria are thought to encode bioactive small molecules and proteins that play an intimate role in human health and disease. Here, we report on the creation and functional screening of a multigenomic library constructed using genomic DNA from 116 bacteria found at diverse sites across the human body. Individual clones were screened for genes capable of conferring NF-κB-inducing activity to Escherichia coli. NF-κB is a useful reporter for a range of cellular processes related to immunity, pathogenesis, and inflammation. Compared to the screening of metagenomic libraries, the ability to normalize input DNA ratios when constructing a multigenomic library should facilitate the more efficient examination of commensal bacteria for diverse bioactivities. Multigenomic screening takes advantage of the growing available resources in culturing and sequencing the human microbiota and generates starting points for more in-depth studies on the mechanisms by which commensal bacteria interact with their human host.The effect of the microbiota on its human host is driven, at least in part, by small-molecule and protein effectors it produces. Here, we report on the use of functional multigenomic screening to identify microbiota-encoded effectors. In this study, genomic DNA from 116 human-associated bacteria was cloned en masse, and the resulting multigenomic library was screened using a nuclear factor-κB reporter (NF-κB) assay. Functional multigenomics builds on the concept of functional metagenomics but takes advantage of increasing advances in cultivating and sequencing human-associated bacteria. Effector genes found to confer NF-κB-inducing activity to Escherichia coli encode proteins in four general categories: cell wall hydrolases, membrane transporters, lipopolysaccharide biosynthetic enzymes, and proteins of unknown function. The compact nature of multigenomic libraries, which results from the ability to normalize input DNA ratios, should simplify screening of libraries using diverse heterologous hosts and reporter assays, increasing the rate of discovery of novel effector genes

Calprotectin influences the aggregation of metal-free and metal-bound amyloid- beta by direct interaction

Proteins from the S100 family perform numerous functions and may contribute to Alzheimer&apos;s disease (AD). Herein, we report the effects of S100A8/S100A9 heterooligomer calprotectin (CP) and the S100B homodimer on metal-free and metal-bound amyloid- (A; A(40) and A(42)) aggregation in vitro. Studies performed with CP-Ser [S100A8(C42S)/S100A9(C3S) oligomer] indicate that the protein influences the aggregation profile for A(40) in both the absence and presence of metal ions [i.e., Zn(ii) and Cu(ii)]. Moreover, the detection of A(40)-CP-Ser complexes by mass spectrometry suggests a direct interaction as a possible mechanism for the involvement of CP in A(40) aggregation. Although the interaction of CP-Ser with A(40) impacts A(40) aggregation in vitro, the protein does not attenuate A-induced toxicity in SH-SY5Y cells. In contrast, S100B has a slight effect on the aggregation of A. Overall, this work supports a potential association of CP with A in the absence and presence of metal ions in AD

Biophysical Examination of the Calcium-Modulated Nickel-Binding Properties of Human Calprotectin Reveals Conformational Change in the EF-Hand Domains and His₃Asp Site

Calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is a host-defense protein that sequesters nutrient transition metals from microbes. Each S100A8/S100A9 heterodimer contains four EF-hand domains and two transition-metal-binding sites. We investigate the effect of Ca­(II) ions on the structure and Ni­(II)-binding properties of human CP. By employing energy dispersive X-ray (EDX) spectroscopy, we evaluate the metal content of Ni­(II)-bound CP-Ser [oligomer of S100A8­(C42S) and S100A9­(C3S)] crystals obtained in the absence and presence of Ca­(II). We present a 2.1 Å resolution crystal structure of Ni­(II)-bound CP-Ser and compare this structure to a reported Ni­(II)- and Ca­(II)-bound CP-Ser structure [Nakashige, T. G., et al. (2017) <i>J. Am. Chem. Soc.</i> <i>139</i>, 8828–8836]. This analysis reveals conformational changes associated with coordination of Ca­(II) to the EF-hands of S100A9 and that Ca­(II) binding affects the coordination number and geometry of the Ni­(II) ion bound to the His₃Asp site. In contrast, negligible differences are observed for the Ni­(II)-His₆ site in the absence and presence of Ca­(II). Biochemical studies show that, whereas the His₆ site has a thermodynamic preference for Ni­(II) over Zn­(II), the His₃Asp site selects for Zn­(II) over Ni­(II), and relatively rapid metal exchange occurs at this site. These observations inform the working model for how CP withholds nutrient metals in the extracellular space

Calprotectin influences the aggregation of metal-free and metal-bound amyloid-β by direct interaction

Proteins from the S100 family perform numerous functions and may contribute to Alzheimer's disease (AD). Herein, we report the effects of S100A8/S100A9 heterooligomer calprotectin (CP) and the S100B homodimer on metal-free and metal-bound amyloid-β (Aβ; Aβ 40 and Aβ 42 ) aggregation in vitro. Studies performed with CP-Ser [S100A8(C42S)/S100A9(C3S) oligomer] indicate that the protein influences the aggregation profile for Aβ 40 in both the absence and presence of metal ions [i.e., Zn(ii) and Cu(ii)]. Moreover, the detection of Aβ 40 -CP-Ser complexes by mass spectrometry suggests a direct interaction as a possible mechanism for the involvement of CP in Aβ 40 aggregation. Although the interaction of CP-Ser with Aβ 40 impacts Aβ 40 aggregation in vitro, the protein does not attenuate Aβ-induced toxicity in SH-SY5Y cells. In contrast, S100B has a slight effect on the aggregation of Aβ. Overall, this work supports a potential association of CP with Aβ in the absence and presence of metal ions in AD

Biophysical Examination of the Calcium-Modulated Nickel-Binding Properties of Human Calprotectin Reveals Conformational Change in the EF-Hand Domains and His₃Asp Site

Calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is a host-defense protein that sequesters nutrient transition metals from microbes. Each S100A8/S100A9 heterodimer contains four EF-hand domains and two transition-metal-binding sites. We investigate the effect of Ca­(II) ions on the structure and Ni­(II)-binding properties of human CP. By employing energy dispersive X-ray (EDX) spectroscopy, we evaluate the metal content of Ni­(II)-bound CP-Ser [oligomer of S100A8­(C42S) and S100A9­(C3S)] crystals obtained in the absence and presence of Ca­(II). We present a 2.1 Å resolution crystal structure of Ni­(II)-bound CP-Ser and compare this structure to a reported Ni­(II)- and Ca­(II)-bound CP-Ser structure [Nakashige, T. G., et al. (2017) <i>J. Am. Chem. Soc.</i> <i>139</i>, 8828–8836]. This analysis reveals conformational changes associated with coordination of Ca­(II) to the EF-hands of S100A9 and that Ca­(II) binding affects the coordination number and geometry of the Ni­(II) ion bound to the His₃Asp site. In contrast, negligible differences are observed for the Ni­(II)-His₆ site in the absence and presence of Ca­(II). Biochemical studies show that, whereas the His₆ site has a thermodynamic preference for Ni­(II) over Zn­(II), the His₃Asp site selects for Zn­(II) over Ni­(II), and relatively rapid metal exchange occurs at this site. These observations inform the working model for how CP withholds nutrient metals in the extracellular space