The S100A8/S100A9 heterodimer calprotectin (CP) functions in the host response to pathogens through a mechanism termed "nutritional immunity." CP binds Mn 2+ and Zn 2+ with high affinity and starves bacteria of these essential nutrients. Combining biophysical, structural, and microbiological analysis, we identified the molecular basis of Mn 2+ sequestration. The asymmetry of the CP heterodimer creates a single Mn 2+ -binding site from six histidine residues, which distinguishes CP from all other Mn 2+ -binding proteins. Analysis of CP mutants with altered metal-binding properties revealed that, despite both Mn 2+ and Zn 2+ being essential metals, maximal growth inhibition of multiple bacterial pathogens requires Mn 2+ sequestration. These data establish the importance of Mn 2+ sequestration in defense against infection, explain the broad-spectrum antimicrobial activity of CP relative to other S100 proteins, and clarify the impact of metal depletion on the innate immune response to infection.bacterial pathogenesis | Staphylococcus aureus | antibiotic resistance | protein crystal structure | isothermal titration calorimetry B acterial pathogens are a significant threat to global public health. This threat is compounded by the fact that these organisms are rapidly becoming resistant to all relevant antimicrobials. Of particular note is the recent emergence of antibiotic-resistant strains of Staphylococcus aureus as a leading cause of bacterial infection in the United States (1) and arguably the most important threat to the public health of the developed world. Consequently, the identification of therapeutics to treat bacterial pathogens is paramount to our continued ability to limit this infectious threat.One promising area of potential therapeutic development involves targeting bacterial access to essential transition metals. This strategy is based on the fact that all bacterial pathogens require these nutrient metals to colonize their hosts (2-5). In vertebrates, the bacterial need for nutrient transition metals is counteracted by the sequestration of these metals by the host. This limitation of essential nutrients, termed "nutritional immunity," is a potent defense against infection (6). Although a variety of metals is required for microbial growth, studies of nutritional immunity have been primarily restricted to the struggle for iron (Fe) between host and pathogen (7-9).An innate immune factor, calprotectin (CP), is abundant in neutrophils and plays a key role in nutritional immunity. CP can be found at sites of infection in excess of 1 mg/mL and is required for the control of a number of medically relevant bacteria and fungi including S. aureus, Candida albicans, and Aspergillus fumigates (10-14). The antimicrobial activity of CP is due to the chelation of the essential nutrients Zn 2+ (Zn) and Mn 2+ (Mn), which results in bacterial metal starvation and is reversed by the addition of these metals in excess (11, 13). Moreover, CP-deficient mice have increased microbial burdens following systemic challenge, und...

Summary By sequestering manganese and zinc, the neutrophil protein calprotectin plays a crucial role in host defense against bacterial and fungal pathogens. However, the essential processes disrupted by calprotectin remain unknown. We report that calprotectin enhances the sensitivity of Staphylococcus aureus to superoxide through inhibition of manganese-dependent bacterial superoxide defenses thereby increasing superoxide levels within the bacterial cell. Superoxide dismutase activity is required for full virulence in a systemic model of S. aureus infection and disruption of staphylococcal superoxide defenses by calprotectin augments the antimicrobial activity of neutrophils promoting in vivo clearance. Calprotectin mutated in two transition metal binding sites and therefore defective in binding manganese and zinc does not inhibit microbial growth, unequivocally linking the antimicrobial properties of calprotectin to metal chelation. These results suggest that calprotectin contributes to host defense by rendering bacterial pathogens more sensitive to host immune effectors and reducing bacterial growth.

SummaryVertebrates sequester iron from invading pathogens, and conversely, pathogens express a variety of factors to steal iron from the host. Recent work has demonstrated that in addition to iron, vertebrates sequester zinc and manganese both intracellularly and extracellularly to protect against infection. Intracellularly, vertebrates utilize the ZIP/ZnT families of transporters to manipulate zinc levels, as well as Nramp1 to manipulate manganese levels, respectively. Extracellularly, the S100 protein calprotectin sequesters manganese and potentially zinc to inhibit microbial growth. To circumvent these defenses, bacteria possess high affinity transporters to import specific nutrient metals. Limiting the availability of zinc and manganese as a mechanism to defend against infection expands the spectrum of nutritional immunity and further establishes metal sequestration as a key defense against microbial invaders.

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Acinetobacter baumannii is an important nosocomial pathogen that accounts for up to 20 percent of infections in intensive care units worldwide. Furthermore, A. baumannii strains have emerged that are resistant to all available antimicrobials. These facts highlight the dire need for new therapeutic strategies to combat this growing public health threat. Given the critical role for transition metals at the pathogen-host interface, interrogating the role for these metals in A. baumannii physiology and pathogenesis could elucidate novel therapeutic strategies. Toward this end, the role for calprotectin- (CP)-mediated chelation of manganese (Mn) and zinc (Zn) in defense against A. baumannii was investigated. These experiments revealed that CP inhibits A. baumannii growth in vitro through chelation of Mn and Zn. Consistent with these in vitro data, Imaging Mass Spectrometry revealed that CP accompanies neutrophil recruitment to the lung and accumulates at foci of infection in a murine model of A. baumannii pneumonia. CP contributes to host survival and control of bacterial replication in the lung and limits dissemination to secondary sites. Using CP as a probe identified an A. baumannii Zn acquisition system that contributes to Zn uptake, enabling this organism to resist CP-mediated metal chelation, which enhances pathogenesis. Moreover, evidence is provided that Zn uptake across the outer membrane is an energy-dependent process in A. baumannii. Finally, it is shown that Zn limitation reverses carbapenem resistance in multidrug resistant A. baumannii underscoring the clinical relevance of these findings. Taken together, these data establish Zn acquisition systems as viable therapeutic targets to combat multidrug resistant A. baumannii infections.