Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens

Abstract

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...

Association of Metal Homeostasis and (p)ppGpp Regulation in the Pathophysiology of Enterococcus faecalis

“…The biological significance of this observation is underscored by the high incidence of opportunistic infections in indi- viduals with high levels of circulating free Fe, as seen in patients with hemochromatosis and other iron-related disorders (17). The biological significance of Mn is much less understood, but data from studies from the Skaar laboratory and others that were performed using calprotectin and Mn transport mutants indicate that chelation of Mn is biologically (and medically) important (35,36). Notably, depletion of both metals in the laboratory media, mimicking the metal restriction found in host tissues, completely halted cell growth of the (p)ppGpp 0 strain, stressing the overlapping functions of these two metals.…”

The Human Antimicrobial Protein Calgranulin C Participates in Control of Helicobacter pylori Growth and Regulation of Virulence

“…(39,65,66). S100 proteins have recently been recognized as important players in host metal sequestration strategies that contribute to nutritional immunity (18,20,22). Calprotectin is the best-characterized of these proteins and has been shown to inhibit the growth of numerous bacteria and fungi (22,25).…”

“…However, recent work demonstrates that proteins isolated from human neutrophil cytosol, including calprotectin (but not S100A12), become carbonylated in the presence of reactive oxygen species generated by oxidative burst (67). This oxidative modification occurred at the S100A9 Cys2 and six histidine residues which have been associated with chelation of manganese and zinc (20). Interestingly, carbonylation of these residues inhibited the bacteriostatic activity of calprotectin, indicating that this posttranslational modification could be modulating the metal-binding nutritional immunity capacity of this protein (67).…”

“…This mechanism of antimicrobial activity constitutes a form of nutritional immunity. Numerous pathogens, including Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Escherichia coli, Borrelia burgdorferi, Listeria monocytogenes, Candida albicans, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus lugdunensis, Enterococcus faecalis, Pseudomonas aeruginosa, and Shigella flexneri, have been shown to be susceptible to the antimicrobial activity of the S100A8/S100A9 heterodimer calprotectin (19)(20)(21)(22)(23)(24)(25)(26). Our recent work demonstrated that calprotectin represses the growth and virulence of H. pylori by sequestering nutrient zinc (27).…”

Impact of Dental Plaque Biofilms in Periodontal Disease: Management and Future Therapy

“…These ions are essential for usual microbial functioning; thus, calprotectin is a growth inhibitory type of host defense [50].…”