Nutrient Metal Sequestration by Calprotectin Inhibits Bacterial Superoxide Defense, Enhancing Neutrophil Killing of Staphylococcus aureus

Abstract

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.

Salivary metals, age, and gender correlate with cultivable oral Candida carriage levels

“…Saliva contains many proteins involved in nutritional immunity [5,10,12]. Calprotectin is the classic example of a metal-sequestering protein [10] that binds manganese, zinc, and iron with nanomolar to picomolar affinity, and inhibits microbial growth by disrupting superoxide dismutase function [13,14]. Salivary amylase [15], ferritin [16], Histatin 5 [17], gustin [18], and lactoferrin (functioning synergistically with calprotectin) [19] are all metal binding proteins expected to have a role in nutritional immunity.…”

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

“…Two binding sites for Zn (and presumably other transition metals) have been identified at the dimer interface in crystal structures of Zn-bound S100B, S100A7, and S100A12 (15)(16)(17). Site-directed mutagenesis has been used to confirm that Zn and Mn are bound in the corresponding sites in CP (13). Although the inhibition of bacterial growth via metal sequestration has been established for CP, the individual contributions of Mn and Zn limitation to this process are not known (11,13,18).…”

“…Although the inhibition of bacterial growth via metal sequestration has been established for CP, the individual contributions of Mn and Zn limitation to this process are not known (11,13,18). This uncertainty arises because of an inability to separate the effects attributable to the binding of Mn vs. the binding of Zn (11,13). Knowledge of the specific Mn-and Zndependent processes within bacteria that are inhibited by CP is important for understanding cellular physiology and identifying new effective antimicrobial therapeutics that exploit these targets.…”

“…Site-directed mutagenesis has been used to confirm that Zn and Mn are bound in the corresponding sites in CP (13). Although the inhibition of bacterial growth via metal sequestration has been established for CP, the individual contributions of Mn and Zn limitation to this process are not known (11,13,18). This uncertainty arises because of an inability to separate the effects attributable to the binding of Mn vs. the binding of Zn (11, 13).…”

“…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)(11)(12)(13)(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, underscoring the significance of Mn and Zn chelation to defense against infection (11).…”