Combatting Bacteria by Manganese Sequestration
By: Carol A. Rouzer, VICB Communications
Published: March 4, 2013
Binding of Mn2+ by the antibacterial protein calprotectin is critical to its ability to inhibit bacterial growth by “nutritional immunity”.
Despite the immense life-saving role that antibiotics have played in fighting infectious diseases, bacterial pathogens remain a major threat to global public health. The threat is exacerbated by the growing emergence of bacterial strains exhibiting resistance to many antibiotics. In fact, antibiotic-resistant Staphylococcus aureus (Figure 1) is currently one of the greatest public health threats in the developed world. Clearly, there is a pressing need for new approaches to antibacterial therapies. Addressing that need are Vanderbilt Institute of Chemical Biology (VICB) members Eric Skaar and Walter Chazin and their laboratories* who reveal the mechanism of action of a key host defense protein, calprotectin (CP) [S. M. Damo, T. E. Kehl-Fie et al. (2013) Proc. Natl. Acad. Sci. U.S.A., published online February 19, DOI:10.1073/pnas.1220341110].
Figure 1. Scanning electron micrograph of methicilin-resistant S. aureus surrounded by cellular debris. Image reproduced through the courtesy of Wikimedia Commons, credit NIAID.
Invading pathogenic bacteria require nutrients, including trace metals such as iron, zinc, and manganese, which they must obtain from the host. Consequently, bacteria have evolved complex and highly efficient transport systems to capture these nutrients, and their hosts have evolved equally effective mechanisms to thwart the bacteria. The sequestration of critical nutrients by host cells as a weapon against bacterial infection is called “nutritional immunity”, a defense mechanism that we are just now beginning to understand and appreciate.
An important component of nutritional immunity is calprotectin (CP), a Mn2+- and Zn2+- binding protein found in neutrophils, a type of white blood cell. Neutrophils are among the first responders in the battle against an invading pathogen. They bring with them large quantities of CP, which can reach concentrations as high as 1 mg/mL at infected sites. CP effectively removes free Zn2+ and Mn2+ from the pathogen’s environment, leading to a selective nutrient starvation. Mice genetically deficient in CP have increased susceptibility to many types of bacterial and fungal pathogens, indicating the protein’s importance to host defenses.
CP belongs to the S100 subfamily of calcium-binding proteins. Most S100 proteins are homodimers that form two Zn2+ binding sites at the interface of the identical subunits. Each Zn2+ binding site comprises two histidine residues from one subunit and a histidine and aspartic acid residue from the second subunit. In contrast, CP is a heterodimer of the subunits S100A8 and S100A9, and its two metal binding sites are not identical. One site, designated S2, is formed from three histidine and one aspartic acid residue as seen in other S100 proteins. The second site, S1, is formed from four histidine residues, two from each subunit. Notably, CP is the only known S100 protein that binds Mn2+ strongly as well as Zn2+, leading the Skaar and Chazin teams to hypothesize that the unique S1 binding site may be responsible for CP’s expanded metal-binding specificity.
To test their hypothesis, the investigators created two mutant forms of CP. In the ΔS1 mutant, all four histidine residues in the S1 metal binding site were mutated to asparagine. In the ΔS2 mutant, the three histidine residues were mutated to asparagine, and the aspartic acid residue was mutated to histidine. Circular dichroism spectroscopy verified that the mutations did not disrupt the structural integrity of the proteins. Indeed, both mutants retained a high affinity for Zn2+. However, only CP ΔS2 retained the ability to bind Mn2+, supporting the hypothesis that the S1 site conferred CP with the ability to bind Mn2+.
Exposure of cultured S. aureus to wild-type, ΔS1, and ΔS2 CP revealed an approximately two-fold increase in the concentration required to inhibit bacterial growth by 50% (IC50) for both mutants. Despite the seemingly similar potencies, the ΔS1 mutant was decidedly less efficacious in its ability to inhibit growth of S. aureus. Even at very high concentrations of the protein, complete growth inhibition could not be achieved. Moreover, whereas addition of Zn2+ and Mn2+ reversed the inhibition of S. aureus growth in the case of CP wild-type and ΔS2, only Zn2+ could reverse the growth inhibitory effect of the ΔS1 mutant.
The data suggested that Mn2+ sequestration plays an important role in bacterial growth inhibition by CP. One mechanism by which this might occur is through the inhibition of superoxide dismutase (SOD) activity. SOD is critical for the scavenging of the highly reactive superoxide anion, and both SODs expressed in S. aureus require Mn2+ for activity. Consistent with this hypothesis, treatment of S. aureus with CP wild-type or ΔS2 led to elevated intracellular levels of superoxide and increased sensitivity of the bacteria to the superoxide anion-generating toxicant paraquat. In contrast, exposure to CP ΔS1 had no effect on superoxide anion levels or paraquat sensitivity in the bacteria. As discussed by the authors, CP’s disruption of SOD activity acts synergistically in vivo with the production of superoxide by pathogen-activated neutrophils that carry CP to the site of the infection.
To better understand CPs ability to bind Mn2+ at the structural level, the investigators acquired an X-ray crystal structure of CP with Mn2+ (Figure 2A). The data revealed that the overall protein was very similar in structure to other S100 proteins and that Mn2+ was bound, as expected, in the S1 site. An unexpected finding, however, was that binding of Mn2+ induced a conformational change in the C-terminal tail of the S100A9 subunit, allowing it to contribute two additional histidine residues to the coordination of the metal ion (Figure 2). As a result, Mn2+ was centered in a nearly perfectly symmetrical octahedral binding site comprising six histidine residues, a structure that efficiently excludes solvent. The authors note that a Mn2+ binding site constructed entirely of histidine residues is unique among known proteins. Exclusive use of nitrogen-containing ligands is inconsistent with the prevailing understanding of the electronic structure of Mn2+, which prefers coordination with oxygen; however, any resulting loss of stability is likely compensated by the optimal hexacoordinate geometry around the ion and the exclusion of solvent.
Figure 2. X-ray crystal structure of CP complexed with Mn2+. (A) Ribbon diagram showing the S100A8 (green) and S100A9 (yellow) subunits. The S1 and S2 sites are labeled, and Mn2+ is shown in purple. (B) Close up of the S1 binding site. (C) Electron density map for Mn2+ and the histidine side chains. Reproduced with permission from S. M. Damo, T. E. Kehl-Fie et al. (2013) Proc. Natl. Acad. Sci. U.S.A., published online February 19, DOI:10.1073/pnas.1220341110. Copyright 2013, S. M. Damo, T. E. Kehl-Fie, et al.
Together the results indicate that the capacity for Mn2+ binding conveyed by the S1 site is critical to the antibacterial activity of CP. This observation is not limited to S. aureus, as indicated by experiments showing strong growth inhibition of many bacterial species by CP wild-type and ΔS2, but not CP ΔS1 (Figure 3). Bacteria contain more Mn2+ binding proteins than eukaryotic cells, suggesting that Mn2+ plays a particularly important role in bacterial metabolism. Thus, Mn2+ acquisition and utilization may be a fruitful target for the future development of novel antimicrobial agents.
Figure 3. Inhibition of growth of the indicated bacteria by wild-type (WT), ΔS1, and ΔS2 CP. Reproduced with permission from S. M. Damo, T. E. Kehl-Fie et al. (2013) Proc. Natl. Acad. Sci. U.S.A., published online February 19, DOI:10.1073/pnas.1220341110. Copyright 2013, S. M. Damo, T. E. Kehl-Fie, et al.
*The VICB notes the contributions of three undergraduate students, co-authors Marilyn Holt, Laura Hench, and Wesley Murphy, who participated in this work through the NSF-sponsored Research Experiences for Undergraduates Program in Chemical Biology at Vanderbilt.