The C-terminal extension of calprotectin mediates zinc chelation and modulates Staphylococcus aureus biomass accumulation

Abstract

Calprotectin (CP) is an S100A8/S100A9 heterodimer that plays an important role in nutritional immunity at the host-microbe interface. CP combats Staphylococcus aureus growth by sequestration of zinc and other trace transition metals; however, questions remain about whether CP antimicrobial activity strictly relies on metal sequestration. Moreover, the precise mechanism for how zinc binds at the two distinct transition metal binding sites of CP is not known. High-resolution X-ray crystal structures reveal tetracoordinate binding in the canonical His₃Asp site and hexacoordinate binding in the His₆ site similar to the binding of manganese and nickel in this site. The S100A9 C-terminal extension (tail) contributes two of the His residues in the His₆ metal-binding site, but measurements of zinc affinity show there is no significant reduction upon mutation of these His residues or deletion of the entire C-terminal tail. Bacterial growth and static biofilm assays show that the His mutations affect S. aureus biomass accumulation differently than loss of the S100A9 C-terminal tail, despite resulting in the same defect in bacterial-CP binding. These results reveal that the S100A9 tail of CP has a role in preventing S. aureus biomass accumulation.

Keywords: Staphylococcus aureus; X‐ray crystallography; biofilm; calprotectin; nutritional immunity; zinc.

FIGURE 1

CP metal‐binding sites are critical for anti‐staphylococcal activity. (a) Growth of S. aureus in 1:1.5::TSB:Buffer conditions with CP, CP*, or CP* without transition metal binding (CP∆His₆ ∆His₃Asp). All cultures were monitored for changes in OD₆₀₀ every 30 min of growth at 37°C, continuously shaking in a total of 150 μL of media. Data represent three biological and eight total replicates, normalized to starting OD₆₀₀; mean and SD. (b) Growth of S. aureus in 1:1.5::TSB:Buffer conditions with and without CP* or variants at a concentration of 250 μg/mL. Data represent three biological and eight total replicates, normalized to starting OD₆₀₀; mean and SD. CP* ∆His₆∆His₃Asp data is shared across (a) and (b), where baseline is re‐normalized to account for differences. (c) Statistical comparison of area under the curve (AUC) values via ordinary one‐way ANOVA with Tukey's multiple comparison test with a single pooled variance. (d) Statistical comparison of endpoint OD₆₀₀ values via ordinary one‐way ANOVA tests with Tukey's multiple comparison test with a single pooled variance, **p < 0.01, ***p < 0.005, ****p < 0.001.

FIGURE 2

X‐ray crystal structures of the Zn²⁺‐bound Ca²⁺‐loaded CP dimer of CP* (PDB ID: (8SJC) and Ca²⁺‐loaded CP* H87C (8SJB). Ribbon diagrams in panels (a) and (b) depict the CP* and CP* H87C dimers with S100A8 colored blue and S100A9 magenta. Ca²⁺ ions are not shown here for clarity, while Zn²⁺ ions are depicted in gray. The complete tetramer with Ca²⁺ ions is shown in Figure S4. The close‐up view of the unique His₆ site in panel (c) highlights the six histidine residues that chelate the Zn²⁺ ion. The close‐up view of the canonical His₃Asp site of the H87C variant (d) shows the residues responsible for chelating the Zn²⁺ ion.

FIGURE 3

Mutation of S100A9 C‐terminal tail has limited effect on CP Zn²⁺ affinity. Determination of zinc binding affinity of CP and variants by a competitive chelator approach. Each binding curve was fit individually, and then the reported apparent K _d was calculated by averaging the individual values obtained for triplicate independent measurements. The Zn affinity of CP* = 0.16 ± 0.07 pM, CP H103/104/105N = 0.21 ± 0.03 pM, CP G102Stop = 0.22 ± 0.07 pM, S100A9 = 0.48 ± 0.04 pM, CP ∆His6∆His3Asp = n/a.

FIGURE 4

Early S. aureus biomass accumulation significantly differs in the absence of the S100A9 C‐terminal tail. Measurement of adherent (a–c) and planktonic (d–f) S. aureus biomass grown under 1:1.5 TSB:Buffer conditions with and without CP* or variants for 12 h. Cultures were either stained with crystal violet for total biomass (a, d), SYPRO Ruby for total protein (b, e), or DAPI for total DNA (c, f). Two biological and 12 total technical replicates were used for each condition. (g) Representative western blot and (h) SYPRO Ruby stained SDS PAGE gel depicting the abundance of S100 proteins in media pre‐inoculation. (i) Representative western blot and (j) SYPRO Ruby stained gel of S100 protein variants treated cultures 12 h after inoculation. Panels (i) and (j) are representative of at least triplicate results (see Figure S5). All statistical comparisons made by ordinary one‐way ANOVA tests with Tukey's multiple comparison test with a single pooled variance, **p < 0.01, ***p < 0.005, ****p < 0.001.