Calprotectin S100A9 calcium-binding loops I and II are essential for keratinocyte resistance to bacterial invasion

Abstract

Epithelial cells expressing calprotectin, a heterodimer of S100A8 and S100A9 proteins, are more resistant to bacterial invasion. To determine structural motifs that affect resistance to bacterial invasion, mutations were constructed in S100A9 targeting the calcium-binding loops I and II (E36Q, E78Q, E36Q,E78Q) and the C terminus (S100A9(1-99) and S100A9(1-112)), which contains putative antimicrobial zinc-binding and phosphorylation sites. The S100A8 and mutated S100A9 encoding plasmids were transfected into calprotectin-negative KB carcinoma cells. All transfected cells (except KB-sham) expressed 27E10-reactive heterodimers. In bacterial invasion assays with Listeria monocytogenes and Salmonella enterica serovar Typhimurium (Salmonella typhimurium), cell lines expressing S100A8 in complex with S100A9E36Q, S100A9E78Q, S100A9(1-99), or S100A9(1-112) mutants or the S100A9(1-114) (full-length) calprotectin resisted bacterial invasion better than KB-sham. When compared with KB-S100A8/A9(1-114), cells expressing truncated S100A9(1-99) or S100A9(1-112) with S100A8 also showed increased resistance to bacterial invasion. In contrast, glutamic acid residues 36 and 78 in calcium-binding loops I and II promote resistance in epithelial cells, because cells expressing S100A9E36Q,E78Q with S100A8 were unable to resist bacterial invasion. Mutations in S100A9 E36Q, E78Q were predicted to cause loss of the calcium-induced positive face in calprotectin, reducing interactions with microtubules and appearing to be crucial for keratinocyte resistance to bacterial invasion.

FIGURE 1.

Structure of S100A8, S100A9, and mutations in selected functional domains. A, amino acid sequences of S100A8 and S100A9. Each subunit contains two EF-hands with helix-loop-helix motifs linked by a hinge region and flanked by N- and C-terminal domains. Calcium-binding loops are in boxes. Putative zinc-binding domains are highlighted in gray. The phosphorylation site is boldface and underlined. Source, NCBI Entrez protein P05109 (A8) and P06702 (A9) (38). B, full-length S100A9 (S100A9_1–114) and S100A9 mutant constructs, including C-terminal domain deletions (S100A9_1–112 and S100A9_1–99) and amino acid substitutions in the calcium-binding loops (S100A9_E36Q, S100A9_E78Q, and S100A9_E36Q,E78Q).

FIGURE 2.

mAb 27E10 reactivity in KB-S100A8/A9 mutants. Monolayers of KB-sham transfectant (A), KB-S100A8/A9_1–114 (B), KB-S100A8/A9_1–112 (C), KB-S100A8/A9_1–99 (D), KB-S100A8/A9_E36Q (E), KB-S100A8/A9_E78Q (F), and KB-S100A8/A9_E36Q,E78Q (G) were fixed with 4% paraformaldehyde and stained as described under “Experimental Procedures.” Monolayers were washed and then permeabilized with 0.2% Triton X-100 for 2 min. Monolayers were incubated with mAb 27E10 for 1 h, followed by Alexa Fluor 568-conjugated goat anti-mouse IgG for 1 h at room temperature, which stains calprotectin red. The inset in A shows enhanced green fluorescent protein expressed in KB-sham cells. The experiments were performed three times with similar results. Scale bar, 5 μm.

FIGURE 3.

Calprotectin production in S100A9 mutants. Calprotectin complex in KB-S100A8/A9_1–112 (A), KB-S100A8/A9_1–99 (B), KB-S100A8/A9_E36Q (C), KB-S100A8/A9_E78Q (D), and KB-S100A8/A9_E36Q,E78Q (E) were estimated using a sandwich ELISA as described under “Experimental Procedures.” KB-sham and KB-S100A8/A9_1–114 cells were used as negative and positive controls. Values are means ± S.E. (n ≥ 3; ^*, p < 0.05; ^**, p < 0.001). For each mutant, at least 10 clones were tested. Each experiment was performed in four replicates. The results are representative clones from each mutant.

FIGURE 4.

Analysis of S100A8 and mutated S100A9 in KB transfectants. To analyze the heterodimeric complexes, the cell lysates (1 mg of protein) from KB-sham, KB-S100A8/A9_1–114, KB-S100A8/A9_1–112, KB-S100A8/A9_1–199, KB-S100A8/A9_E36Q, KB-S100A8/A9_E78Q, and KB-S100A8/A9_E36Q,E78Q were co-immunoprecipitated (IP) using mAb 27E10. The immunoprecipitated proteins were then separated by 15% SDS-PAGE and either silver-stained or electroblotted onto nitrocellulose paper (A) and detected with anti-S100A8 (B) and anti-S100A9 antibodies (C), as described under “Experimental Procedures.” Cell lysates were also directly analyzed for S100A8 (D) and S100A9 (E) by Western blots (WB) as described under “Experimental Procedures.” Actin expression was used as protein loading control (lower panel in D).

FIGURE 5.

C-terminal deletion of S100A9 increases resistance to Listeria and Salmonella invasion. KB-sham, KB-S100A8/A9_1–114, KB-S100A8/A9_1–112, and KB-S100A8/A9_1–99 were analyzed for bacterial invasion using an antibiotic protection assay as described under “Experimental Procedures.” Monolayers were incubated with L. monocytogenes ATCC 10403S (A) or S. typhimurium ATCC 14028 (B) at an m.o.i. of 100:1 and 1:1, respectively, for 2 h. Each experiment was performed in triplicate wells. Values are means ± S.E. of viable intracellular bacteria, relative to KB-sham (100%) from at least three independent experiments. C, immunofluorescence staining for intracellular and extracellular Listeria in KB-sham, KB-S100A8/A9_1–114, KB-S100A8/A9_1–112, and KB-S100A8/A9_1–99 transfectants. Monolayers were incubated with L. monocytogenes for 2 h. The intracellular bacteria were enumerated and reported as means ± S.E. relative to KB-sham (100%). The results shown are from three independent experiments (^*, p < 0.05; ^**, p < 0.01; #, p < 0.001).

FIGURE 6.

Calcium-binding loops of S100A9 and epithelial cell resistance to Listeria and Salmonella invasion. KB-sham, KB-S100A8/A9_1–114, KB-S100A8/A9_E36Q, KB-S100A8/A9_E378Q, and KB-S100A8/A9_E36Q,E78Q were analyzed for bacterial invasion using an antibiotic protection assay. Monolayers were incubated with L. monocytogenes ATCC 10403S (A) or S. typhimurium ATCC 14028 (B) at an m.o.i. of 100:1 and 1:1, respectively, for 2 h. Each experiment was performed in triplicate wells. Values are means ± S.E. of viable intracellular bacteria, relative to KB-sham (100%), from at least three independent experiments. C, immunofluorescence staining for intracellular and extracellular Listeria in KB-sham, KB-S100A8/A9_1–114, KB-S100A8/A9_E36Q, KB-S100A8/A9_E78Q, and KB-S100A8/A9_E36Q,E78Q transfectants. Monolayers were incubated with L. monocytogenes for 2 h. The intracellular bacteria were enumerated and reported as means ± S.E. relative to KB-sham (100%) as in the legend of Fig. 5. The results shown are from three independent experiments (^**, p < 0.01; #, p < 0.001).

FIGURE 7.

Amino acid substitutions in the first and second calcium-binding loops of S100A9 decrease epithelial resistance to Listeria binding. S100A9 C-terminal deletion mutants (KB-S100A8/A9_1–112 and KB-S100A8/A9_1–99) (A) and S100A9 calcium-binding loop mutants (KB-S100A8/A9_E36Q, KB-S100A8/A9_E78Q, KB-S100A8/A9_E36Q,E78Q) (B) were incubated with L. monocytogenes ATCC 10403S for up to 1 h. Nonadherent bacteria were washed out, and the monolayers were fixed with 4% paraformaldehyde. KB-sham and KB-S100A8/A9_1–114 were used as negative and positive controls, respectively. Adherent bacteria were stained with specific antibodies and counted as described under “Experimental Procedures.” Values are means ± S.E. from three independent experiments. (^*, p < 0.05; ^**, p < 0.01).

FIGURE 8.

A representation of the changes in calprotectin structure and charge resulting from S100A9_E36Q,E78Q mutations. The ribbon diagram of calcium-free and calcium-bound calprotectin, both wild-type and the mutant, is presented in A, D, and G, and the corresponding calculated charged molecular surface is shown in B, E, and H. The surface of S100A9 is shown in C, F, and I; the view was obtained by rotating the charged molecular surface of calprotectin 90° on the z axis, revealing the S100A9 underside. The location of C-terminal tail of S100A9 has not been resolved by crystallography and has been omitted from this model. A–C, model structure of calcium-free calprotectin. D–F, calcium-bound form of calprotectin, based on PDB code 1XK4 (4). G–I, S100A8 calcium-bound structure combined with the calcium-free S100A9 structure based upon the E36Q,E78Q mutations. Color key: S100A8, yellow; S100A9, green; calcium, pink; positively charged surface, blue; negatively charged surface, red; and hydrophobic surface, white.