Calprotectin S100A9 Structural Motifs Regulate Epithelial Resistance to Bacterial Invasion

Calprotectin, a heterodimeric complex of S100A8 and S100A9, has broad antimicrobial activity against bacteria and fungi. Suggested to confer antimicrobial function, S100A9 has a C-terminal polypeptide extension unique among S100 family proteins. The calcium-binding domains of calprotectin may also affect antimicrobial activity, since calcium-induced conformational changes can affect interactions of S100 proteins with targeted intracellular signaling molecules. When expressing calprotectin, epithelial cells become more resistant to bacterial invasion. Objective: To determine the structural motifs of S100A9 in the presence of S100A8, which affect resistance to bacterial invasion. Methods: In vitro site-directed mutagenesis of S100A9 was used to ablate calcium-binding sites (E36Q, E78Q and E36Q + E78Q). Deletion mutagenesis truncated the C-terminus of S100A9 at residues 100-114 (including a zinc-binding domain) or 113-114 (phosphorylation site at threonine-113). Using the pIRES and pIRES-EGFP expression system (Clontech), we transfected S100A8 and mutated S100A9 encoded plasmids into KB cells. Calprotectin- or mutant-expressing KB cells were selected for resistance to G418 sulfate and expression of green fluorescent protein. Heterodimer formation was confirmed by reaction of cytosol with anti-human calprotectin complex Mab 27E10. Stable mutants were tested for bacterial invasion using an antibiotic protection assay and confirmed by double immunofluorescence staining for invaded Listeria. Results: Clones with S100A9 C-terminal region deletions expressed 27E10-reactive heterodimers and showed fewer intracellular bacteria than sham transfectants or S100A8/S100A9 clones. Ablation of the phosphorylatable thr-113 or the zinc-binding motif showed fewer invaded Listeria than S100A8/S100A9. Mutants with mutated S100A9 calcium-binding domains showed increased numbers of invaded bacteria. Conclusions: Calcium-binding domains of S100A9 may control resistance to bacterial invasion. In contrast, the C-terminal domain of S100A9 in the calprotectin complex does not appear to control resistance to bacterial invasion.

Supported by NIH/NIDCR R01DE11831 and a scholarship from the Royal Thai Government.