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