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 S100A9 E36Q , S100A9 E78Q , 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 S100A9 E36Q,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.

Bacterial Surface Protein L Binds and Inactivates Neutrophil Proteins S100A8/A9

“…Furthermore, the proteins bind arachidonic acid, and depletion of arachidonic acid has also been suggested as a mechanism of bacterial growth inhibition (49,50). Observations suggesting that S100A8/A9 contributes to mucosal innate immunity are that the complex inhibits invasion of epithelial cells by bacterial pathogens (51), and a recent study showing that S100A9 is crucial for keratinocyte resistance to invasion by Listeria monocytogenes and Salmonella typhimurium (52). In the present study, we provide evidence for another and more direct antibacterial mechanism.…”

Augmentation of Epithelial Resistance to Invading Bacteria by Using mRNA Transfections

“…Calprotectin is a heterodimeric complex of calcium-binding proteins S100A8 (MRP8 or calgranulin A; 10.8 kDa) and S100A9 (MRP14 or calgranulin B; 13.2 kDa) (6). S100A8 and S100A9 are members of the S100 family of proteins (17).…”

“…S100 family members are characterized by their two EF-hand calcium-binding motifs; these proteins are involved in cell growth, cell differentiation, cell cycle progression, cell survival, protein phosphorylation, transcription, cancer development, and inflammatory diseases (18). Calprotectin shows broad-spectrum antimicrobial activity against Candida albicans and bacteria, including Capnocytophaga sputigena, Escherichia coli, Staphylococcus epidermidis, Listeria monocytogenes, S. Typhimurium, and Porphyromonas gingivalis (4,6,(19)(20)(21). After stable transfection to express the calprotectin complex, an epithelial cell line (KB) showed increased resistance to invasion by Listeria monocytogenes and Salmonella enterica serovar Typhimurium (6).…”

“…The structural basis for the contribution of S100A8/A9 to resistance to invasion resides, at least in part, in the integrity of the S100A9 calcium-binding EF hands (6). When expressed in the cell in complex with S100A8, S100A9 E36Q and E78Q mutants showed increased bacterial invasion and were predicted to cause loss of the calcium-induced positive face in the S100A8/A9 complex (6).…”

Association of high levels of α-defensins and S100A proteins with Candida mannan detection in bronchoalveolar lavage fluid of preterm neonates

“…In our previous study (21), a significantly higher absolute number of neutrophils was found in BALF samples of the pneumonia group than in the α-Defensins and S100A proteins in BALF Articles nonpneumonia group, but the BALF samples were collected in most cases on the 5th d of life, whereas the samples were all collected in this study within the first 48 h of life. S100A8 and S100A9 proteins form the heterodimer calprotectin, with demonstrated antimicrobial activity against numerous microorganisms, particularly fungi (26,(29)(30)(31)(32)). However, when tested alone, neither S100A8 nor S100A9 showed activity in the Candida growth assay (31).…”