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Calprotectin S100A9 structural domains regulate epithelial cell resistance to bacterial invasion

Posted on:2009-08-13Degree:Ph.DType:Dissertation
University:University of MinnesotaCandidate:Champaiboon, ChantrakornFull Text:PDF
GTID:1444390002992334Subject:Biology
Abstract/Summary:
Calprotectin, a complex of S100A8/S100A9, increases oral keratinocyte resistance to bacterial invasion. Calcium possibly induces conformational change of S100 proteins leading to signaling downstream effectors and S100A9 has the unique extended C-terminus, therefore, the calcium-binding and the C-terminal domains of S100A9 are suggested to determine calprotectin functions. The S100A9 structural motifs that confer calprotectin-induced resistance are yet unknown. To determine which the structural motifs of S100A9 in complex with S100A8 affect resistance to bacterial invasion, the S100A9 calcium-binding sites were ablated by site-directed mutagenesis and the C-terminus was truncated to delete the zinc-binding/arachidonic acid domain and phosphorylation site. Constructs to express the mutated S100A9 proteins with S100A8 were then transfected into KB cells and production of calprotectin complex was verified by reaction with the complex specific Mab 27E10. Stable mutants were tested for bacterial invasion by Listeria monocytogenes and Salmonella enterica serovar Typhimurium using an antibiotic protection assay and confirmed by double-immunofluorescence (IF) staining for invaded Listeria. Listeria binding to KB cells was quantified for up to 60 min, surface expression of calprotectin was estimated by IF and flow cytometry, and calprotectin mobilization to cytoskeleton was observed by IF. The result showed that S100A8 complexed with all mutants of S100A9. S100A9 C-terminus deletion transfectants resulted in fewer intracellular Listeria and Salmonella than the sham or intact S100A8/S100A9 cells (p<0.01). Ablated S100A9 C-terminus cells showed fewer invaded bacteria than positive control (p<0.05), while mutated S100A9 calcium-binding domain and the sham transfectants showed similar number of invaded bacteria. Listeria binding on KB cells and invasion appeared directly related. Surface calprotectin was undetectable, suggesting that binding only indirectly involved calprotectin. Furthermore, calprotectin mobilized to tubulin in a calcium-dependent manner, which was reversed by BAPTA-AM, an intracellular calcium chelator. In contrast, disruption of S100A9 calcium-binding domains inhibited calcium-dependent calprotectin mobilization. In conclusion, within KB cells, calprotectin mobilization to tubulin and epithelial resistance to invasion by bacteria appear to depend on the calcium-binding domains of S100A9.
Keywords/Search Tags:S100A9, Invasion, Calprotectin, Resistance, Domains, KB cells, S100A8, Complex
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