Introduction: E. coli septicemia with cardiovascular dysfunction is among most frequent causes of human mortality however, underlying molecular mechanisms remain unknown. We recently described a fatal case of uro-sepsis with E. coli, bearing invasion-ligand Dr-adhesin, resulting in myocardial necrosis. Here we investigated an unrecognized mechanism of pathogen mediated cardiac cell invasion as a contributing factor to cardiovascular pathology and patient mortality.
Methods: The experiments on human cardiac fibroblasts (HCF) showed E. coli colonization and invasion. However, the primary cell receptor for Dr adhesin, CD55, was undetectable. Searching for an alternative receptor we focused on matrix protein TSP-1 which is elevated in heart during infection. We over-expressed TSP1 in HCF cells (HCF-TSP1) and challenged with Dr+ E. coli. Bacterial binding/invasion to HCF-TSP1 and wild-type (WT) HCF were compared and evaluated. To rule out the possibility of fibroblast invasion due to presence of CD55 we performed Western blotting with CD55 antibody. Analysis of TSP-1 and CD55 epitopes was done to look for bacteria binding domains.
Results: A dose-dependent TSP-1-E. coli binding assay in triplicates showed bacteria-HCF specific interactions. HCF-TSP1 had 13±4 bacteria bound per cell and invasion level of 2000/well which was up to 2.5 fold higher compared to WT-HCF. Both immunostaining and immunoblotting showed presence of TSP1 and absence of CD55 in HCF-TSP1. The interaction between purified/recombinant TSP1 coated to microtiter plates showed dose-dependent attachment of E. coli which increased with TSP1 concentrations. Sequence analysis of TSP-1 and CD55-SCR-3 revealed multiple 12AA CD55-like sequences within TSP-1. CD55-like epitopes identified on TSP1 shared up to 80% identity with the CD55 functional epitopes required for complement inhibition, Dr+ E. coli binding and invasion.
Conclusions: Here we identified cardiac fibroblasts as target for uro-septic E. coli invasion. We discovered TSP-1 as the receptor facilitating Dr+ E. coli invasion process and identified previously unrecognized CD55-like sequences within TSP-1 which in CD55 account for functional epitopes allowing E. coli attachment, invasion and complement inhibition.