Induction of human-fetal-membrane remodeling in-vitro by the alpha hemolysin of Escherichia coli

Abstract

Introduction: Almost 80% of urinary tract infections during pregnancy are caused by uropathogenic strains of Escherichia coli. Alpha-hemolysin, toxin secreted by them, has a fundamental role in this pathology development. Considering that urinary tract infections are related with premature rupture of fetal membranes, we proposed to evaluate the effects that alpha-hemolysin induces on human-fetal-membranes. Methods: Thirteen fetal membranes obtained from elective cesarean sections (>37 weeks) were mounted in a transwell-device generating two independent chambers. To mimic an ascendant-urinary-tract infection, membranes were incubated with different concentrations of pure alpha-hemolysin from the choriodecidual side during 24h. Extensive histological analyses were performed and transepithelial electrical-resistance were determined. Cell viability, metalloproteinase activity and cyclooxygenase-2- gene expression was estimated by lactate-dehydrogenase–release assay, zymography and RT-qPCR, respectively. Finally, four fetal membranes were treated with hemolysin preincubated with polyclonal anti-hemolysin antibodies. Cell viability and metalloproteinase activity were monitored. Results: After 24 h of treatment, fetal membranes evidenced a structural damage and a decrease in membrane resistance that progressed as the concentration of alpha hemolysin increased. While the amniotic-epithelial-layer remained practically unaffected, the chorion cells manifested an increase in vacuolization and necrosis. In addition, the extracellular matrix exhibited collagen-fiber disorganization, a marked decrease in fiber content, and became thicker in presence of the toxin. Cyclooxigenase-2 expression and metalloproteinase activity were also higher in the treated groups than in untreated ones. Finally, a preincubation of hemolysin with specific antibodies prevented the cytotoxicity on the chorion cells and the increase in metalloproteinase activity. Discussion: Hemolysin induces structural and molecular changes associated with the remodeling of human-fetalmembranes in-vitro.