Effects of the E.Coli exotoxin alpha-hemolysin on human chorioamniotic membranes

PUCCI MOLINERIS MELISA; BARBERO HUGO; ACCIALINI PAULA; FARINA MARIANA; PELINSKI PABLO ; HERLAX VANESA

Ciudad Autónoma de Buenos Aires

Congreso; Congress of International Federation of Placenta Associations 2019 (IFPA2019); 2019

Objectives: Premature rupture of chorioamniotic membranes is associated to urinary tract infections in pregnant women. Therefore, the main aim of this study is to evaluate the effect of alpha-hemolysin (HlyA), an exotoxin secreted by uropathogenic E.Coli, in remodeling of chorioamniotic extracellular matrix in vitro.Methods: HlyA was purified from a WAM1824 E.coli culture by isoelectric precipitation.Chorioamniotic membranes were obtained from deliveries by elective cesarean section (>37 weeks). All included women had uneventful pregnancies, without evidence of active labor and with no signs of infection.Membrane discs (8mm) were cultured in presence of 0-50nM HlyA for 3 and 24h. We evaluated membrane integrity by LDH and MTT assays and Masson´s trichrome stain to stablish work conditions (n=3).Then, explants (18mm) were cut to be placed on a Transwell system to form two independent chambers and insured with linen thread. To simulate an ascending infection explants were incubated in control conditions, with 5nm/50nm HlyA, and 50nM pro-HlyA (inactive form) in the chorion-side during 24h, (n=3). We evaluated membrane integrity by hematoxylin-eosin staining on paraffin-embedded tissue sections and LDH assay on culture medium. Collagen levels were tested by Masson´s trichrome stain, metalloprotease (MMP) activity by zimography, and cyclooxygenase-2 (COX2) expression by RT-qPCR.ResultsUntreated discs remain intact in culture until 48h. Instead, explant of fetal membrane incubated with 5 and 50nM HlyA during 24h showed a detrimental effect in mitochondrial activity and collagen level without LDH release. HlyA induced a structural alteration and a decrease in collagen fibers content in mounted explants. Moreover, an increment in COX-2 expression and metalloproteinase activity were observed in a HlyA concentration dependence. Instead, Pro-HlyA-treated explants remained similar to control.ConclusionE.Coli HlyA toxin by itself is capable to trigger events related to remodeling of chorioamniotic extracellular matrix in vitro. This observations suggest that the blockeage of HlyA may contribute to diminish damage on fetal membrane