However, SDS-PAGE revealed the presence of at least two bands with molecular masses between 70 and 100 kDa, which suggested that the SEPEC cytotoxic factor is a heterodimeric protein complex with a molecular mass of ~150 kDa (Fig.
The [CD.sub.50] of purified SEPEC cytotoxic factor (40 [micro]g/mL) was previously described by Puerner and Borenfreund.
The SEPEC cytotoxic factor causes elongation and loss of intercellular junctions in Vero (African green monkey kidney epithelial) cells (data not shown), same as observed in HUVEC cells (Fig.
Furthermore, the culture supernatant of SEPEC 15 obtained before 4h or after 6 h of culture showed negligible or no cytotoxicity in HUVEC cells (data not shown).
The measurement of the transendothelial electrical resistance (TEER) showed that the SEPEC cytotoxic factor, which caused elongation and loss of intercellular junctions in HUVEC, also increased the cell monolayer permeability by ~46% within 30 min.
In addition to increasing the HUVEC monolayer permeability, the purified SEPEC cytotoxic factor facilitated the translocation of bacteria across the monolayer (transcytosis).
For the transcytosis assay, bacterial suspensions (MOI = 200) of SEPEC 15 or E.
coli was accompanied by an increase in monolayer permeability (seen as a decrease in the TEER), we suggest that the cytotoxic factor secreted by SEPEC 15 facilitates its passage through the endothelial barrier by a paracellular pathway following alterations in the functional intactness of the intercellular junctions.