CHARACTERIZATION OF THE EXPRESSION AND PRODUCTION OF BIOLOGICALLY ACTIVE SHIGA TOXIN 2 (STX2) IN EUKARYOTIC CELLS

BRUBALLA, ANDREA .; FERNANDEZ BRANDO, ROMINA J; PALERMO, MARINA S.; DE BRASI CARLOS; MUÑOZ, MANUEL; PINEDA, GONZALO EZEQUIEL; RAMOS, MARIA VICTORIA

Buenos Aires

Congreso; Reunion conjunta de sociedades de biociências; 2017

The cardinal element virulence of Shiga toxins (Stx)-producing E. coli (STEC) is the production of Stx, which constitute an AB5 toxin. We previously reported the existence of a putative eukaryotic promoter- like sequence (pr1), located upstream of Stx2-A subunit gene. The aim of this work was to characterize Stx2 subunits expression by eukaryotic cells after transfection with a prokaryotic plasmid carrying the Stx2 gene under its own promoter (pStx2). To determine the relevance of the putative promoter sequence, pr1, on Stx2 expression, pDpr1Stx2 plasmid was constructed. 293T cells were transfected with pStx2, total RNA was purifiedand specific mRNA was quantified by RT-qPCR. For both subunits, data revealed similar amounts of mRNA using either oligo (dT) or the corresponding subunit-specific primer (Sub A: Ct specific primer: 22.95, Ct oligo (dT): 21.97, Ct random primer: 27.93; Sub B: Ct specific primer: 28.27, Ct oligo (dT): 25.73 Ct random primer: 28.2). Vero cells, as a representative Stx2-susceptible cell line, were incubated with supernatants from 293T cells transfected with pStx2 (SNpStx) or pDpr1Stx2 (SN-pDpr1) to evaluate Stx2-cytotoxic activity. SN-pStx showed cytotoxicity on Vero cells. In contrast, no cytotoxicity was observed in SN-pr1 (% Vero viability with 1/32 Dilution of SN, SN-Stx2=50.29∓2.12; SN- pDpr1=123.81∓6.47 (p