INFLUENCE OF LER OVEREXPRESSION ON THE PATHOGENICITY OF A SHIGA TOXIN-PRODUCING Escherichia coli O157:H7 (EHEC) STRAIN

GOMEZ, MARTIN; RAMOS, MARIA VICTORIA; PALERMO, MARINA SANDRA; FERNANDEZ BRANDO, ROMINA J; PINEDA, GONZALO EZEQUIEL; GALLY, DAVID; BRUBALLA, ANDREA; MCATEER , SEAN

Buenos Aires

Congreso; Reunion conjunta de sociedades de biociências; 2017

INFLUENCE OF LER OVEREXPRESSION ON THE PATHOGENICITY OF A SHIGA TOXIN-PRODUCING Escherichia coli O157:H7 (EHEC) STRAINRomina Jimena Fernández Brando (1), Martin Gomez (1), Andrea Bruballa (1), Gonzalo Pineda (1), Maria Victoria Ramos (1), Sean McAteer (2), David Gally (2), Marina Palermo (1)(1) Laboratorio de Patogénesis e Inmunología de Procesos Infecciosos, Instituto de Medicina Experimental (IMEX)- CONICET, Academia Nacional de Medicina, (2) Division of Infection and Immunity, The Roslin Institute, University ofEdinburghAlthough the production of Shiga toxin (Stx) by enterohemorragic Escherichia coli (EHEC) determines Hemolytic Uremic Syndrome onset, factors that modulate intestinal colonization are key components in pathogenesis and host mucosal immune response. Type III secretion system (T3S) is essential for colonization and is encoded on the locus of enterocyte effacement island (LEE). The first operonencodes its own regulator Ler, which controls the transcription of the other four operons (LEE2-5). The aim of this work was to study whether Ler overexpression, with the subsequent overexpression of LEE 2-5, determines an increase on EHEC pathogenesis. To do this we transformed a human-isolated EHEC strain (125/99)with a plasmid containing the ler sequence under the control of an IPTG-inducible promotor (125pLer) and also with the empty plasmid (125pW) as a control. We tested the expression of T3S proteins (EspB/D) by SDS-PAGE and Western blot (WB), and the adhesión to intestinal epithelial cells (HCT-8 and Caco-2). We also analyzed Stx2 production by ELISA, as T3S proteins are crossregulated bythe Stx prophage. We observed an increased expression of Esp- B/D on 125pLer by SDS-PAGE and WB. Besides, 125pLer strain showed an increased adhesion to HCT-8 and Caco-2 cells (% adherence HCT-8= 125pLer: 66.7±28.9, 125pW: 15.7±3.8; Caco-2= 125pLer: 33.7±3.2, 125pW: 12.3±4.0; ANOVA p˂0.05). Stx2 production was dependent on the time of pLer induction, i.e. the more time of pLer induction, the more Stx2 production ((mean±SD, μg/ ml) no induction= 125pLer: 0.108±0.000, pW: 0.132±0.022; 3.0 h= 125pLer: 0.171±0.013, 125pW: 0.123±0.004; t-test p˂0.05; 3.5 h= 125pLer: 0.807±0.127, 125pW: 0.349±0.001, t-test p˂0.05). We conclude that Ler overexpression determines the increased production of T3S proteins (EspB/D) together with an increased adherence tointestinal epithelial cells and Stx2 production. All these parameters could determine an increased pathogenicity in vivo.