Recombinant forms of Shiga Toxin 2: activity and antigenicity

GERHARDT ELIZABETH; CATALDI ANGEL; IBARRA CRISTINA

Buenos Aires

Congreso; I Congreso Internacional de Zoonosis y Enfermedades Emergentes y VII Congreso Argentino de Zoonosis; 2011

Asociación Argentina de Zoonosis

@font-face { font-family: "Arial"; }@font-face { font-family: "Times"; }p.MsoNormal, li.MsoNormal, div.MsoNormal { margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: "Times New Roman"; }div.Section1 { page: Section1; } @font-face { font-family: "Arial"; }@font-face { font-family: "Times"; }p.MsoNormal, li.MsoNormal, div.MsoNormal { margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: "Times New Roman"; }div.Section1 { page: Section1; } @font-face { font-family: "Arial"; }@font-face { font-family: "Times"; }p.MsoNormal, li.MsoNormal, div.MsoNormal { margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: "Times New Roman"; }div.Section1 { page: Section1; } @font-face { font-family: "Arial"; }p.MsoNormal, li.MsoNormal, div.MsoNormal { margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: "Times New Roman"; }div.Section1 { page: Section1; } Poster destacado en el área “Experimental en Zoonosis” @font-face { font-family: "Arial"; }p.MsoNormal, li.MsoNormal, div.MsoNormal { margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: "Times New Roman"; }div.Section1 { page: Section1; } Enterohemorragic Escherichia coli (EHEC), and among them the prototypic serotype O157:H7, are zoonotic bacteria responsible for intestinal disease and hemolytic uremic syndrome (HUS), a serious systemic complication which particularly affects children. The main virulence factor of EHEC is the Shiga toxin (Stx2). This toxin inhibit protein synthesis and after entering a cell, functions as an N-glycosidase, cleaving a specific adenine nucleotide from the 28S RNA of the 60S subunit of the ribosome, halting protein synthesis. Stx2 is an AB type toxin formed by one A subunit (were the active site is) and five B subunits (AB5). The goal of this work was to obtain two recombinant forms one inactive with the two subunits fused (fused) and other active with the A and B subunit genes being transcribed as an operon (active). For that the genes were in silico designed and synthesized by whole gene synthesis with no restriction steps. Main characteristics of the two rStx2 are: 1) an active rStx2 composed of A subunit lacking the signal sequence and with a 6xHis tail in the N-terminus of B subunit; and 2) fused contains a 6xHis tail in the N-terminus of A subunit, deleted signal sequence, a mutated active site and a fusion of the C-terminal of A subunit to the N-ter of B-subunit (rStx2AfusB). Both rStx2 were purified by affinity chromatography on nickel columns. To determine the rStx2 purity, the eluted fractions were immunoblotted using a polyclonal anti-Stx2 antibody. The first eluted fraction of purified rStx2AB showed two bands of approximately 32 kDa and 7 kDa corresponding to A and B subunits whereas rStx2AfusB showed one band of approximately 32 kDa. The cytotoxicity of rStx2 was assayed on Vero cells. We found that 3 ng/ml of purified rStx2AB was enough to inhibit 50% of Vero cell viability. On the contrary, 3 ng/ml of purified rStx2AfusB did not affect the Vero cells. In conclusion, rStx2AB is a non-secreted protein with an AB5 structure that shows cytotoxic activity on Vero cells. The rStx2AfusB is a non-secreted protein without activity in Vero cells. These two rStx2 are easy to produce and could be effective tools for physiopathological studies and immunodiagnosis assays.