In vivo formation of hybrid toxins comprising Shiga toxin and the Shiga-like toxin and role of the B subunit in localization and cytotoxic activity

Abstract

Shiga toxin, Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) are cell-associated cytotoxins that kill both Vero cells and HeLa cells, whereas Shiga-like toxin II variant (SLT-IIv) is an extracellular cytotoxin that is more cytotoxic for Vero cells than for HeLa cells. The basis for these differences in cytotoxin localization and host cell specificity were examined in this study. The A and B subunit genes of Shiga toxin and the SLTs were recombined by two methods so that hybrid toxins would be formed in vivo. Complementation of heterologous subunits was accomplished by cloning the individual A and B subunit genes of SLT-I, SLT-II, and SLT-IIv on plasmid vectors of different incompatibility groups so that they could be maintained in double transformants of Escerichia coli. In addition, six operon fusions were constructed so that the A and B subunit genes of Shiga toxins. SLT-II, and SLT-IIv could be expressed as a single operon. The activities of the hybrid cytotoxin, were assessed in three ways:(i) level of cytotoxicity, (ii) ratio of HeLa to Vero cell cytotoxicity and (iii) ratio of extracellular to cell-associated cytotoxicity. Neither the A subunit of Shiga toxin nor SLT-I associated with a heterologous B subunit to form an active cytotoxin. However, in all other cases the hybrid molecules formed by subunit complementation or operon fusion were cytotoxic. Furthermore, the cytotoxic specificity and localization of the hybrid cytotoxins always corresponded to the activities of the native toxin possessing the same B subunit.