A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins

The large clostridial cytotoxins (LCTs) constitute a group of high molecula r weight clostridial cytotoxins that inactivate cellular small GTP-binding proteins. We demonstrate that a novel LCT (TcdB-1470) from Clostridium diff icile strain 1470 is a functional hybrid between "reference" TcdB-10463 and Clostridium sordellii TcsL-1522. It bound to the same specific receptor as TcdB-10463 but glucosylated the same GTP-binding proteins as TcsL-1522. Ah three toxins had equal enzymatic potencies but were equally cytotoxic only when micro injected. When applied extracellularly TcdB-1470 and TcdB-10463 were considerably more potent cytotoxins than TcsL-1522. The small GTP-bin ding protein R-Ras was identified as a target for TcdB-1470 and also for Te st-1522 but not for TcdB-10463. R-Ras is known to control integrin-extracel lular matrix interactions from inside the cell. Its glucosylation may be a major determinant for the cell rounding and detachment induced by the two R -Ras-attacking toxins. In contrast, fibroblasts treated with TcdB-10463 wer e arborized and remained attached, with phosphotyrosine containing structur es located at the cell-to-cell contacts and beta(3)-integrin remaining at t he tips of cellular protrusions. These components were absent from cells tr eated with the R-Ras-inactivating toxins. The novel hybrid toxin will broad en the utility of the LCTs for clarifying the functions of several small GT Pases, now including also R-Ras.