Abstract: Multiple Myeloma (MM) is currently an incurable cancer of plasma B cells often characterized by overproduction of abnormally high quantities of a patient-specific, clonotypic immunoglobulin (Ig) "M-protein". The M-protein is expressed on the cell membrane and secreted into the blood. We previously showed that Ligand-Toxin Conjugates (LTC) incorporating the ribosome-inactivating Ricin-A toxin were very effective in specific cytolysis of the anti-ligand antibody bearing target cells used as models for MM. Here, we report on the incorporation of the membrane-disruptive Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis (Bti) into LTCs targeted to murine myeloma cells. Proteolytically activated Cyt1Aa was conjugated chemically or genetically through either its amino or carboxy termini to the major peptidic epitope VHFFKNIVTPRTP (p87-99) of the Myelin Basic Protein (MBPp). The recombinant fusion-encoding genes were cloned and expressed in acrystalliferous Bti through the shuttle vector pHT315. Both chemically conjugated and genetically fused LTCs were toxic to anti-MBPp-expressing murine hybridoma cells but the recombinant conjugates were more active. LTCs comprising the Cyt1Aa toxin might be useful anticancer agents. Being a membrane-acting toxin, Cyt1Aa is not likely to induce development of resistant cell lines.
Abstract: The gene cyt2Ba of Bacillus thuringiensis subsp. israelensis was cloned for expression, together with p20, in an acrystalliferous strain. The large hexagonal crystals formed were composed of Cyt2Ba, which facilitated its purification. Crystal solubilization in the presence of endogenous proteases (with spores and cell debris) enabled quick and simple procedure to obtain rather pure and active toxin species by cleavage between amino acid residues 34 and 35, most likely by a camelysin-like protease that was discovered in association with activated Cyt2Ba. The product of this cleavage displayed haemolytic activity comparable to that of exogenously activated Cyt2Ba. The sequence of this putative protease shares high homology with the cell envelope-bound metalloprotease (camelysin) of the closely related species Bacillus cereus.
Abstract: The larvicidal activity of Bacillus thuringiensis subsp. israelensis against dipteran larvae is determined by four major polypeptides of the parasporal crystalline body produced during sporulation. Cyt1Aa shows the lowest toxicity when used alone but is the most synergistic with any of the other proteins. The sequence of the plasmid pBtoxis, which contains all the toxin genes in this subspecies, revealed a new cyt-like coding sequence named cyt1Ca. In addition to the Cyt-like region, the predicted Cyt1Ca contained an extra domain at the C terminus, which appeared to be a beta-trefoil carbohydrate-binding motif, as found in several ricin-like toxins. The gene was PCR-amplified from pBtoxis and cloned in several vectors, allowing high-level expression in Escherichia coli. Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae of Aedes aegypti of Cyt1Ca in recombinant E. coli cells was compared with that of Cyt1Aa and Cyt2Ba, and the ability of these proteins to enhance the activity of Cry4Aa was assessed. Although Cyt2Ba appeared able to interact with Cry4Aa, no activity for Cyt1Ca was observed, even when produced in truncated form. Furthermore, in contrast to Cyt1Aa, Cyt1Ca did not lyse sheep erythrocytes, and it was not bactericidal to the host cell.
