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Pseudomonas aeruginosa exoenzyme S ADP-ribosylates p21ras and several related proteins. ADP-ribosylation of p21ras does not alter interactions with guanine nucleotides. The ras-related GTP-binding proteins, including Rab3, Rab4, Ral, Rap1A, and Rap2, are also substrates; given these results, we propose a model for the role of exoenzyme S in pathogenesis.

Clostridium botulinum type C and D strains produce exoenzyme C3, which ADP-ribosylates the Rho protein, a 21-kDa regulatory GTP-binding protein. In a previous work, we demonstrated that the C3 gene is encoded by bacteriophages C and D of C. botulinum by using DNA-DNA hybridizations with oligonucleotides deduced from the C3 protein N-terminal sequence. The C3 coding gene was cloned and sequenced, but its upstream DNA region could not be studied because of its instability in Escherichia coli. In this work, the upstream DNA region of the C3 gene was directly amplified by the polymerase chain reaction and sequenced. The C3 gene encodes a polypeptide of 251 amino acids (27,823 Da) consisting of a 40-amino-acid signal peptide and a mature protein of 211 amino acids (23,546 Da). The C3 mature protein was expressed in E. coli under the control of the trc promoter. The recombinant polypeptide obtained was recognized by C3 antibodies and ADP-ribosylated the Rho protein. The C3 gene nucleotide sequence is identical on C and D phage DNAs. At the amino acid sequence level, no similarity was found among C3, other ADP-ribosylating toxins, or tetanus or botulinal A, C1, and D neurotoxins.

By screening possible ADP-ribosyltransferase activities in culture supernatants from various Clostridium species, we have found one Clostridium difficile strain (CD196) (isolated in our laboratory) that is able to produce, in addition to toxins A and B, a new ADP-ribosyltransferase that was shown to covalently modify cell actin as Clostridium botulinum C2 or Clostridium perfringens E iota toxins do. The molecular weight of the CD196 ADP-ribosyltransferase (CDT) was determined to be 43 kilodaltons, and its isoelectric point was 7.8. No cytotoxic activity on Vero cells or lethal activity upon injection in mice was associated with this enzyme. CDT was neither related to C. difficile A or B toxins nor to C. botulinum C2 toxin component I. However, Vero cells cultivated in the presence of C. difficile B toxin had a lower amount of actin able to be ADP-ribosylated by CDT or C2 toxin in vitro. Antibodies raised against CDT reacted by immunoblot analysis with a 43-kilodalton protein of C. perfringens type E culture supernatant producing the iota toxin.

Exoenzyme S, which had been thought to be unselective, catalyzes the ADP-ribosylation of only a subset of cellular proteins. The intermediate filament protein vimentin is one of the more abundant substrates. Disassembled vimentin, and proteolytic fragments of vimentin that cannot form filaments, is more readily ADP-ribosylated than is filamentous vimentin.

Toxic shock syndrome toxin is already known to induce the production of interleukin-1 (IL-1) by preparations of monocytes and macrophages that are presumably contaminated with other types of cells. The response is enhanced by increasing the density of such monocytes, suggesting that the monocyte's response to toxic shock syndrome toxin may be augmented by its interaction with some other cell. Nevertheless, we now show that several human and murine macrophagelike cell lines (U937, J774, P388D1, and WEHI 3) produce IL-1 when exposed to toxic shock syndrome toxin, and therefore the basic response does not require the presence of cells of other lineages. The cultured cells generally produce less IL-1 than do monocytes, but considerably more IL-1 is induced from cells that have undergone a terminal differentiation as a result of exposure to 1 alpha,25-dihydroxyvitamin D3. High concentrations of cultured cels suppress the production of IL-1; this effect is apparently not due to long-lived inhibitors of IL-1 production or of IL-1 activity, but may be due to a short-lived inhibitor of production.

The Escherichia coli heat-labile enterotoxin (LT) was found to have the same subunit structure as cholera toxin, namely, one A subunit and five B subunits. Reaction with a bisimidate generated all the possible cross-linked derivatives of A5B: B,2B ... 5B and A, AB ... A5B. The isolated B component, coligenoid, contained five B subunits and showed some tendency of polymerize: with a bisimidate it became covalently connected into the set B ... 5B with lesser amounts of 6B ... 10B, etc. The subunit formulas of two independently prepared samples of LT were both proved to be A5B by cross-linking, but their B pentamers migrated at different rates on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, indicating that they have different conformations. The faster (R) form could be converted to a diffuse slower (C) form by incubating it at 50 degrees C or at 37 degrees C with 0.2 M galactose, which is the terminal sugar of ganglioside GM1, the natural receptor for LT. Cholera toxin resembled the R form more than the C form of LT.