David M. Lyerly

Infection and Immunity Mediated by the Carbohydrate Recognition Domain of the Entamoeba histolytica Gal/GalNAc Lectin

Entamoeba histolytica causes invasive amebiasis, a major parasitic disease of the developing world, whose primary symptoms are liver abscess and colitis. All strains of E. histolytica express a 260-kDa surface Gal/GalNAc lectin that is antigenically conserved and immunogenic. The lectin is required for adherence to human intestinal epithelial cells and contact-dependent killing of immune effector cells. By expression cloning, the carbohydrate recognition domain (CRD) was identified within the lectin heavy-subunit cysteine-rich region. Of interest for a hepatic parasite, the CRD had sequence identity to the receptor-binding domain of hepatocyte growth factor (HGF) and competed with HGF for binding to the c-Met HGF receptor. In an animal model of invasive disease, immunization with the CRD inhibited liver-abscess formation, yet in humans, a naturally acquired immune response against the CRD did not persist.

Vaccination against lethalClostridium difficile enterocolitis with a nontoxic recombinant peptide of toxin A

Toxin A ofClostridium difficile has a complex series of repeating units, each 20 or 30 amino acids in length, located at the COOH-terminus of the molecule. In the following study, we found that antiserum against a nontoxic recombinant peptide comprising 33 of the 38 repeating units neutralized the enterotoxic and cytotoxic activity of the toxin and that hamsters vaccinated with the recombinant peptide were partially protected againstC. difficile disease.

Cloning and Expression of the Toxin B Gene of Clostridium difficile

Results from our cloning studies on toxin A indicated that the gene for toxin B resided approximately 1 kb upstream of the toxin A gene. Clone pCD19, which contains the 5′-end of the toxin A gene and a small open reading frame, was found to contain 1.2 kb of DNA which, when subcloned, expressed a nontoxic peptide that reacted with toxin B antibodies. The rest of the toxin B gene was located on the 6.8 kb cloned fragment of plasmid pCD19L. The two fragments overlapped 0.8 kb. Lysates containing protein expressed by the 6.8 fragment were cytotoxic and lethal, and were neutralized by toxin B antibody. The two fragments were ligated to give the complete toxin B gene. The protein expressed by the complete gene was cytotoxic and lethal, and showed complete immunological identity with toxin B. Further analysis of the expressed protein and the toxin B gene confirmed our earlier findings showing that toxin B has a molecular weight of 240,000 or greater.

Nonspecific binding of mouse monoclonal antibodies toClostridium difficile toxins A and B

Several recent reports have described large numbers of monoclonal antibodies that cross-react with toxins A and B ofClostridium difficile; this suggests that the toxins share major epitopes. Our results show that monoclonal antibodies (MAb) against other antigens bind nonspecifically to both toxins. Therefore, we believe that the cross-reacting MAb bind by this manner and not by a true immune reaction.

Molecular Biology of the Clostridium difficile Toxins

Publisher Summary It causes a less severe, but more common, form of the disease termed antibiotic associated diarrhea (ADD). Clostridium difficile is now recognized as the primary cause of ADD in the United States. Toxins are the major virulence factors in C. difficile disease. Protection against toxins A and B are consistently found in fecal specimens from patients and experimental animals with the disease. Effective vaccination of experimental animals against the disease requires protection against both toxins. Most of the research on C. difficile disease has focused on purification and characterization of the molecular and biological properties of toxins A and B. Protein fractions attributed to purified toxin erroneously showed lower molecule weight proteins when analyzed by gel electrophoresis. Cloning, sequencing and expression of the toxin genes in Escherichia coli established that toxins A and B are the largest known bacterial toxins and are each composed of a single polypeptide. The toxins covalently modify low molecular weight guanine triphosphate (GTP)-binding proteins that regulate the actin microfilaments. A number of other clostridial enzymes modify the Rho family of proteins by adenosine diphosphate(ADP)-ribosylation.

Characteristics of the Toxins of Clostridium Difficile

The production of toxin by Clostridium difficile was first described by Hall and O’Toole (1935) who noted that subcutaneous injections of cell-free filtrates into guinea pigs caused edema, respiratory arrest, and death. These observations were confirmed two years later by Snyder (1937). Almost 40 years later, Hafiz and Oakley (1976) reported that 5 of 30 strains of C. difficile which they isolated produced very small amounts of toxin that was lethal and necrotizing. They did not study the toxin further, in part because their strains produced only 4 mouse lethal doses per ml. Then, in the late 1970’s, the results from a series of studies implicated C. difficile as the causative agent of antibiotic-associated colitis and these findings resulted in the initiation of a number of studies on the toxins of the organism (Bartlett et al. 1977a, 1978 a, c, d; Browne et al. 1977a; Larson et al. 1977, 1978; Larson and Price, 1977; Rifkin et al. 1977, 1978a; Chang et al. 1978a, b; George et al. 1978a, c; Katz et al. 1978; Allo et al. 1979; Willey and Bartlett, 1979; Rehg, 1980).