Clostridium difficile is a relatively """"""""new"""""""" pathogen that causes colitis in some patients receiving antibiotic therapy. The organism and its toxins occur in about 15% of patients with diarrhea in hospitals and that is a lot of patients. If untreated some of these patients would progress to Pseudomembranous Colitis, which has a high mortality rate. The disease is commonly acquired in hospitals because the organism can only grow in the colon when the normal flora has been essentially eliminated by large doses of antibiotics. Hospitals provide an ideal environment for the spread of a spore-forming organism that causes diarrhea. The organism produces two potent toxins. Toxin A is an enterotoxin that may be responsible for the major symptoms of the disease. Toxin B is a very potent cytotoxin. We have shown that toxin A binds to a Galalpha1-3Galbeta1-4G1cNAc trisaccharide via multiple binding sites that occur on this huge toxin. The repeated binding structures will be analyzed in detail by the construction of synthetic peptides and by cloning the repeats into both E. coli and C. perfringens. We will determine whether this trisaccharide is the receptor that allows toxin A to initiate the enterotoxicity and whether this is the receptor to which the toxin binds in adult human colonic tissue. Adults may vary in the occurrence of this trisaccharide in their colons and this could account for differences in susceptibility to the toxins. We also will determine whether babies have this receptor; about half of the babies born in hospitals get colonized with this organism as """"""""normal flora"""""""" without apparent ill effects and we want to know why the toxins do not kill them. The trisaccharide is not degraded by human intestinal enzymes so we may be able to treat the disease by giving large amounts of the receptor attached to latex beads. We have found that a lectin that binds to this trisaccharide prevents the action of toxin A so this may be useful in therapy. A monoclonal antibody specific for this carbohydrate receptor also is available and will be tested. As the active site of the toxin is sequenced we will attempt to determine the mechanism of action by comparison to sequences of other toxins and enzymes. Such hints as to the mechanisms will be confirmed by collaborations with research groups working in the respective areas. As the sequence of toxin B becomes available we will analyze it in the same manner. We will continue our collaborative efforts on the mechanism of action of the toxins and continue to be a source of pure toxins and specific antibodies.
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