TxeR, a sigma factor that directs Clostridium difficile RNA polymerase to recognize the promoters of two major toxin genes, was shown to stimulate its own synthesis. Whether expressed in C. difficile, Clostridium perfringens, or Escherichia coli, TxeR stimulated transcription of fusions of the txeR promoter region to reporter genes. As is the case for the tox genes, txeR expression was responsive to the cellular growth phase and the constituents of the medium. That is, the level of expression in broth culture was low during the exponential growth phase, but rapidly increased as cells approached the stationary phase. In the presence of excess glucose, expression from the txeR promoter was repressed. The results support a model for toxin gene expression in which synthesis of TxeR is induced by specific environmental signals. The increased level of TxeR then permits high-level expression of the toxin genes.

During the past decade, strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in animals and humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (M r , ϳ20,000). Single specific primer-PCR was used to clone a portion of the B. fragilis enterotoxin gene. The recombinant protein expressed by the cloned gene fragment reacted with monospecific antibodies to B. fragilis enterotoxin by enzyme-linked immunosorbent assay and immunoblot analysis. The deduced amino acid sequence revealed a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of metalloproteases termed metzincins. Sequence comparisons showed close identity to matrix metalloproteases (e.g., human fibroblast collagenase) within the zinc-binding and Met-turn region. Purified enterotoxin contained 1 g-atom of Zn 2؉ per molecule and hydrolyzed gelatin, azocoll, actin, tropomyosin, and fibrinogen. The enterotoxin also underwent autodigestion. The N-terminal amino acid sequences of two autodigestion products were identical to the deduced amino acid sequence of the recombinant enterotoxin and revealed cleavage at Cys-Leu and Ser-Leu peptide bonds. Gelatinase (type IV collagenase) activity comigrated with the toxin when analyzed by gel fractionation and zymography, indicating that protease activity is due to the enterotoxin and not to a contaminating protease(s). Optimal proteolytic activity occurred at 37؇C and pH 6.5. Primary proteolytic cleavage sites in actin were identified, revealing cleavage at Gly-Met and Thr-Leu peptide bonds. Enzymatic activity was inhibited by metal chelators but not by inhibitors of other classes of proteases. Additionally, cytotoxic activity of the enterotoxin on human carcinoma HT-29 cells was inhibited by acetoxymethyl ester EDTA. The metalloprotease activity of the enterotoxin suggests a possible mechanism for enterotoxicity and may have additional implications in the study of disease caused by B. fragilis.

EMBL accession no. X53138 Clostridium difficile, the causative agent of antibiotic-associated pseudomembranous colitis, produces two toxins, A and B (1).

No abstract

We have previously shown that vaccination with purified Entamoeba histolytica Gal/GalNAc lectin or recombinant subunits can protect mice from intestinal amebiasis upon intracecal challenge. In this study, we demonstrated with adoptive-transfer experiments that this lectin vaccine protection is mediated by T cells but not serum. The cell-mediated immune (CMI) response was characterized by significant gamma interferon (IFN-␥), interleukin 12 (IL-12), IL-2, IL-10, and IL-17 production. To move toward a human vaccine, we switched to a recombinant protein and tested a range of adjuvants and routes appropriate for humans. We found that subcutaneous delivery of LecA with IDRI's adjuvant system EM014 elicited a potent Th1-type CMI profile and provided significant protection, as measured by culture negativity (79% efficacy); intranasal immunization with cholera toxin provided 56% efficacy; and alum induced a Th2-type response that protected 62 to 68% of mice. Several antibody and CMI cytokine responses were examined for correlates of protection, and prechallenge IFN-␥ ؉ or IFN-␥-, IL-2-, and tumor necrosis factor alpha-triple-positive CD4 cells in blood were statistically associated with protection. To test the role of IFN-␥ in LecA-mediated protection, we neutralized IFN-␥ in LecA-immunized mice and found that it abrogated the protection conferred by vaccination. These data demonstrate that CMI is sufficient for vaccine protection from intestinal amebiasis and reveal an important role for IFN-␥, even in the setting of alum.The enteric protozoan parasite Entamoeba histolytica is the causative pathogen of amebic dysentery and liver abscess that affects millions of people worldwide. Bangladeshi children experience a 40% annual incidence of E. histolytica infection (24), and evidence of prior E. histolytica infection can be detected in 8.4% of the general population in Mexico (6). Despite the availability of effective antibiotics, the World Health Organization estimates that up to 100,000 deaths occur annually, highlighting the need for alternate approaches to control amebiasis. One approach is to develop a vaccine to prevent intestinal infection (26).Several vaccine candidates for amebiasis have been proposed (48), including the serine-rich E. histolytica protein, peroxiredoxin, the EhCP112 molecule, and the galactose/N-acetyl-Dgalactosamine-inhibitable lectin (Gal/GalNAc lectin). A significant body of work has focused on the latter: vaccination with either parasite-purified Gal/GalNAc lectin (10,29,32,38,40) or recombinant lectin subunits has provided protection in rodent models against amebic liver abscess and amebic colitis (29,37,46,47,53). Although these results are encouraging, two limitations remain. First, in most of these vaccine studies, the adjuvants and delivery routes are not compatible with eventual use in humans. Second, the mechanisms of amebiasis vaccine-mediated protection are still not fully understood. For instance, in the intestinal model, there was an association between the presence of an antiparasit...