Cloning of Clostridium difficile toxin B gene and demonstration of high N-terminal homology between toxin A and B

High titered Clostridium sordellii lethal toxin antiserum, cross-reactive with C. difficile cytotoxin B (ToxB), was used to isolate toxB fragments from a C. difficile expression library. Recombinant clones containing toxB fragments of the 5 and 3 end were isolate. A 2.5-kb HincII fragment of chromosomal DNA overlaps both groups of clones. A partial restriction map of the total toxB gene is presented. The gene is positioned upstream of utxA and toxA toxB has a size of 6.9kb, corresponding to a 250-kDa polypeptide. A partial sequence of the 5 end of toxB was determined. The sequence contains 398 bp upstream of toxB with a putative Shine-Dalgarno box (AGGAGA) and 609 bp of the toxB open reading frame. The N-terminal 203 amino acids of ToxB were compared with the N-terminal amino acids of the enterotoxin A (ToxA). A homology of 64% of the residues was detected, which proves the relatedness of ToxA and ToxB of C. difficile.Abbreviations PBS phosphate-buffered saline - ToxA C. difficile enterotoxin A - ToxB C. difficile cytotoxin B - toxB gene encoding ToxB - HT C. sordellii hemorrhagic toxin - LT C. sordellii lethal toxin     

Foot Infection by Clostridium sordellii: Case Report and Review of 15 Cases in France

We report a case of foot infection by Clostridium sordellii and review 15 human infections registered at a Reference Center in France during the period 1998 to 2011. All strains were found nontoxigenic, lacking the lethal toxin gene coding for TcsL. Like Clostridium septicum, several C. sordellii infections were associated with intestinal neoplasms.     

Upregulation of the Host SLC11A1 Gene by Clostridium difficile Toxin B Facilitates Glucosylation of Rho GTPases and Enhances Toxin Lethality

Pseudomembranous enterocolitis associated with Clostridium difficile infection is an important cause of morbidity and mortality in patients being treated with antibiotics. Two closely related large protein toxins produced by C. difficile, TcdA and TcdB, which act identically but at different efficiencies to glucosylate low-molecular-weight Rho GTPases, underlie the microbe''s pathogenicity. Using antisense RNA encoded by a library of human expressed sequence tags (ESTs), we randomly inactivated host chromosomal genes in HeLa cells and isolated clones that survived exposure to ordinarily lethal doses of TcdB. This phenotypic screening and subsequent analysis identified solute carrier family 11 member 1 (SLC11A1; formerly NRAMP1), a divalent cation transporter crucial to host defense against certain microbes, as an enhancer of TcdB lethality. Whereas SLC11A1 normally is poorly expressed in human cells of nonmyeloid lineage, TcdB increased SLC11A1 mRNA abundance in such cells through the actions of the RNA-binding protein HuR. We show that short hairpin RNA (shRNA) directed against SLC11A1 reduced TcdB glucosylation of small Rho GTPases and, consequently, toxin lethality. Consistent with the previously known role of SLC11A1 in cation transport, these effects were enhanced by elevation of Mn²⁺ in media; conversely, they were decreased by treatment with a chelator of divalent cations. Our findings reveal an unsuspected role for SLC11A1 in determining C. difficile pathogenicity, demonstrate the novel ability of a bacterial toxin to increase its cytotoxicity, establish a mechanistic basis for these effects, and suggest a therapeutic approach to mitigate cell killing by C. difficile toxins A and B.     

Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile

A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A−B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A−B−, and A−B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A−B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A−B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections.     

New method to generate enzymatically deficient Clostridium difficile toxin B as an antigen for immunization

The family of the large clostridial cytotoxins, encompassing Clostridium difficile toxins A and B as well as the lethal and hemorrhagic toxins from Clostridium sordellii, monoglucosylate the Rho GTPases by transferring a glucose moiety from the cosubstrate UDP-glucose. Here we present a new detoxification procedure to block the enzyme activity by treatment with the reactive UDP-2', 3'-dialdehyde to result in alkylation of toxin A and B. Alkylation is likely to occur in the catalytic domain, because the native cosubstrate UDP-glucose completely protected the toxins from inactivation and the alkylated toxin competes with the native toxin at the cell receptor. Alkylated toxins are good antigens resulting in antibodies recognizing only the C-terminally located receptor binding domain, whereas formaldehyde treatment resulted in antibodies recognizing both the receptor binding domain and the catalytic domain, indicating that the catalytic domain is concealed under native conditions. Antibodies against the native catalytic domain (amino acids 1 through 546) and those holotoxin antibodies recognizing the catalytic domain inhibited enzyme activity. However, only antibodies against the receptor binding domain protected intact cells from the cytotoxic activity of toxin B, whereas antibodies against the catalytic domain were protective only when inside the cell.     

Presence ofClostridium difficile toxin in guinea pigs with penicillin-associated colitis

Cecal filtrates from guinea pigs treated with penicillin contained a toxin which produced cytotoxic changes in HeLa cell cultures and was lethal to guinea pigs when administered intracecally. The cytotoxicity could be neutralized byClostridium difficile andC.sordellii antitoxins,but not by other clostridial antitoxins. Rabbit immunization with toxic cecal extracts produced antibody which neutralized the cytotoxicity of guinea pig cecal extracts, of stool extracts from humans with antibiotic-associated colitis and of culture supernatant fluids ofC.difficile. Treatment with vancomycin reduced the number of deaths and increased the survival time of penicillin-treated animals. No cytotoxin was present in cecal extracts from these guinea pigs. Gram-negative bacteremia was present in half the penicillin-treated animals, the sick ones as well as the healthy ones. Treatment with vancomycin did not decrease the incidence of bacteremia. Gram-negative bacteremia and changes in fecal flora were observed in some antibiotic-treated guinea pigs; all diseased animals, however, contained this cytotoxin.C.difficile was isolated from cecal contents of sick animals and these isolates produced the cytotoxinin vitro. The results suggest thatC. difficile toxin can cause antibiotic-associated colitis in guinea pigs.     

Comparison of the premier toxin A and B assay and the TOX A/B II assay for diagnosis of Clostridium difficile infection

Clostridium difficile causes nosocomial diarrhea and is responsible for complications such as pseudomembranous colitis, megacolon, and perforation. Using 442 stool specimens, we compared the sensitivities and specificities of the Premier toxin A and B (Meridian Bioscience, Inc.) and C. difficile TOX A/B II (TechLab, Inc., Blacksburg, VA) immunoassays in the Virology Department of the Kaiser Permanente Regional Reference Laboratories. The Premier toxin A and B assay demonstrated a higher sensitivity (97.44%) and a higher positive predictive value (79.17%) than the C. difficile TOX A/B II assay (87.18% and 75.56%, respectively), while assay specificities and negative predictive values were similar. We also performed experiments using serially diluted, purified toxin A and B antigens to understand the basis for assay differences. The two assays’ toxin A antibodies detected toxin A at comparable levels. Preliminary results indicated that the toxin B antibody in the Premier toxin A and B assay could detect toxin B at a concentration of 125 pg/100 μl, while the toxin B antibody in the C. difficile TOX A/B II assay could not detect toxin B below a concentration of 250 pg/100 μl. Therefore, the Premier toxin A and B assay provides greater sensitivity than the C. difficile TOX A/B II assay, perhaps due to a superior detection ability of its toxin B antibody.     

Expression of the large clostridial toxins is controlled by conserved regulatory mechanisms

The clostridia cause many human and animal diseases, resulting in significant morbidity and mortality. Host damage results from the action of potent exotoxins, an important group of which is the large clostridial toxins (LCTs) produced by Clostridium difficile, Clostridium sordellii, Clostridium perfringens and Clostridium novyi. Knowledge of the structure and function of these toxins has been attained, however, apart from C. difficile, the regulatory pathways that control LCT production remain largely unknown. Here we show that LCT production in C. sordellii and C. perfringens is temporally regulated and repressed by glucose in a similar manner to C. difficile. Furthermore, we show that the TpeL-encoding gene of C. perfringens is located in an uncharacterized Pathogenicity Locus (PaLoc), along with accessory genes predicted to encode a bacteriophage holin-type protein and a TcdR-family alternative sigma factor, TpeR. Inactivation of tpeR demonstrated that TpeR is critical for C. perfringens TpeL production, in a similar manner to C. difficile TcdR and C. sordellii TcsR, but cross-complementation showed that TpeR is not functionally interchangeable with TcdR or TcsR. Although conserved mechanisms are employed by the clostridia to control LCT production there are important functional differences that distinguish members of the TcdR-family of clostridial alternative sigma factors.     

The N-Terminal Part of the Enzyme Component (C2I) of the Binary Clostridium botulinum C2 Toxin Interacts with the Binding Component C2II and Functions as a Carrier System for a Rho ADP-Ribosylating C3-Like Fusion Toxin

The binary actin–ADP-ribosylating Clostridium botulinum C2 toxin consists of the enzyme component C2I and the binding component C2II, which are separate proteins. The active component C2I enters cells through C2II by receptor-mediated endocytosis and membrane translocation. The N-terminal part of C2I (C2IN), which consists of 225 amino acid residues but lacks ADP-ribosyltransferase activity, was identified as the C2II contact site. A fusion protein (C2IN-C3) of C2IN and the full-length C3-like ADP-ribosyltransferase from Clostridium limosum was constructed. The fusion protein C2IN-C3 ADP-ribosylated Rho but not actin in CHO cell lysates. Together with C2II, C2IN-C3 induced complete rounding up of CHO and HeLa cells after incubation for 3 h. No cell rounding was observed without C2II or with the original C3-like transferase from C. limosum. The data indicate that the N-terminal 225 amino acid residues of C2I are sufficient to cause the cellular uptake of C. limosum transferase via the binding component of C2II, thereby increasing the cytotoxicity of the C3-like exoenzyme several hundred-fold.     

Role of fecal Clostridium difficile load in discrepancies between toxin tests and PCR: is quantitation the next step in C. difficile testing?

Direct tests for Clostridium difficile are 30–50?% more sensitive than tests for C. difficile toxins but the reasons for this discrepancy are incompletely understood. In addition to toxin degradation and strain differences, we hypothesized that C. difficile concentration could be important in determining whether toxins are detected in fecal samples. We performed standard curves on an FDA-approved real-time PCR test for the C. difficile tcdB gene (Xpert C. difficile/Epi, Cepheid) during a prospective comparison of a toxin immunoassay (Meridian Premier), PCR and toxigenic culture. Immunoassay-negative, PCR-positive samples were retested with a cell cytotoxin assay (TechLab). Among 107 PCR-positive samples, 46 (43.0?%) had toxins detected by immunoassay and an additional 18 (16.8?%) had toxin detected by the cytotoxin assay yielding 64 (59.8?%) toxin-positive and 43 (40.2?%) toxin-negative samples. Overall, toxin-negative samples with C. difficile had 10¹–10⁴ fewer DNA copies than toxin-positive samples and most discrepancies between toxin tests and PCR were associated with a significant difference in C. difficile quantity. Of the toxin-positive samples, 95?% had ≥4.1 log₁₀ C. difficile tcdB DNA copies/mL; 52?% of immunoassay-negative samples and 70?% of immunoassay and cytotoxin negative samples had <4.1 log₁₀ C. difficile tcdB DNA copies/mL. These findings suggest that fecal C. difficile concentration is a major determinant of toxin detection and C. difficile quantitation may add to the diagnostic value of existing test methods. Future studies are needed to validate the utility of quantitation and determine the significance of low concentrations of C. difficile in the absence of detectable toxin.     

Identification of functional antigenic segments of toxic shock syndrome toxin 1 by differential immunoreactivity and by differential mitogenic responses of human peripheral blood mononuclear cells, using active toxin fragments.

When toxic shock syndrome toxin 1 was subjected to papain hydrolysis, two serologically active fragments of 16.3 kilodaltons (16K fragment) and 12.4 kilodaltons (12K fragment) were generated, whereas a third fragment of 9.7 kilodaltons (10K fragment) was inactive. The biologic activities of the fragments were evaluated in vitro by determining their ability to promote nonspecific proliferation of human peripheral blood mononuclear cells. The 12K fragment was significantly (P less than or equal to 0.013) more stimulatory than the 16K fragment. When human peripheral blood mononuclear cells were preincubated for a period of 24 h with various concentrations of the 16K fragment, followed by incubation with a constant amount (2 x 10(-2) ng/ml) of whole toxin, the level of DNA synthesis induced by the holotoxin was reduced by approximately 60% when compared with that of controls exposed to whole toxin alone. The 12K fragment did not demonstrate a similar blocking effect. Immunoblots of the toxic shock syndrome toxin 1 digest, which were exposed to monoclonal antibodies (MAbs) developed against native toxin, depicted the presence of two different antigenic regions (epitopes). One MAb, 8-5-7, which has been shown previously to inhibit the biologic activity of the holotoxin in vitro and in vivo, reacted primarily with the 12K fragment. A second MAb, 10-6-1, that did not neutralize interleukin-1 production reacted primarily with the 16K fragment. On the basis of the differential mitogenic responses and the identification of heterologous epitopes, it was concluded that the functional region of the holotoxin can be partitioned into at least two functional segments encompassed between amino acid residues 53 and 87 and between amino acid residues 88 and 194 on the polypeptide chain.     

Comparison of enzyme immunoassays and rapid diagnostic tests for <Emphasis Type="Italic">clostridium difficile</Emphasis> glutamate dehydrogenase and toxin a + B to toxinogenic culture on a highly selective chromogenic medium

To compare Clostridium. (C.) difficile toxin A/B and glutamate dehydrogenase (GDH) enzyme immunoassays or rapid diagnostic tests to toxinogenic culture on recently described highly selective agar plates. Five hundred consecutive samples sent in for C. difficile diagnostics were tested by toxin A/B enzyme immunoassay (EIA) and rapid diagnostic test (RDT), GDH EIA and RDT, and culture on chromID C. difficile plates for 48 hrs, with toxin testing from culture if the toxin EIA from feces was negative. Samples with discordant results from EIA and RDT were submitted to C. difficile-specific 16S rRNA gene and tcdB PCR. Ninety-two, 88, 31, and 37 samples were positive by GDH EIA, GDH RDT, toxin A/B EIA, and toxin A/B RDT respectively. Seventy-four samples were positive by culture, 54 culture-positive samples were subjected to repeat toxin testing, with an additional 29 samples positive. Thus, there were 60 C. difficile toxin A/B positive samples in total (12 %). Single-step screening with GDH EIA, GDH RDT, toxin A/B EIA, and toxin A/B RDT would have missed seven (12 %), 11 (18 %), 29 (48 %) or 27 (45 %) of all positive samples respectively. Single-step screening with GDH or toxin A/B tests from feces misses a significant proportion of patients compared to toxinogenic culture, putting these patients at risk from undiagnosed C. difficile infection. More data are needed to establish the clinical significance of a positive toxinogenic culture result in the absence of detectable toxin A/B in feces.     

Enzyme immunoassay for detection ofClostridium difficile toxins a and b in patients with antibiotic-associated diarrhoea and colitis

A sandwich enzyme immunoassay (ELISA) was developed to detectClostridium difficile toxins A and B in stools from patients with antibiotic associated diarrhoea and colitis. Immune serum to crudeClostridium difficile toxin and non-immune serum were coated onto polystyrene microtiter plates to act as capture antibodies; toxins A and B in human stools were detected by antibodies from rabbits immunized with purified toxins A and B. The ELISA for toxin B showed cross-reactions withClostridium bifermentans andClostridium sordellii and lacked diagnostic sensitivity in clinical samples. The ELISA for toxin A showed no cross-reactions with other clostridiae investigated and was positive in 33 % (62/189) of patients with antibiotic associated diarrhoea. This compared with 38 % (71/189) positive in the tissue culture assay forClostridium difficile cytotoxin. With a predictive value of 96 % in clinical specimens, the ELISA for toxin A constitutes a sensitive and specific tool for diagnosis ofClostridium difficile associated diarrhoea and colitis.     

Agents That Inhibit Rho, Rac, and Cdc42 Do Not Block Formation of Actin Pedestals in HeLa Cells Infected with Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including Clostridium difficile toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.     

Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (V_HH) library raised against TcdB, we identified two V_HH antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD₅₀) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.     

Neutralization of hemolytic and mouse lethal activities ofC. perfringensalpha-toxin need simultaneous blockade of two epitopes by monoclonal antibodies

Three murine monoclonal antibodies (MAbs 3B4, 1E8, 1F9) were produced by fusion of X63-Ag8.653 myeloma cells and splenocytes of mice immunized with glutaraldehyde-inactivated alpha-toxin ofClostridium perfringens.All MAbs belonged to the immunoglobulin G (IgG) class and possessed a kappa light chain. All the MAbs were specific for alpha-toxin ofC. perfringensas demonstrated by immunoblotting experiments performed with culture supernatants ofC. perfringens,C. bifermentans,C. sordellii, andBacillus cereus.Competition analysis in an ELISA revealed that the MAbs recognized different epitopes on the alpha-toxin molecule. In an immunoblot assay based on a recombinant protein expressed inEscherichia coli, the binding site of MAb 1E8 but not those of MAbs 3B4 and 1F9 were mapped to the COOH-terminal fragment of alpha-toxin (aa 248–370). To prove the neutralizing potential of the MAbs, alpha-toxin was preincubated with MAbs and subsequently tested for its lecithinase activity in an egg yolk diffusion turbidity (EYDT) assay, its hemolytic activity in a hemolysis test, and its lethal effect on mice after intraperitoneally administration. When the MAbs were tested individually, neutralization was only seen in the EYDT assay, where MAb 3B4 completely abolished the lecithinase activity of alpha toxin. However, when MAbs 3B4 and 1E8 were used in combination, they acted synergistically and inhibited the lysis of rabbit erythrocytesin vitro.The same mixture of MAbs was also able to completely neutralize the lethal effect of three LD₅₀of alpha-toxin on Balb/c mice. Our results suggest that the alpha-toxin molecule contains several domains which are differently involved in the various activities of the toxin. We conclude that the hemolytic domain(s) of alpha-toxin is (are) identical with or very closely located to the domain(s) that cause the mouse lethal effect. The lecithinase activity may be involved in the mechanisms of hemolysis and mouse lethality but appears not to be the only determinant.     

Protective Efficacy Induced by Recombinant Clostridium difficile Toxin Fragments

Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.     

Sensitive and Selective Culture Medium for Detection of Environmental Clostridium difficile Isolates without Requirement for Anaerobic Culture Conditions

Effective and easy-to-use methods for detecting Clostridium difficile spore contamination would be useful for identifying environmental reservoirs and monitoring the effectiveness of room disinfection. Culture-based detection methods are sensitive for detecting C. difficile, but their utility is limited due to the requirement of anaerobic culture conditions and microbiological expertise. We developed a low-cost selective broth medium containing thioglycolic acid and l-cystine, termed C. difficile brucella broth with thioglycolic acid and l-cystine (CDBB-TC), for the detection of C. difficile from environmental specimens under aerobic culture conditions. The sensitivity and specificity of CDBB-TC (under aerobic culture conditions) were compared to those of CDBB (under anaerobic culture conditions) for the recovery of C. difficile from swabs collected from hospital room surfaces. CDBB-TC was significantly more sensitive than CDBB for recovering environmental C. difficile (36/41 [88%] versus 21/41 [51%], respectively; P = 0.006). C. difficile latex agglutination, an enzyme immunoassay for toxins A and B or glutamate dehydrogenase, and a PCR for toxin B genes were all effective as confirmatory tests. For 477 total environmental cultures, the specificity of CDBB-TC versus that of CDBB based upon false-positive yellow-color development of the medium without recovery of C. difficile was 100% (0 false-positive results) versus 96% (18 false-positive results), respectively. False-positive cultures for CDBB were attributable to the growth of anaerobic non-C. difficile organisms that did not grow in CDBB-TC. Our results suggest that CDBB-TC provides a sensitive and selective medium for the recovery of C. difficile organisms from environmental samples, without the need for anaerobic culture conditions.     

Identification of Functional Domains of Bordetella Dermonecrotizing Toxin

Bordetella dermonecrotizing toxin (DNT) stimulates the assembly of actin stress fibers and focal adhesions by deamidating Gln63 of the small GTPase Rho. To clarify the functional and structural organization of DNT, we cloned and sequenced the DNT gene and examined the functions of various DNT mutants. Our analyses of the nucleotide and amino acid sequences revealed that the start codon of the DNT gene is a GTG triplet located 39 bp upstream of the reported putative initiation ATG codon; consequently, DNT contains an additional 13 amino acids at its N-terminal end. All of the N-terminally truncated mutants were found to modify Rho. The shortest fragment of DNT possessing the Rho modification activity consists of amino acids from Ile1176 to the C-terminal end. This fragment overlaps the region homologous to Escherichia coli cytotoxic necrotizing factors (CNFs), which show activity similar to that of DNT. The introduction of a mutation at Cys1305 located in the highly conserved region between CNFs and DNT eliminated the activity, indicating that this domain is the catalytic center of DNT. The N-terminal fragment (1 to 531) of DNT failed to modify Rho but reduced the DNT-induced polynucleation in MC3T3-E1 cells when simultaneously added with the holotoxin, suggesting competitive inhibition in the receptor-binding or internalizing step. Our finding that DNT consists of an N-terminal receptor-binding and/or internalizing domain and a C-terminal catalytically active domain may facilitate analysis of the overall action of the toxin on the mammalian target cells.     

Antibodies to Recombinant Clostridium difficile Toxins A and B Are an Effective Treatment and Prevent Relapse of C. difficile-Associated Disease in a Hamster Model of Infection

Clostridium difficile causes antibiotic-associated diarrhea and colitis in humans through the actions of toxin A and toxin B on the colonic mucosa. At present, broad-spectrum antibiotic drugs are used to treat this disease, and patients suffer from high relapse rates after termination of treatment. This study examined the role of both toxins in pathogenesis and the ability of orally administered avian antibodies against recombinant epitopes of toxin A and toxin B to treat C. difficile-associated disease (CDAD). DNA fragments representing the entire gene of each toxin were cloned, expressed, and affinity purified. Hens were immunized with these purified recombinant-protein fragments of toxin A and toxin B. Toxin-neutralizing antibodies fractionated from egg yolks were evaluated by a toxin neutralization assay in Syrian hamsters. The carboxy-terminal region of each toxin was most effective in generating toxin-neutralizing antibodies. With a hamster infection model, antibodies to both toxins A and B (CDAD antitoxin) were required to prevent morbidity and mortality from infection. In contrast to vancomycin, CDAD antitoxin prevented relapse and subsequent C. difficile reinfection in the hamsters. These results indicate that CDAD antitoxin may be effective in the treatment and management of CDAD in humans.