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Biomedical subjects

D M Lyerly

Publications and source records attributed to D M Lyerly.

At least 37 records · Page 2Linked to original sources
PubMed1995

The enterotoxin of Bacteroides fragilis is a metalloprotease.

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), approximately 20,000). 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 Zn2+ 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 degrees 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.

Actins↗
PubMed1995

Proteolytic activity of the Bacteroides fragilis enterotoxin causes fluid secretion and intestinal damage in vivo.

Strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in farm animals and humans during the past decade. Our laboratory has purified and characterized this enterotoxin as a single polypeptide (M(r), approximately 20,000). Recently, we used PCR to clone and sequence the enterotoxin gene from B. fragilis and showed that it exhibits significant homology with extracellular metalloproteases. Further studies showed that the purified enterotoxin has protease activity. To further characterize the role of this enterotoxin in diarrheal disease, we studied the histological and pathological effects of highly purified B. fragilis enterotoxin in lamb, rabbit, and rat ligated intestinal loops. When the enterotoxin was injected into ligated ileal and colonic loops, there was significant tissue damage and subsequent fluid accumulation. The fluid response in the ileum was greater in lambs than in rabbits and rats, whereas the fluid response in the colon was greater in rabbits than in lambs and rats. Analysis of the intestinal fluid elicited by the enterotoxin revealed an accumulation of chloride and sodium as well as albumin and total protein. Histological examination revealed mild necrosis of epithelial cells, crypt elongation, villus attenuation, and hyperplasia. There was extensive detachment and rounding of surface epithelial cells and an infiltration of neutrophils. Enterotoxic activity was inhibited by the metal chelators EDTA and 1,10-phenanthroline; to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to the chelated enterotoxin. Our results indicate that the enterotoxin elicits a significant fluid response subsequent to tissue damage in the small and large intestine. These data further support the idea that this enterotoxin is an important virulence factor in B. fragilis-associated diarrhea.

Animals↗
PubMed1995

Evaluation of formalin-inactivated Clostridium difficile vaccines administered by parenteral and mucosal routes of immunization in hamsters.

Clostridium difficile produces toxins that cause inflammation, necrosis, and fluid in the intestine and is the most important cause of nosocomial antibiotic-associated diarrhea and colitis. We evaluated C. difficile antigens as vaccines to protect against systemic and intestinal disease in a hamster model of clindamycin colitis. Formalin-inactivated culture filtrates from a highly toxigenic strain were administered by mucosal routes (intranasal, intragastric, and rectal) with cholera toxin as a mucosal adjuvant. A preparation of culture filtrate and killed whole cells was also tested rectally. The toxoid was also tested parenterally (subcutaneously and intraperitoneally) and by a combination of three intranasal immunizations followed by a combined intranasal-intraperitoneal boost. Serum antibodies against toxins A and B and whole-cell antigen were measured by enzyme-linked immunosorbent assay, neutralization of cytotoxic activity, and bacterial agglutination. The two rectal immunization regimens induced low antibody responses and protected only 20% of hamsters against death and 0% against diarrhea. The intragastric regimen induced high antibody responses but low protection, 40% against death and 0% against diarrhea. Hamsters immunized by the intranasal, intraperitoneal, and subcutaneous routes were 100% protected against death and partially protected (40, 40, and 20%, respectively) against diarrhea. Among the latter groups, intraperitoneally immunized animals had the highest serum anticytotoxic activity and the highest agglutinating antibody responses. Hamsters immunized intranasally and revaccinated intraperitoneally were 100% protected against both death and diarrhea. Protection against death and diarrhea correlated with antibody responses to all antigens tested. The results indicate that optimal protection against C. difficile disease can be achieved with combined parenteral and mucosal immunization.

Administration, Intranasal↗
PubMed1994

Mutagenesis of the Clostridium difficile toxin B gene and effect on cytotoxic activity.

Toxins A and B of Clostridium difficile are large cytotoxic proteins that share several unusual structural features, including four conserved cysteines, a potential nucleotide binding site, a hydrophobic region, and a series of contiguous repeating units at the carboxyl terminus. In the following study, we developed a series of toxin B mutants with altered properties in each of these features and examined the effect of the mutation on cytotoxic activity. Altering conserved cysteines to serine resulted in a 90% reduction in activity, whereas altering a histidine residue located in the potential nucleotide binding site to glutamine resulted in a 99% reduction. Removing the repeating units lowered the activity by 90% whereas removing the repeating units plus a conserved cysteine located just upstream of the units reduced the activity by more than five logs, resulting in an inactive toxin. Deleting the internal hydrophobic region had a similar effect. Our findings demonstrate that these conserved features appear to be important for expression of cytotoxic activity.

Amino Acid Sequence↗
PubMed1994

Production of antisera against the enterotoxin of Bacteroides fragilis and their use in a cytotoxicity neutralization assay of HT-29 cells.

To study the enterotoxin of Bacteroides fragilis, the colon carcinoma cell line HT-29 was used in a standard cytotoxicity assay. We produced high-titer neutralizing antisera in rabbits and goats against both crude and purified toxin and developed a cytotoxicity neutralization assay for use in confirming enterotoxin activity in culture filtrates and stools. The neutralization titers of the antisera on the colon carcinoma cell line HT-29 ranged from 1,600 to 2,400. In an antibody screening enzyme-linked immunosorbent assay, titers ranged from 10(4) to 10(5). The antisera produced against the highly purified toxin also neutralized the enterotoxic activity of the toxin and were monospecific by immunoelectrophoresis.

Animals↗
PubMed1994

Characterization of enterotoxigenic Bacteroides fragilis by a toxin-specific enzyme-linked immunosorbent assay.

Within the past decade, certain strains of Bacteroides fragilis have been associated with diarrhea in humans and cytotoxic activity on certain colon carcinoma cell lines. An enzyme-linked immunosorbent assay (ELISA) for detecting the enterotoxin of B. fragilis in cultures and stools was developed by using high-titer monospecific goat and rabbit antitoxins in an indirect format. The lower limit of detection for purified toxin was approximately 0.05 micrograms/ml; the linear range was from 0.05 to 10 microgram/ml. Using the ELISA to screen cultures of toxigenic and nontoxigenic strains of B. fragilis, we observed 100% correlation with 16 known toxigenic strains which had various cytotoxic activities on HT-29 cells. In addition, we found 6 of 62 previously untested strains also to be positive in both assays. Stability studies revealed that although the cytotoxic activities of crude and purified toxin preparations incubated at elevated temperatures were rapidly lost, the ELISA responses were not significantly reduced. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis showed that the purified toxin autodigested to several stable peptides. Studies on partially purified membranes from the toxigenic strains revealed the presence of several membrane-associated components which were noncytotoxic but strongly immunoreactive in the ELISA. Preliminary studies with spiked feces indicated that the ELISA may be useful for screening not only cultures for the enterotoxigenic B. fragilis but also stool specimens. Ongoing studies are focusing on determining the nature of the toxin's apparent proteolytic capabilities and investigating the feasibility of using the ELISA on stool specimens from healthy and diarrheic humans.

Animals↗
PubMed1994

Vascular and glomerular effects of Clostridium difficile toxin A peptide on the isolated rat kidney.

Toxin A peptide from Clostridium difficile caused damage and secretion in the intestinal mucosa. These effects are mediated in part by pro-inflammatory substances. In order to evaluate and compare the biologic effect of toxin A on renal vascular, glomerular and tubular functions, we studied this toxin in isolated rat kidneys. Isolated kidneys from adult male Wistar rats (260-320 g) were perfused with Krebs-Henseleit solution containing 60 mg/ml dialyzed bovine serum albumin. We studied the effect of toxin A peptide (3.2 x 10(-6) M, injected into perfusate) on glomerular filtration rate (GFR), urinary flow rate (UF) and total sodium reabsorption (TNa+, %). All experiments were preceded by a 30-min basal period, and in another group of kidneys the time course of the variables was followed without toxin infusion for unpaired control. Toxin A (TxA) reduced the perfusion pressure (PP), from PPcontrol/30min = 124.89 +/- 1.91 to PPTxA/120min = 88.13 +/- 5.1 mmHg (N = 6, P < 0.01) with a maximal effect at 120 min after toxin infusion. TxA also caused a significant decrease in GFR with maximal effect at 90 min after toxin infusion (GFRcontrol/30min = 0.53 +/- 0.05 to GFRTxA/90min = 0.30 + 0.05 ml min-1g-1; N = 6, P < 0.01). TxA did not alter renal tubular sodium transport when compared with a control without toxin infusion. In addition, toxin-treated kidneys caused a time-dependent increase in urinary flow from UFcontrol/30min = 0.16 +/- 0.08 to UFTxA/120min = 0.35 +/- 0.1 ml min-1g-1 (N = 6, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Purification and characterization of Clostridium difficile glutamate dehydrogenase.

Recombinant Clostridium difficile glutamate dehydrogenase (L-glutamate:NAD oxidoreductase, EC 1.4.1.2) was purified 177-fold to electrophoretic homogeneity with a 62% recovery through a four-step procedure involving gel filtration and ion-exchange and dye affinity chromatography. The approximate molecular weights of the native enzyme by gel filtration and subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were consistent with a hexameric structure for the purified enzyme. The enzyme-catalyzed glutamate oxidation was an NAD-dependent sequential process in which NADP could not be substituted as coenzyme. Several dinucleotide analogs of NAD structurally altered in either the pyridine or the purine moiety were observed to function as coenzymes when substituted for NAD. Nicotinamide mononucleotide did not serve as a coenzyme for glutamate oxidation. Product inhibition by NADH was competitive with respect to NAD. In deadend inhibition studies, adenosine diphosphoribose was shown to be an effective coenzyme-competitive inhibitor.

Chromatography, Affinity↗
PubMed1993

Production of monoclonal antibodies against Clostridium difficile cytotoxin using immunosorbent binding bioassay procedure.

In the following study, a novel screening approach was used to develop monoclonal antibodies specific for toxin B of Clostridium difficile. The approach, which consisted of an immunosorbent binding bioassay (ISBBA), is based on antigen immunocapture by monoclonal antibodies and detection of biological activity. Our results showed ISBBA, which uses unpurified antigen, to be more sensitive than the neutralization assay and ELISA for the detection of toxin B antibody.

Animals↗
PubMed1993

Purification and characterization of alpha-toxin produced by Clostridium novyi type A.

Our study describes the production, purification, and properties of alpha-toxin from Clostridium novyi type A 19402. The bacterium produced maximal amounts of alpha-toxin when grown at 37 degrees C for 72 h in dialysis flask cultures containing brain heart infusion supplemented with 0.75% Tween 80 and 2% glycerol. The alpha-toxin was purified by precipitation with polyethylene glycol 6000, followed by chromatography on Q-Sepharose, phenyl-agarose, and Mono-Q. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the toxin exhibited a single band with an M(r) of 200,000. The toxin also exhibited a single immunoprecipitin arc by crossed immunoelectrophoresis with antiserum against crude toxin. It was stable when stored at 4 degrees C and also following exposure to buffers with pHs in the range of 4 to 7. The toxin had a minimum lethal dose in mice of 5 to 10 ng, caused rounding of a variety of cells in tissue culture, and was negative in the rabbit ileal loop assay. The cytotoxic activity was inhibited by agents that affect receptor-mediated processes, and the toxin was less active on a CHO mutant cell line that is defective in endosomal acidification. The analysis of the amino acid composition revealed an unusually high proline content. The N-terminal sequence is Met-Leu-Ile-Thr-Arg-Glu-Gln-Leu-Met-Lys.

Amino Acid Sequence↗
PubMed1992

Cytotoxicity of Clostridium difficile toxin A for human colonic and pancreatic carcinoma cell lines.

The use of bacterial exotoxins may constitute novel adjuncts to treatment of gastrointestinal tract malignancies. Clostridium difficile toxin A was evaluated for its cytotoxic effect in vitro on 24 human cell lines and strains including carcinomas of the colon, pancreas, prostate, lung, breast, and lymphoid malignancies, as well as nonmalignant tissues. All nine colon and five pancreas cell lines were extraordinarily sensitive to the cytotoxic effect of Clostridium difficile toxin A at very low concentrations. This effect, which occurred rapidly and was dose dependent, was observed in all cells of seven colon and two pancreas cell lines at concentrations as low as 1-5 ng/ml (10(-12) to 10(-11) M), whereas cells derived from other sites required 60 to greater than 500 ng/ml to achieve an equivalent effect. The data suggest that Clostridium difficile toxin A may have potential therapeutic value in the treatment of some gastrointestinal tract cancers.

Bacterial Toxins↗
PubMed1992

Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile.

This study was undertaken to examine toxin production by Clostridium difficile 8864, a naturally occurring isolate that has been reported to produce toxin B in the absence of toxin A. To date, this is the only strain of C. difficile reported to produce only one of the toxins. The results of our initial studies with antibodies against toxins A and B confirmed these observations. Toxin B from strain 8864 and from VPI strain 10463, a strain that produces high levels of both toxin A and toxin B, was purified to homogeneity by sequential anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Ultrogel AcA22, and immunoadsorption chromatography, and their toxic activities were compared. Our results showed that toxin B from strain 8864 and toxin B from C. difficile VPI strain 10463 were comparable in their cytotoxic activities and that the 8864 toxin B was more lethal. In addition, we observed that toxin B from strain 8864 was weakly enterotoxic, which may explain the ability of this strain to cause intestinal disease in hamsters treated with antibiotics. Analysis with specific antibodies showed that the toxin B molecules from these strains were highly related but contained distinct epitopes. The results of hybridization studies with probes specific for the toxin B gene of VPI strain 10463 demonstrated differences between the toxin B genes of the two strains. In addition, probes specific for the toxin A gene of VPI strain 10463 showed that strain 8864 contains a region which shows identity with the 5' end of the toxin A gene but not the region of the gene which encodes a hydrophobic region and the repeating units.

Bacterial Proteins↗
PubMed1992

Purification and characterization of an enterotoxin from Bacteroides fragilis.

An enterotoxin produced by Bacteroides fragilis was purified to homogeneity and characterized as to its biological activity and basic molecular properties. Toxin preparations were prepared by growing B. fragilis VPI 13784 in brain heart infusion broth to early stationary phase, immediately precipitating the culture supernatant fluid with 70% ammonium sulfate, and stabilizing the precipitate with the protease inhibitor TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone). The toxin was sequentially purified by anion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on phenyl-agarose, and high-resolution ion-exchange chromatography on Mono Q. The toxin appeared homogeneous as judged by polyacrylamide gel electrophoresis. The estimated molecular weight of the highly purified toxin as determined by gel filtration chromatography on Superose-12 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 19,000. It has an isoelectric point of approximately 4.5 and is stable at pHs 5 to 10. The purified toxin is stable at -20 and 4 degrees C and upon freeze-drying, but it is unstable at temperatures above 55 degrees C. It is sensitive to proteinase K and Streptomyces protease but is resistant to trypsin and chymotrypsin. The activity of the purified toxin is neutralized by antiserum to a toxigenic strain of B. fragilis but not by antiserum to nontoxigenic strains. N-terminal amino acid analysis reveal an unambiguous sequence of Ala-Val-Pro-Ser-Glu-Pro-Lys-Thr-Val-Tyr-Val-Ile-Xxx-Leu-Arg-Glu-Asn-Gly- Ser-Thr . The highly purified toxin induced a strong fluid accumulation response in the lamb ileal-loop assay as well as a cytotoxic response (cell rounding) on HT-29 colon carcinoma cells. Thus, the purified toxin can cause both enterotoxic and cytotoxic activities.

Animals↗
PubMed1991

Passive immunization of hamsters against disease caused by Clostridium difficile by use of bovine immunoglobulin G concentrate.

Gestating Holstein cows were vaccinated with Clostridium difficile toxoid prepared from the culture filtrate of a strain that produces high levels of toxins A and B and other antigens. A bovine immunoglobulin G (IgG) concentrate was prepared from colostrum collected at parturition. The results of our studies showed that hamsters treated prophylactically with the hyperimmune bovine IgG concentrate were protected against C. difficile disease. These results suggest that orally administered hyperimmune bovine IgG specific for C. difficile culture filtrate may be useful in prophylaxis against C. difficile disease.

Animals↗
PubMed1991

Identification of the latex test-reactive protein of Clostridium difficile as glutamate dehydrogenase.

Computer analysis showed that the gene encoding the latex test-reactive protein of Clostridium difficile exhibited high levels of homology with glutamate dehydrogenases from various sources. Further analysis demonstrated that the recombinant protein possessed glutamate dehydrogenase activity. Our results show that the protein that reacts in commercial latex tests for C. difficile is a glutamate dehydrogenase.

Amino Acid Sequence↗