Browsing by Author "Wilkins, Tracy D."

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    Activation of acetate in acetate-grown Methanosarcina thermophila: purificationm and characterization of acetate kinase
    Aceti, David John (Virginia Tech, 1980-04-05)
    Extracts of acetate-grown Methanosarcina thermoghila were assayed for the presence of enzymes which might catalyze a proposed activation of acetate as the initial step in the pathway of methanogenesis from acetate by that organism. Acetate kinase and phosphate acetyltransferase activities of 4.9 and 49 μmoles of product/min/mg protein, respectively, were detected. Acetate kinase was purified 102- fold to a specific activity of 656 μmoles ADP formed/min/mg protein and was essentially homogeneous by denaturing gel electrophoresis. The native enzyme (Mr 94,000) was an α₂ homodimer with a subunit Mr of 53,000. Activity was optimal between pH 7.0 and 7.4 and was stable to heating at 70°C for 15 min. The apparent Km for acetate was 22 mM (Vmax = 668 μmoles ADP/min/mg protein) and 2.8 mM for ATP (Vmax = 777 pmoles ADP/min/ protein). The enzyme phosphorylated propionate at 602 of the rate with acetate but was unable to use formate. TTP, ITP, UTP, GTP, and CTP replaced ATP as the phosphoryl donor to acetate. One of several divalent cations was required for activity; the maximum rate was obtained with Mn2+.
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    Anaerobic microbe transport assembly and method of using
    (United States Patent and Trademark Office, 1976-02-17)
    Disclosure is made of a novel assembly for transporting anaerobic microbes from clinical patient to laboratory. The assembly provides a convenient and reliable means of maintaining microbial viability in the critical period between collection and deposition in a culturing environment.
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    Antibody for C.difficile
    (United States Patent and Trademark Office, 1989-11-07)
    Monoclonal antibody to toxin A of C.difficile has been prepared and is used in an assay for toxin A of C.difficile.
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    A biochemical and physiological characterization of coenzyme F420-reducing hydrogenase from Methanobacterium formicicum
    Baron, Stephen Francis (Virginia Polytechnic Institute and State University, 1988)
    The coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme formed aggregates (1,000 kd) of a coenzyme F₄₂₀-active monomer (109 kd) composed of 1 each of a, β, and γ subunits (43.6, 36.7, xy and 28.8 kd, respectively). It contained 1 mol of FAD, 1 mol of nickel, 12-14 mols of iron, and 11 mols of acid-labile sulfide per mol of the 109 kd species, but no selenium. The amino acid sequence I---P--R-EGH-----EV was conserved in the N-terminus of a subunit of the enzyme and the largest subunits of nickel-containing hydrogenases from Methanobacterium thermoautotrophicum, Desulfovibrio baculatus, and Desulfovibrio gigag. FAD dissociated from the coenzyme F42O-reducing hydrogenase during reactivation with H2 and coenzyme F₄₂₀, unless KCl was present, yielding coenzyme F₄₂₀-inactive apoenzyme. The hydrogenase catalyzed H₂ production at a rate 3-fold less than that for H2 uptake. Specific antiserum inhibited the coenzyme F₄₂₀ dependent activity but not the methyl viologen-dependent activity of the purified enzyme. Cell extract of M. formicicum contained a coenzyme F₄₂₀-mediated formate hydrogenlyase system. Formate hydrogenlyase activity was reconstituted with coenzyme F₄₂₀-reducing hydrogenase, coenzyme F₄₂₀-reducing formate dehydrogenase, and coenzyme F₄₂₀, all purified from M. formicicum. The reconstituted system required FAD for maximal activity (kinetic Kd= 4 μM). without FAD, the formate dehydrogenase and hydrogenase rapidly lost coenzyme F₄₂₀-dependent activity relative to methyl viologen-dependent activity. Immunoadsorption of the formate dehydrogenase or hydrogenase from cell extract greatly reduced formate hydrogenlyase activity; addition of the purified enzymes restored activity. Formate hydrogenlyase activity of cell extract and the reconstituted system was reversible. The coenzyme F₄₂₀-reducing hydrogenase and formate dehydrogenase of M. formicicum were shown to be located at the cytoplasmic membrane using immunogold labeling of thin sectioned, Lowicryl-embedded cells. Neither enzyme was released from whole cells by osmotic shock treatment.
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    Biochemistry and genetics of the pathway for the anaerobic degradation of aromatic compounds by Eubacterium oxidoreducens
    Haddock, John David (Virginia Tech, 1990-08-05)
    The biochemical pathway for the anaerobic degradation of gallate, pyrogallol and phloroglucinol by Eubacterium oxidoreducens was investigated. Phloroglucinol reductase was purified 90-fold, from the soluble fraction of cell extract, to electrophoretic homogeneity. The enzyme was an α₂ homodimer with a native Mr of 78,000, did not contain metals or cofactors and was specific for phloroglucinol and NADPH with a Km of 800 μM and 6.7 μM respectively at pH 6.8. The Km for phloroglucinol decreased with increasing pH. The enzyme catalyzed reaction was reversible and the equilibrium constant was 9.6. Dihydroresorcinol was a competitive inhibitor of the reverse reaction (Ki = 756 μM). Dihydrophloroglucinol produced in cell extract with H₂ as the reductant was identical to the compound produced by sodium borohydride reduction of phloroglucinol as shown by 1H NMR spectroscopy. The ¹³C NMR spectrum was consistent with the structural assignment of dihydrophloroglucinol. The mechanism of the proposed enzymatically catalyzed reaction is proposed to involve transfer of a hydride equivalent from NADPH to the carbonyl carbon of the phloroglucinol dianion. Mutant strains of E. oxidoreducens that showed no gallate decarboxylase or dihydrophloroglucinol hydrolase activity were isolated after mutagenesis with ethylmethane sulfonate and emichment with ampicillin. The decarboxylase deficient mutants were unable to grow on gallate while pyrogallol and phloroglucinol supported growth. The hydrolase deficient mutants were unable to grow on any aromatic substrates and converted gallate to pyrogallol and dihydrophloroglucinol. The conversion of gallate to non-aromatic intermediates by cell extract of the wild-type stain was dependent on the presence of 1,2,3,5-benzenetetrol for the conversion of pyrogallol to phloroglucinol and on formate for the reduction of phloroglucinol to dihydrophloroglucinol. Transhydroxylase activity involved in the conversion of pyrogallol to phloroglucinol was induced by growth on aromatic substrates. The formate dehydrogenase was located in the soluble fraction of cell extract, and activity was protected from oxygen inactivation by sodium azide. The Km for formate and NADP was 290 μM and 140 μM respectively at pH 7.5. The pH optimum for activity was 7.5 and maximum activity was observed at a temperature of 50°C.
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    Characterization and Molecular Analysis of Fragilysin: The Bacteroides fragilis Toxin
    Obiso, Richard J. Jr. (Virginia Tech, 1997-05-06)
    Bacteroides fragilis is a gram negative, anaerobic rod, that is a member of the normal colonic microflora of most mammals, and it is the anaerobe most commonly isolated from human soft tissue infections. During the past decade, strains of B. fragilis that produce an enterotoxin have been implicated as the cause of diarrhea in a number of animals, including humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (Mr~ 20,600) that causes rapid morphological changes in human colon carcinoma cell lines, particularly, HT-29. This dissertation research began in 1993 with the purpose of determining how this enterotoxin, termed fragilysin, causes diarrhea. The deduced amino acid sequence revealed a signature zinc binding consensus motif (His-Glu-Xx-Xxx-His-Xxx-Xxx-Gly-Xxx-Xxx-His/Met) characteristic of metalloproteinases. Sequence analysis showed close identity with metalloproteinases within the zinc-binding and Met-turn regions. Purified fragilysin contained 1 gram atom of zinc per molecule, and it hydrolyzed a number of proteins, including gelatin. Optimal proteolytic activity occurred at 37° C and pH 6.5. Activity was inhibited by metal chelators but not by inhibitors of other classes of proteinases. When fragilysin is injected into ligated ileal and colonic loops of animals, there is significant tissue damage and a subsequent dose dependent fluid response. 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; and, to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to chelated toxin. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). Furthermore, there is a direct effect on the tight junction proteins. Fragilysin appears to cause diarrhea by proteolytically degrading the paracellular barrier of epithelial cells. Fragilysin is a recently discovered virulence factor that could contribute to the pathogenesis of B. fragilis in both intestinal and soft tissue infections. This research was supported by a Public Health Service grants AI 322940 and AI 32940-03 from the National Institute of Allergy and Infectious Diseases, and by the Commonwealth of Virginia project 6127250
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    Characterization of Mycobacterium avium cytoplasmic membrane proteins with an emphasis on the major cytoplasmic membrane protein
    Carlisle, Glenn E. (Virginia Tech, 1991-05-16)
    Proteins of the cytoplasmic membrane of Mycobacterium avium were investigated to identify those which were: (1)intrinsic or extrinsic, (2) attached to the cell wall, (3)surface accessible and (4) excreted. In addition sera containing anti-cytoplasmic membrane proteins were obtained and preliminary purification of the cytoplasmic membrane protein was attempted. The predominating cytoplasmic membrane protein of 31,000 daltons (MCMP) was found to be intrinsic, attached to the cell wall and possibly surface accessible. The MCMP was not excreted, even in media in which the MCMP is not found in the cytoplasmic membrane. Other cytoplasmic membrane proteins were also found to be intrinsic; a few were likely to be extrinsic based upon their separation from the membrane in sucrose gradients. Cytoplasmic membrane proteins of 66, 000, 115, 000 and 129 dalton were surface accessible as judged by I 125-Iodobead labeling. Antisera against the HCMP and other cytoplasmic membrane proteins was obtained and will be useful in further cytoplasmic membrane protein characterization. Acetone precipitation of a cytoplasmic membrane preparation was performed to partially purify the MCMP. The data from this study can be used for the development of serodiagnostic reagents for detecting mycobacterial infection.
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    Characterization of the carbohydrate receptors of the Clostridium difficile enterotoxin
    Tucker, Kenneth D. (Virginia Tech, 1990)
    Clostridium difficile causes pseudomembranous colitis in humans and a similar ileocecitis in hamsters. This organism can colonize the intestines after antibiotic therapy disrupts the normal intestinal microflora. Once established in the intestines, the organism causes disease by producing two toxins, designated toxin A and toxin B. Only toxin A is active on intestinal epithelium, thus toxin A is the cause of the initial tissue damage in the intestines. In order for a toxin to affect a cell, it must first bind to the cell. Toxin A has been shown to bind to Galα1- 3Galβ 1-4GIcNAc on the intestinal epithelium of hamsters. I provide evidence that toxin A can use this trisaccharide as a functional receptor on cell lines, and that the expression of the carbohydrate receptor increases the sensitivity of the cells to toxin A. Furthermore, the intestinal epithelium of infant hamsters bound less toxin A at 37C than did the adult tissue, and infants are less sensitive to the disease caused by C. difficile than are adults. This provides further evidence that the activity of toxin A is increased by the binding of the toxin to Galα1-3Galβ1- 4GlcNAc. Even though Galα1-3Galβ 1-4GlcNAc was a receptor for toxin A on animal cells, it probably is not a receptor for toxin A in humans, because people do not normally express this carbohydrate. Instead, I found that toxin A bound to the carbohydrate antigens designated I, X, and Y, which are present on the intestinal epithelium of humans. These carbohydrates could be receptors for toxin A. The possible significance of these receptors is discussed.
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    Characterization of the genes and gene products of the acetate-activating enzymes and a novel iron-sulfur flavoprotein from Methanosarcina thermophila strain TM-1
    Latimer, Matthew T. (Virginia Tech, 1993-10-05)
    The genes encoding the acetate kinase and phosphotransacetylase enzymes from Methanosarcina thermophila were isolated from a genomic library on a fifteen kilobase fragment The genes are located adjacent to one another, with the phosphotransacetylase gene (pta) directly upstream of the acetate kinase gene (ack). The two genes were sequenced, along with a third Open Reading Frame (designated orfY). The orfY gene appears to encode a novel protein whose physiological function has yet to be determined.
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    Clostridium difficile toxins A and B: exploring the possible mechanism of action
    Jefferson, Kimberly Kay (Virginia Tech, 1995-01-05)
    Clostridium difficile is a common cause of antibiotic-associated diarrhea and occasionally causes the life-threatening disease pseudomembranous colitis. The pathogenicity of the organism has been attributed to the production of two large exotoxins, toxin A (308,000 daltons) and toxin B (269,000 daltons). Toxin A is a powerful enterotoxin and is generally thought to play the more important role in the pathology of the disease. Toxin B may exert its effect after the initial tissue damage by toxin A. Both toxins cause rounding of mammalian culture cells by disrupting the cytoskeletal system. The similar biological activities and high percentage of sequence homology between the two toxins suggest that they have a similar mechanism of action. I found that purified preparations of both toxins cleave skeletal muscle actin at a single site, producing a 38,000 dalton actin fragment, and that the toxins are capable of autodigestion. The proteolytic activity may be involved in the mechanism of action of the toxins. I also analyzed an aberrant strain of C. difficile which reportedly lacked the gene for toxin B. Such a strain would be very useful for the study of the mechanism of toxin A. I concluded however, that the strain contained the genes for both toxin A and toxin B. The toxin genes and resulting proteins appear, however, to be slightly different from those of other strains.
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    Detection, isolation and purification of Clostridium difficile toxin A with toxin receptors
    (United States Patent and Trademark Office, 1992-03-24)
    A method is provided for detecting the presence of C. difficile toxin A. Stool or other appropriate specimen is contacted with a reagent containing the human X, Y or I-antigens, each of which is a specific receptor for toxin A. The reagent may be intact cells, cell membranes, membrane fractions containing any of these antigens, glycoconjugates, as well as the purified oligosaccharide antigen per se. Binding of toxin A is determined by conventional assay techniques. The method may also be used to isolate and purify toxin A. Conversely, immobilized toxin A may be used to detect, isolate, or purify biological materials of interest expressing the X, Y or I antigens.
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    Development of defined media for motility and for growth of Spirillum volutans, with special reference to biological monitoring of pollutants and to obligate microaerophilism of bacteria
    Bowdre, Jean Handy (Virginia Tech, 1975-05-05)
    Two investigations were pursued in this study. One was a possible application of the motility of Spirillum volutans to detection of pollutants in industrial effluent for the purpose of in-plant biological monitoring. The other was a nutritional study of the organism with emphasis on its obligate microaerophilism. In the pollution monitoring project, flagellar uncoordination in S. volutans was studied as a bioindicator of toxicity. Uncoordination is produced by a variety of agents. Cells displaying normal motility show frequent reversal of direction, accompanied by reversal of orientation of their bipolar fascicles of flagella. In uncoordinated cells, the flagella at opposite poles oppose each other and the cell cannot swim, although the flagella remain active. A defined motility test medium was devised in an attempt to maximize the sensitivity of the uncoordination response to potential pollutants. Suspensions of S. volutans in this medium responded to a variety of agents, including metal ions (1-3 ppm), cetyl pyridinium chloride (1 ppm), hydrazine (10 ppm), i-naphthol (3 ppm), and others. Effective concentrations ranged up to several per cent for the alcohols tested. The response was immediate.
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    Effect of Autoregulated TxeR on the Expression of Clostridium difficile Toxins
    Barroso, Lisa Ann (Virginia Tech, 1999-12-01)
    Clostridium difficile is a major nosocomial pathogen responsible for causing pseudomembranous colitis. It is estimated that 25% of antibiotic-associated diarrhea is due to C. difficile. These diseases result from intestinal tissue damage caused by two of the largest known bacterial toxins, A and B. Molecular studies of the C. difficile toxins have identified a 19.6 kb toxigenic element that contains both toxin genes flanked by three small open reading frames (ORFs). The focus of this study is to elucidate the function of the ORF, designated txeR, which is located at the beginning of the toxigenic element. The deduced amino acid sequence of txeR predicts a 22-kDa protein that contains a helix-turn-helix motif characteristic of DNA binding regulatory proteins. To determine if the protein TxeR regulates expression from the toxA, toxB, and txeR promoters, gene fusions were constructed that contained the various promoter regions and a reporter gene. The immunodominant region of toxin A located at the carboxy-terminus, termed the repeating units (ARU), was selected as the reporter gene. Expression studies were performed in Escherichia coli host strains. Levels of ARU expression were measured by enzyme-linked immunosorbent assay using an ARU-specific monoclonal antibody. Expression levels of ARU from the toxin B promoter region with TxeR supplied on the same plasmid (in cis) or on a different plasmid (in trans) were determined. In cis, ARU levels were 50-fold higher than strains without txeR. In trans, expression of ARU from the toxin B promoter region increased over 800-fold. When TxeR was supplied in trans to a toxin A promoter region-ARU fusion, expression levels of ARU increased over 500-fold. To test for autoregulation, TxeR was supplied in trans to the txeR promoter region fused to ARU. The effect was an increase of ARU expression up to 20-fold over background. These results suggest that TxeR is a trans-acting regulator that stimulates expression of the C. difficile toxins and is subjected to autoregulation.
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    Effects of anti-inflammatory drugs on fever and neutrophilia induced by Clostridium difficile toxin B
    Cardoso, R. A.; Melo Fihlo, A. A.; Melo, M. C. C.; Lyerly, D. M.; Wilkins, Tracy D.; Lima, A. A. M.; Ribeiro, R. A.; Souza, G. E. P. (Hindawi Publishing Corporation, 1996-06)
    This study investigated the ability of Clostridium difficile toxin B, isolated from the VPI 10463 strain, to induce fever and neutrophilia in rats. Intravenous injection of toxin B (0.005-0.5 mu g/kg) evoked a dose-dependent increase in body temperature. The febrile response to 0.5 mu g/kg of the toxin started in 2.5 h, peaked at 5 h, and subsided fully within 24 h. Toxin B also induced a dose-dependent neutrophilia. Pretreatment with indomethacin (2 mg/kg, i.p.) did not affect the neutrophilia induced by toxin B, but significantly reduced the febrile response measured 4 to 8 h after toxin B injection. Dexamethasone (0.5 mg/kg) also markedly diminished the febrile response induced by toxin B. These results show that Clostridium difficile toxin B induced a febrile response susceptible to inhibition by dexamethasone and indomethacin. Furthermore, they suggest that prostaglandins are not involved in the neutrophilia caused by this toxin.
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    Evaluation of a Monoclonal-based EIA for the Detection of Giardia lamblia and the Identification of the Antigen
    Boone, James Hunter M.S. (Virginia Tech, 1998-05-05)
    I. A number of commercial enzyme immunoassay (EIA) tests are available for the diagnosis of giardiasis. In a time of rising health-care costs, there is a need for diagnostic tests that are rapid, specific, sensitive and inexpensive. In the first phase of this study, I developed a monoclonal-based EIA, the GIARDIA TEST, with these qualities in mind. This assay's performance characteristics were determined by a comparison study using conventional ova and parasite examination, immunofluorescence antibody test (IFA) and other commercial EIA tests. Studies were done in-house at TechLab, Inc. and at various U.S. medical facilities. Results were statistically analyzed to determine sensitivity (ability of the assay to detect a positive result), specificity (amount of cross-reactivity), predictive positive value (the confidence in a positive result), predictive negative value (the confidence in a negative result) and overall correlation with the reference assay. II. There remain many questions to be answered about the various antigens produced by Giardia lamblia and how they can be utilized as diagnostic markers. In the second phase of this study, I identified and partially characterized the antigen (Ct7 Ag) that reacts with the Ct7 monoclonal antibody (MAb). This MAb is an IgM class mouse immunoglobin that is utilized by the GIARDIA TEST and by an immunofluorence antibody test (IFA) which detects Giardia cysts in water and feces. The results of this study will provide physicians and researchers with detailed information about the Ct7 Ag and why it is a useful marker for giardiasis.
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    Experimental anaerobic bacterial infections in mice
    Walker, Clay B. (Virginia Tech, 1977-06-15)
    The development of a technique for producing a pure Bacteroides fragilis infection in mice is described. The infection produces large subcutaneous abscesses at the site of bacterial injection in approximately 90% of the mice. The abscess can be seen as an obvious swelling within 5-7 days post-injection. The infection was initiated by injection of pure cultures grown in a semisolid medium. Similar infections were also produced with pure cultures of B. distasonis, B. ovatus, B. thetaiotaomicron, and B. vulgatus.
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    Formulation of improved media for isolation and cultivation of Campylobacter fetus
    George, Hugh A. (Virginia Tech, 1977-12-15)
    Campylobacter fetus, a microaerophilic, Gram-negative rod, is a well-known cause of contagious abortion and infertility in cattle and sheep and is gaining increasing recognition as an opportunistic human pathogen. In the past, the unusual oxygen requirements of the organism have complicated its recovery from clinical sources; optimum recovery necessitates the use of special gas mixtures, vacuum pumps, etc., not routinely used in most laboratories. In this study, the stimulatory effects of compounds found to enhance aerotolerance and growth of C. fetus were tested for 62 strains of C. fetus, representing each subspecies, to test the desirability of supplementing conventional media with these additives. Brucella agar supplemented with 0.025% (each) FeS04·7H20, sodium bisulfite, and pyruvic acid (FBPA agar) supported growth of 82% of the strains tested under simulated candle jar condtions. Brucella broth supplemented with 0.2% FeS04 ·7H20, 0.025% sodium bisulfite, and 0.050% pyruvic acid (FBPB broth) supported growth of 61 of 62 strains at 21% 02, 2.5% CO2 with static incubation. Therefore, FBPA agar and FBPB broth are recommended for the isolation and cultivation of C. fetus. Although isolation from clinical sources is still dictated to some extent by the oxygen tension used for cultivation, improved recovery may be expected regardless of equipment or facilities available. Another compound found to enhance aerotolerance of C. fetus was SOD. This finding supports the hypothesis that the stimulatory effect of the media additives results from a direct action on the culture medium by degrading toxic derivatives of oxygen, such as the superoxide radical and hydrogen peroxide.
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    Glutamate Dehydrogenase Is Highly Conserved among Clostridium difficile Ribotypes
    Carman, R. J.; Wickham, K. N.; Chen, L.; Lawrence, A. M.; Boone, J. H.; Wilkins, Tracy D.; Kerkering, Thomas M.; Lyerly, D. M. (American Society for Microbiology, 2012-02-01)
    gluD was highly conserved and glutamate dehydrogenase (GDH) was readily expressed in vitro by all 77 Clostridium difficile ribotypes assayed. All ribotypes, including ARL 002, ARL 027, and ARL 106, were reactive in assays that detect C. difficile GDH.
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    Identification and characterization of a receptor for Clostridium Difficile enterotoxin
    Krivan, Howard C. (Virginia Polytechnic Institute and State University, 1986)
    Clostridium difficile and its toxins are implicated as the cause of pseudomembranous colitis in patients undergoing antibiotic therapy. Very little information is known about how these toxins bind to cells and cause disease. In an attempt to understand how these toxins work this dissertation research was undertaken to determine whether a receptor for C. difficile enterotoxin (toxin A) exists in the brush border membranes (BBMs) of the hamster, an animal known to be extremely sensitive to the action of the toxin. Toxin A was the only antigen adsorbed by the BBMs from the culture filtrate of C. difficile. Erythrocytes from various animal species were also examined for binding activity. Only rabbit erythrocytes could bind the toxin, and the cells agglutinated. A binding assay based on an enzyme-linked immunosorbent assay method for quantifying C. difficile toxin A was used to compare binding of the toxin to hamster BBMS, rabbit erythrocytes, and to BBMs from rats, which are less susceptible to the action of C. difficile toxin A than hamsters. Results of this comparison indicated the following order of toxin binding frequency: rabbit erythrocytes > hamster BBMs > rat BBMs. Binding of toxin A to BBMS from hamsters at 37°C was comparable to what has been observed with cholera toxin, but binding was enhanced at 4°C. A similar binding phenomenon was observed with rabbit erythrocytes. Examination of the cell surfaces of hamster BBMs and rabbit erythrocytes with lectins and specific glycosidases revealed a high concentration of terminal α-linked galactose. Treatment of both membrane types with α-galactosidase destroyed the binding activity. The glycoprotein, calf thyroglobulin, also bound the toxin and inhibited toxin binding to cells. An efficient, single-step method for isolation of highly purified toxin A from C. difficile culture filtrate has been developed based on toxin association with calf thyroglobulin. Toxin A did not bind to human erythrocytes from blood group A, B, or O donors. However, after fucosidase treatment of human erythrocytes, only blood group B erythrocytes could bind the toxin. This indicated that toxin A was likely binding to Ga1α1-3Ga1ß1-4GlcNAc, a carbohydrate sequence also found on calf thyroglobulin and rabbit erythrocytes. All of the results indicate that hamster BBMs contain a carbohydrate binding site for toxin A that has at least a Ga1α1—3Ga1ßl-4GlcNAc nonreducing terminal sequence.
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    The localization of two epitopes recognized by the monoclonal antibody PCG-4 on toxin A of Clostridium difficile
    Frey, Steven M. (Virginia Tech, 1990-12-05)
    Clostridium difficile causes pseudomenbranous colitis (PMC) and diarrhea in humans. Toxigenic strains of C. difficile produce two toxins. Toxin A is an enterotoxin and cytotoxin, and toxin B is a potent cytotoxin. The gene encoding toxin A has been sequenced and was shown to possess a 2.5 kb region, containing 38 similar repeating amino acid sequences, at the 3' -end of the gene. This region of the toxin A gene codes for the carbohydrate-binding portion of the toxin. The monoclonal antibody PCG-4 (MAb) binds to this portion of toxin A and neutralizes its enterotoxic activity. In addition, this monoclonal antibody has been shown to immunoprecipitate toxin A, suggesting that the MAb PCG-4 is binding to two or more similar epitopes on the toxin. The goal of this research project was to identify the neutralizing epitopes recognized by the MAb PCG-4 on the surface of the toxin A. To map the epitopes bound by the MAb PCG-4, a series of overlapping deletion clones were constructed from a 4.7 kb fragment from the 3'-end of the toxin A gene. The recombinant polypeptides expressed by these clones were tested for reactivity with the MAb PCG-4. By comparing the overlapping polypeptides, defined as either PCG-4 reactive or nonreactive, I localized the PCG-4 epitope to a 44-amino acid sequence situated between the amino acid residues 2098-2141 of toxin A A similarity search of the toxin with the 44-amino acid sequence containing the PCG-4 epitope revealed the presence of two other possible PCG-4 epitopes located between the amino acid residues 2355-2398 and 2459-2502. However, subsequent cloning experiments showed that only the region located between the amino acid residues 2355-2398 contained a PCG-4 reactive epitope. The identification of two similar epitopes within the toxin's structure explains how this monoclonal antibody is able to immunoprecipitate toxin A in the absence of subunits. Furthermore, I found that small recombinant polypeptides, containing the PCG-4 epitope lost reactivity with this monoclonal antibody following denaturation, suggesting that the epitopes recognized by this monoclonal antibody are conformationally dependent.