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

被引:26
作者
Genth, H [1 ]
Selzer, J [1 ]
Busch, C [1 ]
Dumbach, J [1 ]
Hofmann, F [1 ]
Aktories, K [1 ]
Just, I [1 ]
机构
[1] Univ Freiburg, Inst Pharmakol & Toxikol, D-79104 Freiburg, Germany
关键词
D O I
10.1128/IAI.68.3.1094-1101.2000
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
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 mere protective only when inside the cell.
引用
收藏
页码:1094 / 1101
页数:8
相关论文
共 46 条
[1]   BOTULINUM ADP-RIBOSYLTRANSFERASE C-3 - PURIFICATION OF THE ENZYME AND CHARACTERIZATION OF THE ADP-RIBOSYLATION REACTION IN PLATELET MEMBRANES [J].
AKTORIES, K ;
ROSENER, S ;
BLASCHKE, U ;
CHHATWAL, GS .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1988, 172 (02) :445-450
[2]  
ALLO M, 1979, GASTROENTEROLOGY, V76, P351
[3]   NUCLEOTIDE-SEQUENCE OF CLOSTRIDIUM-DIFFICILE TOXIN-B GENE [J].
BARROSO, LA ;
WANG, SZ ;
PHELPS, CJ ;
JOHNSON, JL ;
WILKINS, TD .
NUCLEIC ACIDS RESEARCH, 1990, 18 (13) :4004-4004
[4]   A COMPARATIVE BIOCHEMICAL, PHARMACOLOGICAL AND IMMUNOLOGICAL STUDY OF CLOSTRIDIUM-NOVYI ALPHA-TOXIN, CLOSTRIDIUM-DIFFICILE TOXIN-B AND C-SORDELLII LETHAL TOXIN [J].
BETTE, P ;
OKSCHE, A ;
MAULER, F ;
VONEICHELSTREIBER, C ;
POPOFF, MR ;
HABERMANN, E .
TOXICON, 1991, 29 (07) :877-887
[5]   A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins [J].
Busch, C ;
Hofmann, F ;
Selzer, J ;
Munro, S ;
Jeckel, D ;
Aktories, K .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (31) :19566-19572
[6]   A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins [J].
Chaves-Olarte, E ;
Löw, P ;
Freer, E ;
Norlin, T ;
Weidmann, M ;
von Eichel-Streiber, C ;
Thelestam, M .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (16) :11046-11052
[7]  
CIESIELSKITRESKA J, 1991, EUR J CELL BIOL, V56, P68
[8]   CONJOINT TIME-BUDGETING - INVESTIGATING BEHAVIORAL ACCOMMODATION IN MARRIAGE [J].
CLARKE, DD ;
ALLEN, CMB ;
SALINAS, M .
JOURNAL OF SOCIAL AND PERSONAL RELATIONSHIPS, 1986, 3 (01) :53-69
[9]   Mucosal and systemic immunogenicity of a recombinant, non-ADP-ribosylating pertussis toxin: Effects of formaldehyde treatment [J].
Cropley, I ;
Douce, G ;
Roberts, M ;
Chatfield, S ;
Pizza, M ;
Marsili, I ;
Rappuoli, R ;
Dougan, G .
VACCINE, 1995, 13 (17) :1643-1648
[10]   MOLECULAR CHARACTERIZATION OF THE CLOSTRIDIUM-DIFFICILE TOXIN-A GENE [J].
DOVE, CH ;
WANG, SZ ;
PRICE, SB ;
PHELPS, CJ ;
LYERLY, DM ;
WILKINS, TD ;
JOHNSON, JL .
INFECTION AND IMMUNITY, 1990, 58 (02) :480-488