Comparison of wild type with recombinant Clostridium difficile toxin A

被引:14
作者
Gerhard, R [1 ]
Burger, S [1 ]
Tatge, H [1 ]
Genth, H [1 ]
Just, I [1 ]
Hofmann, F [1 ]
机构
[1] Hannover Med Sch, Inst Toxicol, D-30625 Hannover, Germany
关键词
glucosyltransferase; cytochrome c-release; enzyme-deficient toxin A; cytotoxicity; RhoA;
D O I
10.1016/j.micpath.2004.12.002
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
Toxins A and B from Clostridium difficile are single-chain proteins of 308,000 and 270,000 Da, respectively. They possess transferase activity to monoglucosylate proteins of the Rho GTPase family whereby Rho, Rac, and Cdc42 are the canonical substrates. For application of these toxins as specific Rho GTPase inhibitors the highest possible purity is of crucial interest. We, therefore, expressed recombinant His-tagged toxin A using the Bacillus megaterium, expression system. Specific antisera raised against the native toxin A from C. difficile and the recombinant toxin, respectively, showed identical sensitivity and specificity in Western blot and ELISA analyses towards both toxins. By comparison of both toxins in functional studies we showed that the recombinant toxin was about two times more cytotoxic than the native toxin, and the glucosyltransferase-activity of the recombinant toxin was even 10-fold increased. However, recombinant toxin A showed one essential difference to the classically purified one. The reported transferase-independent effect of toxin A to release cytochrome c from isolated mitochondria was not exhibited by the recombinant toxin A. This putative mitochondrial effect decreased with increased purity of toxin A, and was absent with recombinant toxin, strongly suggesting an clostridial contamination responsible. In summary, we tested the recombinant toxin A to be at least an adequate substitute for the native toxin, bearing the advantage of a rapid single-step purification and the absence of biological active contaminations. (c) 2005 Elsevier Ltd. All rights reserved.
引用
收藏
页码:77 / 83
页数:7
相关论文
共 28 条
[1]   Mechanism of Clostridium difficile toxin A-induced apoptosis in T84 cells [J].
Brito, GAC ;
Fujji, J ;
Carneiro, BA ;
Lima, AAM ;
Obrig, T ;
Guerrant, RL .
JOURNAL OF INFECTIOUS DISEASES, 2002, 186 (10) :1438-1447
[2]   Expression of recombinant Clostridium difficile toxin A using the Bacillus megaterium system [J].
Burger, S ;
Tatge, H ;
Hofmann, F ;
Genth, H ;
Just, I ;
Gerhard, R .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2003, 307 (03) :584-588
[3]   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
[4]   Clostridium difficile toxin A causes early damage to mitochondria in cultured cells [J].
D, HE ;
Hagen, SJ ;
Pothoulakis, C ;
Chen, M ;
Medina, ND ;
Warny, M ;
LaMont, JT .
GASTROENTEROLOGY, 2000, 119 (01) :139-+
[5]   The enzymatic domain of Clostridium difficile toxin A is located within its N-terminal region [J].
Faust, C ;
Ye, BX ;
Song, KP .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1998, 251 (01) :100-105
[6]   The complete receptor-binding domain of Clostridium difficile toxin A is required for endocytosis [J].
Frisch, C ;
Gerhard, R ;
Aktories, K ;
Hofmann, F ;
Just, I .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2003, 300 (03) :706-711
[7]   Monoglucosylation of RhoA at threonine 37 blocks cytosol-membrane cycling [J].
Genth, H ;
Aktories, K ;
Just, I .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (41) :29050-29056
[8]   New method to generate enzymatically deficient Clostridium difficile toxin B as an antigen for immunization [J].
Genth, H ;
Selzer, J ;
Busch, C ;
Dumbach, J ;
Hofmann, F ;
Aktories, K ;
Just, I .
INFECTION AND IMMUNITY, 2000, 68 (03) :1094-1101
[9]   Clostridium difficile toxin A triggers human colonocyte IL-8 release via mitochondrial oxygen radical generation [J].
He, D ;
Sougioultzis, S ;
Hagen, S ;
Liu, J ;
Keates, S ;
Keates, AC ;
Pothoulakis, C ;
Lamont, JT .
GASTROENTEROLOGY, 2002, 122 (04) :1048-1057
[10]   Chimeric clostridial cytotoxins: Identification of the N-terminal region involved in protein substrate recognition [J].
Hofmann, F ;
Busch, C ;
Aktories, K .
INFECTION AND IMMUNITY, 1998, 66 (03) :1076-1081