Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Atypon full text link Atypon Free PMC article
Full text links

Actions

.2001 Oct;69(10):6004-11.
doi: 10.1128/IAI.69.10.6004-6011.2001.

Characterization of the enzymatic component of the ADP-ribosyltransferase toxin CDTa from Clostridium difficile

Affiliations

Characterization of the enzymatic component of the ADP-ribosyltransferase toxin CDTa from Clostridium difficile

I Gülke et al. Infect Immun.2001 Oct.

Abstract

Certain strains of Clostridium difficile produce the ADP-ribosyltransferase CDT, which is a binary actin ADP-ribosylating toxin. The toxin consists of the binding component CDTb, which mediates receptor binding and cellular uptake, and the enzyme component CDTa. Here we studied the enzyme component (CDTa) of the toxin using the binding component of Clostridium perfringens iota toxin (Ib), which is interchangeable with CDTb as a transport component. Ib was used because CDTb was not expressed as a recombinant protein in Escherichia coli. Similar to iota toxin, CDTa ADP-ribosylates nonmuscle and skeletal muscle actin. The N-terminal part of CDTa (CDTa1-240) competes with full-length CDTa for binding to the iota toxin binding component. The C-terminal part (CDTa244-263) harbors the enzyme activity but was much less active than the full-length CDTa. Changes of Glu428 and Glu430 to glutamine, Ser388 to alanine, and Arg345 to lysine blocked ADP-ribosyltransferase activity. Comparison of CDTa with C. perfringens iota toxin and Clostridium botulinum C2 toxin revealed full enzyme activity of the fragment Ia208-413 but loss of activity of several N-terminally deleted C2I proteins including C2I103-431, C2I190-431, and C2I30-431. The data indicate that CDTa belongs to the iota toxin subfamily of binary actin ADP-ribosylating toxins with respect to interaction with the binding component and substrate specificity. It shares typical conserved amino acid residues with iota toxin and C2 toxin that are suggested to be involved in NAD-binding and/or catalytic activity. The enzyme components of CDT, iota toxin, and C2 toxin differ with respect to the minimal structural requirement for full enzyme activity.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Characterization of CDTa. In vitro ADP-ribosylation of actin with 1 (▪), 5 (●), and 10 ng (▴) of CDTa was performed for 15 min at 37°C with cytosolic actin from platelets (50 μg of protein) (A) or purified rabbit skeletal muscle actin (1 μg of protein) (B) and [32P]NAD as a cosubstrate. Labeled proteins were detected by SDS-PAGE and phosphorimaging. The percentage of modified actin is shown. The 15-min value for 10 ng of CDTa was taken as 100%. The inset in panel A shows the Coomassie blue staining of purified recombinant CDTa.
FIG. 2
FIG. 2
(A) Highly conserved amino acid residues in the catalytic centers of the ADP-ribosyltransferases C2I fromC. botulinum, Ia fromC. perfringens, and CDTa fromC. difficile. Numbers indicate the positions of amino acid residues. The conserved residues are given in boldface. (B) Amino acid sequences of rCDTa (wild type) and the respective mutants. The exchanged amino acid residues are given in boldface.
FIG. 3
FIG. 3
SDS-PAGE and immunoblot analysis of mutant CDTa proteins. CDTa proteins were expressed inE. coli as GST fusion proteins and purified as described in the text. Proteins (500 ng of each) were subjected to SDS–12.5% PAGE and stained with Coomassie blue (A) or blotted onto nitrocellulose and detected with an antiserum againstC. spiroforme ADP-ribosyltransferase and peroxidase-coupled protein A (B). Lanes 1, rCDTa; lanes 2, E428Q; lanes 3, E430Q; lanes 4, S388A; lanes 5, T389V; lanes 6, S390A; lanes 7, R345K; lanes 8, R346K.
FIG. 4
FIG. 4
In vitro ADP-ribosylation of actin with rCDTa and mutant CDTa proteins. ADP-ribosylation of cytosolic actin from platelets (50 μg of protein) (A) and purified rabbit skeletal muscle actin (1 μg of protein) (B) was performed for 15 min at 37°C with [32P]NAD and the various CDTa proteins (100 ng of each). Labeled proteins were detected by SDS-PAGE and phosphorimaging. The autoradiogram is shown. Lanes 1, rCDTa; lanes 2, E428Q; lanes 3, E430Q; lanes 4, S388A; lanes 5, T389V; lanes 6, S390A; lanes 7, R345K; lanes 8, R346K.
FIG. 5
FIG. 5
Time courses of actin ADP-ribosylation by rCDTa (▪) and CDTa mutant proteins E428Q (▾), E430Q (▴), S388A (●), and R345K (♦). ADP-ribosylation was done with platelet cytosol (1 μg of protein) (A) and with purified rabbit muscle actin (1 μg of protein) (B). Proteins were incubated with [32P]NAD, rCDTa (100 ng of CDTa in panel A and 25 ng of CDTa in panel B), and 1 μg of CDTa mutant protein for 1, 5, 10, 15, and 30 min at 37°C. Labeled proteins were detected by SDS-PAGE, and the amount of radioactively labeled actin was quantified by phosphorimaging. The value for the 30-min incubation of the actin with rCDTa was taken as 100%. Shown are results from a representative experiment, which was repeated at least three times with similar results.
FIG. 6
FIG. 6
Cytotoxic effects of rCDTa and mutant CDTa on Vero cells. Subconfluent Vero cells were incubated in complete medium with the indicated protein at 37°C, and pictures were taken after 4.5 h. For controls, cells were incubated without any toxin (A), with Ib (300 ng/ml) alone (B), or with rCDTa (500 ng/ml) alone (C). The toxin treatments were as follows: rCDTa (D), E428Q (E), E430Q (F), S388A (G), T389V (H), S390A (I), R345K (J), and R346K (K). Samples in panels D to K contained 200 ng of each protein per ml together with 300 ng of Ib per ml.
FIG. 7
FIG. 7
In vitro [32P]ADP-ribosylation of Vero cell lysates after treatment of intact cells with Ib and rCDTa and mutant CDTa proteins. Cells were treated for 4.5 h with the toxins (300 ng of Ib per ml and 200 ng of the CDTa protein per ml) at 37°C and lysed. Lysates (50 μg of protein each) were incubated with 50 ng of CDTa and [32P]NAD for 15 min at 37°C. Proteins were subjected to SDS–12.5% PAGE, and labeled actin was detected by phosphorimaging. Lane 1, control cells; lane 2, cells treated with rCDTa; lane 3, cells treated with E428Q; lane 4, cells treated with E430Q; lane 5, cells treated with S388A; lane 6, cells treated with T389V; lane 7, cells treated with S390A; lane 8, cells treated with R345K; lane 9, cells treated with R346K.
FIG. 8
FIG. 8
Characterization of CDTa and CDTa244–463 proteins. Proteins were expressed inE. coli, purified as described in the text, and cleaved with thrombin. (A) Proteins were subjected to SDS–12.5% PAGE and stained with Coomassie blue. (B) Time course of ADP-ribosylation of actin from platelet cytosol (50 μg of protein) by full-length CDTa (5 ng) (▪) and CDTa244–463 (100 ng [●] and 1 μg [▴]). (C) Time course of ADP-ribosylation of actin from rabbit skeletal muscle (1 μg of protein) by full-length CDTa (5 ng) and CDTa244–463 (100 ng and 1 μg). The value for the 15-min incubation with full-length CDTa was taken as 100%. Panels B and C show results from representative individual experiments. The experiments were done three times.
FIG. 9
FIG. 9
ADP-ribosylation of actin by C-terminal fragments of Ia. (A) Coomassie blue-stained GST-Ia fragments after SDS-PAGE. The numbers give the amino acid residues of Ia. (B) In vitro ADP-ribosylation of actin from platelet cytosol (50 μg of protein) with various GST-Ia proteins. Labeled proteins were detected by SDS-PAGE and phosphorimaging. The autoradiogram is shown.
FIG. 10
FIG. 10
Influence of CDTa1–240 on cytotoxic effects of Ib plus CDTa on Vero cells. Subconfluently growing Vero cells were incubated in complete medium with 300 ng of Ib per ml with CDTa (300 ng/ml) and with or without a 400-fold excess of CDTa1–240. For a control, cells were treated without toxin. After 8 h, pictures were taken.
See this image and copyright information in PMC

References

    1. Aktories K, Bärmann M, Ohishi I, Tsuyama S, Jakobs K H, Habermann E. Botulinum C2 toxin ADP-ribosylates actin. Nature. 1986;322:390–392. - PubMed
    1. Aktories K, Just I. GTPases and actin as targets for bacterial toxins. In: Dickey B F, Birnbaumer L, editors. GTPases in biology I. Berlin-Heidelberg, Germany: Springer; 1993. pp. 87–112.
    1. Aktories K, Koch G. Modification of actin and of Rho proteins by clostridial ADP-ribosylating toxins. In: Iglewski B, Moss J, Tu A T, Vaughan M, editors. Microbial toxins and virulence factors in disease. New York, N.Y: Marcel Dekker; 1995. pp. 491–520.
    1. Aktories K, Sehr P, Just I. Actin-ADP-ribosylating toxins: cytotoxic mechanism of Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. In: Aktories K, editor. Bacterial toxins. Weinheim, Germany: Chapman & Hall; 1997. pp. 93–101.
    1. Aktories K, Wille M, Just I. Clostridial actin-ADP-ribosylating toxins. Curr Top Microbiol Immunol. 1992;175:97–113. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources

Full text links
Atypon full text link Atypon Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2026 Movatter.jp