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Chapter 16 : Glucosylating and Deamidating Bacterial Protein Toxins

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Glucosylating and Deamidating Bacterial Protein Toxins, Page 1 of 2

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Abstract:

The family of large clostridial cytotoxins comprises toxins A and B, the lethal and the hemorrhagic toxins from , the α-toxin from , and various toxin isoforms mainly produced by . Differential glucosylation is used to measure the modification of Rho GTPases in intact cells. Toxin-modified Rho GTPase in intact cells blocks subsequent toxin-catalyzed labeling of Rho proteins in the cell lysate upon addition of UDP[C]glucose. Covalent modification by bacterial protein toxins not only inhibits but also activates Rho GTPases. Measurement of the GTP hydrolysis by cytotoxic necrotizing factor (CNF)1-treated RhoA reveals an inhibition of the intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activity, indicating that deamidation of Gln-63 forms a constitutively active Rho protein. The modification of Rho GTPases by dermonecrotizing toxin (DNT) occurs predominantly with the GDP-bound form of Rho, while the modification of Rho by CNFs is not nucleotide dependent. Like deamidation of Gln-63, transglutamination of Gln-63 inhibits GTP hydrolase activity of Rho proteins, although the precise function of the addition of primary amines onto Rho GTPases is not completely understood. Deamidation and transglutamination by CNFs or DNT change the migration of GTPases by SDS-PAGE. This change in migration occurs with RhoA but not Rac or Cdc42 and depends on the deamidation of Glu-63 and, therefore, is also observed with the recombinant RhoA-Q63E. Mass spectrometric analysis of proteolytic peptides of Rho GTPases allows the detection of the 1-Da shift caused by CNF1-induced deamidation of Gln-63 of RhoA (Gln-61 of Rac and Cdc42).

Citation: Aktories K. 2003. Glucosylating and Deamidating Bacterial Protein Toxins, p 229-244. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch16

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Figures

Image of Figure 1
Figure 1

Scheme of the primary structure of toxin B, the prototype of large clostridial cytotoxins. Toxin B consists of three domains: (i) N-terminal enzyme domain harboring glucosyltransferase activity; (ii) small hydrophobic domain in the middle of the toxin, which is most likely involved in toxin translocation; and (iii) C-terminal part consisting of polypeptide repeats suggested to be involved in receptor binding. The minimal length of the glucosyltransferase enzyme domain includes the 546 N-terminal amino acids. The conserved DXD motif is essential for transferase activity and likely to be involved in divalent cation and/or sugar nucleotide binding. The conserved Trp-102 is also important for nucleotide binding.

Citation: Aktories K. 2003. Glucosylating and Deamidating Bacterial Protein Toxins, p 229-244. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch16
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Image of Figure 2
Figure 2

Cytotoxic effects of toxin B on NIH3T3 fibroblasts.

Citation: Aktories K. 2003. Glucosylating and Deamidating Bacterial Protein Toxins, p 229-244. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch16
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Image of Figure 3
Figure 3

Rho GTPases are modified by various bacterial protein toxins. Large clostridial cytotoxins glucosylate Rho subfamily proteins at Thr-37 (RhoA) or at the equivalent Thr-35 (Rac, Cdc42). C3 transferases, including Clostridium botulinum C3 exoenzyme, ADP-ribosylate RhoA at Asn-41. CNFs from Escherichia coli and the DNT from Bordetella sp. deamidate and transglutaminate, respectively, Gln-63 of RhoA and Gln-61 of Rac and Cdc42 (2).

Citation: Aktories K. 2003. Glucosylating and Deamidating Bacterial Protein Toxins, p 229-244. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch16
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Image of Figure 4
Figure 4

Toxins A and B from modify GDP-bound RhoA. In GTPbound RhoA, the hydroxyl group of Thr-37 coordinates a divalent cation directed into the protein and is not available for glucosylation by the toxins. In GDP-bound RhoA, the hydroxyl group of Thr-37 is directed to the solvent and is accessible for glucosylation by the toxins.

Citation: Aktories K. 2003. Glucosylating and Deamidating Bacterial Protein Toxins, p 229-244. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch16
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Figure 5

Functional consequences of the glucosylation of Rho GTPases. Inhibition of the interaction of glucosylated Rho with effectors is the most important effect of glucosylation. In addition, glucosylation inhibits Rho activation by guanine nucleotide exchange factors (GEF), the Rho cycling because glucosylated Rho locates at the membrane and does not bind to the guanine nucleotide dissociation inhibitor (GDI), and the stimulation of the GTP hydrolase activity by GTPase-activating proteins (GAP).

Citation: Aktories K. 2003. Glucosylating and Deamidating Bacterial Protein Toxins, p 229-244. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch16
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Figure 6

Structural comparison of CNF1 from with the DNT from species. Alignment analysis identified a short region of high sequence identity at the C terminus. Indicated are the catalytical amino acids cysteine (C; Cys-1292 of DNT and Cys-866 of CNF1) and histidine (H, His-1307 of DNT and His-881 of CNF1). The minimal domain, possessing enzyme activity, is given as ΔCNF and ΔDNT, respectively.

Citation: Aktories K. 2003. Glucosylating and Deamidating Bacterial Protein Toxins, p 229-244. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch16
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Image of Figure 7
Figure 7

Cytotoxic effects of CNF1 on fibroblasts. CNF1 increases formation of lamellipodia, filopodia, and stress fibers.

Citation: Aktories K. 2003. Glucosylating and Deamidating Bacterial Protein Toxins, p 229-244. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch16
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Image of Figure 8
Figure 8

CNF treatment of RhoA induces a change in the migration of the GTPase on SDS-PAGE to an apparent larger molecular size. Deamidation of RhoA by CNF results in an increase in size of 1 Da. This can be analyzed by MALDI-TOF mass spectrometry. RhoA modified by CNF1 is digested by trypsin and subsequently the peptides formed are analyzed by mass spectrometry. One peptide, corresponding to amino acids 52 to 68, shows an increase in size of 1 Da, caused by deamidation of Gln-63.

Citation: Aktories K. 2003. Glucosylating and Deamidating Bacterial Protein Toxins, p 229-244. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch16
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Image of Figure 9
Figure 9

Deamidation or transglutamination of RhoA at Glu-63 by CNF and DNT, respectively, blocks the GTP hydrolysis stimulated by GAP. Rho-Q63E is constitutively active and induces activation of effectors, which eventually cause formation of stress fibers and a large array of other effects.

Citation: Aktories K. 2003. Glucosylating and Deamidating Bacterial Protein Toxins, p 229-244. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch16
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References

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1. Bishop, A. L.,, and A. Hall. 2000. Rho GTPases and their effector proteins. Biochem. J. 348:241255.
2. Boquet, P.,, P. Munro,, C. Fiorentini,, and I. Just. 1998. Toxins from anaerobic bacteria: specificity and molecular mechanisms of action. Curr. Opin. Microbiol. 1:6674.
3. Busch, C.,, and K. Aktories. 2000. Microbial toxins and the glucosylation of Rho family GTPases. Curr. Opin. Struct. Biol. 10:528535.
4. Farrell, R. J.,, and J. T. LaMont. 2000. Pathogenesis and clinical manifestations of Clostridium difficile diarrhea and colitis. Curr. Top. Microbiol. Immunol. 250:109125.
5. Fiorentini, C.,, and M. Thelestam. 1991. Clostridium difficile toxin A and its effects on cells. Toxicon 29:543567.
6. Just, I.,, J. Selzer,, M. Wilm,, C. Von Eichel-Streiber,, M. Mann,, and K. Aktories. 1995. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375:500503.
7. Lemichez, E.,, G. Flatau,, M. Bruzzone,, P. Boquet,, and M. Gauthier. 1997. Molecular localization of the Escherichia coli cytotoxic necrotizing factor CNF1 cell-binding and catalytic domains. Mol. Microbiol. 24:10611070.
8. Masuda, M.,, L. Betancourt,, T. Matsuzawa,, T. Kashimoto,, T. Takao,, Y. Shimonishi,, and Y. Horiguchi. 2000. Activation of Rho through a cross-link with polyamines catalyzed by Bordetella dermonecrotizing toxin. EMBO J. 19:521530.
9. Schmidt, G.,, P. Sehr,, M. Wilm,, J. Selzer,, M. Mann,, and K. Aktories. 1997. Gln63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor 1. Nature 387:725729.
10. Von Eichel-Streiber, C.,, P. Boquet,, M. Sauerborn,, and M. Thelestam. 1996. Large clostridial cytotoxins—a family of glycosyltransferases modifying small GTP-binding proteins. Trends Microbiol. 4:375382.

Tables

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Table 1

Second substrates (cosubstrates) and protein substrates of the large clostridial cytotoxins

Citation: Aktories K. 2003. Glucosylating and Deamidating Bacterial Protein Toxins, p 229-244. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch16

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