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Variant toxin B and a functional toxin A produced by Clostridium difficile C34
FEMS Microbiology Letters 198 (2001) 171^176
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Variant toxin B and a functional toxin A produced by Clostridium di¤cile C34 Markus Mehlig a , Michael Moos a , Veit Braun a , Bettina Kalt a , David E. Mahony b , Christoph von Eichel-Streiber a; * a
Verfu«gungsgeba«ude fu«r Forschung und Entwicklung, Institut fu«r Medizinische Mikrobiologie und Hygiene, Johannes Gutenberg-Universita«t, 55101 Mainz, Germany b Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada B3H 4H7 Received 29 January 2001 ; received in revised form 11 March 2001; accepted 15 March 2001
Abstract A particular property of Clostridium difficile strain C34 is an insertion of approximately 2 kb in the tcdA-C34 gene that does not hinder expression of a fully active TcdA-C34 molecule. Intoxication with TcdA-C34 induced an arborized appearance in eukaryotic cells (D-type cytopathic effect); intoxication with TcdB-C34 induced a spindle-like appearance of cells (S-type cytopathic effect). Inactivation of GTPases with purified toxins revealed that Rho, Rac, Cdc42, and Rap are substrates of TcdA-C34. The variant cytotoxin TcdB-C34 inactivated Rho, Rac, Cdc42, Rap, Ral, and R-Ras. Hence, this is the first `S-type' cytotoxin which inactivates both Rho and R-Ras, and is coexpressed with a `D-type' enterotoxin. Our results support the hypothesis that R-Ras is a key GTPase related to the S-type cytopathic effect and suggest that induction of a S-type cytopathic effect dominates induction of the D-type cytopathic effect. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Large clostridial cytotoxin; Cytopathic e¡ect; Rho; R-Ras; Glucosylation ; GTPase
1. Introduction Toxin-producing Clostridium di¤cile strains are known to cause antibiotic-associated diarrhea and pseudomembranous colitis in man [1]. Such strains produce two major virulence factors, an enterotoxin TcdA and a cytotoxin TcdB, both encoded by the pathogenicity locus [2]. TcdA and TcdB are large clostridial cytotoxins that glucosylate the Ras and Rho subfamilies of GTPases [3]. Variations in toxin genes of di¡erent strains are common and useful for di¡erentiation [4], whereas the cytopathic e¡ects of the toxins are less heterogeneous. Following intoxication of eukaryotic cells, D-type and S-type cytopathic e¡ects with a clear di¡erence in actin staining have been reported [5]. The D-type cytopathic e¡ect is characterized by an arborized appearance of the cells whereas a spindle-like appearance is typical of the S-type
* Corresponding author. Tel. : +49 (6131) 3930020; Fax: +49 (6131) 3933364; E-mail : [email protected]
cytopathic e¡ect. The S-type cytopathic e¡ect is exclusively induced by variant cytotoxins, such as TcdB-1470 and TcdB-8864 [6,7], or by the lethal toxin TcsL of Clostridium sordellii. Due to deletions and frameshift mutations no functional enterotoxins are expressed in strains 1470 and 8864 [6,8,9]. In strains which coexpress the enterotoxin and the cytotoxin, both induce a D-type cytopathic e¡ect as reported for reference strain 10463 [10^12]. Recently, Chaves-Olarte et al. [11] suggested that the inactivation of the small GTP binding protein R-Ras is correlated with the S-type cytopathic e¡ect. Similarly, Schmidt and Aktories [13] proposed that inactivation of small GTPases of the Rho subfamily (e.g., Rho, Rac and Cdc42) is correlated with a D-type cytopathic e¡ect. Both interpretations explained the observation that none of the known large clostridial cytotoxins inactivated both R-Ras and Rho. With the clinical isolate C34 we present the ¢rst strain coexpressing a variant `S-type cytotoxin' TcdB-C34 and a fully functional `D-type enterotoxin' TcdA-C34. Moreover, TcdB-C34 inactivates both R-Ras and Rho, and a proposed dominance of R-Ras inactivation on the observed cytopathic e¡ect is discussed.
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 1 4 3 - 4
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2. Materials and methods 2.1. Bacterial strains, chemicals and media C. di¤cile strain 10463 was a gift of N.M. Sullivan (Virginia Polytechnic Institute, Blacksburg, VA, USA). C. di¤cile C34 is a clinical isolate from Halifax, NS, Canada, C. di¤cile strain 8864 was obtained from the Swedish type culture collection (strain 20309), C. di¤cile strain 1470 from Michel Delme¨e (Unite¨ de Microbiologie, Universite¨ de Louvain, Brussels, Belgium) and C. sordellii RE1522 from H.-P. Schau (Thu«ringer Medizinal-, Lebensmittel- und Veterina«runtersuchungsamt, Erfurt, Germany). All clostridial strains were grown in an anaerobic chamber (Don Whitley Scienti¢c, Shipley, UK) using brain heart infusion (Difco, Augsburg, Germany) or Wilkins^Chalgren broth (Oxoid, Wesel, Germany). Chemicals and media were purchased from Merck (Darmstadt, Germany), Sigma (Heidelberg, Germany), Roth (Karlsruhe, Germany), Qiagen (Hilden, Germany), and Gibco BRL Life Technologies (Burlington, ON, Canada). Oligonucleotides were ordered from MWG-Biotech (Ebersberg, Germany). 2.2. Genetic analysis To characterize the pathogenicity locus of C. di¤cile strain C34, 10 PCR ampli¢cations representing the entire pathogenicity locus were performed [8] (Fig. 3A) in a Hybaid Omnigene Cycler (MWG-Biotech). TcdB-C34 cluster 1^2, representing the catalytically active part of the cytotoxin, was ampli¢ed [14] and cloned into plasmid pCR2.1 using the TOPO TA cloning kit in accordance with the manufacturer's instructions (Invitrogen, Groningen, The Netherlands). Sequencing was done with a LICOR 4000L automatic DNA sequencer from MWG-Biotech and the Thermosequenase Cycle Sequencing Kit from Amersham Pharmacia Biotech (Freiburg, Germany). Primers used for sequencing were IRD-800-labelled (MWG-Biotech). Two independent clones were sequenced to guarantee the reliability of the PCR-based sequence. The sequence data of tcdB-C34 cluster 1^2 are available in the EMBL database under accession number AJ294944. Computer-based secondary structure analysis was done with Protean from DNASTAR Software. The Chou^Fasman and the Garnier^Robson secondary structure prediction algorithms were used to determine the secondary structure of the proteins.
Rho, Rac, Cdc42, Ras, Rap, Ral, and R-Ras as target proteins. Radiolabeled proteins were separated by SDS^ PAGE and the bands were visualized by PhosphoImager analysis (Molecular Dynamics, Krefeld, Germany). 2.4. Cell culture and scanning electron microscopy Cytotoxicity of C. di¤cile toxins and culture supernatants were tested on Chinese hamster ovary cells (CHO cells) as described elsewhere [7]. Additionally, human foreskin cells (FSK cells) were used to detect cytotoxicity. The cells were grown as monolayers in 96-well tissue culture plates (Costar, Cambridge, MA, USA) in a humidi¢ed incubator under an atmosphere of 5% CO2 . The growth medium consisted of minimum essential medium (MEM, Gibco BRL) containing streptomycin, penicillin and 10% fetal bovine serum. Maintenance medium used in the cytotoxicity assay consisted of MEM supplemented with 1% fetal calf serum. For scanning electron microscopy, the FSK cells were grown as monolayers on glass coverslips placed on the bottom of glass vials containing MEM as described above. The culture £uids were removed and crude toxin, at a dilution of 1:1000 in MEM medium containing 1% fetal calf serum, was applied to the monolayers. After incubation for 5 h and 21 h, the coverslips were removed from the vials, placed in 2.5% glutaraldehyde solution in 0.1 M sodium cacodylate bu¡er (pH 7.4) for 1 h, osmicated with 1% osmium tetroxide, dehydrated in a series of ethanol concentrations and dried in a Balzer
2.3. Protein preparation and glucosyltransferase activity C. di¤cile toxins TcdA and TcdB and C. sordellii TcsL were prepared as described previously [15]. Purity of toxin preparations was determined by SDS^PAGE. Glucosyltransferase reactions were performed as described by Wagenknecht-Wiesner et al. [14], using recombinant GTPases
Fig. 1. Scanning electron microscopy of FSK cells incubated with or without toxin at 37³C in an atmosphere of 5% CO2 . A: FSK control cells without toxin added and processed for electron microscopy 5 h after the addition of MEM medium. B: FSK cells after 5 h pre-treatment with TcdB-10463. C: FSK cells after 5 h pre-treatment with TcdB-C34. D: Single FSK cell exposed to TcdB-C34 for 21 h prior to processing for electron microscopy.
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critical point drier using CO2 as the drying £uid. Following sputter coating with gold palladium, the samples were viewed with a Cambridge S150 stereoscan scanning electron microscope. 3. Results
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present in TcdB-1470 and TcdB-8864. Both methods predict a di¡erent secondary structure of TcdB-C34 cluster 1^ 2. Since a correlation between the inactivation of R-Ras and the induction of the S-type cytopathic e¡ect was recently suggested [11], we also tested R-Ras as a substrate.
3.1. Cytotoxic e¡ects on cultured cells caused by TcdA-C34 and TcdB-C34 Intoxication of cultured CHO and FSK cells with puri¢ed TcdA-C34 resulted in a D-type cytopathic e¡ect (data not shown), as typically seen following application of C. di¤cile TcdA-10463 and TcdB-10463 [10,12]. In contrast to reference toxin TcdB-10463, TcdB-C34 induced an Stype cytopathic e¡ect on CHO and FSK cells (Fig. 1C,D). An S-type cytopathic e¡ect was also induced by the variant cytotoxins TcdB-1470 and TcdB-8864 [6,7]. 3.2. Analysis of TcdA and TcdB of strain C34 SDS^PAGE analysis of the puri¢ed proteins revealed that TcdA-C34 and TcdA-10463 have comparable sizes of approximately 308 kDa [16] (Fig. 2A). TcdB-C34 and TcdB-10463 have sizes of about 270 kDa (Fig. 2A). To prove that TcdA-C34 and TcdB-C34 are functional glycosyltransferases their catalytic potentials were tested. The glucosyltransferase activity of TcdA-C34 was tested on a variety of small GTP binding proteins of the Ras and Rho subfamilies. These experiments demonstrated that GTPases Rho, Rac, Cdc42, and Rap are substrates for TcdA-C34 [16] (Fig. 2B). Thus, the substrates of TcdAC34 and the reference toxin TcdA-10463 are identical [10,17]. When TcdB-C34 was tested for its glucosyltransferase activity it inactivated Rho, Rac, Cdc42, Rap, and Ral (Fig. 2B). A comparison of TcdB cluster 1^2 of strains 1470, 8864, and C34 revealed four unique amino acid residues in the sequence of strain C34 (Fig. 3B). At position 3 of TcdB-C34 cluster 1^2, the neutral amino acid leucine is replaced by the polar amino acid serine. Polar amino acids are involved in the formation of hydrogen bonds which stabilize the protein structure. At position 177 the cyclic amino acid proline is situated in TcdB-C34 instead of the polar amino acid glutamine. Due to a missing free hydrogen, proline cannot be involved in the formation of hydrogen bonds. The neutral amino acid valine is present in TcdB-C34 at position 192 instead of the neutral amino acid alanine. At position 356 the polar amino acid asparagine is situated in TcdB-C34 whereas the negatively charged aspartic acid occupies this position in TcdB1470 and TcdB-8864. A computer-based analysis points out di¡erences in the secondary structure of TcdB-C34 cluster 1^2. The analysis, carried out with two separate algorithms, demonstrates that the proline at position 177 in TcdB-C34 cluster 1^2 disrupts an K-helix which is
Fig. 2. Size comparison of puri¢ed toxins and analysis of their glucosyltransferase activity. A: FPLC-puri¢ed TcdA and TcdB from C. di¤cile strains VPI 10463 (2 Wg of each toxin) and C34 (1 Wg of each toxin) were separated on a 7.5% SDS^PAGE gel and subsequently stained with Coomassie blue. The molecular mass of the toxin bands is indicated at the side. B: Radioactive [14 C]glucose labeling of substrates Rho, Rac, Cdc42, Ras, Rap, and Ral was analyzed with TcdA-C34 and TcdB-C34 as the glucosyltransferase. Note that Ras and Ral are not modi¢ed by TcdA-C34. Only the small GTPase Ras is not a substrate of TcdB-C34. C: Proof of R-Ras labeling by the variant toxins TcdBC34, TcdB-1470, and TcdB-8864, and the lethal toxin of C. sordellii, TcsL-1522. As expected, R-Ras is not modi¢ed by TcdB-10463 and TcdA-C34.
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Fig. 3. Map of the pathogenicity locus of C. di¤cile C34. A: Partial map of the C. di¤cile C34 pathogenicity locus (shaded in gray). The tcdA^D genes are shown as open boxes with arrows indicating the direction of gene transcription. The adjacent ORFs cdu1 and cdd1 are located outside the pathogenicity locus. Relative positions of PCR products covering the pathogenicity locus are depicted above the map. Primers and conditions used for PCR ampli¢cations have been presented elsewhere [8]. Insertions in the pathogenicity locus are depicted as triangles below the map together with comparative ampli¢cations of PL2 and A1 [16] of C34 and the reference strain VPI 10463. PCR products are shown after separation on a 1% agarose gel (M: GeneRuler 1-kb DNA ladder in kb from top to bottom: 10, 8, 6, 5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.75). B: Comparison of the amino acid sequence of TcdB cluster 1^2 between the variant strains C34, 1470 and 8864. The catalytically active part of the cytotoxins is situated in cluster 1^2 [14]. The di¡ering amino acid residues are depicted in larger ovals, the ¢rst and last amino acids of the TcdB cluster 1^2 are shown in a smaller gray oval. Black dots indicate the spaces between the residues. Exact positions of the varying residues are displayed at the top bar.
As shown in Fig. 2C, puri¢ed TcdB-C34, TcdB-1470 and TcdB-8864 ^ and additionally TcsL-1522 ^ modi¢ed R-Ras, whereas toxins TcdB-10463 and TcdA-C34 did not. 3.3. The pathogenicity locus of C. di¤cile C34 PCR products covering the pathogenicity locus of C. di¤cile C34 were generated and compared with corresponding products obtained from the reference strain 10463 (Fig. 3A). The sizes of all PCR fragments of strains C34 and 10463 were identical (data not shown), except for the two fragments PL2-C34 and A1-C34 [16] (Fig. 3A). The PL2 fragment of strain C34 is approximately 150 bp larger compared to PL2 of the reference strain. It covers the upstream region of the pathogenicity locus. A similar PL2 fragment is present in a number of C. di¤cile isolates
including the variant strains 1470 and 8864 [4]. Amplicon A1-C34, which encodes the catalytic domain of toxin TcdA, is about 2000 bp larger than A1-10463 [16]. No similar insertion into the pathogenicity locus ^ particularly into the tcdA gene ^ has been found in any of the 400 isolates from C. di¤cile strain collections available to us [4,16]. The properties of the large clostridial cytotoxins of strain C34 reported herein are summarized in Table 1 and compared to data known for large clostridial cytotoxins produced by strains 1470, 8864 and 10463. 4. Discussion C. di¤cile C34 is a pathogenic clinical isolate which was collected in the course of a nosocomial diarrhea study.
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Table 1 Properties of large clostridial cytotoxins prepared from di¡erent C. di¤cile strains Isolate
C34
1470
8864
10463
tcdA gene
Insertion [2]
Deletion [8]
Reference type [22]
tcdB gene Protein size TcdA Protein size TcdB TcdA substrates TcdB substrates
RFLPsa 308 kDab 270 kDab Rho, Rac, Cdc42, Rapb Rho, Rac, Cdc42, Rap, Ral, R-Rasb D-typeb S-typeb
Deletion and frameshift mutation [9] RFLPs [9] not expressed [9] 270 kDa [7] ^ Rac, Cdc42, Rap, Ral, R-Ras [11] ^ S-type [7]
RFLPs [8] not expressed [6,8] 270 kDa [6,8] ^ Rac, Cdc42, Rap, Ral [19], R-Rasb ^ S-type [6]
Reference type [22] 308 kDa [15] 270 kDa [15] Rho, Rac, Cdc42, Rap [10] Rho, Rac, Cdc42 [10,17]
Cytopathic e¡ect TcdA Cytopathic e¡ect TcdB a b
D-type [10] D-type [10^12]
Data not shown. Data presented in this work.
Strain C34 attracted our attention because TcdB-C34 induces an S-type cytopathic e¡ect typically observed after intoxication of cells with variant cytotoxins like TcdB1470 and TcdB-8864 [6,7] (Fig. 1C,D). Usually, C. di¤cile large clostridial cytotoxins cause a D-type cytopathic e¡ect like that of the reference strain 10463 [7] (Fig. 1B). In contrast to C. di¤cile strains 1470 and 8864, which both have deletions in their tcdA genes [8,9], C34 carries an insertion of 1975 bp in tcdA [16] (Fig. 3A). This insertion was shown to be a complex ribozyme precisely spliced from the premature mRNA [16]. In consequence the TcdA protein is of regular size and displays a substrate speci¢city and cytopathic e¡ect identical to the reference enterotoxin TcdA-10463 [10] (Fig. 2 and Table 1). Thus, strain C34 is the ¢rst C. di¤cile strain that expresses a variant TcdB and a functional TcdA of the reference type. It has been suggested before that a correlation between the inactivation of R-Ras and the S-type cytopathic e¡ect [11], and between Rho modi¢cation and the D-type cytopathic e¡ect [13] should exist. Knowing that endogenous R-Ras is a regulator of integrin activation [18], it was shown that inactivation of R-Ras leads to the loss of integrin adhesiveness to the extracellular matrix [11,18]. A disassembly of focal adhesion complexes is a further result of integrin inactivation, thus the manifestation of the Stype cytopathic e¡ect was hypothesized to result from RRas inactivation [11]. Chaves-Olarte et al. [11] further speculate that inactivation of Rho induces a partial disassembly of the focal adhesion complexes with the integrins remaining active and in place, resulting in a D-type cytopathic e¡ect. Similarly Schmidt and Aktories [13] hypothesized that induction of the D-type cytopathic e¡ect is correlated with inactivation of small GTPases of the Rho subfamily (Rho, Rac and Cdc42). In view of these hypotheses we analyzed the substrate speci¢city of TcdB-C34. It is the ¢rst known variant cytotoxin that induces an S-type cytopathic e¡ect while modifying Rho in addition to other small GTPases known to be substrates of the variant toxins TcdB-1470 and TcdB8864 [8,11,19] (Fig. 2B,C and Table 1). Furthermore, we demonstrate in this study that inactivation of the GTPase
R-Ras is a feature shared by TcdB-C34, TcdB-8864, TcdB-1470, and TcsL-1522 [11] (Fig. 2C and Table 1). Analysis of the amino acid sequence of TcdB-C34 cluster 1^2 reveals four unique residues compared to TcdB1470 and TcdB-8864 (Fig. 3B). Recently, the importance of two conserved regions within the catalytically active part of large clostridial cytotoxins was discussed [20,21]. A conserved tryptophan residue at position 102 and the existence of a conserved DXD motif at position 286^288 (TcdB-10463) was shown to be essential for the enzymatic activity of large clostridial cytotoxins. These conserved regions are also present in TcdB-C34, but the varying amino acid residues are not in close proximity (Fig. 3B). The computer-based prediction of a di¡erent secondary structure probably re£ects the unique substrate speci¢city of TcdB-C34. Our observation that the Rho-modifying cytotoxin TcdB-C34 induces an S-type cytopathic e¡ect supports the hypothesis of Chaves-Olarte et al. [11]. Considering the hypothesis of Schmidt and Aktories [13], the activity of toxin TcdB-C34 demonstrates that inactivation of GTPases belonging to the Rho subfamily (e.g., Rho, Rac and Cdc42) does not necessarily result in a D-type cytopathic e¡ect. In this case the S-type cytopathic e¡ect induced by the variant cytotoxin TcdB-C34 dominates over the D-type cytopathic e¡ect. The present study points out that strain C34 is the ¢rst C. di¤cile isolate coexpressing a D-type-inducing TcdA with an S-type-inducing TcdB molecule. Moreover, with the aid of TcdB-C34 ^ the ¢rst large clostridial cytotoxin to modify Rho and R-Ras ^ we could substantiate the hypothesis of Chaves-Olarte et al. [11] that R-Ras is a key GTPase related to the S-type cytopathic e¡ect. Our data suggest that induction of an S-type cytopathic e¡ect dominates induction of the D-type cytopathic e¡ect. Acknowledgements The work of the laboratory of C.v.E. was supported by Grant Ei206/9-1 from the Deutsche Forschungsgemein-
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schaft. Some of the data reported here will be presented in the PhD Thesis of M.M. C.v.E. expresses special gratitude to the Johannes Gutenberg-Universita«t in Mainz for providing laboratory space in the Verfu«gungsgeba«ude fu«r Forschung und Entwicklung. D.E.M. acknowledges the past support of the National Health Research and Development Program of Canada (Grant 6603-1252-54) when strain C34 was isolated. References [1] Knoop, F.C., Owens, M. and Crocker, I.C. (1993) Clostridium di¤cile: clinical disease and diagnosis. Clin. Microbiol. Rev. 6, 251^265. [2] Braun, V., Hundsberger, T., Leukel, P., Sauerborn, M. and von Eichel-Streiber, C. (1996) De¢nition of the single integration site of the pathogenicity locus in Clostridium di¤cile. Gene 181, 29^38. [3] von Eichel-Streiber, C., Boquet, P., Sauerborn, M. and Thelestam, M. (1996) Large clostridial cytotoxins ^ a family of glycosyltransferases modifying small GTP-binding proteins. Trends Microbiol. 4, 375^382. [4] Rupnik, M., Avesani, V., Janc, M., von Eichel-Streiber, C. and Delmee, M. (1998) A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium di¤cile isolates. J. Clin. Microbiol. 36, 2240^2247. [5] Giry, M., Popo¡, M.R., von Eichel-Streiber, C. and Boquet, P. (1995) Transient expression of RhoA, -B, and -C GTPases in HeLa cells potentiates resistance to Clostridium di¤cile toxins A and B but not to Clostridium sordellii lethal toxin. Infect. Immun. 63, 4063^ 4071. [6] Torres, J.F. (1991) Puri¢cation and characterisation of toxin B from a strain of Clostridium di¤cile that does not produce toxin A. J. Med. Microbiol. 35, 40^44. [7] von Eichel-Streiber, C., Meyer zu Heringdorf, D., Habermann, E. and Sartingen, S. (1995) Closing in on the toxic domain through analysis of a variant Clostridium di¤cile cytotoxin B. Mol. Microbiol. 17, 313^321. [8] Soehn, F., Wagenknecht-Wiesner, A., Leukel, P., Kohl, M., Weidmann, M., von Eichel-Streiber, C. and Braun, V. (1998) Genetic rearrangements in the pathogenicity locus of Clostridium di¤cile strain 8864 ^ implications for transcription, expression and enzymatic activity of toxins A and B. Mol. Gen. Genet. 258, 222^232. [9] von Eichel-Streiber, C., Zec-Pirnat, I., Grabnar, M. and Rupnik, M. (1999) A nonsense mutation abrogates production of a functional enterotoxin A in Clostridium di¤cile toxinotype VIII strains of serogroups F and X. FEMS Microbiol. Lett. 178, 163^168.
[10] Chaves-Olarte, E., Weidmann, M., von Eichel-Streiber, C. and Thelestam, M. (1997) Toxins A and B from Clostridium di¤cile di¡er with respect to enzymatic potencies, cellular substrate speci¢cities, and surface binding to cultured cells. J. Clin. Invest. 100, 1734^1741. [11] Chaves-Olarte, E., Low, P., Freer, E., Norlin, T., Weidmann, M., von Eichel-Streiber, C. and Thelestam, M. (1999) A novel cytotoxin from Clostridium di¤cile serogroup F is a functional hybrid between two other large clostridial cytotoxins. J. Biol. Chem. 274, 11046^ 11052. [12] Donta, S.T., Sullivan, N. and Wilkins, T.D. (1982) Di¡erential e¡ects of Clostridium di¤cile toxins on tissue-cultured cells. J. Clin. Microbiol. 15, 1157^1158. [13] Schmidt, G. and Aktories, K. (1998) Bacterial cytotoxins target Rho GTPases. Naturwissenschaften 85, 253^261. [14] Wagenknecht-Wiesner, A., Weidmann, M., Braun, V., Leukel, P., Moos, M. and von Eichel-Streiber, C. (1997) Delineation of the catalytic domain of Clostridium di¤cile toxin B-10463 to an enzymatically active N-terminal 467 amino acid fragment. FEMS Microbiol. Lett. 152, 109^116. [15] von Eichel-Streiber, C., Harperath, U., Bosse, D. and Hadding, U. (1987) Puri¢cation of two high molecular weight toxins of Clostridium di¤cile which are antigenically related. Microb. Pathogen. 2, 307^ 318. [16] Braun, V., Mehlig, M., Moos, M., Rupnik, M., Kalt, B., Mahony, D.E. and von Eichel-Streiber, C. (2000) A chimeric ribozyme in Clostridium di¤cile combines features of group I introns and insertion elements. Mol. Microbiol. 36, 1447^1459. [17] Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M. and Aktories, K. (1995) Glucosylation of Rho proteins by Clostridium di¤cile toxin B. Nature 375, 500^503. [18] Zhang, Z., Vuori, K., Wang, H., Reed, J.C. and Ruoslahti, E. (1996) Integrin activation by R-ras. Cell 85, 61^69. [19] Muller, S., von Eichel-Streiber, C. and Moos, M. (1999) Impact of amino acids 22^27 of Rho-subfamily GTPases on glucosylation by the large clostridial cytotoxins TcsL-1522, TcdB-1470 and TcdB8864. Eur. J. Biochem. 266, 1073^1080. [20] Busch, C., Hofmann, F., Selzer, J., Munro, S., Jeckel, D. and Aktories, K. (1998) A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins. J. Biol. Chem. 273, 19566^19572. [21] Busch, C., Hofmann, F., Gerhard, R. and Aktories, K. (2000) Involvement of a conserved tryptophan residue in the UDP-glucose binding of large clostridial cytotoxin glycosyltransferases. J. Biol. Chem. 275, 13228^13234. [22] von Eichel-Streiber, C., Laufenberg-Feldmann, R., Sartingen, S., Schulze, J. and Sauerborn, M. (1992) Comparative sequence analysis of the Clostridium di¤cile toxins A and B. Mol. Gen. Genet. 233, 260^268.
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Variant toxin B and a functional toxin A produced by Clostridium di¤cile C34 Markus Mehlig a , Michael Moos a , Veit Braun a , Bettina Kalt a , David E. Mahony b , Christoph von Eichel-Streiber a; * a
Verfu«gungsgeba«ude fu«r Forschung und Entwicklung, Institut fu«r Medizinische Mikrobiologie und Hygiene, Johannes Gutenberg-Universita«t, 55101 Mainz, Germany b Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada B3H 4H7 Received 29 January 2001 ; received in revised form 11 March 2001; accepted 15 March 2001
Abstract A particular property of Clostridium difficile strain C34 is an insertion of approximately 2 kb in the tcdA-C34 gene that does not hinder expression of a fully active TcdA-C34 molecule. Intoxication with TcdA-C34 induced an arborized appearance in eukaryotic cells (D-type cytopathic effect); intoxication with TcdB-C34 induced a spindle-like appearance of cells (S-type cytopathic effect). Inactivation of GTPases with purified toxins revealed that Rho, Rac, Cdc42, and Rap are substrates of TcdA-C34. The variant cytotoxin TcdB-C34 inactivated Rho, Rac, Cdc42, Rap, Ral, and R-Ras. Hence, this is the first `S-type' cytotoxin which inactivates both Rho and R-Ras, and is coexpressed with a `D-type' enterotoxin. Our results support the hypothesis that R-Ras is a key GTPase related to the S-type cytopathic effect and suggest that induction of a S-type cytopathic effect dominates induction of the D-type cytopathic effect. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Large clostridial cytotoxin; Cytopathic e¡ect; Rho; R-Ras; Glucosylation ; GTPase
1. Introduction Toxin-producing Clostridium di¤cile strains are known to cause antibiotic-associated diarrhea and pseudomembranous colitis in man [1]. Such strains produce two major virulence factors, an enterotoxin TcdA and a cytotoxin TcdB, both encoded by the pathogenicity locus [2]. TcdA and TcdB are large clostridial cytotoxins that glucosylate the Ras and Rho subfamilies of GTPases [3]. Variations in toxin genes of di¡erent strains are common and useful for di¡erentiation [4], whereas the cytopathic e¡ects of the toxins are less heterogeneous. Following intoxication of eukaryotic cells, D-type and S-type cytopathic e¡ects with a clear di¡erence in actin staining have been reported [5]. The D-type cytopathic e¡ect is characterized by an arborized appearance of the cells whereas a spindle-like appearance is typical of the S-type
* Corresponding author. Tel. : +49 (6131) 3930020; Fax: +49 (6131) 3933364; E-mail : [email protected]
cytopathic e¡ect. The S-type cytopathic e¡ect is exclusively induced by variant cytotoxins, such as TcdB-1470 and TcdB-8864 [6,7], or by the lethal toxin TcsL of Clostridium sordellii. Due to deletions and frameshift mutations no functional enterotoxins are expressed in strains 1470 and 8864 [6,8,9]. In strains which coexpress the enterotoxin and the cytotoxin, both induce a D-type cytopathic e¡ect as reported for reference strain 10463 [10^12]. Recently, Chaves-Olarte et al. [11] suggested that the inactivation of the small GTP binding protein R-Ras is correlated with the S-type cytopathic e¡ect. Similarly, Schmidt and Aktories [13] proposed that inactivation of small GTPases of the Rho subfamily (e.g., Rho, Rac and Cdc42) is correlated with a D-type cytopathic e¡ect. Both interpretations explained the observation that none of the known large clostridial cytotoxins inactivated both R-Ras and Rho. With the clinical isolate C34 we present the ¢rst strain coexpressing a variant `S-type cytotoxin' TcdB-C34 and a fully functional `D-type enterotoxin' TcdA-C34. Moreover, TcdB-C34 inactivates both R-Ras and Rho, and a proposed dominance of R-Ras inactivation on the observed cytopathic e¡ect is discussed.
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 1 4 3 - 4
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2. Materials and methods 2.1. Bacterial strains, chemicals and media C. di¤cile strain 10463 was a gift of N.M. Sullivan (Virginia Polytechnic Institute, Blacksburg, VA, USA). C. di¤cile C34 is a clinical isolate from Halifax, NS, Canada, C. di¤cile strain 8864 was obtained from the Swedish type culture collection (strain 20309), C. di¤cile strain 1470 from Michel Delme¨e (Unite¨ de Microbiologie, Universite¨ de Louvain, Brussels, Belgium) and C. sordellii RE1522 from H.-P. Schau (Thu«ringer Medizinal-, Lebensmittel- und Veterina«runtersuchungsamt, Erfurt, Germany). All clostridial strains were grown in an anaerobic chamber (Don Whitley Scienti¢c, Shipley, UK) using brain heart infusion (Difco, Augsburg, Germany) or Wilkins^Chalgren broth (Oxoid, Wesel, Germany). Chemicals and media were purchased from Merck (Darmstadt, Germany), Sigma (Heidelberg, Germany), Roth (Karlsruhe, Germany), Qiagen (Hilden, Germany), and Gibco BRL Life Technologies (Burlington, ON, Canada). Oligonucleotides were ordered from MWG-Biotech (Ebersberg, Germany). 2.2. Genetic analysis To characterize the pathogenicity locus of C. di¤cile strain C34, 10 PCR ampli¢cations representing the entire pathogenicity locus were performed [8] (Fig. 3A) in a Hybaid Omnigene Cycler (MWG-Biotech). TcdB-C34 cluster 1^2, representing the catalytically active part of the cytotoxin, was ampli¢ed [14] and cloned into plasmid pCR2.1 using the TOPO TA cloning kit in accordance with the manufacturer's instructions (Invitrogen, Groningen, The Netherlands). Sequencing was done with a LICOR 4000L automatic DNA sequencer from MWG-Biotech and the Thermosequenase Cycle Sequencing Kit from Amersham Pharmacia Biotech (Freiburg, Germany). Primers used for sequencing were IRD-800-labelled (MWG-Biotech). Two independent clones were sequenced to guarantee the reliability of the PCR-based sequence. The sequence data of tcdB-C34 cluster 1^2 are available in the EMBL database under accession number AJ294944. Computer-based secondary structure analysis was done with Protean from DNASTAR Software. The Chou^Fasman and the Garnier^Robson secondary structure prediction algorithms were used to determine the secondary structure of the proteins.
Rho, Rac, Cdc42, Ras, Rap, Ral, and R-Ras as target proteins. Radiolabeled proteins were separated by SDS^ PAGE and the bands were visualized by PhosphoImager analysis (Molecular Dynamics, Krefeld, Germany). 2.4. Cell culture and scanning electron microscopy Cytotoxicity of C. di¤cile toxins and culture supernatants were tested on Chinese hamster ovary cells (CHO cells) as described elsewhere [7]. Additionally, human foreskin cells (FSK cells) were used to detect cytotoxicity. The cells were grown as monolayers in 96-well tissue culture plates (Costar, Cambridge, MA, USA) in a humidi¢ed incubator under an atmosphere of 5% CO2 . The growth medium consisted of minimum essential medium (MEM, Gibco BRL) containing streptomycin, penicillin and 10% fetal bovine serum. Maintenance medium used in the cytotoxicity assay consisted of MEM supplemented with 1% fetal calf serum. For scanning electron microscopy, the FSK cells were grown as monolayers on glass coverslips placed on the bottom of glass vials containing MEM as described above. The culture £uids were removed and crude toxin, at a dilution of 1:1000 in MEM medium containing 1% fetal calf serum, was applied to the monolayers. After incubation for 5 h and 21 h, the coverslips were removed from the vials, placed in 2.5% glutaraldehyde solution in 0.1 M sodium cacodylate bu¡er (pH 7.4) for 1 h, osmicated with 1% osmium tetroxide, dehydrated in a series of ethanol concentrations and dried in a Balzer
2.3. Protein preparation and glucosyltransferase activity C. di¤cile toxins TcdA and TcdB and C. sordellii TcsL were prepared as described previously [15]. Purity of toxin preparations was determined by SDS^PAGE. Glucosyltransferase reactions were performed as described by Wagenknecht-Wiesner et al. [14], using recombinant GTPases
Fig. 1. Scanning electron microscopy of FSK cells incubated with or without toxin at 37³C in an atmosphere of 5% CO2 . A: FSK control cells without toxin added and processed for electron microscopy 5 h after the addition of MEM medium. B: FSK cells after 5 h pre-treatment with TcdB-10463. C: FSK cells after 5 h pre-treatment with TcdB-C34. D: Single FSK cell exposed to TcdB-C34 for 21 h prior to processing for electron microscopy.
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critical point drier using CO2 as the drying £uid. Following sputter coating with gold palladium, the samples were viewed with a Cambridge S150 stereoscan scanning electron microscope. 3. Results
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present in TcdB-1470 and TcdB-8864. Both methods predict a di¡erent secondary structure of TcdB-C34 cluster 1^ 2. Since a correlation between the inactivation of R-Ras and the induction of the S-type cytopathic e¡ect was recently suggested [11], we also tested R-Ras as a substrate.
3.1. Cytotoxic e¡ects on cultured cells caused by TcdA-C34 and TcdB-C34 Intoxication of cultured CHO and FSK cells with puri¢ed TcdA-C34 resulted in a D-type cytopathic e¡ect (data not shown), as typically seen following application of C. di¤cile TcdA-10463 and TcdB-10463 [10,12]. In contrast to reference toxin TcdB-10463, TcdB-C34 induced an Stype cytopathic e¡ect on CHO and FSK cells (Fig. 1C,D). An S-type cytopathic e¡ect was also induced by the variant cytotoxins TcdB-1470 and TcdB-8864 [6,7]. 3.2. Analysis of TcdA and TcdB of strain C34 SDS^PAGE analysis of the puri¢ed proteins revealed that TcdA-C34 and TcdA-10463 have comparable sizes of approximately 308 kDa [16] (Fig. 2A). TcdB-C34 and TcdB-10463 have sizes of about 270 kDa (Fig. 2A). To prove that TcdA-C34 and TcdB-C34 are functional glycosyltransferases their catalytic potentials were tested. The glucosyltransferase activity of TcdA-C34 was tested on a variety of small GTP binding proteins of the Ras and Rho subfamilies. These experiments demonstrated that GTPases Rho, Rac, Cdc42, and Rap are substrates for TcdA-C34 [16] (Fig. 2B). Thus, the substrates of TcdAC34 and the reference toxin TcdA-10463 are identical [10,17]. When TcdB-C34 was tested for its glucosyltransferase activity it inactivated Rho, Rac, Cdc42, Rap, and Ral (Fig. 2B). A comparison of TcdB cluster 1^2 of strains 1470, 8864, and C34 revealed four unique amino acid residues in the sequence of strain C34 (Fig. 3B). At position 3 of TcdB-C34 cluster 1^2, the neutral amino acid leucine is replaced by the polar amino acid serine. Polar amino acids are involved in the formation of hydrogen bonds which stabilize the protein structure. At position 177 the cyclic amino acid proline is situated in TcdB-C34 instead of the polar amino acid glutamine. Due to a missing free hydrogen, proline cannot be involved in the formation of hydrogen bonds. The neutral amino acid valine is present in TcdB-C34 at position 192 instead of the neutral amino acid alanine. At position 356 the polar amino acid asparagine is situated in TcdB-C34 whereas the negatively charged aspartic acid occupies this position in TcdB1470 and TcdB-8864. A computer-based analysis points out di¡erences in the secondary structure of TcdB-C34 cluster 1^2. The analysis, carried out with two separate algorithms, demonstrates that the proline at position 177 in TcdB-C34 cluster 1^2 disrupts an K-helix which is
Fig. 2. Size comparison of puri¢ed toxins and analysis of their glucosyltransferase activity. A: FPLC-puri¢ed TcdA and TcdB from C. di¤cile strains VPI 10463 (2 Wg of each toxin) and C34 (1 Wg of each toxin) were separated on a 7.5% SDS^PAGE gel and subsequently stained with Coomassie blue. The molecular mass of the toxin bands is indicated at the side. B: Radioactive [14 C]glucose labeling of substrates Rho, Rac, Cdc42, Ras, Rap, and Ral was analyzed with TcdA-C34 and TcdB-C34 as the glucosyltransferase. Note that Ras and Ral are not modi¢ed by TcdA-C34. Only the small GTPase Ras is not a substrate of TcdB-C34. C: Proof of R-Ras labeling by the variant toxins TcdBC34, TcdB-1470, and TcdB-8864, and the lethal toxin of C. sordellii, TcsL-1522. As expected, R-Ras is not modi¢ed by TcdB-10463 and TcdA-C34.
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Fig. 3. Map of the pathogenicity locus of C. di¤cile C34. A: Partial map of the C. di¤cile C34 pathogenicity locus (shaded in gray). The tcdA^D genes are shown as open boxes with arrows indicating the direction of gene transcription. The adjacent ORFs cdu1 and cdd1 are located outside the pathogenicity locus. Relative positions of PCR products covering the pathogenicity locus are depicted above the map. Primers and conditions used for PCR ampli¢cations have been presented elsewhere [8]. Insertions in the pathogenicity locus are depicted as triangles below the map together with comparative ampli¢cations of PL2 and A1 [16] of C34 and the reference strain VPI 10463. PCR products are shown after separation on a 1% agarose gel (M: GeneRuler 1-kb DNA ladder in kb from top to bottom: 10, 8, 6, 5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.75). B: Comparison of the amino acid sequence of TcdB cluster 1^2 between the variant strains C34, 1470 and 8864. The catalytically active part of the cytotoxins is situated in cluster 1^2 [14]. The di¡ering amino acid residues are depicted in larger ovals, the ¢rst and last amino acids of the TcdB cluster 1^2 are shown in a smaller gray oval. Black dots indicate the spaces between the residues. Exact positions of the varying residues are displayed at the top bar.
As shown in Fig. 2C, puri¢ed TcdB-C34, TcdB-1470 and TcdB-8864 ^ and additionally TcsL-1522 ^ modi¢ed R-Ras, whereas toxins TcdB-10463 and TcdA-C34 did not. 3.3. The pathogenicity locus of C. di¤cile C34 PCR products covering the pathogenicity locus of C. di¤cile C34 were generated and compared with corresponding products obtained from the reference strain 10463 (Fig. 3A). The sizes of all PCR fragments of strains C34 and 10463 were identical (data not shown), except for the two fragments PL2-C34 and A1-C34 [16] (Fig. 3A). The PL2 fragment of strain C34 is approximately 150 bp larger compared to PL2 of the reference strain. It covers the upstream region of the pathogenicity locus. A similar PL2 fragment is present in a number of C. di¤cile isolates
including the variant strains 1470 and 8864 [4]. Amplicon A1-C34, which encodes the catalytic domain of toxin TcdA, is about 2000 bp larger than A1-10463 [16]. No similar insertion into the pathogenicity locus ^ particularly into the tcdA gene ^ has been found in any of the 400 isolates from C. di¤cile strain collections available to us [4,16]. The properties of the large clostridial cytotoxins of strain C34 reported herein are summarized in Table 1 and compared to data known for large clostridial cytotoxins produced by strains 1470, 8864 and 10463. 4. Discussion C. di¤cile C34 is a pathogenic clinical isolate which was collected in the course of a nosocomial diarrhea study.
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Table 1 Properties of large clostridial cytotoxins prepared from di¡erent C. di¤cile strains Isolate
C34
1470
8864
10463
tcdA gene
Insertion [2]
Deletion [8]
Reference type [22]
tcdB gene Protein size TcdA Protein size TcdB TcdA substrates TcdB substrates
RFLPsa 308 kDab 270 kDab Rho, Rac, Cdc42, Rapb Rho, Rac, Cdc42, Rap, Ral, R-Rasb D-typeb S-typeb
Deletion and frameshift mutation [9] RFLPs [9] not expressed [9] 270 kDa [7] ^ Rac, Cdc42, Rap, Ral, R-Ras [11] ^ S-type [7]
RFLPs [8] not expressed [6,8] 270 kDa [6,8] ^ Rac, Cdc42, Rap, Ral [19], R-Rasb ^ S-type [6]
Reference type [22] 308 kDa [15] 270 kDa [15] Rho, Rac, Cdc42, Rap [10] Rho, Rac, Cdc42 [10,17]
Cytopathic e¡ect TcdA Cytopathic e¡ect TcdB a b
D-type [10] D-type [10^12]
Data not shown. Data presented in this work.
Strain C34 attracted our attention because TcdB-C34 induces an S-type cytopathic e¡ect typically observed after intoxication of cells with variant cytotoxins like TcdB1470 and TcdB-8864 [6,7] (Fig. 1C,D). Usually, C. di¤cile large clostridial cytotoxins cause a D-type cytopathic e¡ect like that of the reference strain 10463 [7] (Fig. 1B). In contrast to C. di¤cile strains 1470 and 8864, which both have deletions in their tcdA genes [8,9], C34 carries an insertion of 1975 bp in tcdA [16] (Fig. 3A). This insertion was shown to be a complex ribozyme precisely spliced from the premature mRNA [16]. In consequence the TcdA protein is of regular size and displays a substrate speci¢city and cytopathic e¡ect identical to the reference enterotoxin TcdA-10463 [10] (Fig. 2 and Table 1). Thus, strain C34 is the ¢rst C. di¤cile strain that expresses a variant TcdB and a functional TcdA of the reference type. It has been suggested before that a correlation between the inactivation of R-Ras and the S-type cytopathic e¡ect [11], and between Rho modi¢cation and the D-type cytopathic e¡ect [13] should exist. Knowing that endogenous R-Ras is a regulator of integrin activation [18], it was shown that inactivation of R-Ras leads to the loss of integrin adhesiveness to the extracellular matrix [11,18]. A disassembly of focal adhesion complexes is a further result of integrin inactivation, thus the manifestation of the Stype cytopathic e¡ect was hypothesized to result from RRas inactivation [11]. Chaves-Olarte et al. [11] further speculate that inactivation of Rho induces a partial disassembly of the focal adhesion complexes with the integrins remaining active and in place, resulting in a D-type cytopathic e¡ect. Similarly Schmidt and Aktories [13] hypothesized that induction of the D-type cytopathic e¡ect is correlated with inactivation of small GTPases of the Rho subfamily (Rho, Rac and Cdc42). In view of these hypotheses we analyzed the substrate speci¢city of TcdB-C34. It is the ¢rst known variant cytotoxin that induces an S-type cytopathic e¡ect while modifying Rho in addition to other small GTPases known to be substrates of the variant toxins TcdB-1470 and TcdB8864 [8,11,19] (Fig. 2B,C and Table 1). Furthermore, we demonstrate in this study that inactivation of the GTPase
R-Ras is a feature shared by TcdB-C34, TcdB-8864, TcdB-1470, and TcsL-1522 [11] (Fig. 2C and Table 1). Analysis of the amino acid sequence of TcdB-C34 cluster 1^2 reveals four unique residues compared to TcdB1470 and TcdB-8864 (Fig. 3B). Recently, the importance of two conserved regions within the catalytically active part of large clostridial cytotoxins was discussed [20,21]. A conserved tryptophan residue at position 102 and the existence of a conserved DXD motif at position 286^288 (TcdB-10463) was shown to be essential for the enzymatic activity of large clostridial cytotoxins. These conserved regions are also present in TcdB-C34, but the varying amino acid residues are not in close proximity (Fig. 3B). The computer-based prediction of a di¡erent secondary structure probably re£ects the unique substrate speci¢city of TcdB-C34. Our observation that the Rho-modifying cytotoxin TcdB-C34 induces an S-type cytopathic e¡ect supports the hypothesis of Chaves-Olarte et al. [11]. Considering the hypothesis of Schmidt and Aktories [13], the activity of toxin TcdB-C34 demonstrates that inactivation of GTPases belonging to the Rho subfamily (e.g., Rho, Rac and Cdc42) does not necessarily result in a D-type cytopathic e¡ect. In this case the S-type cytopathic e¡ect induced by the variant cytotoxin TcdB-C34 dominates over the D-type cytopathic e¡ect. The present study points out that strain C34 is the ¢rst C. di¤cile isolate coexpressing a D-type-inducing TcdA with an S-type-inducing TcdB molecule. Moreover, with the aid of TcdB-C34 ^ the ¢rst large clostridial cytotoxin to modify Rho and R-Ras ^ we could substantiate the hypothesis of Chaves-Olarte et al. [11] that R-Ras is a key GTPase related to the S-type cytopathic e¡ect. Our data suggest that induction of an S-type cytopathic e¡ect dominates induction of the D-type cytopathic e¡ect. Acknowledgements The work of the laboratory of C.v.E. was supported by Grant Ei206/9-1 from the Deutsche Forschungsgemein-
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schaft. Some of the data reported here will be presented in the PhD Thesis of M.M. C.v.E. expresses special gratitude to the Johannes Gutenberg-Universita«t in Mainz for providing laboratory space in the Verfu«gungsgeba«ude fu«r Forschung und Entwicklung. D.E.M. acknowledges the past support of the National Health Research and Development Program of Canada (Grant 6603-1252-54) when strain C34 was isolated. References [1] Knoop, F.C., Owens, M. and Crocker, I.C. (1993) Clostridium di¤cile: clinical disease and diagnosis. Clin. Microbiol. Rev. 6, 251^265. [2] Braun, V., Hundsberger, T., Leukel, P., Sauerborn, M. and von Eichel-Streiber, C. (1996) De¢nition of the single integration site of the pathogenicity locus in Clostridium di¤cile. Gene 181, 29^38. [3] von Eichel-Streiber, C., Boquet, P., Sauerborn, M. and Thelestam, M. (1996) Large clostridial cytotoxins ^ a family of glycosyltransferases modifying small GTP-binding proteins. Trends Microbiol. 4, 375^382. [4] Rupnik, M., Avesani, V., Janc, M., von Eichel-Streiber, C. and Delmee, M. (1998) A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium di¤cile isolates. J. Clin. Microbiol. 36, 2240^2247. [5] Giry, M., Popo¡, M.R., von Eichel-Streiber, C. and Boquet, P. (1995) Transient expression of RhoA, -B, and -C GTPases in HeLa cells potentiates resistance to Clostridium di¤cile toxins A and B but not to Clostridium sordellii lethal toxin. Infect. Immun. 63, 4063^ 4071. [6] Torres, J.F. (1991) Puri¢cation and characterisation of toxin B from a strain of Clostridium di¤cile that does not produce toxin A. J. Med. Microbiol. 35, 40^44. [7] von Eichel-Streiber, C., Meyer zu Heringdorf, D., Habermann, E. and Sartingen, S. (1995) Closing in on the toxic domain through analysis of a variant Clostridium di¤cile cytotoxin B. Mol. Microbiol. 17, 313^321. [8] Soehn, F., Wagenknecht-Wiesner, A., Leukel, P., Kohl, M., Weidmann, M., von Eichel-Streiber, C. and Braun, V. (1998) Genetic rearrangements in the pathogenicity locus of Clostridium di¤cile strain 8864 ^ implications for transcription, expression and enzymatic activity of toxins A and B. Mol. Gen. Genet. 258, 222^232. [9] von Eichel-Streiber, C., Zec-Pirnat, I., Grabnar, M. and Rupnik, M. (1999) A nonsense mutation abrogates production of a functional enterotoxin A in Clostridium di¤cile toxinotype VIII strains of serogroups F and X. FEMS Microbiol. Lett. 178, 163^168.
[10] Chaves-Olarte, E., Weidmann, M., von Eichel-Streiber, C. and Thelestam, M. (1997) Toxins A and B from Clostridium di¤cile di¡er with respect to enzymatic potencies, cellular substrate speci¢cities, and surface binding to cultured cells. J. Clin. Invest. 100, 1734^1741. [11] Chaves-Olarte, E., Low, P., Freer, E., Norlin, T., Weidmann, M., von Eichel-Streiber, C. and Thelestam, M. (1999) A novel cytotoxin from Clostridium di¤cile serogroup F is a functional hybrid between two other large clostridial cytotoxins. J. Biol. Chem. 274, 11046^ 11052. [12] Donta, S.T., Sullivan, N. and Wilkins, T.D. (1982) Di¡erential e¡ects of Clostridium di¤cile toxins on tissue-cultured cells. J. Clin. Microbiol. 15, 1157^1158. [13] Schmidt, G. and Aktories, K. (1998) Bacterial cytotoxins target Rho GTPases. Naturwissenschaften 85, 253^261. [14] Wagenknecht-Wiesner, A., Weidmann, M., Braun, V., Leukel, P., Moos, M. and von Eichel-Streiber, C. (1997) Delineation of the catalytic domain of Clostridium di¤cile toxin B-10463 to an enzymatically active N-terminal 467 amino acid fragment. FEMS Microbiol. Lett. 152, 109^116. [15] von Eichel-Streiber, C., Harperath, U., Bosse, D. and Hadding, U. (1987) Puri¢cation of two high molecular weight toxins of Clostridium di¤cile which are antigenically related. Microb. Pathogen. 2, 307^ 318. [16] Braun, V., Mehlig, M., Moos, M., Rupnik, M., Kalt, B., Mahony, D.E. and von Eichel-Streiber, C. (2000) A chimeric ribozyme in Clostridium di¤cile combines features of group I introns and insertion elements. Mol. Microbiol. 36, 1447^1459. [17] Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M. and Aktories, K. (1995) Glucosylation of Rho proteins by Clostridium di¤cile toxin B. Nature 375, 500^503. [18] Zhang, Z., Vuori, K., Wang, H., Reed, J.C. and Ruoslahti, E. (1996) Integrin activation by R-ras. Cell 85, 61^69. [19] Muller, S., von Eichel-Streiber, C. and Moos, M. (1999) Impact of amino acids 22^27 of Rho-subfamily GTPases on glucosylation by the large clostridial cytotoxins TcsL-1522, TcdB-1470 and TcdB8864. Eur. J. Biochem. 266, 1073^1080. [20] Busch, C., Hofmann, F., Selzer, J., Munro, S., Jeckel, D. and Aktories, K. (1998) A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins. J. Biol. Chem. 273, 19566^19572. [21] Busch, C., Hofmann, F., Gerhard, R. and Aktories, K. (2000) Involvement of a conserved tryptophan residue in the UDP-glucose binding of large clostridial cytotoxin glycosyltransferases. J. Biol. Chem. 275, 13228^13234. [22] von Eichel-Streiber, C., Laufenberg-Feldmann, R., Sartingen, S., Schulze, J. and Sauerborn, M. (1992) Comparative sequence analysis of the Clostridium di¤cile toxins A and B. Mol. Gen. Genet. 233, 260^268.
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