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Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile
FEMS Microbiology Letters 186 (2000) 307^312
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Production of actin-speci¢c ADP-ribosyltransferase (binary toxin) by strains of Clostridium di¤cile Simon Stubbs a
a;
*, Maja Rupnik b , Maryse Gibert c , Jon Brazier a , Brian Duerden a , Michel Popo¡ c
Anaerobe Reference Unit, Department of Medical Microbiology and Public Health Laboratory, University Hospital of Wales, Cardi¡ CF4 4XW, UK b Department of Biology, University of Ljubljana, Ljubljana, Slovenia c Unite¨ des Toxines Microbiennes, Institut Pasteur, Paris, France Received 6 March 2000; received in revised form 24 March 2000; accepted 27 March 2000
Abstract In addition to the two large clostridial cytotoxins (TcdA and TcdB) certain strains of Clostridium difficile produce an actin-specific ADPribosyltransferase, or binary toxin. PCR reactions were developed to detect genes encoding the enzymatic (cdtA) and binding (cdtB) components of the binary toxin and 170 representative strains were tested to assess the prevalence of the toxin. Positive PCR results (n = 59) were confirmed by immunoblotting and ADP-ribosyltransferase assay. PCR ribotype and toxinotype (restriction fragment length polymorphism analysis of genes for TcdA and TcdB) correlated with possession of binary toxin genes. All strains with cdtA and cdtB belonged to toxin-variable toxinotypes and five toxin-producing groups of strains have been described according to the presence or absence of TcdA, TcdB and binary toxin. Result indicate that ca. 6.4% of toxigenic isolates of C. difficile referred to the Anaerobe Reference Unit from UK hospitals have cdtA and cdtB genes. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Clostridium di¤cile ; Binary toxin; ADP-ribosyltransferase
1. Introduction Clostridia produce various toxins that induce alterations in the actin cytoskeleton. These cytotoxins have been classi¢ed into at least three groups; two of them, the C3-like toxins and the binary toxins, are ADP-ribosyltransferases, whilst the large clostridial cytotoxins (LCT) are glucosyltransferases [1^4]. The primary virulence factors produced by strains of Clostridium di¤cile belong to the LCT group, and are known as toxin A (TcdA) and toxin B (TcdB) [2,5]. TcdA and TcdB are similar in structure and activity, and have been reported to be monoglucosyltransferases that modify the low-molecular-mass GTP-binding proteins of the Rho and Ras subfamily using UDP-glucose as a cosubstrate [6^8]. One strain of C. di¤cile (CD196) has been shown to produce an actin-speci¢c ADP-ribosyltransferase (binary
* Corresponding author. Tel. : +44 (1222) 742378; Fax: +44 (1222) 742161; E-mail : [email protected]
toxin) which exhibits activity and structure similar to C. perfringens S-toxin [9]. The genes for the binary toxin have been identi¢ed and the enzymatic (cdtA) and binding components (cdtB) characterised [10]. It has been suggested that other strains may produce binary toxin, and that it may be an additional virulence factor [9,10]. Various typing schemes have been developed to study the epidemiology of C. di¤cile infection and determine the similarity of strains associated with disease. A library of distinct `types' of C. di¤cile, based upon polymerase chain reaction (PCR) ribotyping [11,12], and a toxinotyping scheme, based upon PCR-restriction fragment length polymorphism (RFLP) analysis of the LCT genes [13], have been reported. Certain `types' of C. di¤cile have been described that have signi¢cant changes in LCT genes whilst others lack enterotoxic activity but exhibit cytotoxicity [12^15]. One such strain (1470), belonging to serogroup F (toxinotype VIII, ribotype 017), has been shown to possess a cytotoxin that is a functional hybrid between TcdB and the C. sordellii lethal toxin [6,16]. The cytotoxin from another TcdA-negative strain, CCUG 20309 (strain 8864), has been reported to be equally cyto-
0378-1097 / 00 / $20.00 ß 2000 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 0 ) 0 0 1 6 2 - 2
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toxic but more lethal than the TcdB from the type strain of C. di¤cile [17]. Typing schemes have highlighted a number of epidemiologically important factors regarding the possible clonality and spread of C. di¤cile and there is some evidence to suggest that they can provide a reliable indication of virulence potential [12,13]. The aims of the present study were to detect and assess the prevalence of binary toxin genes in C. di¤cile. The study also investigated variation in binary toxin genes and examined the relationship between the possession of binary toxin genes, changes in LCT genes and ribotype.
72³C for 1 min 20 s. Nucleic acid preparations and PCR were done in triplicate. 2.4. Partial sequencing of binary toxin genes
2. Materials and methods
PCR products, generated from 11 representative strains of selected ribotypes, were cleaned with QIAquick-spin PCR clean-up columns (Qiagen Ltd., Crawley, West Sussex, UK) and sequenced with the ABI-Prism Dye Terminator Cycle Sequencing kit (Perkin-Elmer, Warrington, UK). Sequences for short cdtA products from strains R10456 and IS51, and short cdtB products from strains R8637, IS81, R10456 and IS51 have been assigned EMBL accession numbers AJ238324, AJ238325 and AJ237817^ AJ237820, respectively.
2.1. Strains, detection of TcdA and TcdB and nucleic acid preparation
2.5. SDS^PAGE, Western blotting and ADP-ribosyltransferase assay
Strains (n = 170) of C. di¤cile, belonging to 95 ribotypes [12] and 14 toxinotypes [13], have been analysed. Strains pre¢xed R were obtained from the Anaerobe Reference Unit (ARU) library [12]; other strains were obtained from NCTC, ATCC and CCUG. Enterotoxin and cytotoxin production were determined with the Tox A TEST immunoassay (TechLab, BioConnections, Leeds, UK) and Vero cell cytotoxicity [18]. Crude template nucleic acid was prepared using Chelex-100 (Bio-Rad, Hemel Hempstead, UK) [11].
Representative `type' strains (n = 13) from selected ribotypes [12] and toxinotypes [13] were cultured in brain heart infusion broth for 48 h and protein was precipitated from culture supernate with ammonium sulfate [20]. Supernatant protein (30 Wg) was electrophoresed in SDS^PAGE gels (10% acrylamide) and transferred onto nitrocellulose membranes [21]. Membranes were blocked in phosphate-bu¡ered saline containing dried milk (5%) for 1 h and incubated overnight at room temperature with rabbit immunoglobulin (1 in 5000 dilution) raised against enzymatic (Ia) and binding components (Ib) of C. perfringens S-toxin [20]. Bound antibody was detected with peroxidase-labelled protein A and the Signal Plus kit (Pierce Chemical Co., Rockford, IL, USA). For ADP-ribosyltransferase assay, the supernatant protein (10 Wg) was incubated at 37³C for 1 h in 50 Wl of a solution of 50 mM triethanolamine^HCl, pH 7.5, 5 mM MgCl2 , 10mM dithiothreitol, 10 mM thymidine containing brain extract as a source of actin (10 Wg) and [32 P]NAD (106 cpm per reaction). Protein was precipitated with 20 Wl of a solution of bovine serum albumin (1 mg ml31 ) and SDS (10% w/v) and 0.5 ml of trichloroacetic acid (10% w/v); reactions were incubated on ice for 1 h. The precipitate was immobilised on GFC ¢lters (Millipore, Watford, UK), washed twice in 10 ml of trichloroacetic acid (10% w/v), dried and counted for radioactivity.
2.2. PCR ribotyping and toxinotyping PCR ribotyping and toxinotyping were performed according to methods described previously [11,13]. Dendrograms were produced with the cluster correlation algorithm by the unweighted pair group method using arithmetic averages (UPGMA) and GelCompar 4.0 (Applied Maths, Kortrijk, Belgium). 2.3. PCR for binary toxin genes The primers Tim6 and Struppi6 amplify the cdd3 gene from all C. di¤cile strains [19] and were used as a positive PCR control. Primers designed to amplify regions of cdtA and cdtB were as follows: cdtApos 5P-TGAACCTGGAAAAGGTGATG-3P (position, cdtA 507^526); cdtArev 5P-AGGATTATTTACTGGACCATTTG-3P (position, cdtA 882^860); cdtBpos 5P-CTTAATGCAAGTAAATACTGAG-3P (position, cdtB 368^389); cdtBrev 5P-AACGGATCTCTTGCTTCAGTC-3P (position, cdtB 878^858). Template nucleic acid (5 Wl) was added to a PCR mixture (total 50 Wl; 50 mM KCl, 10 mM Tris^ HCl pH 9.0, 0.1% Triton X-100, 1.5 mM MgCl2 , 200 WM each dNTP, 0.15 WM each primer, 1 U Taq polymerase (Promega, Southampton, UK)). Reactions were subjected to 30 cycles of 94³C for 45 s, 52³C for 1 min and
3. Results 3.1. Detection of cdtA and cdtB by PCR PCR reactions with nucleic acid from CD196 (ribotype 027, toxinotype III; Table 1) resulted in products (622, 375 and 510 bp) with sequences corresponding to the cdd3, cdtA and cdtB genes (Fig. 1). PCR results for a further 169 strains belonging to 95 ribotypes and 14 tox-
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Table 1 PCR ribotype, toxinotype and results of PCR detection of cdtA and cdtB for strains of C. di¤cile PCR ribotypea
Number of strains tested
Toxinotypea
PCR detection (cdtA/cdtB)
PCR ribotypea
Number of strains tested
Toxinotypea
PCR detection (cdtA/cdtB)
001 002 003 004 005 006 007 008 009 010 011 012 013 014 015 016 017 018 019 020 021 022 023 024 025 026 027 028 029 030 031 032 033 034 035 036 037 038 039 040 041 042 043 044 045 046 047
6 3 1 1 4 1 1 1 2 2 1 3 1 3 1 1 4 1 4 3 1 1 8 1 1 2 2 1 1 1 1 1 2 5 1 1 1 1 1 1 1 1 1 1 6 2 1
0 0 I 0 0 0 0 0 NTb NT 0 0 0 0 0 0 VIII 0 IX 0 0 0 IV 0 0 NT III NT 0 NT NT NT XIc III NT X 0 NT NT NT NT 0 0 0 VI 0 VIII
3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 +/+ 3/3 3/3 3/3 +/+ 3/3 3/3 3/3 +/+ 3/3 3/3 3/3 3/3 3/3 +/+ +/+ 3/3 +/+ 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 +/+ 3/3 3/3
048 049 050 052 053 054 055 056 057 058 059 060 061 063 064 066 069 070 072 073 075 076 077 078 080 081 082 083 085 086 087 088 090 092 093 096 097 100 102 103 106 107 110 111 115 116 119 122
1 1 1 1 1 1 1 2 1 4 1 1 1 4 1 4 1 1 1 1 2 2 2 11 3 2 1 1 2 2 2 2 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 1
0 0 0 0 0 0 0 XIIc 0 IV IV NT 0 IV/VI 0 V/VI NT XIIIc 0 NT III 0 0 V III 0 NT 0 NT 0 0 NT 0 0 0 0 0 NT I II 0 0 VIII XIVc 0 0 NT XIVc
3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 +/+ +/+ 3/3 3/3 +/+ 3/3 +/+ 3/3 3/3 3/3 3/3 +/+ 3/3 3/3 +/+ +/+ 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 +/+ 3/3 3/3 3/3 +/+
a
See [15^17] for complete PCR ribotype and toxinotype data. NT: non-toxigenic strains (tcdA- and tcdB-negative). c New toxinotype (to be described elsewhere). b
inotypes are summarised in Table 1. PCR indicated the presence of cdtA and cdtB in 59 strains belonging to 16 ribotypes and nine toxinotypes. 3.2. Correlation between ribotype, tcdA/tcdB toxinotype and binary toxin PCR A correlation between binary toxin PCR results, toxino-
type and ribotype was observed (Table 1). All strains within a ribotype (and toxinotype) consistently produced results that indicated the presence or absence of cdtA and cdtB. Only strains belonging to variant toxinotypes [13] that have signi¢cant changes in LCT genes when compared to strain VPI 10463 (toxinotypes III, IV, V, VI, VII, IX, X, XI and XIV) possessed binary toxin genes. The tcdA-negative, tcdB-positive strains (including strain
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Fig. 1. Individual PCR products for (a) cdtA and (b) cdtB generated with representative strains: CD196, R6786, R8637, IS51, CCUG 20309, R5989, IS93 and IS58 (lanes 1^8, respectively). Lanes L refer to 100-bp ladder (300^700 bp).
1470) belonging to variant toxinotype VIII did not possess binary toxin genes. All strains in toxinotypes 0, I, II, XII and XIII that have only minor changes in tcdA and tcdB when compared to VPI 10463 do not possess cdtA and cdtB. Comparative analysis of ribotype pro¢les from certain binary toxin PCR-positive strains highlighted a similarity in pro¢les (Fig. 2). Of the 2246 strains constituting the Anaerobe Reference Unit C. di¤cile collection [12], 1902 strains produce TcdA and TcdB, and 123 strains (5.5% of total and 6.4% of toxigenic strains) have been assigned to the 16 binary toxin-positive ribotypes. Strains of these ribotypes have been isolated from patients in 18 of the 42 hospitals that have referred isolates to the Anaerobe Reference Unit. Strains have also been isolated from patients in the USA and Europe, and from veterinary and environmental sources. 3.3. Sequence characterisation of short region of binary toxin genes Similarity values for short regions of cdtA and cdtB and predicted amino acid sequences are shown in Table 2. Sequences of cdtA (327 bp) for 11 strains clustered into three groups containing strains with identical DNA sequence: group 1a, CD196, CCUG 20309, R8637, IS81 and IS93; group 2a, IS51, IS58, R6786 and R7605 ; group 3a, R10456 and R5989. Translated sequence resulted in two groups, one containing CD196, CCUG 20309, R8637, IS81, IS93, IS58, R6786, IS51, R7605 and another group of R5989 and R10456. The short amino acid se-
Fig. 2. UPGMA dendrogram depicting similarities in PCR ribotype patterns for strains of C. di¤cile possessing cdtA and cdtB.
quences had 84^87% similarity with analogous regions of Ia of C. perfringens and the C. spiroforme toxin (Sa). DNA and translated protein sequences for cdtB (451 bp) clustered the 11 strains into ¢ve groups (Fig. 3) with identical sequence: group 1b, CD196 and CCUG 20309 ; group 2b, IS51, IS58, R6786 and R7605 ; group 3b R10456 and R5989 ; group 4b, R8637 ; group 5b, IS81 and IS93. The short amino acid sequences had 64^66% similarity with analogous regions of Ib of C. perfringens and the C. spiroforme toxin (Sb). 3.4. Western blotting, immunodetection and ADP-ribosyltransferase assay Expression of the binary toxin genes for 13 representative strains was assessed immunologically and activity measured by ADP-ribosyltransferase assay (Table 3). Seven PCR-positive strains reacted with anti-Ia and anti-Ib. Strains IS58, R6786, IS51 and R7605 (belonging to sequence groups 2a/2b and ribotypes 033, 045, 066 and 078, respectively) were positive by PCR but failed to react with anti-Ia, anti-Ib or both. These strains grouped together when analysing cdtA and cdtB sequences and ribotype patterns (Figs. 2 and 3). All PCR-positive strains, with the exception of IS51, produced signi¢cant actin-speci¢c ADP-ribosylating activity. The two strains that were negative in PCR reactions failed to react with anti-Ia and anti-Ib and did not pro-
Table 2 Comparison of partial nucleotide and protein sequences of cdtA and cdtB from 11 strains of C. di¤cile Strain
CD196 and CCUG20309 R8637 IS81 and IS93 R10456 and R5989 IS51, IS58, R6786 and R7605
Similarity (%) CD196 and CCUG20309
R8637
IS81 and IS93
R10456 and R5989
^ 99.8 99.6 98.7 98.4
100.0 (100)a ^ 99.3 (98.6)b 98.4 (98.0)b 98.2 (96.0)b
100.0 (100)a 100.0 (100)a ^ 98.7 (99.3)b 98.4 (97.4)b
97.9 97.9 97.9 ^ 98.4
(99.3)b (99.3)b (98.6)b (96.6)b
(97.3)a (97.3)a (97.3)a (98.0)b
IS51, IS58, R6786 and R7605 98.8 99.8 99.8 97.9 ^
(100)a (100)a (100)a (97.3)a
a Similarity values (%) for a 327-bp (109-aa) region of the cdtA gene (amino acid similarity values in parentheses) from 11 strains (within ¢ve groupings) of C. di¤cile. b Similarity values (%) for a 451-bp (149-aa) region of the cdtB gene.
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strain of C. di¤cile (CD196) was ¢rst reported in 1988 [9]. In the present study 170 representative strains, which have been characterised previously by ribotyping [11,12] and PCR-RFLP analysis of tcdA and tcdB (toxinotyping) [13], were analysed and 59 strains yielded PCR products with primers for binary toxin components. A correlation was observed between ribotype, toxinotype and possession of cdtA and cdtB, and this relationship permitted the reliable prediction of which strains possess binary toxin. Expression of CDTa and CDTb by strains representing 13 ribotypes was assessed with antibodies raised to the Stoxin of C. perfringens (anti-Ia and anti-Ib), and also by ADP-ribosyltransferase assay. The culture supernate from most PCR-positive strains reacted with anti-Ia and antiIb, but four strains (IS58, R6786, IS51 and R7605) exhibited ambiguous results. Analysis of short DNA and predicted amino acid sequences of binary toxin components revealed that these four strains group peripheral to CD196 and other binary toxin-producing strains (Fig. 3, Table 2). These di¡erences in sequence could explain the lack of reaction with anti-Ia or anti-Ib [9] and actin-speci¢c ADP-ribosyltransferase assays support this theory since only one PCR-positive strain (IS51) failed to yield activity. Binary toxin genes were observed only in strains that possess some part of the pathogenicity locus containing the genes for TcdA and TcdB (Tables 1 and 2). The binary toxin was not detected in C. di¤cile VPI 10463 [9] or in the large proportion of strains with similar LCT toxinotypes [13]. Interestingly, binary toxin genes were only detected in strains that have signi¢cant changes in the genes for TcdA and TcdB when compared to VPI 10463 ^ the so called variant toxinotypes [13]. Toxinotype VIII is the only
Fig. 3. UPGMA dendrogram depicting similarities in predicted protein sequences for a short region (150 aa) of cdtB from strains of C. di¤cile.
duce ADP-ribosylating activity. Strain IS58 does not produce active TcdA or TcdB, but toxinotyping results indicate that a short region of the tcdA gene is present. IS58 did not react with anti-Ib. However, this strain gave cdtA/ cdtB PCR products, reacted with anti-Ia and had ADPribosyltransferase activity indicating the presence of binary toxin. 4. Discussion It has been generally accepted that C. di¤cile produces two major toxins (TcdA and TcdB) that belong to the LCT family. The production of another toxin, an actinspeci¢c ADP-ribosyltransferase (binary toxin), by a single
Table 3 PCR ribotype, production of large clostridial cytotoxins TcdA and TcdB, toxinotype, and binary toxin detection by PCR, immunoblotting and ADP-ribosyltransferase assay for 13 strains of C. di¤cile PCR ribotypea
Type straina
LCT analysis Toxinotypeb
001 019 027 034 075 023 058 045 066 078 017 036 033
R8366 R8637 CD196 IS81 IS93 R5989 R10456 R6786 IS51 R7605 R7404 CCUG 20309 IS58
0 IX III III III IV IV VI VI V VIII X XId
Binary toxin analysis Enterotoxin TcdA
+ + + + + + + + + + 3 3 3
Cytotoxin TcdB
+ + + + + + + + + + + + 3
PCR
Western blot analysis
cdtA
cdtB
CDTa (using anti-Ia)
CDTb (using anti-Ib)
3 + + + + + + + + + 3 + +
3 + + + + + + + + + 3 + +
3 + + + + + + 3 3 3 3 + +
3 + + + + + + 3 + 3 3 + 3
a
PCR ribotype and strain data are detailed in [12]. Toxinotyping of toxin genes tcdA and tcdB is detailed in [13]. c Signi¢cant ADP-ribosyltransferase activity compared to control. d Strain does not produce detectable enterotoxin or cytotoxin but contains part of the tcdA gene. b
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ADP-ribosyltransferase activity (U) 170 1764c 20270c 4732c 2710c 5709c 1350c 1400c 270 1430c 180 7588c 1700c
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group of strains with signi¢cantly changed LCT genes that does not possess the genes for binary toxin. However, strains of toxinotype VIII are TcdA-negative and have been shown to possess a cytotoxin that is a functional hybrid between TcdB and the C. sordellii lethal toxin [6,16]. The results of the present study show that C. di¤cile can be divided into at least ¢ve di¡erent toxin-producing groups: (1) LCT producers, (2) LCT and binary toxin producers, (3) TcdB-only producers, (4) TcdB and binary toxin producers and (5) binary toxin-only producers. These groupings add further support to the theory that C. di¤cile is divided into stable subpopulations that have evolved from common ancestors [13]. The role of binary toxins in the pathogenesis of intestinal infections is unclear. Strains of C. perfringens that produce binary toxins also produce additional toxins [4]. However, C. spiroforme would seem to produce only binary toxin and is involved in gastrointestinal disease in animals and humans, whilst the C. botulinum C2 toxin has been found to induce haemorrhagic enteritis in animals [3]. In C. di¤cile infections, the binary toxin may act in synergy with LCT to depolymerise the actin cytoskeleton by a complementary mechanism. C. di¤cile CD196 was investigated because of the severity of clinical symptoms [9] and it is conceivable that the production of this additional toxin may exacerbate symptoms. Results indicate that 6.4% of toxigenic isolates referred to the Anaerobe Reference Unit from UK hospitals contain cdtA and cdtB. Strains of the predominant ribotypes acquired nosocomially in the UK [12] do not possess binary toxin, indicating that it does not play a major role in the aetiology of antibiotic-associated diarrhoea. However, binary toxin-producing strains have been isolated from patients in 18 of 42 UK hospitals that refer strains for epidemiological analysis. Binary toxin-positive isolates have also been obtained from patients in the USA and throughout Europe indicating that they are widespread. Acknowledgements The authors would like to thank Tamara Majstorovic (University of Ljubljana) for contributing certain DNA samples and PCR results. References [1] Aktories, K., Selzer, J., Hofmann, F. and Just, I. (1997) Molecular mechanisms of action of Clostridium di¤cile toxins A and B. In: The Clostridia : Molecular Biology and Pathogenesis (Rod, J.I., McClane, B.A., Songer, J.G. and Titball, R.W., Eds.), pp. 393^407. Academic Press, London. [2] von Eichel-Streiber, C., Boquet, C., Sauerborn, M. and Thelestam, M. (1996) Large clostridial cytotoxins ^ a family of glycosyltransferases modifying small GTP-binding proteins. Trends Microbiol. 4, 375^382.
[3] Hatheway, C.L. (1990) Toxigenic clostridia. Clin. Microbiol. Rev. 3, 66^98. [4] Petit, L., Gibert, M. and Popo¡, M.R. (1999) Clostridium perfringens: toxinotype and genotype. Trends Microbiol. 7, 104^110. [5] Thelestam, M., Florin, I. and Chaves-Olarte, E. (1997) Clostridium di¤cile toxins. In: Bacterial Toxins, Tools in Cell Biology and Pharmacology (Aktories, K., Ed.), pp. 141^158. Chapman and Hall, Weinheim. [6] 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. [7] 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. [8] Just, I., Wilm, M., Selzer, J., Rex, G., von Eichel-Streiber, C., Mann, M. and Aktories, K. (1995) The enterotoxin from Clostridium di¤cile (ToxA) monoglucosylates the Rho proteins. J. Biol. Chem. 270, 13932^13936. [9] Popo¡, M.R., Rubin, E.J., Gill, M. and Boquet, P. (1988) Actinspeci¢c ADP-ribosyltransferase produced by a Clostridium di¤cile strain. Infect. Immun. 56, 2299^2306. [10] Perelle, S., Gibert, M., Bourlioux, P., Corthier, G. and Popo¡, M.R. (1997) Production of a complete binary toxin (actin-speci¢c ADPribosyltransferase) by Clostridium di¤cile CD196. Infect. Immun. 65, 1402^1407. [11] O'Neill, G.L., Ogunsola, F.T., Brazier, J.S. and Duerden, B.I. (1996) Modi¢cation of a PCR ribotyping method for application as a routine typing scheme for Clostridium di¤cile. Anaerobe 2, 205^209. [12] Stubbs, S.L.J., Brazier, J.S., O'Neill, G.L. and Duerden, B.I. (1999) PCR targeted to the 16S-23S rRNA gene intergenic spacer region of Clostridium di¤cile and construction of a library consisting of 116 di¡erent ribotypes. J. Clin. Microbiol. 37, 461^463. [13] Rupnik, M., Avensani, 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. [14] Borriello, S.P., Wren, B.W., Hyde, S., Seddon, S.V., Sibbons, P., Krishna, M.M., Tabaqchali, S., Manek, S. and Price, A.B. (1992) Molecular, immunological and biological characterization of a toxin A-negative, toxin B-positive strain of Clostridium di¤cile. Infect. Immun. 60, 4192^4199. [15] Depitre, C., Delmee, M., Avesani, V., L'Haridon, R., Roels, A., Popo¡, M. and Corthier, G. (1993) Serogroup F strains of Clostridium di¤cile produce toxin B but not toxin A. J. Med. Microbiol. 38, 434^441. [16] 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. [17] Lyerly, D.M., Barroso, L.A., Wilkins, T.D., Depitre, C. and Corthier, G. (1992) Characterization of a toxin A-negative, toxin B-positive strain of Clostridium di¤cile. Infect. Immun. 60, 4633^4639. [18] Brazier, J.S. (1993) Role of the laboratory in investigations of Clostridium di¤cile diarrhoea. Clin. Infect. Dis. 16 (Suppl. 4), 228^233. [19] 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. [20] Perelle, S., Gibert, M., Boquet, P. and Popo¡, M.R. (1993) Characterization of Clostridium perfringens iota-toxin genes and expression in Escherichia coli. Infect. Immun. 61, 5147^5156 (Author's correction 63, 4967, 1995). [21] Burnette, W.N. (1981) Western-blotting: electrophoresis transfer of proteins from sodium-dodecyl sulphate polyacrylamide gel to unmodi¢ed nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 115^203.
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Production of actin-speci¢c ADP-ribosyltransferase (binary toxin) by strains of Clostridium di¤cile Simon Stubbs a
a;
*, Maja Rupnik b , Maryse Gibert c , Jon Brazier a , Brian Duerden a , Michel Popo¡ c
Anaerobe Reference Unit, Department of Medical Microbiology and Public Health Laboratory, University Hospital of Wales, Cardi¡ CF4 4XW, UK b Department of Biology, University of Ljubljana, Ljubljana, Slovenia c Unite¨ des Toxines Microbiennes, Institut Pasteur, Paris, France Received 6 March 2000; received in revised form 24 March 2000; accepted 27 March 2000
Abstract In addition to the two large clostridial cytotoxins (TcdA and TcdB) certain strains of Clostridium difficile produce an actin-specific ADPribosyltransferase, or binary toxin. PCR reactions were developed to detect genes encoding the enzymatic (cdtA) and binding (cdtB) components of the binary toxin and 170 representative strains were tested to assess the prevalence of the toxin. Positive PCR results (n = 59) were confirmed by immunoblotting and ADP-ribosyltransferase assay. PCR ribotype and toxinotype (restriction fragment length polymorphism analysis of genes for TcdA and TcdB) correlated with possession of binary toxin genes. All strains with cdtA and cdtB belonged to toxin-variable toxinotypes and five toxin-producing groups of strains have been described according to the presence or absence of TcdA, TcdB and binary toxin. Result indicate that ca. 6.4% of toxigenic isolates of C. difficile referred to the Anaerobe Reference Unit from UK hospitals have cdtA and cdtB genes. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Clostridium di¤cile ; Binary toxin; ADP-ribosyltransferase
1. Introduction Clostridia produce various toxins that induce alterations in the actin cytoskeleton. These cytotoxins have been classi¢ed into at least three groups; two of them, the C3-like toxins and the binary toxins, are ADP-ribosyltransferases, whilst the large clostridial cytotoxins (LCT) are glucosyltransferases [1^4]. The primary virulence factors produced by strains of Clostridium di¤cile belong to the LCT group, and are known as toxin A (TcdA) and toxin B (TcdB) [2,5]. TcdA and TcdB are similar in structure and activity, and have been reported to be monoglucosyltransferases that modify the low-molecular-mass GTP-binding proteins of the Rho and Ras subfamily using UDP-glucose as a cosubstrate [6^8]. One strain of C. di¤cile (CD196) has been shown to produce an actin-speci¢c ADP-ribosyltransferase (binary
* Corresponding author. Tel. : +44 (1222) 742378; Fax: +44 (1222) 742161; E-mail : [email protected]
toxin) which exhibits activity and structure similar to C. perfringens S-toxin [9]. The genes for the binary toxin have been identi¢ed and the enzymatic (cdtA) and binding components (cdtB) characterised [10]. It has been suggested that other strains may produce binary toxin, and that it may be an additional virulence factor [9,10]. Various typing schemes have been developed to study the epidemiology of C. di¤cile infection and determine the similarity of strains associated with disease. A library of distinct `types' of C. di¤cile, based upon polymerase chain reaction (PCR) ribotyping [11,12], and a toxinotyping scheme, based upon PCR-restriction fragment length polymorphism (RFLP) analysis of the LCT genes [13], have been reported. Certain `types' of C. di¤cile have been described that have signi¢cant changes in LCT genes whilst others lack enterotoxic activity but exhibit cytotoxicity [12^15]. One such strain (1470), belonging to serogroup F (toxinotype VIII, ribotype 017), has been shown to possess a cytotoxin that is a functional hybrid between TcdB and the C. sordellii lethal toxin [6,16]. The cytotoxin from another TcdA-negative strain, CCUG 20309 (strain 8864), has been reported to be equally cyto-
0378-1097 / 00 / $20.00 ß 2000 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 0 ) 0 0 1 6 2 - 2
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toxic but more lethal than the TcdB from the type strain of C. di¤cile [17]. Typing schemes have highlighted a number of epidemiologically important factors regarding the possible clonality and spread of C. di¤cile and there is some evidence to suggest that they can provide a reliable indication of virulence potential [12,13]. The aims of the present study were to detect and assess the prevalence of binary toxin genes in C. di¤cile. The study also investigated variation in binary toxin genes and examined the relationship between the possession of binary toxin genes, changes in LCT genes and ribotype.
72³C for 1 min 20 s. Nucleic acid preparations and PCR were done in triplicate. 2.4. Partial sequencing of binary toxin genes
2. Materials and methods
PCR products, generated from 11 representative strains of selected ribotypes, were cleaned with QIAquick-spin PCR clean-up columns (Qiagen Ltd., Crawley, West Sussex, UK) and sequenced with the ABI-Prism Dye Terminator Cycle Sequencing kit (Perkin-Elmer, Warrington, UK). Sequences for short cdtA products from strains R10456 and IS51, and short cdtB products from strains R8637, IS81, R10456 and IS51 have been assigned EMBL accession numbers AJ238324, AJ238325 and AJ237817^ AJ237820, respectively.
2.1. Strains, detection of TcdA and TcdB and nucleic acid preparation
2.5. SDS^PAGE, Western blotting and ADP-ribosyltransferase assay
Strains (n = 170) of C. di¤cile, belonging to 95 ribotypes [12] and 14 toxinotypes [13], have been analysed. Strains pre¢xed R were obtained from the Anaerobe Reference Unit (ARU) library [12]; other strains were obtained from NCTC, ATCC and CCUG. Enterotoxin and cytotoxin production were determined with the Tox A TEST immunoassay (TechLab, BioConnections, Leeds, UK) and Vero cell cytotoxicity [18]. Crude template nucleic acid was prepared using Chelex-100 (Bio-Rad, Hemel Hempstead, UK) [11].
Representative `type' strains (n = 13) from selected ribotypes [12] and toxinotypes [13] were cultured in brain heart infusion broth for 48 h and protein was precipitated from culture supernate with ammonium sulfate [20]. Supernatant protein (30 Wg) was electrophoresed in SDS^PAGE gels (10% acrylamide) and transferred onto nitrocellulose membranes [21]. Membranes were blocked in phosphate-bu¡ered saline containing dried milk (5%) for 1 h and incubated overnight at room temperature with rabbit immunoglobulin (1 in 5000 dilution) raised against enzymatic (Ia) and binding components (Ib) of C. perfringens S-toxin [20]. Bound antibody was detected with peroxidase-labelled protein A and the Signal Plus kit (Pierce Chemical Co., Rockford, IL, USA). For ADP-ribosyltransferase assay, the supernatant protein (10 Wg) was incubated at 37³C for 1 h in 50 Wl of a solution of 50 mM triethanolamine^HCl, pH 7.5, 5 mM MgCl2 , 10mM dithiothreitol, 10 mM thymidine containing brain extract as a source of actin (10 Wg) and [32 P]NAD (106 cpm per reaction). Protein was precipitated with 20 Wl of a solution of bovine serum albumin (1 mg ml31 ) and SDS (10% w/v) and 0.5 ml of trichloroacetic acid (10% w/v); reactions were incubated on ice for 1 h. The precipitate was immobilised on GFC ¢lters (Millipore, Watford, UK), washed twice in 10 ml of trichloroacetic acid (10% w/v), dried and counted for radioactivity.
2.2. PCR ribotyping and toxinotyping PCR ribotyping and toxinotyping were performed according to methods described previously [11,13]. Dendrograms were produced with the cluster correlation algorithm by the unweighted pair group method using arithmetic averages (UPGMA) and GelCompar 4.0 (Applied Maths, Kortrijk, Belgium). 2.3. PCR for binary toxin genes The primers Tim6 and Struppi6 amplify the cdd3 gene from all C. di¤cile strains [19] and were used as a positive PCR control. Primers designed to amplify regions of cdtA and cdtB were as follows: cdtApos 5P-TGAACCTGGAAAAGGTGATG-3P (position, cdtA 507^526); cdtArev 5P-AGGATTATTTACTGGACCATTTG-3P (position, cdtA 882^860); cdtBpos 5P-CTTAATGCAAGTAAATACTGAG-3P (position, cdtB 368^389); cdtBrev 5P-AACGGATCTCTTGCTTCAGTC-3P (position, cdtB 878^858). Template nucleic acid (5 Wl) was added to a PCR mixture (total 50 Wl; 50 mM KCl, 10 mM Tris^ HCl pH 9.0, 0.1% Triton X-100, 1.5 mM MgCl2 , 200 WM each dNTP, 0.15 WM each primer, 1 U Taq polymerase (Promega, Southampton, UK)). Reactions were subjected to 30 cycles of 94³C for 45 s, 52³C for 1 min and
3. Results 3.1. Detection of cdtA and cdtB by PCR PCR reactions with nucleic acid from CD196 (ribotype 027, toxinotype III; Table 1) resulted in products (622, 375 and 510 bp) with sequences corresponding to the cdd3, cdtA and cdtB genes (Fig. 1). PCR results for a further 169 strains belonging to 95 ribotypes and 14 tox-
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Table 1 PCR ribotype, toxinotype and results of PCR detection of cdtA and cdtB for strains of C. di¤cile PCR ribotypea
Number of strains tested
Toxinotypea
PCR detection (cdtA/cdtB)
PCR ribotypea
Number of strains tested
Toxinotypea
PCR detection (cdtA/cdtB)
001 002 003 004 005 006 007 008 009 010 011 012 013 014 015 016 017 018 019 020 021 022 023 024 025 026 027 028 029 030 031 032 033 034 035 036 037 038 039 040 041 042 043 044 045 046 047
6 3 1 1 4 1 1 1 2 2 1 3 1 3 1 1 4 1 4 3 1 1 8 1 1 2 2 1 1 1 1 1 2 5 1 1 1 1 1 1 1 1 1 1 6 2 1
0 0 I 0 0 0 0 0 NTb NT 0 0 0 0 0 0 VIII 0 IX 0 0 0 IV 0 0 NT III NT 0 NT NT NT XIc III NT X 0 NT NT NT NT 0 0 0 VI 0 VIII
3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 +/+ 3/3 3/3 3/3 +/+ 3/3 3/3 3/3 +/+ 3/3 3/3 3/3 3/3 3/3 +/+ +/+ 3/3 +/+ 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 +/+ 3/3 3/3
048 049 050 052 053 054 055 056 057 058 059 060 061 063 064 066 069 070 072 073 075 076 077 078 080 081 082 083 085 086 087 088 090 092 093 096 097 100 102 103 106 107 110 111 115 116 119 122
1 1 1 1 1 1 1 2 1 4 1 1 1 4 1 4 1 1 1 1 2 2 2 11 3 2 1 1 2 2 2 2 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 1
0 0 0 0 0 0 0 XIIc 0 IV IV NT 0 IV/VI 0 V/VI NT XIIIc 0 NT III 0 0 V III 0 NT 0 NT 0 0 NT 0 0 0 0 0 NT I II 0 0 VIII XIVc 0 0 NT XIVc
3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 +/+ +/+ 3/3 3/3 +/+ 3/3 +/+ 3/3 3/3 3/3 3/3 +/+ 3/3 3/3 +/+ +/+ 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 +/+ 3/3 3/3 3/3 +/+
a
See [15^17] for complete PCR ribotype and toxinotype data. NT: non-toxigenic strains (tcdA- and tcdB-negative). c New toxinotype (to be described elsewhere). b
inotypes are summarised in Table 1. PCR indicated the presence of cdtA and cdtB in 59 strains belonging to 16 ribotypes and nine toxinotypes. 3.2. Correlation between ribotype, tcdA/tcdB toxinotype and binary toxin PCR A correlation between binary toxin PCR results, toxino-
type and ribotype was observed (Table 1). All strains within a ribotype (and toxinotype) consistently produced results that indicated the presence or absence of cdtA and cdtB. Only strains belonging to variant toxinotypes [13] that have signi¢cant changes in LCT genes when compared to strain VPI 10463 (toxinotypes III, IV, V, VI, VII, IX, X, XI and XIV) possessed binary toxin genes. The tcdA-negative, tcdB-positive strains (including strain
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Fig. 1. Individual PCR products for (a) cdtA and (b) cdtB generated with representative strains: CD196, R6786, R8637, IS51, CCUG 20309, R5989, IS93 and IS58 (lanes 1^8, respectively). Lanes L refer to 100-bp ladder (300^700 bp).
1470) belonging to variant toxinotype VIII did not possess binary toxin genes. All strains in toxinotypes 0, I, II, XII and XIII that have only minor changes in tcdA and tcdB when compared to VPI 10463 do not possess cdtA and cdtB. Comparative analysis of ribotype pro¢les from certain binary toxin PCR-positive strains highlighted a similarity in pro¢les (Fig. 2). Of the 2246 strains constituting the Anaerobe Reference Unit C. di¤cile collection [12], 1902 strains produce TcdA and TcdB, and 123 strains (5.5% of total and 6.4% of toxigenic strains) have been assigned to the 16 binary toxin-positive ribotypes. Strains of these ribotypes have been isolated from patients in 18 of the 42 hospitals that have referred isolates to the Anaerobe Reference Unit. Strains have also been isolated from patients in the USA and Europe, and from veterinary and environmental sources. 3.3. Sequence characterisation of short region of binary toxin genes Similarity values for short regions of cdtA and cdtB and predicted amino acid sequences are shown in Table 2. Sequences of cdtA (327 bp) for 11 strains clustered into three groups containing strains with identical DNA sequence: group 1a, CD196, CCUG 20309, R8637, IS81 and IS93; group 2a, IS51, IS58, R6786 and R7605 ; group 3a, R10456 and R5989. Translated sequence resulted in two groups, one containing CD196, CCUG 20309, R8637, IS81, IS93, IS58, R6786, IS51, R7605 and another group of R5989 and R10456. The short amino acid se-
Fig. 2. UPGMA dendrogram depicting similarities in PCR ribotype patterns for strains of C. di¤cile possessing cdtA and cdtB.
quences had 84^87% similarity with analogous regions of Ia of C. perfringens and the C. spiroforme toxin (Sa). DNA and translated protein sequences for cdtB (451 bp) clustered the 11 strains into ¢ve groups (Fig. 3) with identical sequence: group 1b, CD196 and CCUG 20309 ; group 2b, IS51, IS58, R6786 and R7605 ; group 3b R10456 and R5989 ; group 4b, R8637 ; group 5b, IS81 and IS93. The short amino acid sequences had 64^66% similarity with analogous regions of Ib of C. perfringens and the C. spiroforme toxin (Sb). 3.4. Western blotting, immunodetection and ADP-ribosyltransferase assay Expression of the binary toxin genes for 13 representative strains was assessed immunologically and activity measured by ADP-ribosyltransferase assay (Table 3). Seven PCR-positive strains reacted with anti-Ia and anti-Ib. Strains IS58, R6786, IS51 and R7605 (belonging to sequence groups 2a/2b and ribotypes 033, 045, 066 and 078, respectively) were positive by PCR but failed to react with anti-Ia, anti-Ib or both. These strains grouped together when analysing cdtA and cdtB sequences and ribotype patterns (Figs. 2 and 3). All PCR-positive strains, with the exception of IS51, produced signi¢cant actin-speci¢c ADP-ribosylating activity. The two strains that were negative in PCR reactions failed to react with anti-Ia and anti-Ib and did not pro-
Table 2 Comparison of partial nucleotide and protein sequences of cdtA and cdtB from 11 strains of C. di¤cile Strain
CD196 and CCUG20309 R8637 IS81 and IS93 R10456 and R5989 IS51, IS58, R6786 and R7605
Similarity (%) CD196 and CCUG20309
R8637
IS81 and IS93
R10456 and R5989
^ 99.8 99.6 98.7 98.4
100.0 (100)a ^ 99.3 (98.6)b 98.4 (98.0)b 98.2 (96.0)b
100.0 (100)a 100.0 (100)a ^ 98.7 (99.3)b 98.4 (97.4)b
97.9 97.9 97.9 ^ 98.4
(99.3)b (99.3)b (98.6)b (96.6)b
(97.3)a (97.3)a (97.3)a (98.0)b
IS51, IS58, R6786 and R7605 98.8 99.8 99.8 97.9 ^
(100)a (100)a (100)a (97.3)a
a Similarity values (%) for a 327-bp (109-aa) region of the cdtA gene (amino acid similarity values in parentheses) from 11 strains (within ¢ve groupings) of C. di¤cile. b Similarity values (%) for a 451-bp (149-aa) region of the cdtB gene.
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strain of C. di¤cile (CD196) was ¢rst reported in 1988 [9]. In the present study 170 representative strains, which have been characterised previously by ribotyping [11,12] and PCR-RFLP analysis of tcdA and tcdB (toxinotyping) [13], were analysed and 59 strains yielded PCR products with primers for binary toxin components. A correlation was observed between ribotype, toxinotype and possession of cdtA and cdtB, and this relationship permitted the reliable prediction of which strains possess binary toxin. Expression of CDTa and CDTb by strains representing 13 ribotypes was assessed with antibodies raised to the Stoxin of C. perfringens (anti-Ia and anti-Ib), and also by ADP-ribosyltransferase assay. The culture supernate from most PCR-positive strains reacted with anti-Ia and antiIb, but four strains (IS58, R6786, IS51 and R7605) exhibited ambiguous results. Analysis of short DNA and predicted amino acid sequences of binary toxin components revealed that these four strains group peripheral to CD196 and other binary toxin-producing strains (Fig. 3, Table 2). These di¡erences in sequence could explain the lack of reaction with anti-Ia or anti-Ib [9] and actin-speci¢c ADP-ribosyltransferase assays support this theory since only one PCR-positive strain (IS51) failed to yield activity. Binary toxin genes were observed only in strains that possess some part of the pathogenicity locus containing the genes for TcdA and TcdB (Tables 1 and 2). The binary toxin was not detected in C. di¤cile VPI 10463 [9] or in the large proportion of strains with similar LCT toxinotypes [13]. Interestingly, binary toxin genes were only detected in strains that have signi¢cant changes in the genes for TcdA and TcdB when compared to VPI 10463 ^ the so called variant toxinotypes [13]. Toxinotype VIII is the only
Fig. 3. UPGMA dendrogram depicting similarities in predicted protein sequences for a short region (150 aa) of cdtB from strains of C. di¤cile.
duce ADP-ribosylating activity. Strain IS58 does not produce active TcdA or TcdB, but toxinotyping results indicate that a short region of the tcdA gene is present. IS58 did not react with anti-Ib. However, this strain gave cdtA/ cdtB PCR products, reacted with anti-Ia and had ADPribosyltransferase activity indicating the presence of binary toxin. 4. Discussion It has been generally accepted that C. di¤cile produces two major toxins (TcdA and TcdB) that belong to the LCT family. The production of another toxin, an actinspeci¢c ADP-ribosyltransferase (binary toxin), by a single
Table 3 PCR ribotype, production of large clostridial cytotoxins TcdA and TcdB, toxinotype, and binary toxin detection by PCR, immunoblotting and ADP-ribosyltransferase assay for 13 strains of C. di¤cile PCR ribotypea
Type straina
LCT analysis Toxinotypeb
001 019 027 034 075 023 058 045 066 078 017 036 033
R8366 R8637 CD196 IS81 IS93 R5989 R10456 R6786 IS51 R7605 R7404 CCUG 20309 IS58
0 IX III III III IV IV VI VI V VIII X XId
Binary toxin analysis Enterotoxin TcdA
+ + + + + + + + + + 3 3 3
Cytotoxin TcdB
+ + + + + + + + + + + + 3
PCR
Western blot analysis
cdtA
cdtB
CDTa (using anti-Ia)
CDTb (using anti-Ib)
3 + + + + + + + + + 3 + +
3 + + + + + + + + + 3 + +
3 + + + + + + 3 3 3 3 + +
3 + + + + + + 3 + 3 3 + 3
a
PCR ribotype and strain data are detailed in [12]. Toxinotyping of toxin genes tcdA and tcdB is detailed in [13]. c Signi¢cant ADP-ribosyltransferase activity compared to control. d Strain does not produce detectable enterotoxin or cytotoxin but contains part of the tcdA gene. b
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ADP-ribosyltransferase activity (U) 170 1764c 20270c 4732c 2710c 5709c 1350c 1400c 270 1430c 180 7588c 1700c
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group of strains with signi¢cantly changed LCT genes that does not possess the genes for binary toxin. However, strains of toxinotype VIII are TcdA-negative and have been shown to possess a cytotoxin that is a functional hybrid between TcdB and the C. sordellii lethal toxin [6,16]. The results of the present study show that C. di¤cile can be divided into at least ¢ve di¡erent toxin-producing groups: (1) LCT producers, (2) LCT and binary toxin producers, (3) TcdB-only producers, (4) TcdB and binary toxin producers and (5) binary toxin-only producers. These groupings add further support to the theory that C. di¤cile is divided into stable subpopulations that have evolved from common ancestors [13]. The role of binary toxins in the pathogenesis of intestinal infections is unclear. Strains of C. perfringens that produce binary toxins also produce additional toxins [4]. However, C. spiroforme would seem to produce only binary toxin and is involved in gastrointestinal disease in animals and humans, whilst the C. botulinum C2 toxin has been found to induce haemorrhagic enteritis in animals [3]. In C. di¤cile infections, the binary toxin may act in synergy with LCT to depolymerise the actin cytoskeleton by a complementary mechanism. C. di¤cile CD196 was investigated because of the severity of clinical symptoms [9] and it is conceivable that the production of this additional toxin may exacerbate symptoms. Results indicate that 6.4% of toxigenic isolates referred to the Anaerobe Reference Unit from UK hospitals contain cdtA and cdtB. Strains of the predominant ribotypes acquired nosocomially in the UK [12] do not possess binary toxin, indicating that it does not play a major role in the aetiology of antibiotic-associated diarrhoea. However, binary toxin-producing strains have been isolated from patients in 18 of 42 UK hospitals that refer strains for epidemiological analysis. Binary toxin-positive isolates have also been obtained from patients in the USA and throughout Europe indicating that they are widespread. Acknowledgements The authors would like to thank Tamara Majstorovic (University of Ljubljana) for contributing certain DNA samples and PCR results. References [1] Aktories, K., Selzer, J., Hofmann, F. and Just, I. (1997) Molecular mechanisms of action of Clostridium di¤cile toxins A and B. In: The Clostridia : Molecular Biology and Pathogenesis (Rod, J.I., McClane, B.A., Songer, J.G. and Titball, R.W., Eds.), pp. 393^407. Academic Press, London. [2] von Eichel-Streiber, C., Boquet, C., Sauerborn, M. and Thelestam, M. (1996) Large clostridial cytotoxins ^ a family of glycosyltransferases modifying small GTP-binding proteins. Trends Microbiol. 4, 375^382.
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