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Revised nomenclature of Clostridium difficile toxins and associated genes

¹University of Ljubljana, Department of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia ^2,8Unité de Génétique Moléculaire Bactérienne² and Unité des Toxines Microbiennes⁸, Institut Pasteur, Paris, France ³Centre for Molecular Microbiology and Infection, Imperial College, London, UK ⁴Hines VA Hospital, Hospital/151 Fifth Avenue & Roosevelt Road, Hines, IL 60141, USA ⁵Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA ⁶Institute of Toxicology, Hannover Medical School, Hannover, Germany ⁷TechLab Inc., Blacksburg, VA, USA ⁹Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Victoria 3800, Australia ¹⁰Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USA ¹¹Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden ¹²London School of Hygiene and Tropical Medicine, University of London, UK ¹³Institute of Medical Microbiology and Hygiene, Johannes Gutenberg University of Mainz, D-55101 Mainz, Germany

Correspondence Maja Rupnik maja.rupnik{at}bf.uni-lj.si

Received July 3, 2004
Accepted August 6, 2004

Several different nomenclatures have been applied to the Clostridium difficile toxins and their associated genes. This paper summarizes the new nomenclature that has been agreed to by the research groups currently active in the field. The revised nomenclature includes C. difficile toxins and other related large clostridial toxins produced by Clostridium sordellii and Clostridium novyi, and corresponding toxin genes, as well as toxin production types of C. difficile strains.

Abbreviation: LCT, large clostridial toxin.

	INTRODUCTION

TOP INTRODUCTION REVISED NOMENCLATURE REFERENCES

 
Clostridium difficile is the causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis. Two large protein cytotoxins, toxin A (TcdA) and toxin B (TcdB), are recognized as the main virulence factors. Some C. difficile strains also produce a third, unrelated toxin (binary toxin CDT).

The molecular analysis of C. difficile toxins started in the 1980s with the first attempts to clone fragments of toxin genes and has progressed with the sequencing of both toxin genes (von Eichel-Streiber & Sauerborn, 1990; von Eichel-Streiber et al., 1990, 1992; Dove et al., 1990; Johnson et al., 1990), with the definition of the region encoding the toxins (Hammond & Johnson, 1995; Braun et al., 1996), and with studies of toxin gene regulation (Moncrief et al., 1997; Hammond et al., 1997; Hundsberger et al., 1997; Dupuy & Sonenshein, 1998; Mani & Dupuy, 2001). Unfortunately, in parallel with developments on the molecular biology and biochemistry of C. difficile, several different nomenclature systems have been applied to the toxins and their associated genes (Table 1).

With an increasing number of research groups working on the molecular biology of C. difficile, or using its toxins as tools in cell biology, and with the imminent finalization of the first C. difficile genome sequence (http://www.sanger.ac.uk/Projects/C_difficile/), the need for a unified nomenclature has become apparent. A similar approach was taken on naming the clostridial neurotoxins, where a proposed unified nomenclature (Niemann, 1992) has resulted in consistent and logical citation in the literature.

At the recent First International C. difficile Symposium (FICDS, Kranjska Gora, Slovenia, May 2004), a round-table discussion on C. difficile toxins and toxin gene nomenclature was held. This paper summarizes the new nomenclature that was agreed to by the research groups currently active in the field.

	REVISED NOMENCLATURE

TOP INTRODUCTION REVISED NOMENCLATURE REFERENCES

 
Large clostridial toxins (LCTs) and toxin genes

C. difficile produces three toxins: toxin A (TcdA), toxin B (TcdB) and binary toxin (CDT). Toxins A and B are similar to one another, and belong, together with three other toxins produced by Clostridium sordellii and Clostridium novyi, to the well-defined group of LCTs (von Eichel-Streiber et al., 1996). The structural genes encoding four of the five LCTs have been sequenced (von Eichel-Streiber & Sauerborn, 1990; von Eichel-Streiber et al., 1990, 1992; Dove et al., 1990; Johnson et al., 1990; Green et al., 1995; Hofmann et al., 1995), and in 1995 a nomenclature for LCTs and their genes was proposed that combines information on the bacterial species and the identity of the toxin (Hofmann et al., 1995): Tcd for C. difficile, Tcs for C. sordellii and Tcn for C. novyi; and TcdA and TcdB for toxin A and toxin B, TcsL and TcsH for toxin LT and HT, and Tcn (Tcn-alpha) for the alpha toxin of C. novyi.

At the Slovenia meeting it was agreed that a unified nomenclature should be based on the tcd–tcs–tcn system (Table 2) as it is recommended that homologous genes present in different organisms receive the same or similar names (Demerec et al., 1966).

The genomic region encoding the C. difficile toxins

A 19 kb region encoding two related toxins produced by C. difficile (toxin A or TcdA and toxin B or TcdB) has been defined as the toxigenic element (Hammond & Johnson, 1995) or as the pathogenicity locus, PaLoc (Braun et al., 1996). As the latter name has been used predominately in recent publications, we suggest accepting it for the new C. difficile nomenclature.

However, it is important to stress that although the use of the term ‘pathogenicity locus’ or ‘PaLoc’ is correct, the term ‘pathogenicity island’ is not an accurate description of this gene region since there is no evidence that the PaLoc region is horizontally acquired and it does not fit the generally accepted definition of a pathogenicity island (Hacker et al., 2004).

C. difficile LCT genes and associated genes

For the C. difficile genes found in the PaLoc the nomenclature shown in Table 2 and Fig. 1 was agreed. The genes for toxins A and B should be called tcdA and tcdB, respectively. The other three genes were previously defined as tcdC, D and E by the von Eichel-Streiber group according to the predicted sizes of the encoded proteins and not according to their relative positions in the PaLoc (Braun et al., 1996). As the names tcdE and tcdC have been used in recent publications by several different groups, these two names will remain.

View larger version (6K): [in this window] [in a new window]   Fig. 1. Revised nomenclature for five genes of the C. difficile pathogenicity locus PaLoc.

However, the majority of papers dealing with the tcdD gene use the name txeR. To be consistent with the tcd nomenclature, which indicates that the gene is a part of the PaLoc, but to show that the gene product is involved in regulation, we have decided to rename this gene tcdR.

C. difficile toxins

Various names have been used for C. difficile toxins in recent years (Table 1), but ‘toxin A’ and ‘toxin B’ are the most prevalent. After the first establishment of the tcd–tcs–tcn nomenclature, the toxins were also called ‘TcdA’ or ‘TcdB'. At the round table it was agreed that both prefixes, ‘Tcd’ and ‘toxin', could be used for the toxins (Table 2).

Designation of strain-specific (variant) toxins and corresponding genes

Many C. difficile strains are known to have toxin genes with marked sequence changes in comparison to the toxin genes from the reference strain VPI 10463, resulting in changed properties of the resultant toxins. If applicable, the strain identification should therefore be added to the toxin or to the toxin gene. However, the number after the locus (e.g. ‘tcdB-1470') is used as a designation of mutation site in standard genetic nomenclature (Demerec et al., 1966). To distinguish between the same genes from different strains the use of subscripts was recommended. Therefore, the designation ‘tcdB₁₄₇₀’ for the gene and ‘TcdB₁₄₇₀’ or ‘toxin B₁₄₇₀’ for the protein should be used, when appropriate.

Other LCTs and corresponding genes

As already mentioned, the tcd–tcs–tcn nomenclature applies to the entire group of related LCTs. Only one change was suggested by the nomenclature discussion group. According to standard rules (Demerec et al., 1966), genes should have a three-letter designation followed by a Latin capital letter (not Greek). Therefore the C. novyi alpha toxin gene was renamed tcnA (Table 2).

Binary toxin

The third known toxin produced by C. difficile, the binary toxin CDT (Perelle et al., 1997), is not related to TcdA and TcdB and the nomenclature of this toxin and the corresponding genes was not discussed. The names currently used are ‘C. difficile binary toxin’ or ‘binary toxin CDT'. The term ‘CDT’ should not be used alone as it could be confused with cytolethal distending toxins produced by several other bacteria. The genes are already designated cdtB for the binding component and cdtA for the enzymic component, and we suggest that these gene names be retained.

Strains – description of toxin production

In early studies, C. difficile strains always produced either both toxins, TcdA and TcdB, or neither of them, making the discrimination between toxigenic and nontoxigenic strains easy. During the last decade, however, this definition has become unclear for two reasons. First, strains producing only one of the toxins have been described and have been repeatedly isolated all over the world (A^–B⁺ or ‘toxin A-negative, toxin B-positive strains'). Secondly, a third toxin (binary toxin CDT) has been found in some C. difficile strains and, although the majority of binary toxin positive strains still produce TcdA and TcdB (A⁺B⁺CDT⁺ strains), up to 2 % are estimated to produce only binary toxin CDT but not TcdA and TcdB (A^–B^–CDT⁺ strains). Therefore, the nomenclature discussion group agreed on the following definition of toxigenic and nontoxigenic strains.

Toxigenic strains must produce at least one of the three known toxins. At the present time, five toxin production patterns can be differentiated (Table 3). Toxin production types should be described as A⁺B⁺, A^–B^–, A^–B⁺, etc. The term ‘toxin-positive’ should be avoided unless it is specifically defined in the text to which of the three toxins it refers. If the production of binary toxin CDT was also tested, toxin production type would be A⁺B⁺CDT⁺, etc. If the toxin designation is based on gene identification only we suggest using the toxin designation in parentheses, with an additional text commentary to indicate the basis of presumptive toxin production, e.g. A⁺B⁺(CDT⁺) based on PCR amplification of cdtB. The term ‘nontoxigenic strain’ should be reserved for strains known not to produce any of these three toxins.

	REFERENCES

TOP INTRODUCTION REVISED NOMENCLATURE REFERENCES

 

Hofmann, F., Herrmann, A., Habermann, E. & von Eichel Streiber, C. (1995). Sequencing and analysis of the gene encoding the -toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile. Mol Gen Genet 247, 670–679.[CrossRef][Medline]