Clostridium perfringens extracellular toxins and enzymes: 20 and counting

Clostridium perfringens is a Gram-positive, anaerobic bacterium that is widely distributed in the environment; it is found in soil and commonly inhabits the gastrointestinal tract of humans and animals^1,2. The ubiquitous nature of this bacterium has resulted in it becoming a major cause of histotoxic and enteric diseases³. The success of C. perfringens as both a pathogen and a commensal bacterium lies in its ability to produce a large number of potent toxins and extracellular enzymes⁴. This diverse toxin repertoire results in a broad range of diseases including gas gangrene, various enterotoxaemias, food poisoning and necrotic enteritis^4–6. Since 2007, six new toxins have been identified, adding to the ever-increasing range of potential C. perfringens virulence determinants. This paper briefly reviews the plethora of toxins and extracellular enzymes produced by C. perfringens, highlighting their importance in disease and strain classification as well as introducing the latest additions to the ever increasing C. perfringens toxin family.

Like many clostridial species, the virulence of C. perfringens is dependent on the production of toxins⁷. Not all toxins are produced by any one strain, instead individual isolates vary in toxin carriage and production⁸. This toxin expression profile forms the basis of a toxinotype classification scheme; in which strains are classified into five toxinotypes (A–E) based on the production of four different toxins (alpha, beta, epsilon and iota)^4,9. This typing scheme is now very much outdated, but it has been useful for classification as the different toxinotypes are often associated with specific diseases^4,10 (Table 1). For example, clostridial myonecrosis and human food poisoning are associated with type A strains, whereas type B, C and D strains are most strongly associated with enteric diseases of livestock⁴.

Table 1. Properties of Clostridium perfringens toxins.

The number of characterised C. perfringens toxins is ever increasing; with more than 20 different toxins and enzymes classified to date, see Table 1^3,5,9,11. With a few important exceptions, these toxins are encoded on large conjugative plasmids^4,10,12–18, which allows for potential toxin gene transfer between different C. perfringens strains in the gastrointestinal tract and may prolong disease¹⁰. C. perfringens utilises chromosomally encoded toxins, such as alpha-toxin and perfringolysin O, during human histotoxic infections or human food poisoning (C. perfringens enterotoxin, CPE)³. However, for reasons that are probably related to disease epidemiology, plasmid-encoded toxins are critical for non-foodborne human gastrointestinal diseases, human enteritis necroticans and gastrointestinal diseases of animals^3,10.

The toxins of C. perfringens can be functionally classified into four broad categories: membrane damaging enzymes, pore-forming toxins, intracellular toxins, and hydrolytic enzymes⁴. Membrane damaging toxins such as alpha-toxin are enzymes that damage target cell membranes through their ability to breakdown the constituents of the mammalian cell membrane¹⁹. The pore-forming toxins comprise the largest toxin category and function to disrupt membrane permeability and ion transport by inserting into the membrane and forming a permeable channel or pore²⁰. This category includes toxins such as perfringolysin O, beta-toxin, CPE, NetB and epsilon-toxin. Intracellular toxins, such as TpeL, BEC and iota-toxin, are internalised into target host cells where they act to disrupt the cellular cytoskeleton²¹. Hydrolytic enzymes, such as sialidases and hyaluronidases, are secreted by C. perfringens and degrade surface associated glycans or glycoproteins^4,22. These enzymes are not essential for disease; however, they may still contribute to the overall virulence of the bacterium²³.

In recent years, the number of characterised C. perfringens toxins has increased significantly. The latest editions to the C. perfringens arsenal are the six novel toxins or putative toxins: NetB, BEC, TpeL, NetE, NetF and NetG.

NetB is a beta-barrel pore-forming toxin and, like many other C. perfringens toxins, it is encoded on large conjugative plasmids^24,25. Since its discovery, NetB-encoding plasmids have been identified in many avian necrotic enteritis isolates; and netB deletion studies have indicated that NetB toxin, is required for the development of necrotic enteritis in chickens²⁶.

BEC is a novel binary toxin, composed of two distinct components, BECa and BECb and it appears to function in a similar fashion to iota-toxin¹⁵, with the BECa component having actin-specific ADP-ribosyltranferase activity. BEC was discovered after two unrelated food poisoning outbreaks that were caused by C. perfringens strains that did not encode CPE; the toxin typically associated with human food poisoning¹⁵. Further studies showed that these strains produced a novel binary toxin, designated as BEC, suggesting that this new toxin was responsible for the enteric symptoms observed during these outbreaks¹⁵.

TpeL is a member of the clostridial monoglycosyltransferase toxin family, which mediate cytotoxic effects through the glycosylation of host cell proteins, and is related to the large toxins, TcdA and TcdB, of Clostridium difficile²⁷. There is no definitive evidence that TpeL is involved in disease; however, it is postulated that this toxin may make a contribution to virulence²⁷.

The most recently characterised toxins are the cytotoxic pore-forming toxin NetF and the putative pore-forming toxins NetE and NetG, which were shown to be encoded on plasmids in isolates causing necrotizing gastroenteritis and necrotizing enterocolitis in foals and dogs²⁸. There is a significant association between NetF-producing strains and enteric disease in these animals; however, the function of NetE and NetG in disease remains to be investigated²⁸.

The production of toxins is essential for C. perfringens-mediated disease and this bacterium utilises an arsenal of different toxins and extracellular enzymes to cause a myriad of diseases. Note that the primary function of these toxins is most likely not to cause disease, but to provide nutrients for the growth of C. perfringens cells, which have limited capability to synthesise amino acids and essential co-factors. Many of the toxins that are crucial contributors to disease, for example NetB, are encoded on conjugative plasmids¹⁰, and therefore may be readily disseminated to other strains. Recent findings support the theory that C. perfringens strains have a tight association with the species in which they cause disease; for example, NetB-producing strains in birds and NetF-producers in foals and dogs. Just about everywhere we look, if there is an unclassified disease from which lots of C. perfringens cells can be isolated, the chances are good that another toxin is waiting to be discovered … stay tuned!