Identification and Characterization of Clostridium perfringens Beta Toxin Variants with Differing Trypsin Sensitivity and In Vitro Cytotoxicity Activity

Abstract

b By producing toxins, Clostridium perfringens causes devastating diseases of both humans and animals. C. perfringens beta toxin (CPB) is the major virulence determinant for type C infections and is also implicated in type B infections, but little is known about the CPB structure-function relationship. Amino acid sequence comparisons of the CPBs made by 8 randomly selected isolates identified two natural variant toxins with four conserved amino acid changes, including a switch of E to K at position 168 (E168K) that introduces a potential trypsin cleavage site into the CPB protein of strain JGS1076. To investigate whether this potential trypsin cleavage site affects sensitivity to trypsin, a primary host defense against this toxin, the two CPB variants were assayed for their trypsin sensitivity. The results demonstrated a significant difference in trypsin sensitivity, which was linked to the E168K switch by using site-directed recombinant CPB (rCPB) mutants. The natural CPB variants also displayed significant differences in their cytotoxicity to human endothelial cells. This cytotoxicity difference was mainly attributable to increased host cell binding rather than the ability to oligomerize or form functional pores. Using rCPB site-directed mutants, differences in cytotoxicity and host cell binding were linked to an A300V amino acid substitution in the strain JGS1076 CPB variant that possessed more cytotoxic activity. Mapping of sequence variations on a CPB structure modeled using related toxins suggests that the E168K substitution is surface localized and so can interact with trypsin and that the A300V substitution is located in a putative binding domain of the CPB toxin. C lostridium perfringens is the etiological agent of numerous devastating diseases of both humans and animals (1). Illness caused by this Gram-positive, spore-forming bacterium is mediated by its extensive toxin arsenal, consisting of ϳ17 toxins. Four of these toxins are used to classify individual strains into one of five types, A to E, depending on which toxins are encoded. By definition, both type B and C strains must produce alpha toxin (CPA) and beta toxin (CPB); type B strains are distinguished from type C strains by also producing epsilon toxin (2, 3).Type B infection is typically confined to animals, including domestic livestock species, where it causes hemorrhagic enteritis and diarrhea, which may be accompanied by severe acute neurologic signs or sudden death (1, 4). Type C strains also cause animal diseases, typically in neonatal animals, which manifest as necrohemorrhagic enteritis and toxemia, resulting in rapid death of the animal (2, 4, 5). In addition to animal diseases, type C strains can infect people, causing enteritis necroticans (known regionally as Darmbrand or pig-bel) (6-9). These human infections, which are endemic in Southeast Asia, result in segmental necrotizing enteritis, which may lead to toxemia and are rapidly fatal without prompt medical intervention. Several predisposing factors for type C infection have been i...

Native or Proteolytically Activated NanI Sialidase Enhances the Binding and Cytotoxic Activity of Clostridium perfringens Enterotoxin and Beta Toxin

“…NanI pretreatment increases cytotoxic and binding activity of CPB. While some C. perfringens type C strains produce CPE, all type C strains must (by definition) produce CPB, as must the type B strains that cause enteritis and enterotoxemia in livestock (20,25,26). To evaluate whether NanI enhances the cytotoxic effects of CPB, human umbilical vein endothelial cells (HUVECs) were pretreated with this sialidase, followed by treatment with purified CPB.…”

“…To quantify the effects of purified NanI or an rNanI species (described below) on the ability of CPB or CPE to induce cytotoxicity in host cells, an LDH release-based cytotoxicity assay was utilized, as described previously (21). Briefly, HUVECs or Caco-2 cells (sensitive to CPB and CPE, respectively [26,43]) were seeded into 24-well plates and grown as described above until confluent monolayers were achieved. Monolayers were rinsed twice with HBSS (Corning) and treated (as described in the figure legends) for 1 h at 37°C with purified NanI, an enriched rNanI species, or a mock enrichment preparation from an equivalent volume of the empty vector-carrying E. coli culture.…”

Pathogenicity and virulence of Clostridium perfringens

“…For this reason, trypsin inhibitors are required for CPB to retain activity in vivo [ 18 ]. CPB variants have been identified that display different trypsin sensitivity and cytotoxic effects in vitro [ 19 ].…”

Non-Digestible Oligosaccharides and Short Chain Fatty Acids as Therapeutic Targets against Enterotoxin-Producing Bacteria and Their Toxins

“…Purified CPB is highly sensitive to protease treatment and is thermolabile [23]. As a member of the β-PFT family, CPB shares sequence homology with other toxins, including C. perfringens delta toxin (43% identity) and several S. aureus toxins, including alpha toxin (28% identity) [24].…”