SummaryClostridium perfringens enterotoxin (CPE) binds to host cell receptors, forming a small complex precursor for two large complexes reportedly having molecular masses of~155 or~200 kDa. Formation of the~155 kDa complex causes a Ca 2+ influx that leads to apoptosis or oncosis. CPE complex composition is currently poorly understood, although occludin was identified in the~200 kDa complex. The current study used heteromer gel shift analysis to show both CPE large complexes contain six CPE molecules. Ferguson plots and size exclusion chromatography re-sized the~155 and~200 kDa complexes as~425-500 kDa and~550-660 kDa respectively. Co-immunoprecipitation and electroelution studies demonstrated both CPE-binding and non-CPEbinding claudins are associated with all three CPE complexes in Caco-2 cells and with small complex and~425-500 kDa complex of claudin 4 transfectants. Fibroblast transfectants expressing claudin 4 or C-terminal truncated claudin 4 were CPE-sensitive and formed the~425 kDa complex, indicating claudininduced cell signalling is not required for CPE action and that expression of a single receptor claudin suffices for~425-500 kDa CPE complex formation.These results identify CPE as a unique toxin that combines with tight junction proteins to form highmolecular-mass hexameric pores and alter membrane permeability.

The Gram-positive pathogen Clostridium perfringens is a major cause of human and veterinary enteric disease largely because this bacterium can produce several toxins when present inside the gastrointestinal tract. The enteric toxins of C. perfringens share two common features: (1) they are all single polypeptides of modest (approximately 25-35 kDa) size, although lacking in sequence homology, and (2) they generally act by forming pores or channels in plasma membranes of host cells. These enteric toxins include C. perfringens enterotoxin (CPE), which is responsible for the symptoms of a common human food poisoning and acts by forming pores after interacting with intestinal tight junction proteins. Two other C. perfringens enteric toxins, epsilon-toxin (a bioterrorism select agent) and beta-toxin, cause veterinary enterotoxemias when absorbed from the intestines; beta- and epsilon-toxins then apparently act by forming oligomeric pores in intestinal or extra-intestinal target tissues. The action of a newly discovered C. perfringens enteric toxin, beta2 toxin, has not yet been defined but precedent suggests it might also be a pore-former. Experience with other clostridial toxins certainly warrants continued research on these C. perfringens enteric toxins to develop their potential as therapeutic agents and tools for cellular biology.

Clostridium perfringens enterotoxin (CPE), a 35-kDa polypeptide, induces cytotoxic effects in the enterocytelike CaCo-2 cell culture model. To identify the mammalian cell death pathway(s) mediating CPE-induced cell death, CaCo-2 cultures were treated with either 1 or 10 g of CPE per ml. Both CPE doses were found to induce morphological damage and DNA cleavage in CaCo-2 cells. The oncosis inhibitor glycine, but not a broadspectrum caspase inhibitor, was able to transiently block both of those pathological effects in CaCo-2 cells treated with the higher, but not the lower, CPE dose. Conversely, a caspase 3/7 inhibitor (but not glycine or a caspase 1 inhibitor) blocked morphological damage and DNA cleavage in CaCo-2 cells treated with the lower, but not the higher, CPE dose. Collectively, these results indicate that lower CPE doses cause caspase 3/7-dependent apoptosis, while higher CPE doses induce oncosis. Apoptosis caused by the lower CPE dose was shown to proceed via a classical pathway involving mitochondrial membrane depolarization and cytochrome c release. As the CPE concentrations used in this study for demonstrating apoptosis and oncosis have pathophysiologic relevance, these results suggest that both oncosis and apoptosis may occur in the intestines during CPE-associated gastrointestinal disease.

SummaryCaCo-2 cells exhibit apoptosis when treated with low doses of Clostridium perfringens enterotoxin (CPE), but develop oncosis when treated with high CPE doses. This study reports that the presence of extracellular Ca 2+ in treatment buffers is important for normal activation of both those cell death pathways in CPE-treated CaCo-2 cells. Normal development of CPE-induced cell death pathway effects, such as morphologic damage, DNA fragmentation, caspase activation, mitochondrial membrane depolarization and cytochrome c release, was strongly inhibited when CaCo-2 cells were CPE-treated in Ca 2+ -free buffers. When treatment buffers contained Ca 2+ , CPE caused a rapid increase in CaCo-2 cell Ca 2+ levels, apparently because of increased Ca 2+ influx through a CPE pore. High CPE doses caused massive changes in cellular Ca 2+ levels that appear responsible for activating oncosis, whereas low CPE doses caused less perturbations in cellular Ca 2+ levels that appear responsible for activating apoptosis. Both CPE-induced apoptosis and oncosis were found to be calmodulin-and calpain-dependent processes. As Ca 2+ levels present in the intestinal lumen resemble those of Ca 2+ -containing treatment buffers used in this study, perturbations in cellular Ca 2+ levels and calpain/ calmodulin-dependent processes are also probably important for inducing enterocyte cell death during CPE-mediated gastrointestinal disease.

Clostridium perfringens type A isolates causing food poisoning have a chromosomal enterotoxin gene (cpe), while C. perfringens type A isolates responsible for non-food-borne human gastrointestinal diseases carry a plasmid cpe gene. In the present study, the plasmid cpe locus of the type A non-food-borne-disease isolate F4969 was sequenced to design primers and probes for comparative PCR and Southern blot studies of the cpe locus in other type A isolates. Those analyses determined that the region upstream of the plasmid cpe gene is highly conserved among type A isolates carrying a cpe plasmid. The organization of the type A plasmid cpe locus was also found to be unique, as it contains IS1469 sequences located similarly to those in the chromosomal cpe locus but lacks the IS1470 sequences found upstream of IS1469 in the chromosomal cpe locus. Instead of those upstream IS1470 sequences, a partial open reading frame potentially encoding cytosine methylase (dcm) was identified upstream of IS1469 in the plasmid cpe locus of all type A isolates tested. Similar dcm sequences were also detected in several cpe-negative C. perfringens isolates carrying plasmids but not in type A isolates carrying a chromosomal cpe gene. Contrary to previous reports, sequences homologous to IS1470, rather than IS1151, were found downstream of the plasmid cpe gene in most type A isolates tested. Those IS1470-like sequences reside in about the same position but are oppositely oriented and defective relative to the IS1470 sequences found downstream of the chromosomal cpe gene. Collectively, these and previous results suggest that the cpe plasmid of many type A isolates originated from integration of a cpe-containing genetic element near the dcm sequences of a C. perfringens plasmid. The similarity of the plasmid cpe locus in many type A isolates is consistent with horizontal transfer of a common cpe plasmid among C. perfringens type A strains.

No abstract

SUMMARYBALB/c mice resolve Leishmania donovani infection in the liver over an 8±12-week period. However, after an initial phase of 2±4 weeks where increases in parasite load are not readily detectable, parasite numbers in the spleen begin to increase reaching maximum levels at 16 weeks postinfection. Thereafter, parasite replication in the spleen is controlled and BALB/c mice maintain this residual parasite load in the spleen for many months, without further increase. We evaluated functions of CD11C + splenic dendritic cells throughout the course of L. donovani infection in the spleen of BALB/c mice. Unlike the dendritic cell (DC)-speci®c antigen DEC-205, CD11C was not up-regulated on macrophages during visceral leishmaniasis. No appreciable impairment of splenic DC functions was observed when this antigen-presenting cell subset was puri®ed from 30-day postinfected mice. Signi®cant impairment in inducing allogeneic mixed lymphocyte reaction (MLR) and presenting L. donovani antigens or keyhole limpet haemocyanin (KLH) to speci®c T cells was observed with CD11C + splenic DC puri®ed from 60-day post-infected mice. Functional impairment of splenic DC at 60 days post-infection correlated with their reduced surface expression of major histocompatibility complex (MHC) class II molecules, impairment of interleukin-12 (IL-12) production and to their ability to suppress interferon-c (IFN-c) production by Leishmania antigenprimed T cells. Of interest, the impairment of splenic DC in presenting Leishmania antigens or KLH to speci®c T cells was corrected at 120 days post-infection, and correlated with their up-regulation of MHC class II expression, IL-12 production, induction of IFN-c by Leishmania antigen-primed T cells and the onset of control over splenic parasite replication in vivo. These results indicate that functional integrity of DC may be important in controlling L. donovani infection.

No abstract