Synergistic Effects of Clostridium perfringens Enterotoxin and Beta Toxin in Rabbit Small Intestinal Loops

Ma, Menglin; Gurjar, Anagha; Theoret, James R.; García, J. Arrazola; Beingesser, Juliann; Freedman, John C.; Fisher, Derek J.; McClane, Bruce A.; Uzal, Francisco A.

doi:10.1128/iai.01848-14

Cited by 35 publications

(41 citation statements)

References 38 publications

(89 reference statements)

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“…The lower cell death caused by the reversed mutant versus the wild-type strains is consistent with results shown in Fig. 4C, indicating that reversal of the tpeL mutation was partial and did not completely restore TpeL production, as is typical for this approach (18,47). When the processed culture supernatants were preincubated with the TpeL-neutralizing antibody, cytotoxicity caused by the wild-type supernatant samples decreased significantly; in contrast, preincubation with preimmune serum did not affect cytotoxicity of this sample, confirming a substantial role for TpeL in the cytotoxic activity of these culture supernatants (Fig.…”

Section: Resultssupporting

confidence: 78%

“…Of note, the earlier studies had challenged animals with TGY cultures of CN3685, which (as now shown in the current study and the study by Carter et al [46]) is not the most favorable culture medium for TpeL production and release. Alternatively, it is possible that TpeL contributes additively or synergistically to CPB action, similar to the recent discovery of CPB and CPE synergism for the enteric pathogenicity of some TpeL-negative type C strains (47).…”

Section: Discussionmentioning

confidence: 99%

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Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

Chen

McClane

2015

Infect Immun

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Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age.

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Section: Resultssupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

Chen

McClane

2015

Infect Immun

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“…However, previous studies (37,(40)(41)(42) have shown that mutations from group II introns inserted in the sense orientation can be partially reversed at the mRNA level by growth at 30°C in the presence of an LtrA-encoding plasmid, such as pJIR750ccpAi, with the frequency of this reversion varying for different intron insertions. Therefore, pJIR750ccpAi was reintroduced into strain ccpAko, creating strain ccpArev, in order for LtrA to remove by splicing under permissive 30°C conditions, the sense-oriented intron from some ccpA mRNA, thus restoring a functional ccpA transcript and CcpA production.…”

Section: Resultsmentioning

confidence: 99%

NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

Freedman

McClane

2015

J Bacteriol

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Clostridium perfringens type D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011, http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, a nanI null mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due to nanI gene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions in codY and ccpA gene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A double codY ccpA null mutant produced even less ETX than a codY or ccpA single null mutant. CcpA bound directly to sequences upstream of the etx or codY start codon, and bioinformatics identified putative CcpA-binding cre sites immediately upstream of both the codY and etx start codons, suggesting possible direct CcpA regulatory effects. A ccpA mutation also decreased codY transcription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects on codY transcription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease. IMPORTANCEClostridium perfringens NanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators. Results obtained using single ccpA or codY null mutants and a ccpA codY double null mutant showed that codY and ccpA regulate ETX production independently of one another but that ccpA also affects codY transcription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulate etx transcription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producing C. perfringens strains, via CcpA and CodY, to upregulate ETX production and cause disease. C lostridium perfringens is an important cause of human and livestock infections, including many diseases originating in the intestines (1, 2). The virulence of C. perfringens is largely attributable to its prolific toxin-producing ability, with different toxins involved in specific diseases (1,3,4). By definition, type D strains must produce both C. perfringens alpha toxin (CPA) and epsilon toxin (ETX). ETX is a class B NIAID priority toxin, since it is one of the most potent ba...

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“…In the feces of diseased people, CPE is present at concentrations up to ϳ100 g/ml (40,41). Supporting their pathophysiologic relevance as an in vivo model of CPE activity, rabbit small intestinal loops consistently respond to challenge with 50 g or more of CPE/ml (41).…”

Section: Figmentioning

confidence: 97%

A Synthetic Peptide Corresponding to the Extracellular Loop 2 Region of Claudin-4 Protects against Clostridium perfringens Enterotoxin In Vitro and In Vivo

et al. 2014

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bClostridium perfringens enterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 ( r claudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such as r claudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE action in vitro and in vivo. Significant protection of Caco-2 cells was also observed using either r claudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since r claudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeated in vivo using a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similar in vivo protection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease.

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Synergistic Effects of Clostridium perfringens Enterotoxin and Beta Toxin in Rabbit Small Intestinal Loops

Cited by 35 publications

References 38 publications

Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

A Synthetic Peptide Corresponding to the Extracellular Loop 2 Region of Claudin-4 Protects against Clostridium perfringens Enterotoxin In Vitro and In Vivo

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