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

2015 ◽  
Vol 83 (6) ◽  
pp. 2369-2381 ◽  
Author(s):  
Jianming Chen ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Toxin Gene ◽  
Type B ◽  
Content Type ◽  
Positive Type ◽  
Natural Production ◽  
Type D ◽  
Clostridial Species ◽  
Beta Toxin

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 byClostridium perfringensthat has received relatively limited study. In response, the current study surveyed carriage of thetpeLgene among differentC. perfringensstrains, 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 duringin vitroculture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production byC. perfringensstrain 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 oftpeL-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.

scholarly journals Sequencing and Diversity Analyses Reveal Extensive Similarities between Some Epsilon-Toxin-Encoding Plasmids and the pCPF5603 Clostridium perfringens Enterotoxin Plasmid

2008 ◽  
Vol 190 (21) ◽  
pp. 7178-7188 ◽  
Author(s):  
Kazuaki Miyamoto ◽  
Jihong Li ◽  
Sameera Sayeed ◽  
Shigeru Akimoto ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Open Reading Frames ◽  
Toxin Gene ◽  
Type B ◽  
Clostridium Perfringens Enterotoxin ◽  
Type D ◽  
Transfer Genes ◽  
Epsilon Toxin ◽  
Beta2 Toxin ◽  
Beta Toxin

ABSTRACT Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-like etx plasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive, cpe-negative type D isolates, while other type D isolates carry different etx plasmids. Sequences upstream of the etx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-like etx plasmid. However, nearly all type B and D isolates have an etx locus with an upstream IS1151, and those etx loci typically reside near a dcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin or etx genes, respectively, onto a common progenitor plasmid.

scholarly journals Cloning and expression most expected antigenic fragment of beta-toxin gene from Clostridium perfringens type B

2016 ◽  
Vol 05 (05) ◽  
pp. 491-494
Author(s):  
Maryam Gholami ◽  
Mohsen Fathi Najafi ◽  
Mohammad Rabbani Khorasgani ◽  
Behjat Majidi
Keyword(s):  
Clostridium Perfringens ◽  
Cloning And Expression ◽  
Toxin Gene ◽  
Type B ◽  
Beta Toxin

scholarly journals Enhanced expression of recombinant beta toxin of Clostridium perfringens type B using a commercially available Escherichia coli strain

2016 ◽  
Vol 83 (1) ◽  
Author(s):  
Fatemah Bakhshi ◽  
Reza Pilehchian Langroudi ◽  
Bahram Golestani Imani
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Clostridium Perfringens ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Toxin Gene ◽  
Type B ◽  
Iptg Induction ◽  
E Coli ◽  
Beta Toxin

Clostridium perfringens beta toxin is only produced by types B and C and plays an important role in many human and animal diseases, causing fatal conditions that originate in the intestines. We compared the expression of C. perfringens type B vaccine strain recombinant beta toxin gene in the Escherichia coli strains RosettaTM(DE3) and BL21(DE3). The beta toxin gene was extracted from pJETβ and ligated with pET22b(+). pET22β was transformed into E. coli strains BL21(DE3) and RosettaTM(DE3). Recombinant protein was expressed as a soluble protein after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction in strain RosettaTM(DE3) but not in BL21(DE3). Expression was optimised by growing recombinant cells at 37 °C and at an induction of 0.5 mM, 1 mM, 1.5 mM IPTG. Expression was evaluated using sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant protein was purified via Ni-NTA and was analysed using western blot. We concluded that E. coli strain RosettaTM(DE3) can enhance the expression of C. perfringens recombinant beta toxin.Keywords: C. perfringens beta toxin (CPB); expression; RosettaTM; BL21

scholarly journals Characterization of Toxin Plasmids in Clostridium perfringens Type C Isolates

2010 ◽  
Vol 78 (11) ◽  
pp. 4860-4869 ◽  
Author(s):  
Abhijit Gurjar ◽  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Toxin Gene ◽  
Gene Encoding ◽  
Positive Type ◽  
Toxin Genes ◽  
Type C ◽  
Beta2 Toxin ◽  
Beta Toxin

ABSTRACT Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasmid borne among a collection of type C isolates. Those analyses revealed that the surveyed type C isolates carry their beta toxin-encoding gene (cpb) on plasmids ranging in size from ∼65 to ∼110 kb. When present in these type C isolates, the beta2 toxin gene localized to plasmids distinct from the cpb plasmid. However, some enterotoxin-positive type C isolates appeared to carry their enterotoxin-encoding cpe gene on a cpb plasmid. The tpeL gene encoding the large clostridial cytotoxin was localized to the cpb plasmids of some cpe-negative type C isolates. The cpb plasmids in most surveyed isolates were found to carry both IS1151 sequences and the tcp genes, which can mediate conjugative C. perfringens plasmid transfer. A dcm gene, which is often present near C. perfringens plasmid-borne toxin genes, was identified upstream of the cpb gene in many type C isolates. Overlapping PCR analyses suggested that the toxin-encoding plasmids of the surveyed type C isolates differ from the cpe plasmids of type A isolates. These findings provide new insight into plasmids of proven or potential importance for type C virulence.

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

2014 ◽  
Vol 82 (7) ◽  
pp. 2958-2970 ◽  
Author(s):  
Menglin Ma ◽  
Abhijit Gurjar ◽  
James R. Theoret ◽  
Jorge P. Garcia ◽  
Juliann Beingesser ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Synergistic Effects ◽  
Fluid Accumulation ◽  
Content Type ◽  
Positive Type ◽  
Intestinal Infections ◽  
Type C ◽  
Small Intestinal ◽  
Beta Toxin ◽  
Enteritis Necroticans

ABSTRACTThe ability ofClostridium perfringenstype C to cause human enteritis necroticans (EN) is attributed to beta toxin (CPB). However, many EN strains also expressC. perfringensenterotoxin (CPE), suggesting that CPE could be another contributor to EN. Supporting this possibility, lysate supernatants from modified Duncan-Strong sporulation (MDS) medium cultures of three CPE-positive type C EN strains caused enteropathogenic effects in rabbit small intestinal loops, which is significant since CPE is produced only during sporulation and sinceC. perfringenscan sporulate in the intestines. Consequently, CPE and CPB contributions to the enteropathogenic effects of MDS lysate supernatants of CPE-positive type C EN strain CN3758 were evaluated using isogeniccpbandcpenull mutants. While supernatants of wild-type CN3758 MDS lysates induced significant hemorrhagic lesions and luminal fluid accumulation, MDS lysate supernatants of thecpbandcpemutants caused neither significant damage nor fluid accumulation. This attenuation was attributable to inactivating these toxin genes since complementing thecpemutant or reversing thecpbmutation restored the enteropathogenic effects of MDS lysate supernatants. Confirming that both CPB and CPE are needed for the enteropathogenic effects of CN3758 MDS lysate supernatants, purified CPB and CPE at the same concentrations found in CN3758 MDS lysates also acted together synergistically in rabbit small intestinal loops; however, only higher doses of either purified toxin independently caused enteropathogenic effects. These findings provide the first evidence for potential synergistic toxin interactions duringC. perfringensintestinal infections and support a possible role for CPE, as well as CPB, in some EN cases.

scholarly journals Epsilon-Toxin Production by Clostridium perfringens Type D Strain CN3718 Is Dependent upon the agr Operon but Not the VirS/VirR Two-Component Regulatory System

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Jianming Chen ◽  
Julian I. Rood ◽  
Bruce A. McClane
Keyword(s):  
Quorum Sensing ◽  
Clostridium Perfringens ◽  
Regulatory System ◽  
Toxin Production ◽  
Broth Culture ◽  
Type B ◽  
Content Type ◽  
Type D ◽  
Two Component ◽  
Epsilon Toxin

ABSTRACT Clostridium perfringens type B and D strains cause enterotoxemias and enteritis in livestock after proliferating in the intestines and producing epsilon-toxin (ETX), alpha-toxin (CPA), and, usually, perfringolysin O (PFO). Although ETX is one of the most potent bacterial toxins, the regulation of ETX production by type B or D strains remains poorly understood. The present work determined that the type D strain CN3718 upregulates production of ETX upon close contact with enterocyte-like Caco-2 cells. This host cell-induced upregulation of ETX expression was mediated at the transcriptional level. Using an isogenic agrB null mutant and complemented strain, the agr operon was shown to be required when CN3718 produces ETX in broth culture or, via a secreted signal consistent with a quorum-sensing (QS) effect, upregulates ETX production upon contact with host cells. These findings provide the first insights into the regulation of ETX production, as well as additional evidence that the Agr-like QS system functions as a global regulator of C. perfringens toxin production. Since it was proposed previously that the Agr-like QS system regulates C. perfringens gene expression via the VirS/VirR two-component regulatory system, an isogenic virR null mutant of CN3718 was constructed to evaluate the importance of VirS/VirR for CN3718 toxin production. This mutation affected production of CPA and PFO, but not ETX, by CN3718. These results provide the first indication that C. perfringens toxin expression regulation by the Agr-like quorum-sensing system may not always act via the VirS/VirR two-component system. IMPORTANCE Mechanisms by which Clostridium perfringens type B and D strains regulate production of epsilon-toxin (ETX), a CDC class B select toxin, are poorly understood. Production of several other toxins expressed by C. perfringens is wholly or partially regulated by both the Agr-like quorum-sensing (QS) system and the VirS/VirR two-component regulatory system, so the present study tested whether ETX expression by type D strain CN3718 also requires these regulatory systems. The agr operon was shown to be essential for signaling CN3718 to produce ETX in broth culture or to upregulate ETX production upon close contact with enterocyte-like Caco-2 cells, which may have pathogenic relevance since ETX is produced intestinally. However, ETX production remained at wild-type levels after inactivation of the VirS/VirR system in CN3718. These findings provide the first information regarding regulation of ETX production and suggest Agr-like QS toxin production regulation in C. perfringens does not always require the VirS/VirR system.

scholarly journals Role of the Agr-Like Quorum-Sensing System in Regulating Toxin Production by Clostridium perfringens Type B Strains CN1793 and CN1795

2012 ◽  
Vol 80 (9) ◽  
pp. 3008-3017 ◽  
Author(s):  
Jianming Chen ◽  
Bruce A. McClane
Keyword(s):  
Quorum Sensing ◽  
Clostridium Perfringens ◽  
Cell Model ◽  
Toxin Production ◽  
Type B ◽  
Wild Type ◽  
Content Type ◽  
Culture Supernatants ◽  
Null Mutants

ABSTRACTClostridium perfringenstype B causes enteritis and enterotoxemia in domestic animals. By definition, these bacteria must produce alpha toxin (CPA), beta toxin (CPB) and epsilon toxin (ETX) although most type B strains also produce perfringolysin O (PFO) and beta2 toxin (CPB2). A recently identified Agr-like quorum-sensing (QS) system inC. perfringenscontrols all toxin production by surveyed type A, C, and D strains, but whether this QS is involved in regulating toxin production by type B strains has not been explored. Therefore, the current study introducedagrBnull mutations into type B strains CN1795 and CN1793. Both type BagrBnull mutants exhibited reduced levels of CPB, PFO, and CPA in their culture supernatants, and this effect was reversible by complementation. The reduced presence of CPB in culture supernatant involved decreasedcpbtranscription. In contrast, theagrBnull mutants of both type B strains retained wild-type production levels of ETX and CPB2. In a Caco-2 cell model of enteritis, culture supernatants of the type BagrBnull mutants were less cytotoxic than supernatants of their wild-type parents. However, in an MDCK cellin vitromodel for enterotoxemic effects, supernatants from theagrBnull mutants or wild-type parents were equally cytotoxic after trypsin activation. Coupling these and previous results, it is now evident that strain-dependent variations exist in Agr-like QS system regulation ofC. perfringenstoxin production. The cell culture results further support a role for trypsin in determining which toxins contribute to disease involving type B strains.

scholarly journals Both Epsilon-Toxin and Beta-Toxin Are Important for the Lethal Properties of Clostridium perfringens Type B Isolates in the Mouse Intravenous Injection Model

2007 ◽  
Vol 75 (3) ◽  
pp. 1443-1452 ◽  
Author(s):  
Mariano E. Fernandez-Miyakawa ◽  
Derek J. Fisher ◽  
Rachael Poon ◽  
Sameera Sayeed ◽  
Vicki Adams ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Clinical Signs ◽  
Toxin Production ◽  
Intestinal Damage ◽  
Type B ◽  
Trypsin Treatment ◽  
Epsilon Toxin ◽  
Type C ◽  
Beta Toxin

ABSTRACT Clostridium perfringens is capable of producing up to 15 toxins, including alpha-toxin (CPA), beta-toxin (CPB), epsilon-toxin (ETX), enterotoxin, beta2-toxin (CPB2), and perfringolysin O. Type B isolates, which must produce CPA, CPB, and ETX, are associated with animal illnesses characterized by sudden death or acute neurological signs, with or without intestinal damage. Type B pathogenesis in ruminants is poorly understood, with some animals showing lesions and clinical signs similar to those caused by either type C or type D infections. It is unknown whether host or environmental conditions are dominant for determining the outcome of type B disease or if disease outcomes are determined by variable characteristics of type B isolates. To help clarify this issue, 19 type B isolates were evaluated for toxin production during late-log-phase growth via quantitative Western blotting and by biological activity assays. Most type B isolates produced CPB levels similar to those produced by type C isolates in vitro and have the potential to produce genotype C-like disease. The lethality of type B isolate supernatants administered intravenously to mice was evaluated with or without prior trypsin treatment, and monoclonal antibody neutralization studies also were performed. Correlation analyses comparing toxin levels in type B supernatants versus lethality and neutralization studies both found that the main contributor to lethality without pretreatment with trypsin was CPB, whereas neutralization studies indicated that CPB and ETX were both important after trypsin pretreatment. At least part of the CPB produced by type B isolates remained active after trypsin treatment. However, the overall lethalities of most supernatants were lower after trypsin pretreatment. Also, there was a significant association between ETX, CPB2, and CPA production in vitro among type B isolates. However, our results suggest that both CPB and ETX are likely the most important contributors to the pathogenesis of C. perfringens type B infections in domestic animals.

scholarly journals CodY Is a Global Regulator of Virulence-Associated Properties for Clostridium perfringens Type D Strain CN3718

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Jihong Li ◽  
Menglin Ma ◽  
Mahfuzur R. Sarker ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Null Mutant ◽  
Intestinal Infection ◽  
Wild Type ◽  
Global Regulator ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Type D ◽  
Epsilon Toxin ◽  
Virulence Properties

ABSTRACT CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection. IMPORTANCE Clostridium perfringens is a major human and livestock pathogen because it produces many potent toxins. C. perfringens type D strains cause intestinal infections by producing toxins, especially epsilon toxin (ETX). Previous studies identified CodY as a regulator of certain virulence properties in other Gram-positive bacteria. Our study now demonstrates that CodY is a global regulator of virulence-associated properties for type D strain CN3718. It promotes production of ETX, attachment of CN3718 vegetative cells or spores to host enterocyte-like Caco-2 cells, and spore germination; the last two effects may assist intestinal colonization. In contrast, CodY represses sporulation. These results provide the first evidence that CodY can function as a global regulator of C. perfringens virulence-associated properties and that this strain coordinately regulates its virulence-associated properties using CodY to increase ETX production, host cell attachment, and spore germination but to repress sporulation, as would be optimal during type D intestinal infection.

scholarly journals Effects of Clostridium perfringens Beta-Toxin on the Rabbit Small Intestine and Colon

2008 ◽  
Vol 76 (10) ◽  
pp. 4396-4404 ◽  
Author(s):  
Jorge E. Vidal ◽  
Bruce A. McClane ◽  
Juliann Saputo ◽  
Jaquelyn Parker ◽  
Francisco A. Uzal
Keyword(s):  
Small Intestine ◽  
Clostridium Perfringens ◽  
Time Course ◽  
Defense Mechanism ◽  
Intestinal Damage ◽  
Loop Model ◽  
Type B ◽  
Ileal Loop ◽  
Type C ◽  
Beta Toxin

ABSTRACT Clostridium perfringens type B and type C isolates, which produce beta-toxin (CPB), cause fatal diseases originating in the intestines of humans or livestock. Our previous studies demonstrated that CPB is necessary for type C isolate CN3685 to cause bloody necrotic enteritis in a rabbit ileal loop model and also showed that purified CPB, in the presence of trypsin inhibitor (TI), can reproduce type C pathology in rabbit ileal loops. We report here a more complete characterization of the effects of purified CPB in the rabbit small and large intestines. One microgram of purified CPB, in the presence of TI, was found to be sufficient to cause significant accumulation of hemorrhagic luminal fluid in duodenal, jejunal, or ileal loops treated for 6 h with purified CPB, while no damage was observed in corresponding loops receiving CPB (no TI) or TI alone. In contrast to the CPB sensitivity of the small intestine, the colon was not affected by 6 h of treatment with even 90 μg of purified CPB whether or not TI was present. Time course studies showed that purified CPB begins to induce small intestinal damage within 1 h, at which time the duodenum is less damaged than the jejunum or ileum. These observations help to explain why type B and C infections primarily involve the small intestine, establish CPB as a very potent and fast-acting toxin in the small intestines, and confirm a key role for intestinal trypsin as an innate intestinal defense mechanism against CPB-producing C. perfringens isolates.

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