Correction for Chen et al., 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

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.

Download Full-text

Identifying the Basis for VirS/VirR Two-Component Regulatory System Control of Clostridium perfringens Beta Toxin Production

Journal of Bacteriology ◽

10.1128/jb.00279-21 ◽

2021 ◽

Author(s):

Iman Mehdizadeh Gohari ◽

Jihong Li ◽

Bruce A. McClane

Keyword(s):

Quorum Sensing ◽

Clostridium Perfringens ◽

Regulatory System ◽

Toxin Production ◽

Start Codon ◽

Binding Motif ◽

Type B ◽

Regulatory Cascade ◽

Two Component ◽

Beta Toxin

Clostridium perfringens toxin production is often regulated by the Agr-like quorum sensing (QS) system signaling the VirS/VirR two-component regulatory system (TCRS), which consists of the VirS membrane sensor histidine kinase and the VirR response regulator. VirS/VirR is known to directly control expression of some genes by binding to a DNA binding motif consisting of two VirR boxes located within 500 bp of the target gene start codon. Alternatively, the VirS/VirR system can indirectly regulate production levels of other proteins by increasing expression of a small regulatory RNA (VR-RNA). Previous studies demonstrated that beta toxin (CPB) production by C. perfringens type B and C strains is positively-regulated by both the Agr-like QS and VirS/VirR TCRS, but the mechanism has been unclear. The current study first inactivated the vrr gene encoding VR-RNA to show that VirS/VirR regulation of cpb expression does not involve VR-RNA. Subsequently, bioinformatic analyses identified a potential VirR binding motif, along with a predicted strong promoter, ∼1.4 kb upstream of the cpb open reading frame (ORF). Two insertion sequences were present between this VirR binding motif/promoter region and the cpb ORF. PCR screening of a collection of strains carrying cpb showed that the presence and sequence of this VirR binding motif/promoter is highly conserved among CPB-producing strains. RT-PCR and a GusA reporter assay showed this VirR binding motif is important for regulating CPB producion. These findings indicate that VirS/VirR directly regulates cpb expression via VirS binding to a VirR binding motif located unusually distant from the cpb start codon. IMPORTANCE Clostridium perfringens beta toxin (CPB) is only produced by type B and C strains. Production of CPB is essential for the pathogenesis of type C-associated infections, which include hemorrhagic necrotizing enteritis and enterotoxemia in both humans and animals. In addition, CPB can synergize with other toxins during C. perfringens gastrointestinal diseases. CPB toxin production is cooperatively regulated by the Agr-like quorum sensing (QS) system and the VirS/VirR two-component regulatory system. This study now reports that the VirS/VirR regulatory cascade directly controls expression of the cpb gene via a process involving a VirR box binding motif located unusually far (∼1.4 kb) upstream of the cpb ORF. This study provides a better understanding of the regulatory mechanisms for CPB production by the VirS/VirR regulatory cascade.

Download Full-text

CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101

Infection and Immunity ◽

10.1128/iai.00855-16 ◽

2017 ◽

Vol 85 (3) ◽

Cited By ~ 9

Author(s):

Jihong Li ◽

John C. Freedman ◽

Daniel R. Evans ◽

Bruce A. McClane

Keyword(s):

Gene Expression ◽

Clostridium Perfringens ◽

Food Poisoning ◽

Type A ◽

Toxin Production ◽

Null Mutant ◽

Wild Type ◽

Content Type ◽

Type D ◽

Epsilon Toxin

ABSTRACT Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY-null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY-null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY-null mutant strain but significantly increased in the SM101 codY-null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation.

Download Full-text

Development and Application of an Oral Challenge Mouse Model for Studying Clostridium perfringens Type D Infection

Infection and Immunity ◽

10.1128/iai.00562-07 ◽

2007 ◽

Vol 75 (9) ◽

pp. 4282-4288 ◽

Cited By ~ 25

Author(s):

Mariano E. Fernandez-Miyakawa ◽

Sameera Sayeed ◽

Derek J. Fisher ◽

Rachael Poon ◽

Vicki Adams ◽

...

Keyword(s):

Mouse Model ◽

Clostridium Perfringens ◽

Clinical Signs ◽

Passive Immunization ◽

Small Animal ◽

Toxin Production ◽

Oral Challenge ◽

Type D ◽

Epsilon Toxin

ABSTRACT Clostridium perfringens type D isolates cause enterotoxemia in sheep, goats, and probably cattle. While the major disease signs and lesions of type D animal disease are usually attributed to epsilon toxin, a class B select agent, these bacteria typically produce several lethal toxins. Understanding of disease pathogenesis and development of improved vaccines are hindered by the lack of a small-animal model mimicking natural disease caused by type D isolates. Addressing this need, we developed an oral challenge mouse model of C. perfringens type D enterotoxemia. When BALB/c mice with a sealed anus were inoculated by intragastric gavage with type D isolates, 7 of 10 type D isolates were lethal, as defined by spontaneous death or severe clinical signs necessitating euthanasia. The lethalities of the seven type D isolates varied between 14 and 100%. Clinical signs in the lethally challenged mice included seizures, convulsions, hyperexcitability, and/or depression. Mild intestinal gas distention and brain edema were observed at necropsy in a few mice, while histology showed multifocal acute tubular necrosis of the kidney and edema in the lungs of most challenged mice that developed a clinical response. When the lethality of type D isolates in this model was compared with in vitro toxin production, only a limited correlation was observed. However, mice could be protected against lethality by intravenous passive immunization with an epsilon toxin antibody prior to oral challenge. This study provides an economical new model for studying the pathogenesis of C. perfringens type D infections.

Download Full-text

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

mBio ◽

10.1128/mbio.00770-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 27

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.

Download Full-text

The production and evaluation of monoclonal antibodies to Clostridium perfringens type D epsilon toxin

Journal of Biological Standardization ◽

10.1016/0092-1157(88)90008-x ◽

1988 ◽

Vol 16 (3) ◽

pp. 207-218 ◽

Cited By ~ 7

Author(s):

C.D.H. Boarer ◽

M.G. Sojka ◽

V.J. White ◽

P.L. Roeder

Keyword(s):

Monoclonal Antibodies ◽

Clostridium Perfringens ◽

Type D ◽

Epsilon Toxin

Download Full-text

Outbreak of Clostridium perfringens type D enterotoxemia in sheep

Semina Ciências Agrárias ◽

10.5433/1679-0359.2019v40n6p2593 ◽

2019 ◽

Vol 40 (6) ◽

pp. 2593

Author(s):

Felipe Masiero Salvarani ◽

Mayane Faccin ◽

Nayra Fernanda de Queiroz Ramos Freitas ◽

Mônica Regina de Matos ◽

Edismair Carvalho Garcia ◽

...

Keyword(s):

Clostridium Perfringens ◽

Clinical Signs ◽

Necrotic Enteritis ◽

Spastic Paralysis ◽

Type D ◽

Laboratory Confirmation ◽

Intestinal Contents ◽

Epsilon Toxin ◽

History Of ◽

Diagnostic Modalities

This work describes the first Brazilian laboratory-confirmed outbreak of enterotoxemia caused by Clostridium perfringens type D in sheep, which occurred in the state of Paraná. We address the epidemiological aspects involved, the diagnostic modalities employed, and the clinical signs and pathological findings observed. Eight healthy pregnant female sheep with no history of vaccination for clostridiosis presented with a history of abrupt feeding changes and neurological manifestations that quickly evolved to illness, coma and death. Four other females with clinical neurological signs were referred to the Veterinary Hospital of the Universidade Federal do Paraná, Palotina Sector. These animals presented with lethargy, motor incoordination, opisthotonus, pedal movements, muscle tremors, spastic paralysis, bruxism, mandibular trismus, sialorrhea, hyperexcitability and the inability to stand. They were examined and euthanized due to the seriousness of the clinical picture with an unfavorable prognosis. We performed gross anatomical and microscopic analyses of the organs and intestinal contents. We also performed bacterial isolation with molecular typing. From the intestinal contents, we detected toxins by means of the seroneutralization technique in mice. At necropsy, we noted pulmonary edema (2/4), necrotizing enteritis (4/4) and hyperemia of the leptomeninges (1/4). Microscopically, we observed lymphohistiocytic interstitial pneumonia, necrotic enteritis associated with the presence of rods, and nephrosis with interstitial lymphohistiocytic nephritis. No significant brain lesions were observed. Using serum neutralization, we identified epsilon toxin in the intestinal contents of all four animals. C. perfringens type D was identified. Based on the history, clinical signs, postmortem findings, and laboratory confirmation of the presence of epsilon toxin, we concluded that C. perfringens type D enterotoxemia caused this outbreak of sheep deaths.

Download Full-text

Pathogenesis and diagnostic features of brain and ophthalmic damage produced by Clostridium perfringens type D epsilon toxin

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638719900190 ◽

2020 ◽

Vol 32 (2) ◽

pp. 282-286 ◽

Cited By ~ 2

Author(s):

John W. Finnie ◽

Mauricio A. Navarro ◽

Francisco A. Uzal

Keyword(s):

Clostridium Perfringens ◽

Vasogenic Edema ◽

Differential Susceptibility ◽

Brain Regions ◽

Cerebral Lesions ◽

Diagnostic Features ◽

Microvascular Damage ◽

Neurologic Disorder ◽

Type D ◽

Epsilon Toxin

Clostridium perfringens type D epsilon toxin (EXT) causes an important neurologic disorder of sheep, goats and, rarely, cattle. The disease can occur in peracute, acute, subacute, and chronic forms. High circulating levels of ETX produce vasculocentric brain lesions, in which microvascular endothelial injury results in diagnostically useful perivascular and intramural extravasations of plasma protein, especially in sheep, and less frequently in goats. With lower toxin doses, a more protracted clinical course tends to occur, particularly in sheep, leading to focal, bilaterally symmetrical, necrotic foci in certain brain regions. Although these morphologic features usually permit the diagnostic pathologist to make a definitive etiologic diagnosis, there are many aspects of the pathogenesis of these cerebral lesions that are not completely understood. ETX has also been shown to produce microvascular damage in the retina of rats, resulting in severe, diffuse vasogenic edema, similar to that found in brains exposed to this neurotoxin. The pathoclisis and vascular theories offer alternative explanations of the differential susceptibility of different brain regions to the same neurotoxic insult.

Download Full-text