Expression, crystallization and preliminary X-ray diffraction studies of recombinant<i>Clostridium perfringens</i>β2-toxin

Gurjar, Anagha; Yennawar, Neela H.; Yennawar, Hemant P.; Rajashankar, Kanagalaghatta R.; Hegde, Narasimha V.; Jayarao, Bhushan M.

doi:10.1107/s1744309107020313

Cited by 5 publications

(4 citation statements)

References 16 publications

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“…That toxin was then crystallized using the batch-under-oil technique. Crystallographic analysis of this CPB2 crystal showed that the triangular prism shaped crystals measuring 200 microns belonged to primitive rhombohedral space group R3 , diffracting up to 2.9 Å [166]. …”

Section: Iota Toxinmentioning

confidence: 99%

Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases

Uzal¹

2013

TOTNJ

141

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Clostridium perfringens is a gram-positive anaerobic rod that is classified into 5 toxinotypes (A, B, C, D, and E) according to the production of 4 major toxins, namely alpha (CPA), beta (CPB), epsilon (ETX) and iota (ITX). However, this microorganism can produce up to 16 toxins in various combinations, including lethal toxins such as perfringolysin O (PFO), enterotoxin (CPE), and beta2 toxin (CPB2). Most diseases caused by this microorganism are mediated by one or more of these toxins. The role of CPA in intestinal disease of mammals is controversial and poorly documented, but there is no doubt that this toxin is essential in the production of gas gangrene of humans and several animal species. CPB produced by C. perfringens types B and C is responsible for necrotizing enteritis and enterotoxemia mainly in neonatal individuals of several animal species. ETX produced by C. perfringens type D is responsible for clinical signs and lesions of enterotoxemia, a predominantly neurological disease of sheep and goats. The role of ITX in disease of animals is poorly understood, although it is usually assumed that the pathogenesis of intestinal diseases produced by C. perfringens type E is mediated by this toxin. CPB2, a necrotizing and lethal toxin that can be produced by all types of C. perfringens, has been blamed for disease in many animal species, but little information is currently available to sustain or rule out this claim. CPE is an important virulence factor for C. perfringens type A gastrointestinal disease in humans and dogs; however, the data implicating CPE in other animal diseases remains ambiguous. PFO does not seem to play a direct role as the main virulence factor for animal diseases, but it may have a synergistic role with CPA-mediated gangrene and ETX-mediated enterotoxemia. The recent improvement of animal models for C. perfringens infection and the use of toxin gene knock-out mutants have demonstrated the specific pathogenic role of several toxins of C. perfringens in animal disease. These research tools are helping us to establish the role ofThis is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

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Section: Iota Toxinmentioning

confidence: 99%

Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases

Uzal¹

2013

TOTNJ

141

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“…Further studies demonstrated the in vitro cytotoxicity of the native CPB2 toxin in different intestinal cell lines; however, currently, its exact role in pathogenesis remains unclear [ 13 , 15 – 20 ]. In the last decades, many studies have been focused on the use of purified recombinant consensus CPB2 expressed in prokaryotic systems for immunological and crystallographic studies [ 21 , 22 ]. Moreover, recombinant consensus CPB2 protein was employed as an antigen to produce antibodies and was subsequently used for the development of an Enzyme-linked Immunosorbent Assay (ELISA) for the detection and quantification of the toxin in the piglet’s intestinal material [ 6 , 7 , 9 , 13 , 23 – 25 ].…”

Section: Introductionmentioning

confidence: 99%

Expression of deleted, atoxic atypical recombinant beta2 toxin in a baculovirus system and production of polyclonal and monoclonal antibodies

et al. 2017

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Background Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. The beta-2 minor toxin (CPB2), comprising atypical and consensus variants, appears to be involved in both human and animal enterotoxaemia syndrome. The exact role of CPB2 in pathogenesis is poorly investigated, and its mechanism of action at the molecular level is still unknown because of the lack of specific reagents such as monoclonal antibodies against the CPB2 protein and/or the availability of a highly purified antigen. Previous studies have reported that purified wild-type or recombinant CPB2 toxin, expressed in a heterologous system, presented cytotoxic effects on human intestinal cell lines. Undoubtedly, for this reason, to date, these purified proteins have not yet been used for the production of monoclonal antibodies (MAbs). Recently, monoclonal antibodies against CPB2 were generated using peptides designed on predicted antigenic epitopes of this toxin.ResultsIn this paper we report, for the first time, the expression in a baculovirus system of a deleted recombinant C-terminal 6xHis-tagged atypical CPB2 toxin (rCPB2Δ1–25-His6) lacking the 25 amino acids (aa) of the N-terminal putative signal sequence. A high level of purified recombinant rCPB2Δ1–25-His6 was obtained after purification by Ni2+ affinity chromatography. The purified product showed no in vitro and in vivo toxicity. Polyclonal antibodies and twenty hybridoma-secreting Mabs were generated using purified rCPB2Δ1–25-His6. Finally, the reactivity and specificity of the new antibodies were tested against both recombinant and wild-type CPB2 toxins.ConclusionsThe high-throughput of purified atoxic recombinant CPB2 produced in insect cells, allowed to obtain monoclonal and polyclonal antibodies. The availability of these molecules could contribute to develop immunoenzymatic methods and/or to perform studies about the biological activity of CPB2 toxin.

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“…CPB2 is an accessory toxin produced by C. perfrignens associated with porcine, equine, and bovine enteritis. While the structure of CPB2 and the identification of residues responsible for its antigenicity and association with the membrane of intestinal epithelial cells are under study [ 67 ], CPB2 has been reported as a pore-forming toxin. Its action mechanism involves forming cation-selective channels approximately 1.4 nm in diameter in lipid bilayers, leading to altered ion flux and increased intestinal permeability [ 34 ].…”

Section: Formation Of Poresmentioning

confidence: 99%

Unveiling the pathogenic mechanisms of Clostridium perfringens toxins and virulence factors

Camargo,

Ramírez,

Kiu

et al. 2024

Emerging Microbes & Infections

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Clostridium perfringens causes multiple diseases in humans and animals. Its pathogenic effect is supported by a broad and heterogeneous arsenal of toxins and other virulence factors associated with a specific host tropism. Molecular approaches have indicated that most C. perfringens toxins produce membrane pores, leading to osmotic cell disruption and apoptosis. However, identifying mechanisms involved in cell tropism and selective toxicity effects should be studied more. The differential presence and polymorphisms of toxin-encoding genes and genes encoding other virulence factors suggest that molecular mechanisms might exist associated with host preference, receptor binding, and impact on the host; however, this information has not been reviewed in detail. Therefore, this review aims to clarify the current state of knowledge on the structural features and mechanisms of action of the major toxins and virulence factors of C. perfringens and discuss the impact of genetic diversity of toxinotypes in tropism for several hosts.

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Expression, crystallization and preliminary X-ray diffraction studies of recombinantClostridium perfringensβ2-toxin

Cited by 5 publications

References 16 publications

Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases

Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases

Expression of deleted, atoxic atypical recombinant beta2 toxin in a baculovirus system and production of polyclonal and monoclonal antibodies

Unveiling the pathogenic mechanisms of Clostridium perfringens toxins and virulence factors

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