Development of a New Bifunctional Fusion Protein of Vaccine Strains Clostridium perfringens Type D and Clostridium septicum Epsilon-alpha Toxin Genes

Clostridium perfringens is currently classified into five types (A, B, C, D, E) based on the different toxins produced. Type A and C are known as the causative agent of enteritis and enterotoxaemia in newborn and young piglets with severe intestinal lesions including edema, hemorrhage and necrosis. A multiplex PCR (mPCR) was developed in order to quickly and early determine the presence of genotypes of C. perfringens based on their genes of cpa, cpb, cpb2 and cpe encoding alpha toxin, beta toxin, beta2 toxin and enterotoxin with predicted products of 324 bp, 196 bp, 107 bp and 257 bp respectively. The detection limit of the mPCR assay was 1 × 103 copies/reaction for each gene. Sequencing of mPCR products performed with clinical samples collected from C. perfringens suspected pigs showed that the mPCR test functioned specifically. In conclusion, the developed mPCR test successfully detected the presence of genes cpa, cpb, cpb2 and cpe in the examined samples. Analysis of the bacteria isolated from field samples of diarrheal piglets collected in this study indicated that C. perfringens carrying gene cpa counted for 96.66% and 3.33% was identified as C. perfringens carrying genes cpa and cpb concurrently. Gene cpe was not found in this study, while gene cpb2 was detected coincidently in 73.33% of the samples with cpa gene. The results indicate that the prevalence of these four toxin genes is cpa, cpb2, cpb and cpe in decending order.

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Epsilon-Toxin Is Required for Most Clostridium perfringens Type D Vegetative Culture Supernatants To Cause Lethality in the Mouse Intravenous Injection Model

Infection and Immunity ◽

10.1128/iai.73.11.7413-7421.2005 ◽

2005 ◽

Vol 73 (11) ◽

pp. 7413-7421 ◽

Cited By ~ 50

Author(s):

Sameera Sayeed ◽

M. E. Fernandez-Miyakawa ◽

Derek J. Fisher ◽

Vicki Adams ◽

Rachael Poon ◽

...

Keyword(s):

Monoclonal Antibody ◽

Clostridium Perfringens ◽

Alpha Toxin ◽

Specific Monoclonal Antibody ◽

Experimental Conditions ◽

Proteolytic Activation ◽

Type D ◽

Injection Model ◽

Epsilon Toxin ◽

Lethal Toxins

ABSTRACT Clostridium perfringens type D enterotoxemias have significant economic impact by causing rapid death of several domestic animal species. Consequently, domestic animals are commonly vaccinated, at varying efficacy, with inactivated type D vegetative supernatants. Improved type D vaccines might become possible if the lethal toxins produced by type D isolates were characterized and the contributions of those toxins to supernatant-induced lethality were established. Therefore, the current study evaluated the presence of lethal toxins in supernatants prepared from late-log-phase vegetative cultures of a large collection of genotype D isolates. Under this growth condition, most genotype D isolates produced variable levels of at least three different lethal toxins, including epsilon-toxin (ETX). To model the rapid lethality of type D enterotoxemias, studies were conducted involving intravenous (i.v.) injection of genotype D vegetative supernatants into mice, which were then observed for neurotoxic distress. Those experiments demonstrated a correlation between ETX (but not alpha-toxin or perfringolysin O) levels in late-log-phase genotype D supernatants and lethality. Consistent with the known proteolytic activation requirement for ETX toxicity, trypsin pretreatment was required for, or substantially increased, the lethality of nearly all of the tested genotype D vegetative supernatants. Finally, the lethality of these trypsin-pretreated genotype D supernatants could be completely neutralized by an ETX-specific monoclonal antibody but not by an alpha-toxin-specific monoclonal antibody. Collectively, these results indicate that, under the experimental conditions used in the present study, ETX is necessary for the lethal properties of most genotype D vegetative supernatants in the mouse i.v. injection model.

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In silico analysis of a chimeric fusion protein as a new vaccine candidate against Clostridium perfringens type A and Clostridium septicum alpha toxins

Comparative Clinical Pathology ◽

10.1007/s00580-020-03136-6 ◽

2020 ◽

Vol 29 (5) ◽

pp. 981-989

Author(s):

Ali Haghroosta ◽

Hossein Goudarzi ◽

Ebrahim Faghihloo ◽

Zohreh Ghalavand ◽

Mohammad Mahdi Ranjbar ◽

...

Keyword(s):

Fusion Protein ◽

Clostridium Perfringens ◽

In Silico ◽

Vaccine Candidate ◽

Fusion Gene ◽

In Silico Analysis ◽

Type A ◽

Gas Gangrene ◽

Clostridium Septicum ◽

Silico Analysis

Abstract In silico analysis is the most important approach to understand protein structure and functions, and the most important problem for designing and producing a fusion construct is producing large amounts of functional protein. Clostridium perfringens type A and Clostridium septicum produce alpha (plc) and alpha toxins respectively. C. perfringens can cause gas gangrene and gastrointestinal diseases. C. septicum can cause traumatic and non-traumatic gas gangrene. The aim of current research was in silico analysis of a chimeric fusion protein against C. perfringens type A and C. septicum alpha toxins. Firstly, the chimeric fusion gene was designed according to nucleotide sequences of C. perfringens type A alpha (KY584046.1) and C. septicum alpha (JN793989.2) toxin genes and then its fusion protein is constructed by amino acid sequences of C. perfringens type A and C. septicum alpha toxins. Secondly, online software was used to determine prediction of secondary and tertiary structures and physicochemical characteristics of the fusion protein. Finally, the validation of the fusion protein was confirmed by Rampage and proSA program. The designed fusion protein has 777 amino acids in length. TASSER server and physicochemical parameters are showed: C-score = − 2.68 and molecular weight = 87.9 KD respectively. Rampage and proSA software revealed the fusion protein is valid. Deposited accession number for the sequence of the fusion gene in the GenBank is MK908396. The designed fusion protein is valid and functional. Thus, the fusion gene could be used for clone and expression in a proper prokaryotic cell and also as a recombinant vaccine candidate.

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Prevalence and Genetic Diversity of Toxin Genes in Clinical Isolates of Clostridium perfringens: Coexistence of Alpha-Toxin Variant and Binary Enterotoxin Genes (bec/cpile)

Toxins ◽

10.3390/toxins11060326 ◽

2019 ◽

Vol 11 (6) ◽

pp. 326 ◽

Cited By ~ 5

Author(s):

Asami Matsuda ◽

Meiji Soe Aung ◽

Noriko Urushibara ◽

Mitsuyo Kawaguchiya ◽

Ayako Sumi ◽

...

Keyword(s):

Genetic Diversity ◽

Clostridium Perfringens ◽

Clinical Isolates ◽

Nucleotide Sequencing ◽

Toxin Gene ◽

Alpha Toxin ◽

Toxin Genes ◽

Food Borne ◽

Putative Origin ◽

High Level

Clostridium perfringens (C. perfringens) is responsible for food-borne gastroenteritis and other infectious diseases, and toxins produced by this bacterium play a key role in pathogenesis. Although various toxins have been described for C. perfringens isolates from humans and animals, prevalence of individual toxins among clinical isolates has not yet been well explored. In the present study, a total of 798 C. perfringens clinical isolates were investigated for prevalence of eight toxin genes and their genetic diversity by PCR, nucleotide sequencing, and phylogenetic analysis. Besides the alpha-toxin gene (plc) present in all the isolates, the most common toxin gene was cpe (enterotoxin) (34.2%), followed by cpb2 (beta2 toxin) (1.4%), netB (NetB) (0.3%), and bec/cpile (binary enterotoxin BEC/CPILE) (0.1%), while beta-, epsilon-, and iota-toxin genes were not detected. Genetic analysis of toxin genes indicated a high level of conservation of plc, cpe, and netB. In contrast, cpb2 was revealed to be considerably divergent, containing at least two lineages. Alpha-toxin among 46 isolates was classified into ten sequence types, among which common types were distinct from those reported for avian isolates. A single isolate with bec/cpile harbored a plc variant containing an insertion of 834-bp sequence, suggesting its putative origin from chickens.

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Molecular Cloning of a New Bi-Functional Fusion Protein of Clostridium perfringens Type A Alpha and Clostridium septicum Alpha Toxin Genes in E. coli

Archives of Clinical Infectious Diseases ◽

10.5812/archcid.95502 ◽

2021 ◽

Vol 15 (6) ◽

Author(s):

Ali Haghroosta ◽

Hossein Goudarzi ◽

Zohreh Ghalavand ◽

Ebrahim Faghihloo ◽

Reza Pilehchian Langroudi

Keyword(s):

Fusion Protein ◽

Clostridium Perfringens ◽

Gel Electrophoresis ◽

Fusion Gene ◽

Gas Gangrene ◽

Alpha Toxin ◽

Protein Fusion ◽

Structural Prediction ◽

E Coli ◽

Fusion Pcr

Background: A synthetic construct bi-functional protein fusion includes two protein domains, or proteins bind by a fragment. The synthetic construct is designed to achieve better characterize and new functionality. Therefore, having proper cells is essential for cloning fusion genes. Clostridium perfringens type A produces the alpha-toxin and can cause gas gangrene and gastrointestinal diseases. C. septicum produces the alpha-toxin and can cause non-traumatic and traumatic gas gangrene. Objectives: The current study aimed to investigate molecular cloning of a new bi-functional fusion protein of C. perfringens alpha (cpa) and C. septicum alpha (csa) toxin genes in E. coli TOP10. In silico analysis was used for the chimeric fusion protein structural prediction. Methods: To produce chimeric fusion protein, the alpha-alpha (α-α) fusion gene was designed according to nucleotide sequences of cpa (KY584046.1) and csa (JN793989.2) genes. Tertiary structural prediction and validation of the fusion protein were determined by online software. In the new synthetic construction, α-α fusion protein genes are bind via the linker AEAAAKEAAAKA. The linker was introduced between the two domains by fusion PCR. The synthetic fusion gene was cloned into the pUC57cloning vector and then transferred into the host cell. Results: Analysis of the chimeric protein fusion is showed using the I-TASSER server as C-score equal to -2.68 as well as Rampage software in order to confirm the geometrical model as a natural like protein. Also, 1.0% agarose gel electrophoresis of fusion PCR product and sequencing analysis revealed a DNA fragment length of 2346 bp. Screening gel electrophoresis showed 996 bp length, which the designed linker was contained in it. Gel electrophoresis of extracted and purified recombinant plasmid (pUC57/αα) showed that a pUC57/αα of 5056 bp. The digested recombinant pUC57/αα showed one 2.3 kb (our fusion gene) band and one 2.7 kb (pUC57) band. Conclusions: This study presented a new approach for the fusion of cpa and csa genes based on the fusion PCR strategy. According to the latest information, this is the first time that α-α fusion gene is designed and cloned into a suitable cloning vector.

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Phenotypic detection and genotyping of Clostridium perfringens associated with enterotoxemia in sheep in the Qassim Region of Saudi Arabia

Veterinary World ◽

10.14202/vetworld.2021.578-584 ◽

2021 ◽

Vol 14 (3) ◽

pp. 578-584

Author(s):

Fehaid Alsaab ◽

Ali Wahdan ◽

Elhassan M. A. Saeed

Keyword(s):

Saudi Arabia ◽

Clostridium Perfringens ◽

Culture Method ◽

Economic Loss ◽

Intestinal Content ◽

Toxin Gene ◽

Rt Pcr ◽

Toxin Genes ◽

Type D ◽

Vitek 2

Background and Aim: Enterotoxemia caused by Clostridium perfringens toxinotypes is an often fatal disease of sheep of all ages, with a substantial economic loss to the sheep industry. This study was conducted to isolate C. perfringens from suspected cases of enterotoxemia in sheep in the central part of the Qassim Region, Saudi Arabia, and to determine the prevalent toxinotype by detecting alpha (cpA), beta (cpB), and epsilon (etX) toxin genes, which might help control this disease locally. Materials and Methods: A total of 93 rectal swabs and intestinal content samples were collected from diseased and animals suspected of having died of enterotoxemia in early 2020. Samples were subjected to bacteriological examination, biochemical analysis of isolates by VITEK 2, and molecular toxinotyping of isolates by LightCycler® real-time polymerase chain reaction (RT-PCR). Results: Our results revealed that only 14 isolates were confirmed by VITEK 2 as being C. perfringens, with excellent identification (probability of 95% and 97%). According to the toxinotyping of isolates by RT-PCR, all 14 isolates possessed both the cpA and etX toxin genes, while the cpB toxin gene was not detected in any of the isolates. Conclusion: Our findings demonstrated that C. perfringens type D was the only toxinotype found in the central part of the Qassim Region in 2020; moreover, according to the culture method, only 15% (14/93) of the suspected cases of enterotoxemia were confirmed to be caused by C. perfringens infection, which highlighted the importance of clinical and laboratory differential diagnosis of enterotoxemia in sheep.

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