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

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.

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

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.

scholarly journals Perfringolysin O Expression in Clostridium perfringens Is Independent of the Upstream pfoR Gene

2002 ◽  
Vol 184 (7) ◽  
pp. 2034-2038 ◽  
Author(s):  
Milena M. Awad ◽  
Julian I. Rood
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Gene Product ◽  
Structural Gene ◽  
Clostridium Perfringens ◽  
Deletion Mutant ◽  
Gas Gangrene ◽  
Alpha Toxin ◽  
Clostridial Myonecrosis ◽  
Perfringolysin O

ABSTRACT The pathogenesis of Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis involves the extracellular toxins alpha-toxin and perfringolysin O. Previous studies (T. Shimizu, A. Okabe, J. Minami, and H. Hayashi, Infect. Immun. 59:137-142, 1991) carried out with Escherichia coli suggested that the perfringolysin O structural gene, pfoA, was positively regulated by the product of the upstream pfoR gene. In an attempt to confirm this hypothesis in C. perfringens, a pfoR-pfoA deletion mutant was complemented with isogenic pfoA+ shuttle plasmids that varied only in their ability to encode an intact pfoR gene. No difference in the ability to produce perfringolysin O was observed for C. perfringens strains carrying these plasmids. In addition, chromosomal pfoR mutants were constructed by homologous recombination in C. perfringens. Again no difference in perfringolysin O activity was observed. Since it was not possible to alter perfringolysin O expression by mutation of pfoR, it was concluded that the pfoR gene product is unlikely to have a role in the regulation of pfoA expression in C. perfringens.

scholarly journals Amentoflavone Attenuates Clostridium perfringens Gas Gangrene by Targeting Alpha-Toxin and Perfringolysin O

2020 ◽  
Vol 11 ◽  
Author(s):  
Shui Liu ◽  
Xiaofeng Yang ◽  
Hong Zhang ◽  
Jian Zhang ◽  
Yonglin Zhou ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Gas Gangrene ◽  
Alpha Toxin ◽  
Perfringolysin O

scholarly journals Synergistic Effects of Alpha-Toxin and Perfringolysin O in Clostridium perfringens-Mediated Gas Gangrene

2001 ◽  
Vol 69 (12) ◽  
pp. 7904-7910 ◽  
Author(s):  
Milena M. Awad ◽  
Darren M. Ellemor ◽  
Richard L. Boyd ◽  
John J. Emmins ◽  
Julian I. Rood
Keyword(s):  
Structural Gene ◽  
Clostridium Perfringens ◽  
Synergistic Effects ◽  
Gas Gangrene ◽  
Alpha Toxin ◽  
Coagulative Necrosis ◽  
Clostridial Myonecrosis ◽  
Perfringolysin O ◽  
Vascular Disruption ◽  
Rapid Spread

ABSTRACT To examine the synergistic effects of alpha-toxin and perfringolysin O in clostridial myonecrosis, homologous recombination was used to construct an alpha-toxin deficient derivative of a perfringolysin O mutant of Clostridium perfringens. The subsequent strain was complemented with separate plasmids that carried the alpha-toxin structural gene (plc), the perfringolysin O gene (pfoA), or both toxin genes, and the resultant isogenic strains were examined in a mouse myonecrosis model. Synergistic effects were clearly observed in these experiments. Infection with the control strain, which did not produce either toxin, resulted in very minimal gross pathological changes, whereas the isogenic strain that was reconstituted for both toxins produced a pathology that was clearly more severe than when alpha-toxin alone was reconstituted. These changes were most apparent in the rapid spread of the disease, the gross pathology of the footpad and in the rate at which the mice had to be euthanatized for ethical reasons. Elimination of both alpha-toxin and perfringolysin O production removed most of the histopathological features typical of clostridial myonecrosis. These effects were restored when the mutant was complemented with the alpha-toxin structural gene, but reconstituting only perfringolysin O activity produced vastly different results, with regions of coagulative necrosis, apparently enhanced by vascular disruption, being observed. Reconstitution of both alpha-toxin and perfringolysin O activity produced histopathology most similar to that observed with the alpha-toxin reconstituted strain. The spreading of myonecrosis was very rapid in these tissues, and coagulative necrosis appeared to be restricted to the lumen of the blood vessels. The results of these virulence experiments clearly support the hypothesis that alpha-toxin and perfringolysin O have a synergistic effect in the pathology of gas gangrene.

scholarly journals Cloning, expression, purification and interaction evaluation of 30 amino acids in C terminus of Clostridium perfringens toxin with its receptor Claudin-4

2019 ◽  
Vol 2 (4) ◽  
pp. 47-55
Author(s):  
Quang Kien Huynh ◽  
An Hoang Nguyen ◽  
Quynh Thi Mong Pham ◽  
Hoan Phuoc Khai Nguyen ◽  
Hieu Van Tran
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Clostridium Perfringens ◽  
Field Effect Transistors ◽  
Oral Vaccine ◽  
Soluble Form ◽  
M Cells ◽  
Gastrointestinal Infections ◽  
E Coli ◽  
C Terminus

Oral vaccine is a strategy being the most interested about treatments of gastrointestinal infections because of many great benefits outweigh conventional injection vaccines. In order to resolve the dispersion of antigens in gastrointestinal surfaces, the immunological tolerance and also be capable to stimulate immune responses effectively, M cells are targeted for antigens delivery. A number of researches reported that 30 amino acids in C terminus of Clostridium perfringens toxin (CPE30) have a high affinity to Claudin-4 receptor presenting on M cells. It is highly indispensable to produce a resource for assessing of CPE30 binding ability so cpe30 gene was cloned into the pET-gfp plasmid by two restriction enzymes BamHI and NdeI on the E. coli DH5α strain. The expression and confirmation of the fusion protein CPE30-GFP which was induced by IPTG in E. coli BL21 (DE3) strain and assessed by SDS-PAGE and Western blot with 6xHis Taq antibody demonstrated that there was the over expression of CPE30 GFP fusion protein in the cytoplasm, mainly in the soluble form. Finally, CPE30-GFP was purified which the purity was approximately 92.3%. In vitro protein interaction measurement using silicon nanowire field-effect transistors (SiNW FETs) showed that CPE30-GFP had a good binding affinity with its receptor Claudin-4 (R4). This result laid the groundwork for the CPE30 interaction study with the M cell in vivo.

scholarly journals Use of Genetically Manipulated Strains of Clostridium perfringens Reveals that Both Alpha-Toxin and Theta-Toxin Are Required for Vascular Leukostasis To Occur in Experimental Gas Gangrene

1999 ◽  
Vol 67 (9) ◽  
pp. 4902-4907 ◽  
Author(s):  
Darren M. Ellemor ◽  
Rebecca N. Baird ◽  
Milena M. Awad ◽  
Richard L. Boyd ◽  
Julian I. Rood ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Type A ◽  
Toxin Production ◽  
Gas Gangrene ◽  
Necrotic Tissue ◽  
Alpha Toxin ◽  
Tissue Necrosis ◽  
Clostridial Myonecrosis ◽  
Leukocyte Aggregation ◽  
Genetically Manipulated

ABSTRACT A hallmark of gas gangrene (clostridial myonecrosis) pathology is a paucity of leukocytes infiltrating the necrotic tissue. The cause of this paucity most likely relates to the observation of leukocyte aggregates at the border of the area of tissue necrosis, often within the microvasculature itself. Infecting mice with genetically manipulated strains of Clostridium perfringens type A (deficient in either alpha-toxin or theta-toxin production) resulted in significantly reduced leukocyte aggregation when alpha-toxin was absent and complete abrogation of leukocyte aggregation when theta-toxin was absent. Thus, both alpha-toxin and theta-toxin are necessary for the characteristic vascular leukostasis observed in clostridial myonecrosis.

scholarly journals Cloning, expression and purification of m-cell specific binding peptide (CO1) fused with GFP

2018 ◽  
Vol 14 (4) ◽  
pp. 599-604
Author(s):  
Nguyen Hoang An ◽  
Dang Tat Truong ◽  
Tran Van Hieu
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Specific Binding ◽  
Soluble Form ◽  
Fluorescent Light ◽  
M Cell ◽  
E Coli ◽  
Monitoring Model ◽  
Overlap Extension

Despite many advantages over injection vaccines such as cost effectiveness, safety and easy to use, and so on, oral vaccines are negligibly concerned. This is mostly because of the availability of vast surface in the gastro-intestinal tract, thereby requiring lot of antigens which could hamper their potential. To circumvent this issue, a novel strategy for targeting antigens to M cells (microfold cells), a minority of cells located in the small intestine for antigen transportation, is utilized by making a fusion protein comprised of an antigen with an M cell specific ligand. Discovered via biopanning, Co1 peptide is a potential ligand because of its small size (12 amino acids) and having an adjuvant capacity. To develop a monitoring model, we fused GFP (green fluorescent protein) as a monitoring marker with Co1 peptide. Initially, co1-gfp fusion gene was created by overlap extension PCR on GFP-encoded vector backbone, then it was incorporated into an expression vector pET22b before transforming into E. coli DH5α. The recombinant vector was screened by PCR method, sequenced and aligned with designed sequences. The in-frame vector was then introduced into E. coli BL21(DE3) for expression by inducing with 0.5mM IPTG. Fusion protein was purified using Ni-affinity chromatography. The results showed that the fused genes were in-frame cloned and completely matched with the designed sequences. SDS-PAGE and Western blot analyses showed Co1-GFP protein expressed in soluble form and could be purified at one-step elution of 500mM imidazole. The purified fusion protein could emit fluorescent light under UV excitation. Collectively, recombinant Co1-GFP fusion protein was successfully produced and its potential applications need to be warranted.

Experimental gas gangrene with food-poisoning Clostridium perfringens type A

1968 ◽  
Vol 14 (6) ◽  
pp. 705-709 ◽  
Author(s):  
A. H. W. Hauschild ◽  
F. S. Thatcher
Keyword(s):  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Type A ◽  
Gas Gangrene ◽  
Sheep Blood Agar ◽  
Alpha Toxin ◽  
Cell Numbers ◽  
Fatal Infection ◽  
Virulent Strains ◽  
Resistant Strains

Classical and food-poisoning strains of Clostridium perfringens type A were tested for their capacity to produce gas gangrene in guinea pigs.The virulence of food-poisoning strains producing heat-sensitive spores and showing beta hemolysis on sheep-blood agar was comparable to that of the classical strains. The most virulent strains of both groups produced fatal infection with only three to five vegetative cells. Of 13 food-poisoning, heat-sensitive strains showing no beta hemolysis, only three were lethal when a minimum of 4 × 104 to 4 × 108 cells was injected. None of the food-poisoning, heat-resistant strains produced fatal infection with cell numbers up to 4 × 108. The groups of strains showed a correlation between virulence and formation of alpha toxin in liquid culture.It is concluded that a number of heat-sensitive, beta-hemolytic strains of C. perfringens may cause gas gangrene as well as food poisoning, and that the current subdivision of C. perfringens type A strains into classical and food-poisoning groups is no longer tenable.

scholarly journals Effects of Clostridium perfringens Alpha-Toxin (PLC) and Perfringolysin O (PFO) on Cytotoxicity to Macrophages, on Escape from the Phagosomes of Macrophages, and on Persistence of C. perfringens in Host Tissues

2004 ◽  
Vol 72 (9) ◽  
pp. 5204-5215 ◽  
Author(s):  
David K. O'Brien ◽  
Stephen B. Melville
Keyword(s):  
Clostridium Perfringens ◽  
Peritoneal Macrophages ◽  
Gas Gangrene ◽  
Polymorphonuclear Cells ◽  
Alpha Toxin ◽  
Phagocytic Cells ◽  
Mouse Muscle ◽  
Perfringolysin O ◽  
Mouse Peritoneal Macrophages ◽  
Host Tissues

ABSTRACT Clostridium perfringens is the most common cause of clostridial myonecrosis (gas gangrene). Polymorphonuclear cells (PMNs) appear to play only a minor role in preventing the onset of myonecrosis in a mouse animal model of the disease (unpublished results). However, the importance of macrophages in the host defense against C. perfringens infections is still unknown. Two membrane-active toxins produced by the anaerobic C. perfringens, alpha-toxin (PLC) and perfringolysin O (PFO), are thought to be important in the pathogenesis of gas gangrene and the lack of phagocytic cells at the site of infection. Therefore, C. perfringens mutants lacking PFO and PLC were examined for their relative cytotoxic effects on macrophages, their ability to escape the phagosome of macrophages, and their persistence in mouse tissues. C. perfringens survival in the presence of mouse peritoneal macrophages was dependent on both PFO and PLC. PFO was shown to be the primary mediator of C. perfringens-dependent cytotoxicity to macrophages. Escape of C. perfringens cells from phagosomes of macrophage-like J774-33 cells and mouse peritoneal macrophages was mediated by either PFO or PLC, although PFO seemed to play a more important role in escape from the phagosome in peritoneal macrophages. At lethal doses (109) of bacteria only PLC was necessary for the onset of myonecrosis, while at sublethal doses (106) both PFO and PLC were necessary for survival of C. perfringens in mouse muscle tissue. These results suggest PFO-mediated cytotoxicity toward macrophages and the ability to escape macrophage phagosomes may be important factors in the ability of C. perfringens to survive in host tissues when bacterial numbers are low relative to those of phagocytic cells, e.g., early in an infection.

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