scholarly journals Construction of an Alpha Toxin Gene Knockout Mutant of Clostridium perfringens Type A by Use of a Mobile Group II Intron

2005 ◽  
Vol 71 (11) ◽  
pp. 7542-7547 ◽  
Author(s):  
Yue Chen ◽  
Bruce A. McClane ◽  
Derek J. Fisher ◽  
Julian I. Rood ◽  
Phalguni Gupta
Keyword(s):  
Clostridium Perfringens ◽  
Gene Knockout ◽  
Antibiotic Resistance Genes ◽  
Group Ii Intron ◽  
Toxin Gene ◽  
No Production ◽  
Alpha Toxin ◽  
Knockout Mutant ◽  
New Strategy ◽  
Group Ii

ABSTRACT In developing Clostridium perfringens as a safe vaccine vector, the alpha toxin gene (plc) in the bacterial chromosome must be permanently inactivated. Disrupting genes in C. perfringens by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the plc gene in C. perfringens ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the plc mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other C. perfringens mutants.

scholarly journals Detection of a Group II Intron without an Open Reading Frame in the Alpha-Toxin Gene of Clostridium perfringens Isolated from a Broiler Chicken

2006 ◽  
Vol 189 (5) ◽  
pp. 1633-1640 ◽  
Author(s):  
Menglin Ma ◽  
Kaori Ohtani ◽  
Tohru Shimizu ◽  
Naoaki Misawa
Keyword(s):  
Clostridium Perfringens ◽  
Broiler Chicken ◽  
Group Ii Intron ◽  
Open Reading Frame ◽  
Start Codon ◽  
Northern Hybridization ◽  
Toxin Gene ◽  
Alpha Toxin ◽  
Reading Frame ◽  
Group Ii

ABSTRACTA DNA insertion of 834 bp, designated CPF-G2Im, was identified within the alpha toxin gene (cpa) ofClostridium perfringensstrain CPBC16ML, isolated from a broiler chicken. Sequence analysis of CPF-G2Im indicated that it was integrated 340 nucleotides downstream of the start codon ofcpa. However, the insertion did not abolish the phospholipase C and hemolytic activities of CPBC16ML. To investigate the expression of its alpha toxin, the intact copy ofcpawas cloned into an expression vector and transformed intoEscherichia coliM15 cells. Immunoblotting analysis showed that the protein expressed from the transformant as well as in the culture supernatant ofC. perfringensstrain CPBC16ML had the expected molecular weight detected in reference strains ofC. perfringens. Northern hybridization and reverse transcriptase PCR (RT-PCR) analysis revealed that the entire CPF-G2Im insertion was completely spliced from thecpaprecursor mRNA transcripts. The sequence of the insertion fragment has 95% and 97% identity to two noncoding regions corresponding to sequences that flank a predicted group II RT gene present in the pCPF4969 plasmid ofC. perfringens. However, an RT was not encoded by the CPF-G2Im fragment. Based on the secondary structure prediction analysis, CPF-G2Im revealed typical features of group II introns. The present study shows that CPF-G2Im is capable of splicing in bothC. perfringensandE. coli. To our knowledge, this is the first report that a group II intron without an open reading frame (ORF) is located in thecpaORF ofC. perfringens.

scholarly journals Quantification of Cell Proliferation and Alpha-Toxin Gene Expression of Clostridium perfringens in the Development of Necrotic Enteritis in Broiler Chickens

2007 ◽  
Vol 73 (21) ◽  
pp. 7110-7113 ◽  
Author(s):  
Weiduo Si ◽  
Joshua Gong ◽  
Yanming Han ◽  
Hai Yu ◽  
John Brennan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Regression Model ◽  
Clostridium Perfringens ◽  
Broiler Chickens ◽  
Toxin Gene ◽  
Necrotic Enteritis ◽  
Lesion Score ◽  
Alpha Toxin ◽  
Toxin Gene Expression

ABSTRACT Cell proliferation and alpha-toxin gene expression of Clostridium perfringens in relation to the development of necrotic enteritis (NE) were investigated. Unlike bacitracin-treated chickens, non-bacitracin-treated birds exhibited typical NE symptoms and reduced growth performance. They also demonstrated increased C. perfringens proliferation and alpha-toxin gene expression that were positively correlated and progressed according to the regression model y = b 0 + b 1 X − b 2 X 2. The average C. perfringens count of 5 log10 CFU/g in the ileal digesta appears to be a threshold for developing NE with a lesion score of 2.

scholarly journals Isolation, identification and toxinotyping of Clostridium perfringens isolated from broilers in Pakistan

2021 ◽  
Vol 58 (04) ◽  
pp. 1367-1372
Author(s):  
Zain Ul Abadeen
Keyword(s):  
Clostridium Perfringens ◽  
Causative Agent ◽  
Broiler Chicken ◽  
Clinical Signs ◽  
Type A ◽  
Toxin Gene ◽  
Necrotic Enteritis ◽  
Alpha Toxin ◽  
Enteric Disease ◽  
Isolation Rate

Necrotic enteritis (NE) is one of the important enteric disease in the poultry industry worldwide, caused by C. perfringens type A. This study describes the isolation, identification, and toxinotyping of C. perfringens in necrotic enteritis affected broiler chicken in Pakistan. A total of 430 intestinal samples from dead carcasses and birds suspected of NE outbreak, in and around Faisalabad, Pakistan were collected from 36 broiler farms which yielded 87 alpha toxin gene (cpa) positive C. perfringens type A isolates. The birds having 4-5 weeks of age, clinical signs, and reared in open (conventional) sheds showed higher C. perfringens isolation rate. The study concluded netB negative C. perfringens type A as a causative agent for NE outbreaks in broiler birds in Faisalabad, Pakistan.

scholarly journals Critical Role of Alpha-Toxin and Protective Effects of Its Neutralization by a Human Antibody in Acute Bacterial Skin and Skin Structure Infections

2016 ◽  
Vol 60 (10) ◽  
pp. 5640-5648 ◽  
Author(s):  
Vien T. M. Le ◽  
Christine Tkaczyk ◽  
Sally Chau ◽  
Renee L. Rao ◽  
Etyene Castro Dip ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Human Monoclonal Antibody ◽  
Critical Role ◽  
The United States ◽  
Toxin Gene ◽  
Human Antibody ◽  
Alpha Toxin ◽  
Wild Type ◽  
Content Type ◽  
Skin Structure

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) causes large-scale epidemics of acute bacterial skin and skin structure infections (ABSSSI) within communities across the United States. Animal models that reproduce ABSSSI as they occur in humans are urgently needed to test new therapeutic strategies. Alpha-toxin plays a critical role in a variety of staphylococcal infection models in mice, but its role in the pathogenesis of ABSSSI remains to be elucidated in rabbits, which are similar to humans in their susceptibility toS. aureussuperantigens and certain bicomponent pore-forming leukocidins. We report here a new rabbit model of ABSSSI and show that those infected with a mutant deficient in expression of alpha-toxin (Δhla) developed a small dermonecrotic lesion, whereas those infected with isogenic USA300 MRSA wild-type or complemented Δhlastrains developed ABSSSI that mimic the severe infections that occur in humans, including the large central dermonecrotic core surrounded by erythema, induration, and marked subcutaneous hemorrhage. More importantly, immunoprophylaxis with MEDI4893*, an anti-alpha-toxin human monoclonal antibody, significantly reduced the severity of disease caused by a USA300 wild-type strain to that caused by the Δhlamutant, indicating that this toxin could be completely neutralized during infection. Thus, this study illustrates a potential high standard for the development of new immunotherapeutic agents in which a toxin-neutralizing antibody provides protection to the same degree achieved with a toxin gene knockout. When MEDI4893* was administered as adjunctive therapy with a subtherapeutic dose of linezolid, the combination was significantly more efficacious than either agent alone in reducing the severity of ABSSSI.

scholarly journals Complete genomic sequence and analysis of β2 toxin gene mapping of Clostridium perfringens JXJA17 isolated from piglets in China

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiu Zeng ◽  
Baosheng Liu ◽  
Jiao Zhou ◽  
Yimin Dai ◽  
Chunsheng Han ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Genomic Sequence ◽  
Antibiotic Resistance Genes ◽  
Close Relation ◽  
Illumina Miseq ◽  
Opportunistic Pathogen ◽  
Toxin Gene ◽  
Circular Chromosome ◽  
Toxin Genes ◽  
Genome Homology

AbstractClostridium perfringens (Cp) is a ubiquitous opportunistic pathogen of humans and animals in the natural environment and animal intestines. The pathogenicity of Cp depends on the production of toxins encoded by genes on the chromosomes or plasmids. In contemporary literature, there is no clear consensus about the pathogenicity of CpA β2 toxin. To analyze the homology of the genome of piglet source CpA and its β2 toxin, we sequenced the whole genome of strain JXJA17 isolated from diarrhea piglets using the Illumina Miseq and Pacbio Sequel platforms. The genome was composed of a circular chromosome with 3,324,072 bp (G + C content: 28.51%) and nine plasmids. Genome and 16S rDNA homology analysis revealed a close relation of the JXJA17 strain with the JGS1495, Cp-06, Cp-16, and FORC_003 strains. These strains were isolated from different samples and belonged to different toxin-types. JXJA17 strain was found to carry two toxin genes (plc and cpb2). In contrast to other Cp strains, the cpb2 of JXJA17 was located on a large plasmid (58 kb) with no co-localization of other toxin genes or antibiotic resistance genes. Analysis of JXJA17 genome homology and its cpb2 would facilitate our further study the relationship between β2 toxin and piglet diarrhea.

Evaluation in broilers of the probiotic properties of Pichia pastoris and a recombinant P. pastoris containing the Clostridium perfringens alpha toxin gene

2012 ◽  
Vol 156 (3-4) ◽  
pp. 448-451 ◽  
Author(s):  
João Rodrigo Gil de los Santos ◽  
Otávio Brod Storch ◽  
Cristina Gevehr Fernandes ◽  
Carlos Gil-Turnes
Keyword(s):  
Pichia Pastoris ◽  
Clostridium Perfringens ◽  
Toxin Gene ◽  
Probiotic Properties ◽  
Alpha Toxin

scholarly journals Recombinant Bacillus subtilis Expressing the Clostridium perfringens Alpha Toxoid Is a Candidate Orally Delivered Vaccine against Necrotic Enteritis

2008 ◽  
Vol 76 (11) ◽  
pp. 5257-5265 ◽  
Author(s):  
Tran H. Hoang ◽  
Huynh A. Hong ◽  
Graeme C. Clark ◽  
Richard W. Titball ◽  
Simon M. Cutting
Keyword(s):  
Bacillus Subtilis ◽  
Clostridium Perfringens ◽  
Vegetative Cell ◽  
Active Agent ◽  
Secretory Iga ◽  
Toxin Gene ◽  
Spore Coat ◽  
Necrotic Enteritis ◽  
Alpha Toxin

ABSTRACT Recombinant Bacillus subtilis endospores have been used to vaccinate against tetanus and anthrax. In this work, we have developed spores that could be used to vaccinate against Clostridium perfringens alpha toxin and that could be used to protect against gas gangrene in humans and necrotic enteritis in poultry. The primary active agent in both cases is alpha toxin. A carboxy-terminal segment of the alpha toxin gene (cpa) fused to the glutathione-S-transferase (GST) gene was cloned in B. subtilis such that the encoded GST-Cpa247-370 polypeptide had been expressed in the following three different ways: expression in the vegetative cell, expression on the surface of the spore coat (fused to the CotB spore coat protein), and a combined approach of spore coat expression coupled with expression in the vegetative cell. Mice immunized orally or nasally with three doses of recombinant spores that carried GST-Cpa247-370 on the spore surface showed the most striking responses. This included seroconversion with anti-Cpa247-370-specific immunoglobulin G (IgG) responses in their sera, a Th2 bias, and secretory IgA responses in saliva, feces, and lung samples. Neutralizing IgG antibodies to alpha toxin were detected using in vitro and in vivo assays, and a toxin challenge established protection. Mice immunized nasally or orally with recombinant spores were protected against a challenge with 12 median lethal doses of alpha toxin. Existing use of spores as competitive exclusion agents in animal feeds supports their use as a potentially economical and heat-stable vaccine for the poultry industry.

The effects of probiotic Bacillus coagulans on the cytotoxicity and expression of alpha toxin gene of Clostridium perfringens type A

Anaerobe ◽  
2019 ◽  
Vol 59 ◽  
pp. 61-67 ◽  
Author(s):  
Amin Kawarizadeh ◽  
Mohammad Tabatabaei ◽  
Saeid Hosseinzadeh ◽  
Mina Farzaneh ◽  
Maryam Pourmontaseri
Keyword(s):  
Clostridium Perfringens ◽  
Bacillus Coagulans ◽  
Type A ◽  
Toxin Gene ◽  
Alpha Toxin

scholarly journals Virulence Gene Regulation by the agr System in Clostridium perfringens

2009 ◽  
Vol 191 (12) ◽  
pp. 3919-3927 ◽  
Author(s):  
Kaori Ohtani ◽  
Yonghui Yuan ◽  
Sufi Hassan ◽  
Ruoyu Wang ◽  
Yun Wang ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Clostridium Perfringens ◽  
Culture Supernatant ◽  
Toxin Production ◽  
Toxin Gene ◽  
Wild Type ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Signal Molecule

ABSTRACT A gram-positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissue. The agr system is known to be important for the regulation of virulence genes in a quorum-sensing manner in Staphylococcus aureus. A homologue for S. aureus agrBD (agrBDSa ) was identified in the C. perfringens strain 13 genome, and the role of C. perfringens agrBD (agrBDCp ) was examined. The agrBDCp knockout mutant did not express the theta-toxin gene, and transcription of the alpha- and kappa-toxin genes was also significantly decreased in the mutant strain. The mutant strain showed a recovery of toxin production after the addition of the culture supernatant of the wild-type strain, indicating that the agrBDCp mutant lacks a signal molecule in the culture supernatant. An agr-virR double-knockout mutant was constructed to examine the role of the VirR/VirS two-component regulatory system, a key virulence regulator, in agrBDCp -mediated regulation of toxin production. The double-mutant strain could not be stimulated for toxin production with the wild-type culture supernatant. These results indicate that the agrBDCp system plays an important role in virulence regulation and also suggest that VirR/VirS is required for sensing of the extracellular signal and activation of toxin gene transcription in C. perfringens.

Sign in / Sign up

Export Citation Format

Share Document