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1.
Antimicrob Agents Chemother ; 59(11): 6855-65, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26282424

ABSTRACT

Bacitracins are mixtures of structurally related cyclic polypeptides with antibiotic properties. They act by interfering with the biosynthesis of the bacterial cell wall. In this study, we analyzed an avian necrotic enteritis strain of Clostridium perfringens that was resistant to bacitracin and produced NetB toxin. We identified a bacitracin resistance locus that resembled a bacitracin resistance determinant from Enterococcus faecalis. It contained the structural genes bcrABD and a putative regulatory gene, bcrR. Mutagenesis studies provided evidence that both bcrA and bcrB are essential for bacitracin resistance, and that evidence was supported by the results of experiments in which the introduction of both the bcrA and bcrB genes into a bacitracin-susceptible C. perfringens strain was required to confer bacitracin resistance. The wild-type strain was shown to contain at least three large, putatively conjugative plasmids, and the bcrRABD locus was localized to an 89.7-kb plasmid, pJIR4150. This plasmid was experimentally shown to be conjugative and was sequenced. The sequence revealed that it also carries a tpeL toxin gene and is related to the pCW3 family of conjugative antibiotic resistance and toxin plasmids from C. perfringens. The bcr genes were located on a genetic element, ICECp1, which is related to the Tn916 family of integrative conjugative elements (ICEs). ICECp1 appears to be the first Tn916-like element shown to confer bacitracin resistance. In summary, we identified in a toxin-producing C. perfringens strain a novel mobile bacitracin resistance element which was experimentally shown to be essential for bacitracin resistance and is carried by a putative ICE located on a conjugative plasmid.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacitracin/pharmacology , Clostridium perfringens/genetics , Clostridium perfringens/drug effects , Conjugation, Genetic/genetics , Genes, Bacterial/genetics , Plasmids/genetics
2.
mBio ; 4(1): e00019-13, 2013 Feb 05.
Article in English | MEDLINE | ID: mdl-23386432

ABSTRACT

Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a ß-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a ß-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.


Subject(s)
Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Clostridium perfringens/chemistry , Clostridium perfringens/pathogenicity , Enterotoxins/chemistry , Enterotoxins/metabolism , Animals , Bacterial Toxins/genetics , Biological Transport , Cations/metabolism , Chickens , Clostridium perfringens/genetics , Crystallography, X-Ray , DNA Mutational Analysis , Enterotoxins/genetics , Erythrocytes/drug effects , Hemolysis , Models, Molecular , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Binding , Protein Conformation , Protein Multimerization
3.
mBio ; 2(5)2011.
Article in English | MEDLINE | ID: mdl-21954306

ABSTRACT

UNLABELLED: The pathogenesis of avian necrotic enteritis involves NetB, a pore-forming toxin produced by virulent avian isolates of Clostridium perfringens type A. To determine the location and mobility of the netB structural gene, we examined a derivative of the tetracycline-resistant necrotic enteritis strain EHE-NE18, in which netB was insertionally inactivated by the chloramphenicol and thiamphenicol resistance gene catP. Both tetracycline and thiamphenicol resistance could be transferred either together or separately to a recipient strain in plate matings. The separate transconjugants could act as donors in subsequent matings, which demonstrated that the tetracycline resistance determinant and the netB gene were present on different conjugative elements. Large plasmids were isolated from the transconjugants and analyzed by high-throughput sequencing. Analysis of the resultant data indicated that there were actually three large conjugative plasmids present in the original strain, each with its own toxin or antibiotic resistance locus. Each plasmid contained a highly conserved 40-kb region that included plasmid replication and transfer regions that were closely related to the 47-kb conjugative tetracycline resistance plasmid pCW3 from C. perfringens. The plasmids were as follows: (i) a conjugative 49-kb tetracycline resistance plasmid that was very similar to pCW3, (ii) a conjugative 82-kb plasmid that contained the netB gene and other potential virulence genes, and (iii) a 70-kb plasmid that carried the cpb2 gene, which encodes a different pore-forming toxin, beta2 toxin. IMPORTANCE: The anaerobic bacterium Clostridium perfringens can cause an avian gastrointestinal disease known as necrotic enteritis. Disease pathogenesis is not well understood, although the plasmid-encoded pore-forming toxin NetB, is an important virulence factor. In this work, we have shown that the plasmid that carries the netB gene is conjugative and has a 40-kb region that is very similar to replication and transfer regions found within each of the sequenced conjugative plasmids from C. perfringens. We also showed that this strain contained two additional large plasmids that were also conjugative and carried a similar 40-kb region. One of these plasmids encoded beta2 toxin, and the other encoded tetracycline resistance. To our knowledge, this is the first report of a bacterial strain that carries three closely related but different independently conjugative plasmids. These results have significant implications for our understanding of the transmission of virulence and antibiotic resistance genes in pathogenic bacteria.


Subject(s)
Bacterial Toxins/genetics , Clostridium perfringens/genetics , Clostridium perfringens/isolation & purification , Drug Resistance, Bacterial , Enterocolitis, Necrotizing/microbiology , Enterotoxins/genetics , Plasmids , Anti-Bacterial Agents/pharmacology , Clostridium perfringens/drug effects , Clostridium perfringens/pathogenicity , Conjugation, Genetic , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Gene Knockout Techniques , Gene Transfer, Horizontal , Humans , Molecular Sequence Data , Mutagenesis, Insertional , Sequence Analysis, DNA
4.
Vet Res ; 41(2): 21, 2010.
Article in English | MEDLINE | ID: mdl-19931005

ABSTRACT

A novel toxin, NetB, has recently been identified in virulent avian Clostridium perfringens isolates and shown to be an essential virulence factor in a clinical necrotic enteritis isolate. To assess whether NetB is more generally associated with avian necrotic enteritis isolates we have screened a range of C. perfringens strains from geographically diverse locations for both the presence and expression of the netB gene. Forty-four isolates were derived from necrotic enteritis disease cases from Australia, Belgium, Denmark and Canada and 55 isolates from healthy chickens from Australia and Belgium. The majority of strains isolated from necrotic enteritis-affected birds were netB positive (70%) and there was an absolute correlation between the presence of netB and in vitro expression of the NetB protein. Only two of the C. perfringens isolates from healthy chickens carried netB. Sequencing of the netB gene from 23 positive isolates showed that NetB is highly conserved, with only one predicted amino acid (A168T) difference, in six isolates, compared to the published sequence. This change did not alter the in vitro activity of the NetB toxin. The gene encoding the recently discovered TpeL toxin was also screened using PCR and only found in a small proportion of NetB-positive isolates from diseased birds. A selection of NetB-negative isolates, originating from diseased birds, was unable to cause disease in a necrotic enteritis induction model. This study provides further evidence that NetB is important in pathogenesis and advances our current understanding of C. perfringens virulence factors in avian necrotic enteritis.


Subject(s)
Bacterial Toxins/metabolism , Clostridium perfringens/metabolism , Enteritis/veterinary , Enterotoxins/metabolism , Gene Expression Regulation, Bacterial/physiology , Poultry Diseases/microbiology , Animals , Bacterial Toxins/genetics , Chickens , Clostridium perfringens/pathogenicity , Enteritis/microbiology , Enterotoxins/genetics , Virulence
5.
Article in Chinese | MEDLINE | ID: mdl-16866150

ABSTRACT

A sandwich ELISA was established with monoclonal antibody as detecting antibody and rabbit anti-Toxoplasma polyclonal antibody as capturing antibody. ABC (avidin-biotin-peroxidase complex)-ELISA and immuno-PCR were also used to detect different concentrations of Toxoplasma antigen. The lowest concentration of antigen to be detected by sandwich ELISA, ABC-ELISA and immuno-PCR was 0. 6 microg/ml, 0. 075 microg/ml and 0.1 ng/ml respectively. The immuno-PCR shows a much higher sensitivity than the other two methods.


Subject(s)
Antigens, Protozoan/analysis , Toxoplasma/immunology , Animals , Antibodies, Monoclonal/immunology , Enzyme-Linked Immunosorbent Assay/methods , Female , Mice , Mice, Inbred BALB C , Polymerase Chain Reaction/methods , Reproducibility of Results
6.
Article in Chinese | MEDLINE | ID: mdl-12567570

ABSTRACT

OBJECTIVE: To investigate the number and mature rate of eggs in gravid proglottids of Taenia solium. METHODS: Ten worms of Taenia solium, expelled from patients, were detected. Eggs were collected from the last 10 gravid proglottids of each worm. RESULTS & CONCLUSION: The egg number in each mature proglottids varied from 3,900 to 126,520, and the mean number was 28,332. The mature rate of eggs was from 7.00% to 36.00% with an average of 29.12%, which was lower than that in proglottids naturally excreted with feces. With suitable temperature and humidity, the proglottids developed continually after excreted out of host body. Two to three days later, the mature rate of their eggs increased to 85%-90%.


Subject(s)
Ovum/physiology , Taenia solium/physiology , Animals , Feces/parasitology , Humans , Ovum/growth & development , Parasite Egg Count
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