An unknown cytotoxin was identified in the culture supernatant of Clostridium perfringens type C. The cytotoxin, named TpeL, which was purified using mAb-based affinity chromatography, had a lethal activity of 62 minimum lethal dose (MLD) mg "1 in mice and a cytotoxic activity of 6.2610 5 cytotoxic units (CU) mg "1 in Vero cells. The nucleotide sequence of TpeL was determined. The entire ORF had a length of 4953 bases, and the same nucleotide sequence was not recorded in the GenBank/EMBL/DDBJ databases. The molecular mass calculated from the deduced amino acid sequence was 191 kDa, and a signal peptide region was not found within the ORF. The deduced amino acid sequence exhibited 30-39 % homology to Clostridium difficile toxins A (TcdA) and B (TcdB), Clostridium sordellii lethal toxin (TcsL) and Clostridium novyi alpha-toxin (TcnA). The amino acid sequence of TpeL is shorter than these toxins, and the homologous region was located at the N-terminal site. Eighteen strains of C. perfringens types A, B and C were surveyed for the presence of the tpeL gene by PCR. The tpeL gene was detected in all type B (one strain) and C strains (five strains), but not in any type A strains (12 strains). TpeL was detected in culture filtrates of the five type C strains by dot-blot analysis, but not in the type B strain. It was concluded that TpeL is a novel toxin similar to the known large clostridial cytotoxins. Furthermore, the data indicated that TpeL is produced by many C. perfringens type C strains.
We have developed a system in which a foreign antigen is delivered and expressed on the surface of an attenuated strain of Erysipelothrix rhusiopathiae YS-1 and have examined the ability of a such recombinant E. rhusiopathiae strain to function as a mucosal vaccine vector. The C-terminal portion, including two repeat regions, R1 and R2, of the P97 adhesin of Mycoplasma hyopneumoniae strain E-1 was successfully translocated and expressed on the E. rhusiopathiae YS-1 cell surface after it was fused to SpaA.1, a cell surface protective antigen of E. rhusiopathiae. BALB/c mice subcutaneously immunized with the E. rhusiopathiae recombinant strains developed specific antibodies against SpaA.1 protein and were protected from lethal challenge with the highly virulent homologous E. rhusiopathiae With the use of recombinant DNA technology, the potential exists to combine antigens within a single microorganism and to produce a vaccine for several different diseases simultaneously. This strategy is particularly attractive for the development of vaccines for veterinary fields in which easy-to-use and cost-effective vaccines are strongly required.Erysipelothrix rhusiopathiae is a gram-positive, facultatively intracellular bacterial pathogen that can cause erysipelas in animals and erysipeloid in humans (21,27). E. rhusiopathiae YS-1 is a stable acapsular mutant that was developed by a novel mechanism with transposon Tn916 (23). Although this strain is highly attenuated, it is able to induce both humoral and cell-mediated immunity in mice, suggesting that the strain may be suitable for use as a recombinant vaccine vector (23).Recent advances in the development of vaccine vectors have demonstrated the importance of localization of the antigen to be expressed in the induction of efficient immune responses. To improve antigen presentation to the immune system, vaccine vectors should be engineered to express the foreign antigens either on the surface (9) or in secreted form (5,20). In gram-positive bacteria, heterologous proteins have been successfully expressed on the bacterial cell surface by using the C-terminal sorting signal including a conserved LPXTG motif, followed by a segment of 15 to 20 hydrophobic amino acids that span the cytoplasmic membrane and a tail of mostly positively charged residues. The conserved C-terminal sorting signal has been shown to be responsible for attachment to the bacterial cell surface and has been found in more than 65 cell surface proteins of many gram-positive bacteria (2, 13). It has been found that heterologous proteins can be directed to the gram-positive bacterial cell surface when fused to the C-terminal sorting signal (4, 15), thus indicating that this engineering technique is applicable to most gram-positive bacteria if they express such surface proteins. A few of the E. rhusiopathiae surface proteins have been identified (21). However, in these proteins, the conserved C-terminal sorting signal has not been identified. We therefore investigated an alternative method by which to deliver ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.