Sayeed Sameera, Li Jihong, McClane Bruce A
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Infect Immun. 2007 May;75(5):2391-8. doi: 10.1128/IAI.02014-06. Epub 2007 Mar 5.
Clostridium perfringens type D isolates are important in biodefense and also cause natural enterotoxemias in sheep, goats, and occasionally cattle. In these isolates, the gene (etx) encoding epsilon-toxin is thought to reside on poorly characterized large plasmids. Type D isolates sometimes also produce other potentially plasmid-encoded toxins, including C. perfringens enterotoxin and beta2 toxin, encoded by the cpe and cbp2 genes, respectively. In the current study we demonstrated that the etx, cpe, and cpb2 genes are carried on plasmids in type D isolates and characterized the toxin-encoding plasmids to obtain insight into their genetic organization, potential transferability, and diversity. Southern blotting of pulsed-field gels showed that the etx gene of type D isolates can be present on at least five different plasmids, whose sizes range from 48 to 110 kb. The etx plasmids also typically carried IS1151 and tcp open reading frames (ORFs) known to mediate conjugative transfer of C. perfringens plasmid pCW3. PCR studies revealed that other than their tcp ORFs, etx plasmids of type D isolates do not carry substantial portions of the conserved or variable regions in the cpe plasmids of type A isolates. Southern blotting also demonstrated that in type D isolates the cpe and cpb2 genes are sometimes present on the etx plasmid. Collectively, these findings confirmed that the virulence of type D isolates is heavily plasmid dependent and indicated that (i) a single type D isolate can carry multiple virulence plasmids, (ii) a single type D virulence plasmid can carry up to three different toxin genes, and (iii) many etx plasmids should be capable of conjugative transfer.
D型产气荚膜梭菌分离株在生物防御中很重要，并且还会在绵羊、山羊以及偶尔在牛中引起自然肠毒血症。在这些分离株中，编码ε毒素的基因（etx）被认为存在于特征不明的大质粒上。D型分离株有时还会产生其他可能由质粒编码的毒素，包括分别由cpe和cbp2基因编码的产气荚膜梭菌肠毒素和β2毒素。在本研究中，我们证明etx、cpe和cbp2基因存在于D型分离株的质粒上，并对编码毒素的质粒进行了表征，以深入了解其遗传组织、潜在可转移性和多样性。脉冲场凝胶的Southern印迹显示，D型分离株的etx基因可以存在于至少五种不同的质粒上，其大小范围为48至110 kb。etx质粒通常还携带已知介导产气荚膜梭菌质粒pCW3接合转移的IS1151和tcp开放阅读框（ORF）。PCR研究表明，除了它们的tcp ORF外，D型分离株的etx质粒不携带A型分离株cpe质粒中保守或可变区域的大部分。Southern印迹还表明，在D型分离株中，cpe和cbp2基因有时存在于etx质粒上。总的来说，这些发现证实了D型分离株的毒力严重依赖于质粒，并表明（i）单个D型分离株可以携带多个毒力质粒，（ii）单个D型毒力质粒可以携带多达三个不同的毒素基因，以及（iii）许多etx质粒应该能够进行接合转移。
