Cloning of the Gene Coding for Staphylococcus hyicus Exfoliative Toxin B and Its Expression in Escherichia coli

Abstract

The Staphylococcus hyicus exfoliative toxin B (SHETB) gene was cloned into pUC118 and expressed in Escherichia coli. The nucleotide sequence of the SHETB gene consists of a coding region of 804 bp specifying a polypeptide of 268 amino acid residues, which included a putative 20-residue signal sequence.Staphylococcus hyicus is the causative agent of exudative epidermitis (EE) in pigs. Amtsberg (1) suggested that the culture filtrate of S. hyicus contains an exotoxin involved in exfoliative activity. Sato et al. (17) isolated such an exotoxin from the culture filtrate of S. hyicus and designated it SHET.Exfoliative toxin (ET)-producing strains of Staphylococcus aureus cause staphylococcal scalded skin syndrome in humans (11). The ET has been divided into two serotypes, ETA and ETB (9).The ET and SHET have some similarities, including their target site (5,17,20) and their molecular weight (7,8,17,18), but they only react with homologous antibodies. The production of ETA is controlled by chromosomal DNA, while that of ETB is controlled by plasmid DNA (12,13,15). S. hyicus P-23, the SHETB-producing strain, harbors the large plasmid (pKUH-1). The plasmid-eliminated substrain of S. hyicus P-23 has lost its toxic activity. From these findings, it appears that SHETB production is controlled by plasmid DNA (H. Sato, T. Tanabe, T. Watanabe, K. Teruya, A. Ohtake, H. Saito, and N. Maehara, Proc. 14th IPVS Cong. Italy, p. 339, 1996).In this study, we cloned the SHETB gene (shetb) on plasmid DNA of S. hyicus P-23 in Escherichia coli and determined the nucleotide sequence and the predicted amino acid sequence.S. hyicus P-23 is a SHETB-producing strain and was isolated from a pig affected with EE (19). E. coli DH5␣ was used as the host strain in cloning experiments. Bacteria were grown in TY broth (6) for S. hyicus and in Luria-Bertani broth (16) for E. coli. Both bacteria were cultured in a Bio-shaker BR-160LF (Taitec Co., Ltd., Tokyo, Japan) at 37°C with shaking operated at 75 oscillations per min for 18 h. The vector plasmid pUC118 (Takara Shuzo Co. Ltd., Tokyo, Japan) was used in the cloning experiments.Isolation of plasmid DNA from S. hyicus and E. coli was carried out by a modification of the method described by O'Reilly et al. (12) and Birnboim and Doly (3), respectively. To purify plasmid DNA from S. hyicus and E. coli, the dye-buoyant density centrifugation was performed in a P65AT rotor (Hitachi Koki Co., Ltd., Tokyo) at 45,000 rpm, and supercoiled DNA was separated from residual chromosomal DNA and nicked plasmid DNA.The large plasmid (pKUH-1) of S. hyicus P-23 was digested to completion with restriction endonucleases EcoRI, BamHI, and HindIII (Nippon Gene Inc., Toyama, Japan), and the resulting fragments were separated by electrophoresis with a 1.0% agarose gel. The transfer of the DNA onto a nylon membrane (Hybond N ϩ ; Amersham Pharmacia Biotech Co. Ltd., Uppsala, Sweden) was performed by alkaline blotting with fixation by a UV cross-linker. The nylon membrane was hybridized to a biotin-labelled ET probe (conserv...

Identification of theStaphylococcus aureus etdPathogenicity Island Which Encodes a Novel Exfoliative Toxin, ETD, and EDIN-B

“…On the other hand, S. hyicus has been shown to produce several ETs (5,41). Of these, genes for two serologically distinct toxins, ShETA and ShETB, have been cloned (54). The orf1 product showed 16% sequence identity to ShETA and 63% sequence identity to ShETB.…”

Cloning and Sequence Analysis of Genes Encoding Staphylococcus hyicu s Exfoliative Toxin Types A, B, C, and D

“…In S. hyicus, the synthesis of SHETB has been shown to be plasmid associated (25,26), and recently the gene encoding SHETB was cloned from a plasmid DNA fraction of S. hyicus strain P-23 (31).…”

Antimicrobial Susceptibilities of Exfoliative Toxigenic and Non-Toxigenic Staphylococcus hyicus Strains in Japan

“…Exfoliative toxins-producing Staphylococcus hyicus (toxigenic strains) have been implicated in causing porcine exudative epidermitis particularly in suckling and weaned pigs [4,9,15]. The isolation rate of toxigenic strains is reportedly four times higher in the pigs with exudative epidermitis than the healthy pigs [8].…”