Cloning and sequence analysis of genes encoding Staphylococcus hyicus exfoliative toxin types A, B, C, and D

Abstract

Exfoliative toxins produced by certain strains of Staphylococcus hyicus mediate exudative epidermitis in pigs. In this study the genes coding for four different exfoliative toxin from S. hyicus (ExhA, ExhB, ExhC, and ExhD) were cloned and sequenced. The coding sequence of the four toxin genes ranged from 816 to 834 bp. The amino acid sequences of these four toxins were homologous to the earlier described exfoliative toxins SHETB from S. hyicus and ETA, ETB, and ETD from Staphylococcus aureus. The homology between the S. hyicus toxins was at the same level as the homology to the exfoliative toxins from S. aureus. The toxins showed similarity to serine proteases, including preservation of the catalytic tract in ExhA, ExhB, and ExhC. However, in ExhD, Asp in the putative catalytic tract was replaced with Glu. The recombinant toxins could be expressed in Escherichia coli, and three of the four toxins were recognized by monoclonal antibodies raised against native exfoliative toxins.

FIG. 1.

Relationships between eight exfoliative toxins from S. hyicus and S. aureus. The phylogenetic tree (neighbor joining method) is based on the amino acid sequences. ExhA, ExhB, ExhC, ExhD, and SHETB are from S. hyicus, while ETA, ETB, and ETD are from S. aureus. No species-specific clustering of the toxin genes was observed.

FIG. 2.

Alignment of the amino acid sequences of ExhA, ExhB, ExhC, ExhD, and SHETB from S. hyicus and ETA, ETB, and ETD from S. aureus. Identical amino acids are shown in red, while grey and yellow indicate six to seven and five identical amino acids, respectively. The putative signal peptide cleavage site around positions 40 is shown in pink. In ETA, His-72, Asp-120, and Ser-195 (ETA numbering) are considered to be involved in the active site of the toxins. These three amino acids are shown in green; His-115 (H), Asp 164 (D), and Ser-239(S). It is noteworthy that Asp-164 in ExhD is replaced with Glu (E).