Functional evidence that the Ser-195 residue of staphylococcal exfoliative toxin A is essential for biological activity

Abstract

The substitution of the serine 195 residue of staphylococcal exfoliative toxin A by a cysteine residue led to a biologically inactive protein. This result is consistent with the hypothesis that exfoliative toxin A could be a protease or a lipase. However, no protease or lipase activity was detected with the native toxin.Exfoliative toxins A and B (ETA and ETB) are produced by 5% of Staphylococcus aureus strains and are the causative agents of the staphylococcal scalded skin syndrome (15). This syndrome is observed mostly in newborns, infants, and young children, but sporadic cases were also reported in immunocompetent patients (11). The lesions consist of intercellular splittings of the epidermis between the stratum spinosum and the stratum granulosum. The mechanism of epidermolysis has been assumed to be the disruption of intercellular junctions (10). Nonetheless, microscopic examinations and the results of several other experiments were consistent with the hypothesis that ETA and ETB had binds to the intracellular proteins profilaggrin and filaggrin (constituents of keratohyalin granules) and to a histonelike protein (16)(17)(18). However, the link between the binding of exfoliative toxin to these intracellular proteins and the intercellular events has not been demonstrated.Recently, two reports described amino acid sequence similarities between both ETA and ETB and serine proteases such as staphylococcal V8 protease and bovine chymotrypsin, suggesting a proteolytic activity for ETA and ETB (1,4). Experiments in which the toxins were labeled with diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride demonstrated the binding of the inhibitor to a peptide containing the Ser-195 residue, which was included in homologous sequences corresponding to the active sites of other serine proteases. However, this binding did not lead to a complete loss of biological activity.In this communication, we report that substitution of this Ser-195 by a cysteine residue by using site-directed mutagenesis led to a mutant protein which had lost any detectable biological activity.The gene for ETA was cloned in Escherichia coli BJ 5183 (5) by hybridization of the DNA library of Staphylococcus aureus 50586 (15) to a 74-mer oligonucleotide constructed from the N-terminal amino acid sequence of ETA. The cloned DNA fragment was a 3.5-kb HindIll-HindIll segment which was inserted into plasmid pUC18. The cloned DNA fragment was then inserted into M13mpl9 and sequenced. The sequence of the coding gene was identical to those already published (8,12), except that Lys-210 was encoded by AAG instead of AAA. Site-directed mutagenesis in M13mpl9 was performed according to a technique previously published (20,21). The Ser-195--Cys was obtained by using the mutation-generating oligonucleotide 5'-CCGGGA CATGTAATGATTTAAAA-3' as a primer. The double-* Corresponding author. stranded DNAs allowed the transformation of E. coli TG1. The single-stranded DNAs obtained from 100 PFU were hybridized on a Hybond-N membrane (Amersham) with the 5' 32P-labele...

Clinical, Microbial, and Biochemical Aspects of the Exfoliative Toxins Causing Staphylococcal Scalded-Skin Syndrome

“…Soon thereafter, Wiley and Rogolsky (278) demonstrated that incubation of purified ET with either egg yolk emulsion or bovine brain sphingomyelin resulted in a dose-dependent release of phosphate, suggesting that ETs have intrinsic phospholipase activity (278). However, it should be noted that other investigators were unable to detect any lipase activity toward triacylglycerol or phospholipids (210).…”

“…Soon thereafter, several papers appeared which further supported this hypothesis. Two groups independently reported that site-directed replacement of the Ser-195 active site of the catalytic triad of ETA resulted in a complete loss of biological activity when excess amounts were injected into newborn mice (210,214).…”

“…Redpath et al used oligonucleotide site-directed mutagenesis to replace the active-site Ser-195 of ETA with glycine and showed that injection of 1 and 10 g of wild-type ETA into 3-day-old Sha-Sha mice gave a positive Nikolsky sign within 2.5 h whereas the mutated ETA had no effect at doses up to 100 g, indicating that Ser-195 is essential for the biological activity of ETA (214). Prevost et al (210) also performed a similar experiment, replacing the Ser-195 with cysteine, and showed that a positive Nikolsky sign was obtained in neonatal mice with as little as 0.1 g of wild-type ETA but not with up to 300 g of mutated ETA.…”

Detection of Staphylococcus hyicus exfoliative toxin genes by dot blot hybridization and multiplex polymerase chain reaction

“…We constructed an ET probe on the basis of the nucleotide sequences coding for the specific amino acid sequences (-GNSGS-) around the active serine residue of the ET molecules (6,8,20,21). Although the ETs have a low homology with each other, this region is highly homologous; all the ETs have this specific sequence (6,8,20,21). We Table 3.…”

New generation of peptide antibiotics.

“…A clear demonstration of a role of a particular proteolytic enzyme in staphylococcal virulence was possible only in the case of epidermolytic toxins in staphylococcal scalded-skin syndrome (Prévost et al, 1991;Redpath et al, 1991). Reportedly, the virulence of V8 serine protease (sspA -) and serine protease-like operon (spl -) knock-outs was not affected significantly in murine abscess infection model or a�er intraperitoneal injection, respectively (Reed et al, 2001;Rice et al, 2001).…”