Despite very low sequence homology between ETA and other trypsin-like serine proteases, the ETA crystal structure, together with biochemical data and site-directed mutagenesis studies, strongly confirms the classification of ETA in the Glu-endopeptidase family. Direct links can be made between the protease architecture of ETA and its biological activity.
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...
Two methods for the detection of exfoliative toxin (ET) from Staphylococcus aureus were compared: (i) a phenotypic assay, electrosyneresis, and (ii) a genotypic assay, staphylococcal DNA hybridization with oligodeoxynucleotide probes. The probes were chosen from the previously determined sequences of serotype A and B of ET, one probe for serotype A and another for serotype B. Strains exhibiting ET production in electrosyneresis always possessed the ET gene(s). Conversely, some strains not exhibiting ET production in electrosyneresis harbored the ET gene(s). The latter strains produced low levels of ET. ET-negative phage group 2 strains of S. aureus as well as tested coagulase-negative staphylococci did not possess the ET gene(s). The sensitivity of the DNA hybridization technique was 106 bacteria or 100 ng of genomic DNA.
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