Crystal structure of Staphylococcus aureus exfoliative toxin D-like protein: Structural basis for the high specificity of exfoliative toxins

Abstract

Exfoliative toxins are serine proteases secreted by Staphylococcus aureus that are associated with toxin-mediated staphylococcal syndromes. To date, four different serotypes of exfoliative toxins have been identified and 3 of them (ETA, ETB, and ETD) are linked to human infection. Among these toxins, only the ETD structure remained unknown, limiting our understanding of the structural determinants for the functional differentiation between these toxins. We recently identified an ETD-like protein associated to S. aureus strains involved in mild mastitis in sheep. The crystal structure of this ETD-like protein was determined at 1.95 Å resolution and the structural analysis provide insights into the oligomerization, stability and specificity and enabled a comprehensive structural comparison with ETA and ETB. Despite the highly conserved molecular architecture, significant differences in the composition of the loops and in both the N- and C-terminal α-helices seem to define ETD-like specificity. Molecular dynamics simulations indicate that these regions defining ET specificity present different degrees of flexibility and may undergo conformational changes upon substrate recognition and binding. DLS and AUC experiments indicated that the ETD-like is monomeric in solution whereas it is present as a dimer in the asymmetric unit indicating that oligomerization is not related to functional differentiation among these toxins. Differential scanning calorimetry and circular dichroism assays demonstrated an endothermic transition centered at 52 °C, and an exothermic aggregation in temperatures up to 64 °C. All these together provide insights about the mode of action of a toxin often secreted in syndromes that are not associated with either ETA or ETB.

Virulence Factors and Phylogeny of Staphylococcus aureus Associated With Bovine Mastitis in Russia Based on Genome Sequences

“…Udder cleft dermatitis is a pathology lacking a clear etiology, but one of the bacterial inducers can be S. aureus. Moreover, some cases of sheep mastitis have been associated with the exfoliative toxin D-like protein (42).…”

Exfoliative Toxins of Staphylococcus aureus

“…The crystal structure of ETA was the first to be determined in atomic detail [51], followed by ETB [52] and by ETD [24] and currently, the atomic coordinates of six ET structures have been deposited with the Protein Data Bank (www.rcsb.org). The crystallographic structures of ETs have revealed much about their mechanisms of action, lack of hydrolytic activity against substrates in the native state, and the susceptibility of certain constituent layers of the epidermis to disruption by ETs.…”

“…In 2002, a new exfoliative toxin (ETD) was identified in a clinical sample of S. aureus [13]. Recent crystallographic studies indicated that the ETD-like protein isolated from ewe mastitis [23] is structurally homologous to ETA and ETB [24]. ETA is codified by the eta gene on chromosomal DNA, carried on the genome on a temperate phage, and ETB by the etb gene on a large plasmid DNA [22,25,26].…”

Exfoliative toxin E, a new Staphylococcus aureus virulence factor with host-specific activity

“…In the present study, we were able to characterize a new S. aureus ET that had initially been identified in S. aureus strains associated with mild mastitis in ewes 7 . It was previously named ETD-like protein because its predicted protein sequence determined from the O46 genome sequence displayed 59% identity with the ETD protein 7,9 . It also significantly differed from the other well-characterized ETA and ETB.…”

“…Of note was the fact that it harbored the typical catalytic site encountered in the other ET proteins described to date. This protein was tentatively referred to as an ETD-like protein, and its crystal structure was determined 9 .…”