Equilibrium of sortase A dimerization on Staphylococcus aureus cell surface mediates its cell wall sorting activity

Abstract

Staphylococcus aureus sortase A (SrtA) transpeptidase is a therapeutically important membrane-bound enzyme in Gram-positive bacteria, which organizes the covalently attached cell surface proteins on the peptidoglycan cell wall of the organism. Here, we report the direct observation of the highly selective homo-dimerization of SrtA on the cell membrane. To address the biological significance of the dimerization towards enzyme function, site-directed mutagenesis was performed to generate a SrtA mutant, which exists as monomer on the cell membrane. We observed that the cell surface display of adhesive proteins in S. aureus cells expressing monomeric SrtA mutant is more prominent than the cells expressing the wild-type enzyme. A cell-based invasion assay was also performed to evaluate the activities of wild-type SrtA and its monomeric mutant as well. Our data demonstrated that S. aureus cells expressing SrtA in monomeric form invade host mammalian cells more efficiently than those expressing wild-type SrtA in dimer-monomer equilibrium. The results suggested that the monomeric form of SrtA is more active than the dimeric form of the enzyme in terms of cell surface display of virulence factors for infection. This is the first study to present the oligomerization of SrtA and its related biological function on the cell membrane. Study of SrtA dimerization has implications for understanding its catalytic mechanism at the cellular level as well as the development of novel anti-infective agents.

Asymmetric Disulfanylbenzamides as Irreversible and Selective Inhibitors of Staphylococcus aureus Sortase A

“…(ii) The active site is predominantly defined by the intrinsic flexibility of the β6/7‐ and β7/8‐loops . (iii) The most active form of SrtA is constituted as a homo‐dimer with a K D =55 μM . (iv) The K M values for both, the LPXTG‐ and Gly n ‐substrates are exceptionally high ( K M = 5.5 mM and 0.14 mM), probably due to the fact that the enzyme and both substrates are spatially co‐localized at the outer membrane yielding high local concentrations .…”

Interrogation of 3D-swapped structure and functional attributes of quintessential Sortase A from Streptococcus pneumoniae

“…Sortase A of S. aureus (SaSrtA) has been shown to exist in a monomerdimer equilibrium in solution [42]. However, functional evaluation of individual species of SaSrtA produced curious results [45]; while in vitro studies showed increased enzyme . Each dimer contains two characteristic 8-stranded beta barrel "sortase fold", but each fold in the dimer is constituted by the combination of beta-strands from both the chains, that is, a complete fold is made through 3D-swapping.…”

Research Progress on the pathogenic mechanism of Streptococcus suis 2

“…Surface proteins at the leucineproline-variable amino acid(X)-threonine-glycine (LPXTG) can be cleaved by Srt and the formation of an amide bond between the carboxyl group of threonine(T) and the amino group of cell-wall cross-bridges (Siegel et al, 2017). The deficiency of srtA in S. aureus severely impedes the anchoring of bacterial surface proteins, thereby significantly reducing their adherence and invasion of host epithelial cells resulting in reduced virulence (Zhu et al, 2016).…”