YycH and YycI Regulate Expression of Staphylococcus aureus Autolysins by Activation of WalRK Phosphorylation

Abstract

Staphylococcus aureus is a facultative pathogen that can encode numerous antibiotic resistance and immune evasion genes and can cause severe infections. Reduced susceptibility to last resort antibiotics such as vancomycin and daptomycin is often associated with mutations in walRK, an essential two-component regulatory system (TCS). This study focuses on the WalK accessory membrane proteins YycH and YycI and their influence on WalRK phosphorylation. Depletion of YycH and YycI by antisense RNA caused an impaired autolysis, indicating a positive regulatory function on WalK as has been previously described. Phosphorylation assays with full-length recombinant proteins in phospholipid liposomes showed that YycH and YycI stimulate WalK activity and that both regulatory proteins are needed for full activation of the WalK kinase. This was validated in vivo through examining the phosphorylation status of WalR using Phos-tag SDS-PAGE with a yycHI deletion mutant exhibiting reduced levels of phosphorylated WalR. In the yycHI knockdown strain, muropeptide composition of the cell wall was not affected, however, the wall teichoic acid content was increased. In conclusion, a direct modulation of WalRK phosphorylation activity by the accessory proteins YycH and YycI is reported both in vitro and in vivo. Taken together, our results show that YycH and YycI are important in the direct regulation of WalRK-dependent cell wall metabolism.

Investigating Extracellular DNA Release in Staphylococcus xylosus Biofilm In Vitro

“…It encodes a member of the two-component regulatory system WalRK. In S. aureus, WalRK positively controls biofilm formation [51] and is essential for cell viability mainly by controlling the transcription of cell wall lysing enzymes [52]. WalKR in S. aureus activated the transcription of nine genes involved in the different steps of cell wall turnover (lytM, atlA, isaA, sceD, ssaA, and four ssaA-related genes) [51].…”

Inferring multilayer interactome networks shaping phenotypic plasticity and evolution

“…QTL Q25261 is located at gene SAOUHSC_00020 ( walR or yycG ) encoding cell envelope biogenesis 65 . walRK phosphorylation activates the expression of YycH and YycI to control cell wall metabolism and biofilm formation in S. aureus 66 , 67 .…”

Membrane Sensor Histidine Kinases: Insights from Structural, Ligand and Inhibitor Studies of Full-Length Proteins and Signalling Domains for Antibiotic Discovery

“…This is particularly appropriate when agonist or antagonist interactions occur within the membrane itself via the transmembrane regions of the kinase. A recent study employing this approach is that of Gajdiss et al (2020) who investigated the modulatory effects of two accessory membrane proteins, YycH and YycI, on the phosphorylation activities of the Staphylococcus aureus WalKR TCS [ 148 ].…”

“…The presence of both accessory proteins together with WalK was required for high level expression of WalKR-expressed genes [ 158 ]. One of the aims of the recent study by Gajdiss et al (2020) of the S. aureus system was to obtain direct evidence for the roles of the YycH and YycI proteins in regulation of WalK activity [ 148 ]. Using purified full-length versions of all three membrane proteins reconstituted into phospholipid liposomes in various combinations, they showed that YycH and YycI stimulated WalK activity, confirming previous studies, and that both YycH and YycI proteins were necessary for full activation of the kinase in vitro, a finding also confirmed by accompanying in vivo studies [ 148 ].…”

“…One of the aims of the recent study by Gajdiss et al (2020) of the S. aureus system was to obtain direct evidence for the roles of the YycH and YycI proteins in regulation of WalK activity [ 148 ]. Using purified full-length versions of all three membrane proteins reconstituted into phospholipid liposomes in various combinations, they showed that YycH and YycI stimulated WalK activity, confirming previous studies, and that both YycH and YycI proteins were necessary for full activation of the kinase in vitro, a finding also confirmed by accompanying in vivo studies [ 148 ]. The two opposite regulatory effects of YycH and YycI proteins on WalK kinase activities identified in S. aureus and B. subtilis and confirmed in this latest study provide an example of how different interaction mechanisms have developed between SHKs and their accessory regulatory proteins amongst different species in order to accommodate their different requirements for controlling and coordinating important cell processes, in this case cell wall remodelling and cell division.…”