Regulated Cleavage of Glycan Strands by the Murein Hydrolase SagB in Staphylococcus aureus Involves a Direct Interaction with LyrA (SpdC)

Abstract

LyrA (SpdC), a homologue of eukaryotic CAAX proteases that act on prenylated substrates, has been implicated in the assembly of several pathways of the envelope of Staphylococcus aureus. We described earlier the Lysostaphin resistance (Lyr) and Staphylococcal protein A display (Spd) phenotypes associated with loss of the lyrA (spdC) gene. However, a direct contribution to the assembly of pentaglycine crossbridges, the target of lysostaphin cleavage in S. aureus peptidoglycan, or of Staphylococcal protein A attachment to peptidoglycan could not be attributed directly to LyrA (SpdC). These two processes are catalyzed by the Fem factors and Sortase A, respectively. To gain insight into the function of LyrA (SpdC), here we use affinity chromatography and LC-MS/MS analysis and report that LyrA interacts with SagB. SagB cleaves glycan strands of peptidoglycan to achieve physiological length. Similar to sagB peptidoglycan, lyrA peptidoglycan contains extended glycan strands. Purified lyrA peptidoglycan can still be cleaved to physiological length by SagB in vitro. LyrA does not modify or cleave peptidoglycan, it also does not modify or stabilize SagB. The membrane bound domain of LyrA is sufficient to support SagB activity but predicted ‘CAAX enzyme’ catalytic residues in this domain are dispensable. We speculate that LyrA exerts its effect on bacterial prenyl substrates, specifically undecaprenol-bound peptidoglycan substrates of SagB, to help control glycan length. Such an activity also explains the Lyr and Spd phenotypes observed earlier. IMPORTANCE Peptidoglycan is assembled on the trans side of the plasma membrane from lipid II precursors into glycan chains that are crosslinked at stem peptides. In S. aureus, SagB, a membrane-associated N-acetylglucosaminidase, cleaves polymerized glycan chains to their physiological length. Deletion of sagB is associated with longer glycan strands in peptidoglycan, altered protein trafficking and secretion in the envelope, and aberrant excretion of cytosolic proteins. It is not clear whether SagB, with its single transmembrane segment, serves as the molecular ruler of glycan chains or whether other factors modulate its activity. Here, we show that LyrA (SpdC), a protein of the CAAX type II prenyl endopeptidase family, modulates SagB activity via interaction though its transmembrane domain.

Staphylococcus aureus susceptibility to complestatin and corbomycin depends on the VraSR two-component system

“…IsaA is a putative lytic transglycosylase able to cleave peptidoglycan ( 49 ). SpdC (formerly LyrA) ( 50 ) forms a complex with the cell-wall hydrolase SagB, and the complex has been proposed to release nascent peptidoglycan strands from the cell membrane to complete integration into the cell-wall matrix ( 51 , 52 ). Last, MGT is a monofunctional transglycosylase that catalyzes the elongation of peptidoglycan chains in a metal ion-dependent manner ( 53 ).…”

“…SpdC is a membrane protein homolog of eukaryotic CAAX proteases that interacts with SagB, a membrane-associated N -acetylglucosaminidase that cleaves glycan strands of peptidoglycan to achieve the physiological length ( 51 – 53 ). By forming a complex, SpdC scaffolds SagB to orient its active site for cleaving glycan strands; thus, spdC and sagB mutants showed similar phenotypes to cell-wall-disrupting agents (resistance to lysostaphin and inhibition of teichoic acids by tunicamycin) ( 52 ). Taking this into account, we next sought to investigate the role of the SpdC/SagB complex in Cm and Cb susceptibility ( Fig.…”

Histidine transport is essential for the growth of Staphylococcus aureus at low pH

“…vraS and vraR coding for the VraSR two-component system, which detects cell-wall stress, were on the list of essential genes. Several peptidoglycan hydrolases were also on the list, including SpdC and SagB, which form a protein complex required for the release of nascent peptidoglycan during daughter cell formation, and IsaA, the immunodominant staphylococcal antigen A, which is a predicted lytic transglycosylase enzyme [26,27,28]. Other pathways linked to the cell wall included those involved in modulating cell surface charge.…”

Quorum-sensing, intra- and inter-species competition in the staphylococci

“…SpdC and SagB (a membrane-bound N -acetylglucosaminidase) form a complex that functions as a peptidoglycan release factor. However, the conserved residues required for CAAX proteolytic activity are not required by SpdC [59, 60].…”