Release of protein A from the cell wall of Staphylococcus aureus

Abstract

Staphylococcal protein A (SpA) is anchored to the cell wall envelope of Staphylococcus aureus by sortase A, which links the threonyl (T) of its C-terminal LPXTG motif to peptidoglycan cross-bridges (i.e., Gly5). SpA binds the Fcγ domains of IgG and protects staphylococci from opsonophagocytic clearance. Moreover, SpA cross-links B-cell receptors to modify host adaptive immune responses. The mechanisms whereby SpA is released from the bacterial surface to access the host's immune system are not known. Here we demonstrate that SpA is released with murein tetrapeptide-tetraglycyl [L-Ala-D-iGln-(SpA-Gly5)L-Lys-D-Ala-Gly4] linked to its C-terminal threonyl. LytN, a cross-wall murein hydrolase, contributes to the release of SpA by removing amino sugars [i.e., N-acetylmuramic acid-N-acetylglucosamine (MurNAc-GlcNAc)] from attached peptidoglycan, whereas LytM, a pentaglycyl-endopeptidase, triggers polypeptide release from the bacterial envelope. A model is proposed whereby murein hydrolases cleave the anchor structure of released SpA to modify host immune responses.

Keywords: Gram-positive bacteria; sortase-anchored protein; surface protein.

Fig. 1.

S. aureus releases protein A from the cell wall envelope. (A) S. aureus Newman WT or sbi, spa, or spa/sbi cultures were centrifuged and the extracellular medium separated with the supernatant (marked as “S”) from the bacterial pellet (marked as “P”). After treatment of the staphylococcal cell wall envelope with lysostaphin, proteins in both fractions were analyzed by immunoblotting with polyclonal antibodies against protein A (αSpA) or sortase A (αSrtA). (B) S. aureus Newman cells were washed in PBS solution, pulse-labeled with [³⁵S]Met/Cys, and mixed with fresh culture media. At timed intervals after labeling (0, 30, 60, 120 min), two aliquots from the culture were precipitated with TCA. One sample was treated with lysostaphin to release sortase (cell wall)-anchored proteins (Left), whereas the other was treated with hot SDS to solubilize released protein A molecules (Right). Radioactive samples were immunoprecipitated with αSpA and analyzed by SDS/PAGE and PhosphorImager. Immunoblotting with αSrtA was used as fractionation control.

Fig. 2.

Structure of released protein A. (A) Protein A was purified by affinity chromatography from the culture medium and cleaved with cyanogen bromide, and C-terminal peptides were isolated by RP-HPLC and identified by Edman degradation (Table S2). (B) C-terminal SpA peptides were subjected to MALDI-TOF MS and ion signals were recorded. (C) Model for the predominant ions signals generated by C-terminal SpA peptides. (D) Observed and predicted (MW) m/z and differentials (Δ) for SpA peptides released by WT S. aureus with their predicted structures.

Fig. 3.

S. aureus murein hydrolases impact the release of protein A. (A) Structure of S. aureus peptidoglycan comprised of the repeating disaccharide N-acetylglucosamine-(β1-4)-N-acetylmuramic acid (GN-MN) with linked wall peptide (Ala-iGln-Lys-Ala) and pentaglycine cross-bridge (Gly₅). Arrowheads identify the cleavage sites of murein hydrolases that act at the cross-wall (Atl, Sle1, and LytN) or that function as glycyl-endopeptidases (LytM). (B) MALDI-TOF MS of C-terminal SpA peptides released from the S. aureus atl mutant. (C) Observed and predicted (MW) m/z ratios and their differentials (Δ) for SpA peptides released by the S. aureus atl mutant with their predicted structures.

Fig. 4.

Cross-wall murein hydrolases shape the structure of released protein A. MALDI-TOF mass spectrometry of C-terminal SpA peptides released from the S. aureus sle1 (A) and lytN (B) mutants. (C) Observed (m/z) and predicted (MW) mass-to-charge ratios and their differentials (Δ) for SpA peptides released by the S. aureus sle1 and lytN mutants with their predicted structures.

Fig. 5.

LytM endopeptidase is required for protein A release from the staphylococcal envelope. (A) S. aureus WT and atl, sle1, lytN, lytM, or atl/sle1/lytN mutant cultures were centrifuged. Proteins in the culture media were separated with the supernatant (marked “S”) from the bacterial pellet (marked “P”). After treatment of the staphylococcal cell wall envelope with lysostaphin, proteins in both fractions were analyzed by immunoblotting with polyclonal antibodies against protein A (αSpA) or sortase A (αSrtA). (B) Immune reactive signals from three different samples analyzed as shown in A were averaged, SEMs determined, and statistical significance analyzed with the two-tailed Student t test. Only the lytM mutant strain released less protein A than WT S. aureus (P = 0.002).

Fig. 6.

SpA release during staphylococcal growth. S. aureus Newman (sbi) cells were washed and diluted into fresh TSB medium to A₆₀₀ 0.05 and incubated with rotation at 37 °C. At 30-min intervals, the absorbance (600 nm) was measured and cfus were enumerated. SpA release was quantified by immunoblotting of culture supernatant samples and recorded as signal intensity divided by cfu (×10⁻³). Experiments were performed in triplicate to calculate average values and SEM (error bars).