Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

Abstract

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.

Fig 1. Cell wall-associated protein A.

Protein A expressed on the surface of S. aureus strains Cowan1, Seattle 1945 (25923), Wood 46, Newman WT and its corresponding deletion mutants, Δsbi, Δspa and Δsbi-Δspa, as measured by flow cytometry with chicken anti-protein A antibody. The values represent the average from three independent experiments. (*P<0.05, **P<0.02, ***P<0.01, ****P<0.001).

Fig 2. Production of extracellular protein A in S. aureus.

The amount of secreted protein A was measured using an antigen-capture ELISA. Commercially available protein A from S. aureus was used to generate the standard curve. The values represent the average from three independent experiments. (*P<0.05, **P<0.02, ***P<0.01, ****P<0.001).

Fig 3

(A) Amino acid composition of sortase A. The amino acid sequence of sortase A in Cowan 1, Seattle 1945, Newman and Wood 46 were aligned. Amino acids from position 155 to the end of the protein are shown. Wood 46 contains a 12bp deletion at the 3’ end of the srtA gene that leads to a corresponding loss of four amino acids at the C-terminus of the protein. (B). Promotor and upstream regulatory region of srtA in Wood 46. Consensus promoter and upstream regulatory sequence from Cowan 1, Seattle 1945 and Newman was aligned with that of Wood 46. The boxes highlight regions with mutations. The transcription start site ATG is indicated as +1.

Fig 4. Expression levels of spa and srtA.

Quantitative real-time PCR was used to measure the amount of (A) spa mRNA and (B) srtA mRNA in log phase bacterial cells. Relative gene expression was calculated using the ΔΔC_T method and expressed as fold change. 16S rRNA was used as the endogenous control. The amount of srtA mRNA in Wood 46 was below the detection limit of the assay. The values represent the average from three independent experiments. (*P<0.05, **P<0.02, ***P<0.01, ****P<0.001). ND- not detected.

Fig 5. Fibrinogen-binding and fibronectin-binding assay.

The amount of sortase A-anchored, LPXTG-motif containing fibrinogen-binding protein (A) and fibronectin-binding protein (B) was measured by flow cytometry using FITC-conjugated fibrinogen and HiLyte Fluor ^TM 488-conjugated fibronectin respectively. The percentage binding was determined relative to highest value obtained for that experiment. The values represent the average from three independent experiments. (*P<0.05, **P<0.02, ***P<0.01, ****P<0.001). (Since the Newman isolate has truncations in both FnBP-A and FnBP-B, it was not tested in the fibronectin binding assay).