beta-Neurexin is a ligand for the Staphylococcus aureus MSCRAMM SdrC

Abstract

Gram-positive bacteria contain a family of surface proteins that are covalently anchored to the cell wall of the organism. These cell-wall anchored (CWA) proteins appear to play key roles in the interactions between pathogenic organisms and the host. A subfamily of the CWA has a common structural organization with multiple domains adopting characteristic IgG-like folds. The identified microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) belong to this subfamily, as does SdrC from S. aureus. However, an interactive host ligand for the putative MSCRAMM SdrC was not previously identified. We have screened a phage display peptide library and identified a peptide sequence found in beta-neurexin that binds SdrC. A synthetic peptide corresponding to the identified sequence as well as a recombinant form of the beta-neurexin 1 exodomain binds SdrC with high affinity and specificity. Furthermore, expression of SdrC on bacteria greatly enhances microbial adherence to cultured mammalian cells expressing beta-neurexin on their surface. Taken together, our experimental results demonstrate that beta-neurexin is a ligand for SdrC. This interaction involves a specific sequence located in the N-terminal region of the mammalian protein and the N(2)N(3) domain of the MSCRAMM. The fact that these two proteins interact when expressed on the appropriate cells demonstrates the functionality of the interaction. Possible implications of this interaction are discussed.

Figure 1. Structural organization of SdrC and recombinant proteins.

(A) Schematic representation of SdrC domain structure. S, signal sequence; A region underlined composed of N1, N2 and N3, B1 and B2, B repeats, R, serine-aspartic acid repeat region; W, wall-spanning fragment; M, transmembrane domain; C, cytoplasmic tail; LPETG, cell wall anchoring motif; VAAPQ, cleavage site; the arrow indicated the cleavage position. Also shown, are the His-tag recombinant SdrC proteins used in this study. (B), Coomassie-stained SDS-PAGE gel of pure recombinant SdrC_A. (C) Coomassie-stained SDS-PAGE gel of the recombinant SdrC_A after 2 weeks storage at 4°C.

Figure 2. Identification of β-neurexins as the potential SdrC-binding partner.

(A), Phage obtained after the third panning round bound specifically to SdrC_N2N3. Fifty random phage clones obtained after the 3^rd round of panning were incubated with immobilized SdrC_N2N3, SdrG_N2N3 and BSA. Shown are the eight phage clones with the highest binding affinity (*p<0.001) for SdrC_N2N3 in comparison with SdrG_N2N3, BSA or fd-tet (insertless phage). (B), Consensus sequence search. Pattern searches with the degenerated consensus (P,T,A)HH(I,M)HHFH(G,R,S,Q,T,A) against a human protein database returned β-neurexin isoforms as SdrC-ligands. Consistent residues of the consensus are highlighted in bold, variable residues are highlighted in gray.

Figure 3. Structural organization of β-neurexins.

(A) A cartoon representation of β-neurexin domain structure. S, signal sequence; NL, neuroligin-binding domain; Glyc, glycosylation domain; TM, transmembrane domain; PDZ, intracellular signaling domain. Also shown, are the recombinant proteins used in this study. (B) Coomassie-stained SDS-PAGE gel of GST-tagged neurexin 1β domains recombinant proteins. Increasing concentrations of SdrC_A (C) and SdrC_N2N3 (D) were incubated with immobilized Nrx_47-255 (black square), Nrx_70–255 (black triangle) or GST (black circle) for 1 hour at room temperature. The apparent K_D (concentrations required for half maximum binding) values were 1.85±0.94×10⁻⁷ M for SdrC_A and 1.26±0.28×10⁻⁷ M for SdrC_N2N3. (E) SdrC_178–496 (0.5 µM) was incubated with increasing concentrations of spNrx_wt peptide (black square) or spNrx_scr peptide (black triangle) at room temperature. After 2 hours, the mixture was incubated with immobilized Nrx_47–255. The values represented here are the mean±SD of triplicates from three experiments.

Figure 4. Determination of equilibrium constant of binding.

(A) SdrC_N2N3 binds specifically to β-neurexin 1. Increasing concentrations of SdrC_N2N3 or ClfB_N2N3 (black circle) were incubated with 10 nM fluorescein-labelled spNrx_wt peptide (black square) or spNrx_scr peptide (black triangle) for 3 hours at room temperature in the dark. Equation ΔP = ΔP_max [protein]/(K_D+[protein]) was used to calculate the equilibrium constant. The values from three experiments returned a K_D = 2.50±0.49×10⁻⁷ M. (B) SdrC_N2N3 (0.5 µM) (black triangle) was incubated with increasing concentration of unlabelled spNrx_wt peptide (white circle) or spNrx_scr peptide (black circle) at room temperature. After 3 hours, the mixture was incubated for another 3 hours at room temperature in the dark with 10 nM fluorescein labeled spNrx_wt peptide. Fluorescence polarization was determined as described above. The values represented here are the mean±SD of triplicates from three experiments.

Figure 5. SdrC mediates S. aureus clinical isolates adherence to Nrxβ expressing cells.

(A) Western immunobloting to detect SdrC expression on the surface of different bacterial strains. S. aureus Newman, DU5988 and clinical strains USA300, MW2, MRSA252 were grown to exponential phase (A1) or stationary phase (A2) in BHI. CWA proteins extracted as described in material and methods were separated on 4–15% gradient SDS-PAGE gradient gels, transferred to nitrocellulose and probed with anti-SdrC serum. (B) Culture supernatants from the above mentioned strains grown to exponential (B1) or stationary phase (B2) were concentrated and separated on 4–15% gradient SDS-PAGE gradient gels after protein A removal. After gel separation the proteins were transferred to nitrocellulose and probed with anti-SdrC serum (B1 and B2). The nitrocellulose blot corresponding to the stationary phase supernatants was stripped and re-probed with anti-SdrC B-repeats antibody (B3). The same blot was stripped again and probed with Nrx_47-255. The binding of Nrx1β was detected with anti-GST serum (B4). A second membrane (B5) was not incubated with Nrx_47-255 and therefore served as control for the primary and secondary sera. (C) L. lactis empty vector, L. lactis SdrC, S. aureus Newman, DU5988 (S. aureus Newman sdrC::Em^r), USA300, MW2 or MRSA252 were incubated with CHOK1 transiently transfected with Nrx1β-mCherry or FLAG Nrx1β-mCherry grown in 24 well plates. Attachment of bacteria was reported as a percentage of total bacteria at the end of incubation. Values shown represent mean±SD of three experiments (*p≤0.001).