The multifunctional Staphylococcus aureus autolysin aaa mediates adherence to immobilized fibrinogen and fibronectin

Abstract

Staphylococci can cause a wide spectrum of infections, including endocarditis, osteomyelitis, and sepsis, which is reflected by the numerous virulence factors they produce, among them a recently identified new class of adhesins, namely, the multifunctional autolysins/adhesins. Here we report the identification and molecular characterization of Aaa, a novel autolysin/adhesin from Staphylococcus aureus. The gene encoding Aaa was cloned from the clinical isolate Staphylococcus aureus 4074. DNA sequence analysis revealed that aaa encodes a deduced protein of 334 amino acids with a predicted molecular mass of 35.8 kDa. Aaa contains three N-terminal repetitive sequences that comprise features of a peptidoglycan-binding domain, the LysM domain. The expression of aaa by Escherichia coli and its subsequent characterization revealed that Aaa possesses bacteriolytic activity as well as adhesive properties, such as binding to extracellular matrix proteins. Real-time biomolecular interaction analysis demonstrated that the interaction of Aaa with fibrinogen, fibronectin, and vitronectin is dose dependent and saturable and occurs with a high affinity. Furthermore, we demonstrate that Aaa binds to the Aalpha and Bbeta chains of fragment D of fibrinogen. Immunofluorescence microscopy revealed that Aaa is located at the cell surface. Finally, an aaa knockout mutant showed reduced adherence to surface-adsorbed fibrinogen and fibronectin, strongly suggesting a role for Aaa in the colonization of host factor-coated polymer surfaces and/or host tissue.

FIG. 1.

(A) Alignment of the deduced amino acid sequences of the repetitive sequences of the autolysin/adhesin Aaa (334 aa) from S. aureus 4074 (Aaa-1 to Aaa-3), Aae (324 aa) from S. epidermidis (Aae-1 to Aae-3), LytE (334 aa) from B. subtilis (LytE-1 to LytE-3), LytF (488 aa) from B. subtilis (LytF-1 to LytF-5), p60 (524 aa) from Listeria ivanovii (P60-1 to P60-3), and N-acetylmuramoyl-l-alanine amidase (641 aa) from E. faecalis (Am-1 to Am-5) (only the first 44 amino acids of the repeats are shown). The consensus sequence indicates the LysM domain that is proposed to have a general peptidoglycan-binding function. Bold letters indicate amino acids that match the consensus sequence. The letters in the second and third line in the consensus sequence show alternative amino acids. (B) Alignment of the deduced C-terminal amino acid sequences of the autolysin/adhesin Aaa from S. aureus 4074, Aae from S. epidermidis (324 aa), the secretory antigen SsaA (257 aa) from S. epidermidis, ORF1 (255 aa) from S. aureus (EMBL database accession number X97985), and the extracellular protein SceB (263 aa) from S. carnosus (EMBL database accession number U96107). Asterisks indicate identical amino acids, colons indicate very similar amino acids, and dots indicate somewhat similar amino acids. Gaps (dashes) were filled in to maximize homologies.

FIG. 2.

Expression and purification of six-His-Aaa. The image shows an SDS-PAGE gel (12.5% separation gel) with crude cell lysates (10 μl) of noninduced E. coli(pQAaa) (lane 1), IPTG-induced E. coli(pQAaa) (lane 2), and six-His-Aaa (1.5 μg) purified from E. coli(pQAaa) (lane 3). The arrow indicates the 35.8-kDa His-tagged Aaa protein purified from E. coli(pQAaa). The same protein band was absent from crude extracts of E. coli(pQAaa) before induction. Marker proteins are shown on the left.

FIG. 3.

Zymographic analysis of bacteriolytic activity of Aaa. The image shows an SDS-PAGE gel of crude cell lysates (10 μl) of noninduced E. coli(pQAaa) (lane 1), IPTG-induced E. coli (pQAaa) (lane 2), six-His-Aaa (1.5 μg) purified from E. coli(pQAaa) (lane 3), and cell surface-associated proteins (10 μl) of S. aureus 4074 (lane 4). The separation gel (12.5%) contained heat-inactivated S. carnosus cells (0.05%) as a substrate for bacteriolytic enzymes. Bacteriolytic activity is visible as a clear zone after incubation in phosphate buffer. The arrows indicate Aaa-associated bacteriolytic activity.

FIG. 4.

Determination of binding of Fg (A, E, F, and G), Fn (B), Vn (C), and BSA (D) to the immobilized autolysin/adhesin Aaa using the BIAcore system. After Aaa was immobilized on the C1 chip surface, Fg, Fn, Vn, and BSA were injected over the chip surface at a flow rate of 30 μl/min. The binding of Fg, Fn, Vn, and BSA was monitored and presented in overlay plots of the sensorgrams (plots of resonance units [RU] versus time). (A) Concentrations of Fg (from bottom to top): 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 700 nM, 800 nM, and 1,200 nM; (B) concentrations of Fn (from bottom to top): 200 nM, 300 nM, 400 nM, 700 nM, 1,200 nM, and 1,600 nM; (C) concentrations of Vn (from bottom to top): 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1,000 nM, 1,100 nM, 1,200 nM, and 1,300 nM; (D) concentrations of BSA (from bottom to top): 0 nM, 100 nM, 500 nM, 1,000 nM, 2,000 nM, 5 μM, and 10 μM; (E) concentrations of Fg and peptide HHLGGAKQAGDV (from bottom to top): 0 nM Fg and 1,000 nM peptide, 100 nM Fg and 1,000 nM peptide, and 100 nM Fg and 0 nM peptide; (F) concentrations of Fg and peptide GRGDSP (from bottom to top): 0 nM Fg and 900 nM peptide, 100 nM Fg and 900 nM peptide, 100 nM Fg and 750 nM peptide, 100 nM Fg and 500 nM peptide, 100 nM Fg and 250 nM peptide, 100 nM Fg and 100 nM peptide, and 100 nM Fg and 0 nM peptide; (G) concentrations of Fg and peptide FYQVLNMPNLNA (from bottom to top): 0 nM Fg and 900 nM peptide, 100 nM Fg and 900 nM peptide, 100 nM Fg and 500 nM peptide, 100 nM Fg and 250 nM peptide, 100 nM Fg and 100 nM peptide, 100 nM Fg and 50 nM peptide, and 100 nM Fg and 0 nM peptide.

FIG. 5.

Detection of Aaa at the cell surface by immunofluorescence microscopy. Cells of the protein A-deficient strain S. aureus Wood 46 grown overnight in BHI broth were incubated with the anti-Aaa antiserum raised in rabbits (A) or with preimmune serum (B). Bound antibodies were detected with fluorescein-conjugated anti-rabbit IgG antibodies. Cells were viewed with a fluorescence microscope. Wood 46 cells reacted with the anti-Aaa antiserum, indicating the cell surface location of Aaa. In comparison, the reaction of the cells with the preimmune serum was much weaker. Magnification, ×1,000.

FIG. 6.

Surface-associated proteins (10 μl) from S. aureus 4074 (lanes 1) and the aaa mutant (lanes 2) were separated by SDS-PAGE (10% separation gel), stained with Coomassie brilliant blue (A), and subjected to zymographic analysis (B) or Western blot analysis (C). A 35.8-kDa protein with bacteriolytic activity (B) that reacted with the anti-Aaa antiserum (C) was missing from the aaa mutant (indicated by an arrow). The sizes of the marker proteins are indicated on the left (A; Bio-Rad prestained marker [M]) and the right (C; MBI Fermentas prestained marker [M]).

FIG. 7.

Adherence of S. aureus 4074 and its aaa mutant to immobilized Fg and Fn adsorbed to polymethylmethacrylate coverslips. [³H]thymidine-labeled S. aureus 4074 or S. aureus aaa (4 × 10⁷ CFU/ml) was incubated with coverslips that were preadsorbed with Fg, Fn, or BSA. Afterwards, the bacterial suspension was removed, the coverslips were washed, the adherent radioactivity was determined, and the CFU were calculated. Experiments were performed three times in quintuplicate. Results are means ± standard errors of the means. Statistical analysis was performed using the Wilcoxon matched-pair signed rank test for nonparametric data. P values of <0.05 were considered to indicate statistically significant differences. *, P < 0.05 for comparison with wild-type strain S. aureus 4074. Black bars, S. aureus 4074; gray bars, aaa mutant.

FIG. 8.

Determination of binding domains of Fg for Aaa by ligand affinity blot analysis. Fragments D (lanes 2) and E (lanes 3) of Fg (nonreduced) and the Fg chains (Aα, Bβ, and γ chains [reduced]) (lanes 1) were separated by SDS-PAGE (10% separation gel) (A) and transferred to nitrocellulose filters that were probed with six-His-Aaa (B) or six-His-dihydrofolate reductase (C) or without a ligand (D), followed by a Ni-NTA-alkaline phosphatase conjugate. The sizes of the marker proteins (MBI Fermentas prestained marker [M]) and the positions of the Aα, Bβ, and γ chains of Fg are indicated.