Characterization of the Modular Design of the Autolysin/Adhesin Aaa from Staphylococcus Aureus

BACKGROUND: Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. CONCLUSIONS/SIGNIFICANCE: We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Functional characterization of the adhesive and invasive properties of <i>S. aureus</i> 4074, 4074<i>aaa</i>, and 4074<i>aaa</i>(pRCK<i>aaa</i>).

<p>A: Complementation of the 4074<i>aaa</i> mutant. SDS-PAGE (I; 10% separation gel) and zymographic analysis (II) of surface-associated proteins (10 µl) of <i>S. aureus</i> 4074 (lane 1), the 4074<i>aaa</i> mutant (lane 2), and the complemented mutant <i>S. aureus</i> 4074<i>aaa</i> (pRCK<i>aaa</i>) (lane 3). The ∼34 kDa band with lytic activity corresponding to Aaa and its ∼28 kDa putative degradation product were missing from the 4074<i>aaa</i> mutant (lane 2) and present in the wild type (lane 1) and the complemented mutant (lane 3). The arrows indicate Aaa-associated bacteriolytic activity. The sizes of marker proteins (kDa) are indicated on the left. B: Adherence of <i>S. aureus</i> 4074, 4074<i>aaa</i>, and 4074<i>aaa</i> (pRCK<i>aaa</i>) to immobilized Fg, Fn, Vn, and EA.hy 26 cells was assessed by ELISA adherence assays. Binding of the 4074<i>aaa</i> mutant to Vn was significantly decreased in comparison to the wild type. In contrast, binding of the 4074<i>aaa</i> mutant to Fg, Fn, and EA.hy 926 cells was not altered. As a negative control, binding to BSA was assessed. Results are shown as the mean of three independent experiments. Statistical significance is marked by asterisks. C: As a more sensitive method, flow cytometry was applied to determine the binding of FITC-labeled, soluble plasma proteins to <i>S. aureus</i> 4074, 4074<i>aaa</i>, and 4074<i>aaa</i> (pRCK<i>aaa</i>). Again, significantly reduced binding of the 4074<i>aaa</i> mutant was only observed to Vn, whereas its binding to Fg and Fn was unchanged. As a negative control, binding to BSA was assessed. The results are shown as the mean of three independent experiments and statistical significance is marked by asterisks. D: The internalization of strains 4074, 4074<i>aaa</i>, and 4074<i>aaa</i> (pRCK<i>aaa</i>) by EA.hy 926 cells was assessed by flow cytometry and computed in relation to the highly invasive strain <i>S. aureus</i> Cowan 1, which was set to 100% internalization. The internalization of the 4074<i>aaa</i> mutant was in the same range as that of the 4074 wild type and the complemented mutant indicating that Aaa does not play a role in internalization. Results are shown as the mean of three independent experiments.</p

Modular design of Aaa and analysis of recombinant fusion proteins.

<p>(A) Schematic model of 6 x His-Aaa, 6 x His-N-Aaa, and 6 x His-C-Aaa. LysM, lysin motif; CHAP, cysteine/histidine-dependent amidohydrolase/peptidase; R1, R2, R3, repeats 1, 2, and 3. (B) SDS-PAGE (15% separation gel) and (C) corresponding zymogram to detect bacteriolytic activity of purified Aaa (lane 1, ∼34 kDa), N-Aaa (lane 2, ∼22 kDa), and C-Aaa (lane 3, ∼13 kDa) against <i>S. carnosus</i> cells. Bacteriolytic activity was visible as a clear zone after staining the gel with methylene blue. Aaa and C-Aaa were bacteriolytically active against <i>S. carnosus</i>, whereas N-Aaa was detected as a precipitation zone presumably indicating binding to, but not cleavage of peptidoglycan. The sizes of the marker proteins (M; kDa) are indicated on the left.</p

Impact of global virulence regulators on <i>aaa</i> expression.

<p>The expression of <i>aaa</i> (A) or <i>atl</i> (B) in the RN6390 wild type and its RN6390<i>agr</i> and RN6390<i>sarA</i> mutants was assessed by real-time PCR analysis. The results are shown as the mean of three independent experiments and statistical significance is marked by asterisks. A: The expression of <i>aaa</i> is significantly increased during the early exponential growth phase (3 h) in the RN6390<i>sarA</i> mutant compared to the wild type. An increased expression of <i>aaa</i> was also observed in the RN6390<i>agr</i> mutant after 6 h, 11 h, and 26 h of growth, which however was not statistically significant. B: The expression of <i>atl</i> in the RN6390<i>agr</i> mutant was higher after 3 h of growth compared to the wild type, which however did not reach statistical significance. In the RN6390<i>sarA</i> mutant, a significantly higher level of <i>atl</i> mRNA was detected after 26 h of growth. C, D: Aaa and Atl protein production was assessed by semi-quantitative SDS-PAGE (10% separation gel) (upper panel) and corresponding zymographic analysis (lower panel) of surface-associated proteins of the RN6390 wild type and the RN6390<i>agr</i> mutant (C) or the RN6390 wild type and the RN6390<i>sarA</i> mutant (D). Aaa and Atl protein production was much more pronounced with the RN6390<i>agr</i> mutant than with the RN6390 wild type after 6 h, 10 h, and 24 h of growth, strongly indicating a negative regulatory effect of <i>agr</i> on the expression of both, <i>aaa</i> and <i>atl</i>. Aaa protein production was more pronounced with the RN6390<i>sarA</i> mutant than with the RN6390 wild type after 3 h, 6 h, and 10 h of growth, indicating a negative regulatory effect of <i>sarA</i> on the expression of <i>aaa</i>.</p

Localization of the functional binding domains within Aaa.

<p>Determination of binding of Fg (A, B, C), Fn (D, E, F), and Vn (G, H, I) to the immobilized Aaa (A, D, G), N-Aaa (B, E, H), and C-Aaa (C, F, I) using the BIAcore system. After Aaa, N-Aaa, and C-Aaa were immobilized on the C1 chip surface, Fg, Fn, and Vn were injected over the chip surface at a flow rate of 50 µl/min. I: Binding of Fg, Fn, and Vn was monitored and presented in an overlay-plot of the sensorgrams (a plot of RU [resonance unit] versus time). A, B, C, concentrations of Fg (from bottom to top): 9.16 pM, 18.3 pM, 36.6 pM, 73.2 pM, 146 pM, 293 pM, 586 pM, 1.17 nM, 2.34 nM, 4.69 nM, 9.38 nM, and 18.8 nM. D, E, F, concentrations of Fn (from bottom to top): 293 pM, 586 pM, 1.17 nM, 2.34 nM, 4.69 nM, 9.38 nM, 18.8 nM, 37.5 nM, 75 nM, 150 nM, and 300 nM. G, H, I, concentrations of Vn (from bottom to top): 1.17 nM, 2.34 nM, 4.69 nM, 9.38 nM, 18.8 nM, 37.5 nM, 75 nM, 150 nM, 300 nM, 600 nM, and 1.2 µM. II: Overlay-plot of the sensorgrams (a plot of RU [resonance unit] versus time) of the binding of 4.7 nM Fg to Aaa, N-Aaa, and C-Aaa (A), 18.8 nM Fn to Aaa, N-Aaa, and C-Aaa (B), and 38 nM Vn to Aaa, N-Aaa, and C-Aaa (C) suggesting a 4∶2∶1 stoichiometry for the Fg-binding to Aaa, N-Aaa, and C-Aaa and a 2∶1∶1 stoichiometry for the Vn-binding to Aaa, N-Aaa, and C-Aaa. The stoichiometry for the binding of Fn to Aaa, N-Aaa, and C-Aaa is not completely clear, but appears to be 2∶1∶1.</p

Expression analysis of <i>atl</i> and <i>aaa</i>.

<p><i>atl</i> and <i>aaa</i> are upregulated in the 4074<i>aaa</i> and SA113<i>atl</i> mutant, respectively (A–D), and upregulation of <i>aaa</i> in the SA113<i>atl</i> mutant correlates with an increased adherence to Fg, Fn, and endothelial cells (E). The expression of the <i>atl</i> and <i>aaa</i> gene was determined by real-time PCR analysis and normalized to the expression of the housekeeping genes <i>gmk</i>, <i>aroE</i>, and <i>gyrB</i>. A: The level of <i>atl</i> mRNA in the 4074<i>aaa</i> mutant was significantly higher after 3 h and 10 h of growth and significantly lower after 6 h of growth compared to the 4074 wild type. Results are shown as the mean of three independent experiments. Statistical significance is indicated by asterisks. B: SDS-PAGE (10% separation gel, I) and semi-quantitative zymographic analysis (II) of surface-associated proteins from the 4074 wild type and the 4074<i>aaa</i> mutant showed increased lytic activity due to Atl (138 kDa) and its cleavage products producing a clearing zone ranging from ∼51 kDa to ∼130 kDa. The arrow indicates Aaa-associated bacteriolytic activity. C: The level of <i>aaa</i> mRNA was significantly and three-fold elevated in SA113<i>atl</i> compared to the wild type in the early exponential growth phase (3 h). Moreover, after 6 h and 11 h, the level of <i>aaa</i> mRNA was slightly higher in the SA113<i>atl</i> mutant, which however was not statistically significant. Results are shown as the mean of three independent experiments. Statistical significance is indicated by asterisks. D: SDS-PAGE (10% separation gel, I) and semi-quantitative zymographic analysis (II) of surface-associated proteins from the SA113 wild type and the SA113<i>atl</i> mutant revealed a more pronounced band with bacteriolytic activity at the molecular size of ∼34 kDa in the SA113<i>atl</i> mutant, reflecting an increased production of Aaa. The arrow indicates Aaa-associated bacteriolytic activity. E: The adhesive properties of the SA113 wild type and the SA113<i>atl</i> mutant were analyzed by ELISA adherence assays. The SA113<i>atl</i> mutant was significantly higher adherent to immobilized Fg, Fn, and EA.hy 926 cells than the SA113 wild type. In contrast, the Vn-binding activity of the SA113<i>atl</i> mutant did not significantly differ from the SA113 wild type. Results are shown as the mean of three independent experiments. Statistical significance is marked by asterisks.</p

Primers used in this study.

<p>Primers used for ¹cloning, ²sequencing, ³real-time PCR analysis.</p