Surface Proteins and the Formation of Biofilms by Staphylococcus aureus

Abstract

Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12-24 hours after stationary phase, and more mature biofilms formed for up to 60 hours after stationary phase. All samples were labeled either by (i) [¹⁵N]glycine and l- [1-¹³C]threonine, or in separate experiments, by (ii) l-[2-¹³C,¹⁵N]leucine. We then measured ¹³C-¹⁵N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in ¹⁵N isotopic enrichments and from the routing of ¹³C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of β-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion.

Graphical abstract

1. Introduction

The inhibition of Staphylococcus aureus biofilm formation and the sterilization of mature biofilms are of great clinical importance. An estimated 50-70% of catheter-related infections, 40-50% prosthetic cardiac-valve infections, and 20-50% of joint-replacement infections are caused by staphylococci biofilms [1], of which S. aureus is the leading pathogen. S. aureus is capable of translocating from a primary site of infection, for example, from a subvenous catheter to secondary sites such as prosthetic devices, implants, cardiac valves, bones, and other surfaces, to form biofilms [2]. The sessile form of S. aureus embedded within a mature biofilm poses a significant health challenge as this form is resistant to most antibiotic therapies and serves as a reservoir for recurring persistent infections.

In general, biofilms are described by three processes: attachment, maturation, and detachment [1]. Biofilm formation is initiated by the attachment of S. aureus to either abiotic or biotic surfaces through wall-teichoic acid and/or receptor-mediated protein interactions. The attached cells grow and mature, and then are encapsulated in a complex extracellular polymeric substance held together by combinations of proteins, polysaccharides, and other biopolymers [3]. During the detachment process, planktonic bacteria are released from the biofilm matrix.

The current challenge in biofilm studies of S. aureus is that the exact nature (composition) of the biofilm is difficult to determine. Although biofilm characterization through a genetic approach, combined with imaging methods, and coupled to biochemical and molecular biology techniques, has provided a wealth of morphological insights [4-7], this strategy does not provide chemical and structural insight at the molecular level. Because the biofilm is a mixture of multiple components and multiple interactions and is inherently complex and heterogeneous, characterization is not amenable to conventional spectroscopic methods.

Our goal is to provide compositional, structural, and functional insights into S. aureus biofilms at the molecular level using solid-state NMR of intact cells, cell walls, and biofilms. We will gain insights into local structure and function in complicated heterogeneous biofilm systems by using a collection of specific labels. This has been our basic strategy over the years in characterizing bacteria [8], plants [9], insects [10], and animal systems [11, 12].

In this report, we focus on the quantitation of surface proteins of S. aureus during the initial stages of biofilm formation. Surface proteins are 50-100-kDa proteins that are attached to the terminal glycyl residues of non-cross-linked S. aureus peptidoglycan bridges (Figure 1) [13]. Their function is to promote binding to each other and a variety of surfaces. Each surface protein has a C-terminal hydrophobic region anchored in the cell membrane that contains a LPXTG signaling motif. Sortase enzyme recognizes this sequence and covalently links the threonine to a bridge terminal glycyl residue (Figure 1). The N-terminal regions of the surface proteins contain leucine-rich repeats which are believed to form a collection of parallel β-strands that act as a scaffold for protein-protein interactions [14].

Figure 1.

(Top) Chemical structure of the peptidoglycan-repeat unit in S. aureus. This unit consists of a disaccharide GlcNAc-MurNAc with a peptide stem sequence L-Ala-d-iso-Gln-L-Lys-d-Ala-d-Ala, and a pentaglycine bridge structure attached to the L-Lys sidechain at the third position of the stem. Cross-links are formed between the amino terminus of a pentaglycine bridge and the C-1 carbon of d-Ala of the fourth position of an adjacent stem. The uncross-linked pentaglycine bridge is a sortase-mediated cell-wall attachment site for surface proteins containing a LPXTG targeting signal. The C-1 of L-threonine of the LPXTG sequence is covalently attached to the amino terminus of an open pentaglycine bridge by sortase A. The inset (far left) shows a schematic representation of the peptidoglycan-repeat unit with the disaccharides as gray-filled circles, amino acids in a pentapeptide stem as open triangles, glycines of a bridge as filled circles, and an attached surface protein (large blue circle). The incorporation of [¹⁵N]glycine and l-[1-¹³C]threonine into peptidoglycan are shown with ¹⁵N and ¹³C represented by blue and red circles, respectively. (Bottom) A schematic representation of the peptidoglycan lattice with 20 repeat units. Eighty percent (16/20) of peptidoglycan-repeat units are shown with a bridge attached, 20% (4/20) with an open bridge (yellow circles), and 25% of the open bridges (1/4) with a surface protein attached. The average peptidoglycan cross-linking is 60% (12/20).

Quantitation of total surface-protein production in biofilms by direct detection of the surface proteins themselves is complicated by multiple degradation pathways, indirect and inaccurate assays, and apparent binding of surface-protein fragments to the bacterial cell wall [15, 16]. We instead base our quantitation of total surface-protein production on solid-state NMR spectra of intact whole cells, isolated cell walls, and intact biofilms of S. aureus grown on media containing either (i) [¹⁵N]glycine (G) and l-[1-¹³C]threonine (T) or (ii) l-[2-¹³C,¹⁵N]leucine. (The leucine ¹⁵N label was not used in these experiments.) The ¹³C,¹⁵N-labeled TG pair is used to quantitate the fraction of peptidoglycan pentaglycyl bridge terminal –NH₂ units covalently linked by sortase enzyme to a surface protein (TG cell-wall links) during biofilm formation, and the leucine ¹³C-label is used to quantitate the increase in leucine-rich β-strand regions available for protein-protein and protein-surface binding in biofilms.

2. Materials and methods

2.1. Preparation of S. aureus whole-cell and biofilm samples

Typically, whole-cell S. aureus (SA113) samples for NMR were grown in 350 ml of enhanced standard medium (ESM) [17] at 37 °C with gentle shaking at 100 rpm. Cells were harvested at the mid-exponential phase by centrifugation at 10,000 g for 10 min at 4 °C. Then a whole-cell pellet was washed two times with water, centrifuged at 10,000 g for 10 min at 4 °C after each wash. The final whole-cell pellet was resuspended in 10 ml of water, flash frozen in liquid nitrogen, and lyophilized to yield 150 to 200 mg powder. The lyophilized powder of whole cells was pressed into cylindrical pellets and packed into a zirconia rotor for NMR measurements.

Biofilm samples were prepared by growing SA113 in ESM supplemented with 1.75% glucose, the condition optimized for maximum biofilm growth determined using a crystal violet assay. The natural abundance amino acids glycine and threonine, or leucine in ESM were respectively replaced by l-[¹³C]threonine and [¹⁵N]glycine or by l-[2-¹³C,¹⁵N]leucine. Following exponential growth, SA113 formed visible cell aggregates and biofilm. After 60 hours growth, cells were harvested by allowing biofilm to precipitate to the bottom of the culture flask. The spent media containing any planktonic cells was carefully decanted prior to centrifugation. The harvested cells were washed once with cold deionized water, flash frozen, and lyophilized.

2.2. Scanning electron microscopy of biofilm

Biofilm slides for scanning electron microscope analysis were prepared by growing SA113 in flat-bottom 6-well polystyrene tissue culture wells (Corning® Costar®) with gentle orbital shaking at 80 rpm. Each well contained 4 ml ESM supplemented with 1.75% glucose with a plastic slide (22 × 22 mm) placed at the bottom of the well for the biofilm attachment. The biofilm coated plastic slides were collected after 12-24 hours (early biofilm) and at 48-60 hours (mature biofilm) growths. Cells in biofilm were fixed with 2.5% glutardialdehyde in 0.06 M phosphate buffer (pH 7.2) for 90 min at room temperature and then washed 4 times (10 min each) in phosphate buffer. Samples were then dehydrated through a series of graded acetone-water (50%, 70%, 90%, and 100% acetone) twice with 10-min incubation for each grade. After critical-point drying (EM CPD300, Leica Microsystems, Wetzlar, Germany) samples were mounted on stubs and sputter coated (EM ACE 600, Leica Microsystems) with 20 nm of iridium. Biofilm was observed in a Versa 3D scanning electron microscope (FEI, Hillsboro, OR, USA).

2.3. Cell-wall isolation

The cell walls were isolated from lyophilized whole cells in a manner similar to that described before [18-21] with minor modifications. The lyophilized whole cells were resuspended in about 10 ml of water and boiled for 20 min to inactivate the cells prior to centrifugation at 3200 g for 30 min at 4 °C. The whole-cell pellet was resuspended in 50 ml of 25 mM cold potassium phosphate buffer (pH 7.0) with 5 mg of DNase I and transferred to a 70 ml bead beater (Biospec Products, Bartlesville, OK) chamber which was two thirds filled with 0.5-mm diameter glass beads. Cells were beat through 10 cycles of 1 min disruption and 1 min rest. Through the beating process, the bead beater chamber was kept in a bigger chamber containing an ice-water mixture to keep the bead beater chamber cold. After the fifth cycle of disruption, the ice-water mixture was refilled. After disruption, the homogenate and beads were filtered with a course sintered glass funnel (25-50 μm) to separate the homogenate (filtrate) from the beads by vacuum filtration. The beads were washed further with 70 ml of 10 mM cold EDTA solution (pH 7.0). The filtrate was centrifuged at 25,000 g for 30 min at 4 °C. The crude cell-wall pellet was resuspended in 20 ml of cold water, added dropwise to 100 ml of boiling 4% sodium dodecyl sulfate (SDS), and boiled for 30 min with constant stirring. The mixture was allowed to cool for 2 h with stirring and then maintained unstirred overnight at room temperature. SDS was removed by centrifugation of the mixture at 38,000 g for 1 h at room temperature, followed by three washes using water with centrifugation after each wash. The resulting pellet was resuspended in 60 ml of 10 mM Tris buffer (pH8.2) containing 5 mg of DNase I, 16 mg trypsin, and 16 mg α-chymotrypsin and incubated at 37 °C with shaking (150 rpm) for 16 h. This mixture was centrifuged at 38,000 g for 1 h at room temperature and washed two times using water with centrifugation after each wash. The final pellet of isolated cell walls was resuspended in about 10 ml of water, flash frozen in liquid nitrogen, and lyophilized. Typically 40 to 50 mg lyophilized powder was obtained. The lyophilized powder of the cell walls was pressed into cylindrical pellets and packed into a zirconia rotor for NMR measurements.

2.4. Solid-state NMR

Experiments were performed at 12 Tesla with a six-frequency transmission-line probe having a 12-mm long, 6-mm inner-diameter analytical coil, and a Chemagnetics/Varian ceramic spinning module. Samples were spun using a thin-wall Chemagnetics/Varian (Fort Collins, CO/Palo Alto, CA) 5-mm outer diameter-zirconia rotor at 7143 Hz, with the speed under active control and maintained to within ±2 Hz. A Tecmag Libra pulse programmer (Houston, TX) controlled the spectrometer. Two-kW American Microwave Technology (AMT) power amplifiers were used to produce radio-frequency pulses for ¹³C (125.7 MHz) and ¹⁵N (50.7 MHz). The ¹H (499.8 MHz) radio-frequency pulses were generated by a 2-kW Creative Electronics tube amplifier driven by a 50-W AMT amplifier. All final-stage amplifiers were under active control [22]. The 7r-pulse lengths were 8 μs for ¹³C and ¹H, and 9 μs for ¹⁵N. Proton-carbon-matched cross-polarization transfers were made in 2 ms at 56 kHz. Proton dipolar decoupling was 100 kHz during N{C} and C{N} REDOR evolution and data acquisition. The S and S₀ alternate-scan strategy compensated for short-term drifts in REDOR experiments. Standard XY-8 phase cycling [23] was used for all refocusing observe-channel π pulses (inserted at the end of each rotor period during dipolar evolution) and dephasing π pulses (inserted in the middle of each rotor period) to compensate for finite pulse imperfections. Typically, CPMAS spectra from 100-mg whole-cell and 50-mg cell-wall samples were the result of the accumulation of 20,000 to 40,000 scans at room temperature with a recycle delay of 2 sec (about 1 day per spectrum). The N{C} REDOR experiments involved 100,000 scans (5 days), and the C{N} REDOR experiments used to generate double differences, 200,000 scans (10 days) for each spectrum.

3. Results and discussion

3.1. Leakage of ¹³C label from threonine

The one-bond 16-T_r N{C} REDOR ΔS/S₀ for the amide-nitrogen signal (80 ppm) of cell walls isolated either from planktonic whole cells or biofilms labeled by [¹⁵N]glycine+ l-[1-¹³C]threonine is about 5% (Figure 2, left and Table 1, first row). If only natural-abundance ¹³C were contributing, we would expect a ΔS/S₀ on the order of 2%. That is, for every 200 carbons in pentaglycine bridges (10 carbons per bridge) of peptidoglycan of isolated cell walls (20 bridges), each glycyl ¹⁵N would have a 1% chance of having a ¹³C carbonyl-carbon nearest neighbor, and a 1% chance of having a ¹³C CH₂-carbon nearest neighbor. (This calculation ignores open bridges which end in glycyl amines and are present in low concentration; see below.) We attribute the observed N{C} ΔS/S₀ of 5.1% to a constant degradative leakage of l-[1-¹³C]threonine to [1-¹³C]glycine [24]. For the cell walls of the planktonic cells, ΔS/S₀ is 4.8% (Table 1, first row, first entry). Hence, the l-[1-¹³C]threonine leakage to [1-¹³C]glycine during growth is at a rate of approximately 2.5 carbonyl carbons per 100 (ΔS/S₀ of 2% + 2.5% = 4.5%). The cell walls from the biofilm samples have an additional contribution to the N{C} ΔS/S₀ which will be detailed in Section 3.4.

Figure 2.

Mature biofilm (48-hour growth) cell walls of S. aureus (SA113) labeled by [¹⁵N]glycine plus l-[1-¹³C]threonine examined by N{C} (left) and C{N} (right) REDOR NMR after 16 rotor periods of dipolar evolution (2.24 ms). Full-echo spectra are shown at the bottom of the figure and REDOR differences (ΔS) at the top. The reference for the nitrogen chemical-shift scale was solid ammonium sulfate. (For a scale based on an external liquid ammonia reference, add 20 ppm.) The reference for the carbon chemical-shift scale was external TMS. Abbreviation: ssb, spinning sideband.

Table 1. Labeling of cell walls by [¹⁵N]Gly + l-[1-¹³C]Thr.

	Planktonic (mid-exponential)	Early Biofilm (12-hour growth)	Mature Biofilm (48-hour growth)
Amide N{C} ΔS/S0	0.048 (exp) 0.045 (model)	0.053 (exp) 0.055 (model)	0.051 (exp) 0.055 (model)
Amine to amide 15N integrated peak intensity (open bridges)	0.038	0.032	0.039
Glycyl C{N} ΔS/S0 (15N-isotopic enrichment)	0.84	0.78	0.72
Carbonyl to glycyl methylene C{N} ΔS/S0	3.2 (exp) 3.5 (model)	3.8 (exp) 4.5 (model)	4.3 (exp) 4.5 (model)

3.2. Open bridges

The full-echo ¹⁵N S₀ for the isolated cell walls of S. aureus (SA113) mature biofilm labeled by [¹⁵N]glycine plus l-[1-¹³C]threonine shows a minor amine-nitrogen peak (Figure 2, bottom left). (S. aureus strain SA113 is ATCC 35556, a restriction-deficient mutant of NCTC 8325, which exhibits strong biofilm-forming tendencies in the presence of excess glucose.) This peak arises from peptidoglycan pentaglycyl bridges that are not cross-linked [19]. The integrated intensity of the amine peak to that of the amide-nitrogen peak (corrected for cross-polarization dynamics) is just under 4% and is constant for cell walls isolated from planktonic and biofilm samples (Table 1, row 2). This means that for every 100 nitrogens in pentaglycine bridges (20 bridges), 16 are involved in cross-links between peptidoglycan peptide stems and 4 are open, or uncross-linked (Figure 1, bottom). Stem cross-linking may be somewhat lower than 80% (16/20) because stems without bridges are possible [19].

3.3. ¹⁵N isotopic enrichment

The one-bond 16-T_r C{N} REDOR ΔS/S₀ for the ¹³C NMR of cell walls isolated either from planktonic whole cells or biofilms labeled by [¹⁵N]glycine+l-[1-¹³C]threonine is dominated by a peptide carbonyl-carbon peak near 170 ppm (and its spinning sidebands) and a smaller methylene-carbon peak near 42 ppm (Figure 2, right). Both ΔS and S₀ methylene-carbon peaks are resolved and arise exclusively from natural-abundance ¹³C. Their ratio therefore determines the ¹⁵N isotopic enrichment which is 84% for the planktonic whole-cell sample; the enrichment decreases with the age of the biofilm samples (Table 1, row 3). We attribute the decrease in enrichment for the biofilm samples to de novo synthesis of glycine by S. aureus as the supply of exogenous labeled glycine decreases [20].

3.4. Threonine-glycine cell-wall linkages

The ratio of the one-bond C{N} carbonyl-carbon ΔS to that of the glycyl methylene-carbon ΔS for cell walls of planktonic cells is 3.2 (Table 1, row 4, first entry). Because the cell-wall isolation removes all cytoplasmic and membrane-associated proteins, the only contributions to this ratio arise from peptidoglycan bridges. We therefore expect a ratio of (1.0+2.5) to 1.0 when we take into account natural-abundance (1.0) and labeled carbonyl-carbon ¹³C (2.5 from the threonine leakage) per 20 bridges, compared to just natural-abundance ¹³C for the CH₂ carbons (1.0). This ratio increases significantly for the biofilm samples, to 3.8 for the early biofilm, and 4.3 for the mature biofilm (Table 1, row 4, second and third entries). The increase occurs because of accumulating sortase-generated TG cell-wall linkages that survive the isolation and digestion procedures even though most of the surface protein itself is lost. The observed mature biofilm carbonyl-to-glycyl-methylene-carbon C{N} ΔS values that reach 4.3 (Table 1, row 4) indicate that 1 TG link (4.3 minus 3.2) is created per 20 bridges so that the expected one-bond C{N} carbonyl-carbon ΔS to that of the glycyl methylene-carbon ΔS for cell walls of mature biofilms is (1.0+2.5+1.0) to 1.0, in reasonable agreement with the 4.3 value that is observed. Thus, one of the four open bridges (per 20 bridges) has been used to attach sortase-A-mediated surface proteins (Figure 1, bottom). The ¹³C-¹⁵N TG link concentration of 1 per 20 bridges in biofilms also increases the expected amide N{C} ΔS/S₀ from 0.045 (Section 3.1) to 0.055 (Table 1, row 1).

3.5. Comparisons of whole-cell and cell-wall labeling using C{N} REDOR double differences

About half of the glycine residues in whole cells of S. aureus are in the cell-wall peptidoglycan bridges and half in the cytoplasm [25-27]. Thus, the planktonic whole-cell carbonyl-carbon C{N} ΔS (Figure 3, bottom left) arises from the peptidoglycan ΔS (3.5 intensity units per 20 bridges which includes label and natural-abundance ¹³C) and membrane-associated surface protein (assumed for now to be 1.0 unit per 20 bridges), and from cytoplasmic glycine (G) or threonine (T) ¹³C directly bonded to glycyl ¹⁵N (Figure 1, bottom). If we assume the frequency of occurrence of the cytoplasmic G or T residues bonded to glycyl ¹⁵N is the same (approximately 4%), then the cytoplasmic C{N} ΔS is dominated by TG linkages because the isotopic enrichment of the threonine carbonyl-¹³C is 99% while that of the glycine carbonyl-¹³C is just 3.5% (including leakage of threonine label to glycine). A cytoplasmic ΔS of about 4 units (per 20 bridges or 100 carbonyl carbons) will result in a total whole-cell planktonic ΔS of 8.5 units (3.5+1.0+4.0), which is a sum of peptidoglycan ΔS (3.5), surface protein (1.0), and cytoplasmic ΔS (4.0). The expected planktonic whole-cell ΔS minus planktonic cell-wall ΔS scaled by the planktonic whole-cell ΔS is therefore (8.5 – 3.5)/8.5= 0.59. The experimental ΔΔS/ΔS value is close at 0.47 (Figure 3, top left, and Table 2, row 1). If there is no membrane-associated surface protein in the planktonic whole cells, the expected ΔΔS/ΔS value is (7.5 - 3.5)/7.5=0.53, a slightly better match to experiment. However, the improvement is small and is not sufficient to conclude that there is no membrane-associated surface protein in the planktonic whole cells. The decision between the presence or absence of membrane-associated surface protein in planktonic whole cells requires the use of another REDOR double difference as discussed in the next section.

Figure 3.

Comparisons of whole-cell and cell-wall labeling of S. aureus by [¹⁵N]glycine plus l-[1-¹³C]threonine using C{N} REDOR double differences (ΔΔS) in which the glycyl methylene carbon ΔS is zeroed by the differencing (see graphical abstract).

Table 2. Comparisons of whole-cell and cell-wall labeling by [¹⁵N]Gly + l-[1-¹³C]Thr using carbonyl-carbon C{N} REDOR double differences.

Comparison	ΔΔS/ΔS
planktonic whole-cell ΔS minus planktonic cell-wall ΔS scaled by planktonic whole-cell ΔS	0.47 (exp) 0.59 (model)
planktonic whole-cell ΔS minus 48-hour biofilm whole-cell ΔS scaled by planktonic whole-cell ΔS	0.18 (exp) 0.12 (model)
48-hour biofilm cell-wall ΔS minus planktonic cell-wall ΔS scaled by planktonic cell-wall ΔS	0.33 (exp) 0.29 (model)

3.6. Schematic model for surface proteins in biofilm formation and REDOR double differences

We show the labeling numbers from the previous two sections in Figure 4 where the top row has scanning electron micrographs of planktonic whole cells, an early biofilm with clear signs of cell-cell aggregation, and a later biofilm with aggregated cells embedded in an extracellular matrix. The bottom row has a model to account for changes in surface proteins during biofilm formation. The surface proteins are membrane associated in the planktonic cells, peptidoglycan bound but not fully deployed in the early-stage biofilm, and peptidoglycan bound and fully deployed in the mature biofilm. The mature biofilm has an enhanced β-strand content and a reduced α-helix content, presumably associated with extracellular leucine-rich regions in cell-cell contacts [14]. The enhanced β-strand determination was made using the α- and β-leucine C-2 carbon chemical shifts (Figure 5), which are separated by about 5 ppm [28].

Figure 4.

Scanning electron micrographs of S. aureus planktonic whole cells and biofilms (top) with a schematic accounting of the state of the surface proteins (bottom). The numbers in parentheses give the counts of directly bonded double-labeled ¹³C-¹⁵N pairs per 100 peptidoglycan bridge carbonyl carbons. (See text for details.)

Figure 5.

Cross-polarization magic-angle spinning ¹³C NMR of whole-cell biofilms of S. aureus labeled by l-[2-¹³C,¹⁵N]leucine with (top) and without (bottom) interrupted proton decoupling. The leucine 2-¹³C label appears in both α-helix and β-strand protein conformations. The sharp peaks near 30 ppm (which largely survive the interrupted decoupling) show that some of the leucine label has been metabolized to acetyl-co-enzyme-A and inserted into lipids. The spectra have been scaled by equal carbonyl-carbon peak intensities (dotted lines).

The cytoplasmic content of the biofilms is assumed to be reduced slightly compared to the planktonic whole-cell value. The numbers in parentheses in Figure 4 give the count of double-labeled ¹³C-¹⁵N direct bonds (per 20 bridges) for each stage of the model and therefore all the C{N} REDOR double differences of Figure 3. (The natural-abundace-¹³C-based contributions are not included in these numbers.) The ΔΔS comparison (planktonic whole cells minus mature biofilm whole cells) scaled by the planktonic whole-cell ΔS is (8.5 – 7.5)/8.5=0.12 for the model and 0.18 observed (Figure 3, left, and Table 2, row 2). A non-zero double difference requires that there be a contribution to the planktonic ΔS from membrane-associated surface protein in the planktonic whole cells. Because all carbonyl-carbon ΔS values were obtained by zeroing the methylene-carbon ΔS, the difference in ¹⁵N isotopic enrichment for planktonic whole cells and mature biofilm whole cells has been taken into account.

The ΔΔS comparison (mature biofilm cell-wall ΔS minus planktonic cell-wall ΔS) scaled by the planktonic cell-wall ΔS is (4.5 – 3.5)/3.5=0.29 for the model and 0.33 observed (Figure 3, right, and Table 2, row 3). Uncertainties in the cytoplasmic content of the biofilms, and in the extent of membrane anchoring of the surface proteins in planktonic whole cells are absent in this important cell-wall double difference. The ΔΔS of one unit (per 20 bridges) is consistent with the determination (based on the carbonyl-to-glycyl-methylene ΔS's) that one of the four open bridges has been used to attach a surface protein in biofilms (Section 3.4). The experimental double difference also shows a 2-ppm shift to low field for ¹³C-¹⁵N pairs in TG linkages compared to those in GG linkages (Figure 3, right, dotted line). This double difference is the first direct detection (with molecular resolution) of peptidoglycan-bound TG linkers to surface proteins.

3.7. Leucine-rich β-strand proximity to tyrosine residues in biofilms

We used ¹³C-¹³C spin diffusion to determine nearest neighbors of the β-strand leucine ¹³C label (Figure 6). After cross-polarization, carbon magnetization is stored along the z-axis by a 90° pulse. The β-strand peak is then selectively inverted and the ¹³C label allowed to couple to nearby natural-abundance ¹³Cs by ¹³C-¹³C spin diffusion during mixing times unaided by coupling to adjacent protons [21]. For mixing times of 200 ms, a range of 2.5 Å is possible, which connects the 2-¹³C leucine label to other carbons in the leucine residue (Figure 6, bottom right). For mixing times of 800 ms, spin diffusion couples the 2-¹³C leucine label to more distant carbons, including aromatic carbons (128 ppm, Figure 6, top right insert). There is usually no phenylalanine aromatic-carbon peak below 130 ppm [29] and the occurrence of tryptophan residues in proteins is small. We therefore tentatively assign the 128-ppm aromatic-carbon peak to tyrosines, which have three ring carbons (γ, δ, δ) with shifts close to 128 ppm [29].

Figure 6.

(Left) Cross-polarization magic-angle spinning ¹³C NMR of a whole-cell mature biofilm (60-hour growth) of S. aureus labeled by l-[2-¹³C,¹⁵N]leucine with z-axis storage and DANTE frequency-selected inversion of the leucine β-strand ¹³C label followed by a mixing period for ¹³C-¹³C spin diffusion (without proton decoupling) of 800 ms. The full-echo spectrum (S₀) is shown at the bottom of the figure, the spectrum with the DANTE inversion (S) in the middle, and the DANTE difference (ΔS = S₀ – S) at the top. Magic-angle spinning was at 6250 Hz. (Right) DANTE difference spectra (×64) after mixing periods of 800 ms (top) and 200 ms (bottom). The spectra have been scaled for equal-intensity leucine carbonyl-carbon peaks (horizontal dotted lines). The insets reveal an aromatic-carbon peak (128 ppm) assigned to tyrosine residues within 5 Å of the leucine β-strand ¹³C label. Abbreviation: ssb, spinning sideband.

3.8. Surface protein mediated biofilm

REDOR NMR analyses of S. aureus whole cells and cell walls have established that 20% of the pentaglycine bridges in cell walls are potential sortase-mediated surface-protein attachment sites. In mature biofilms, approximately one-fourth of these sites have a surface protein attached. That is, for every 20 peptidoglycan-repeat units, one unit has a surface protein attached. Because the cell wall of S. aureus has approximately 20 layers of peptidoglycan, one surface protein per 20 peptidoglycan-repeat units translates into an almost complete cell-wall surface decoration in biofilms by covalently attached proteins (Figure 7, right). That is, there is one surface protein for every surface peptidoglycan repeat unit.

Figure 7.

Schematic representation of the peptidoglycan of S. aureus in a planktonic state (left) and in a mature biofilm (right). The concentrations of open (yellow) and closed (black) pentaglycine bridges and attached surface proteins (multi-colors) reflect the measurements summarized in Tables 1 and 2.

In S. aureus, 21 surface proteins that have a LPXTG targeting signal for sortase-A-mediated cell-wall attachment have been identified by genomic analysis [30]. Of these, 11 are known proteins, mostly adhesins involved in attachment to biotic and abiotic surfaces, cell clumping, endocytosis bacterial uptake, and biofilm formation [31]. The fibronectin-binding proteins (FnBPs) have been recently identified as key cell-wall attached proteins required for the formation of biofilms [32]. Attenuation of FnBPs expression significantly reduces S. aureus biofilm formation on implanted catheters [33]. The deletion of ΔfnbA and ΔfnbB (FnBPs) in S. aureus USA300, a community-acquired methicillin-resistant S. aureus, prevented the formation of biofilm [34].

The majority of cell-wall anchored proteins have high concentrations of β-sheet structures. In the case of FnBPs, 10-11 tandem repeats of a fibronectin-binding domain are rich in β sheets [35]. Our observations of increased surface-protein attachment to peptidoglycan in biofilms (Figure 3), accompanied by an increase in concentration of β-strands in leucine-rich regions (Figure 5), and their proximity to tyrosines and potential O-sulfated binding sites (Figure 6), provide direct experimental molecular parameters associated with sortase-mediated biofilm formation in S. aureus.

Highlights.

• Staphylococcus aureus cell-wall composition has been characterized by solid-state NMR for planktonic cells, young biofilms, and mature biofilms specifically ¹³C and ¹⁵N labeled.

• 20% of cell-wall peptidoglycan pentaglycyl bridges are not cross-linked in planktonic cells and so are potential sortase-mediated surface-protein attachment sites.

• One-fourth of these sites have a surface protein attached in mature biofilms.

• The concentration of β-strands in leucine-rich regions doubles in the transition from planktonic cell to mature biofilm.

• In a mature biofilm, surface proteins attach to each layer of peptidoglycan so that there is one surface protein covalently attached for each surface peptidoglycan repeat unit.

Abbreviations

ESM

enhanced standard medium

REDOR

rotational-echo double-resonance

C{N}

¹³C observe and ¹⁵N dephase

N{C}

¹⁵N observe and ¹³C dephase

S₀

full-echo REDOR signal

S

dephased REDOR signal

ΔS (S₀ – S)

REDOR difference

DANTE

delay and nutation tailored excitation