Structural and functional diversity in Listeria cell wall teichoic acids

Wall teichoic acids (WTAs) are the most abundant glycopolymers found on the cell wall of many Gram-positive bacteria, whose diverse surface structures play key roles in multiple biological processes. Despite recent technological advances in glycan analysis, structural elucidation of WTAs remains challenging due to their complex nature. Here, we employed a combination of ultra-performance liquid chromatography-coupled electrospray ionization tandem-MS/MS and NMR to determine the structural complexity of WTAs from Listeria species. We unveiled more than 10 different types of WTA polymers that vary in their linkage and repeating units. Disparity in GlcNAc to ribitol connectivity, as well as variable O-acetylation and glycosylation of GlcNAc contribute to the structural diversity of WTAs. Notably, SPR analysis indicated that constitution of WTA determines the recognition by bacteriophage endolysins. Collectively, these findings provide detailed insight into Listeria cell wall-associated carbohydrates, and will guide further studies on the structure-function relationship of WTAs.

1-P-(O3] n ( Fig. 1B: only C4 connection to Rbo shown) as the repeating unit, which incorporate the GlcNAc moiety within the polymer chain. The integrated GlcNAc itself may be further decorated with glucose (Glc), galactose (Gal), or with a second GlcNAc residue (19). In contrast, little is known about the linkage unit. To date, only one strain (belonging to type I) has been studied, and it features [4)-Glc-(␤133)-Glc-(␤131)-Gro-3-P-(O34)-ManNAc-(␤134)-GlcNAc-1-P-(O36)-MurNAc] as the linkage unit ( Fig. 1C) (20). The potentially different linkage unit structures from other Listeria serovars remain unsolved. In addition, several structures and modifications from WTA repeating units of the less frequently occurring serovars within the genus Listeria have not been established. As the correlation between WTAs and serovar is clear and their important biological functions are beginning to emerge (5), a comprehensive view of the diverse WTA structures will further strengthen our knowledge of the Listeria cell wall, beyond what has been defined by serotyping alone.

Diversity in Listeria cell wall teichoic acids
approaches also allow for structural elucidation of complex carbohydrates, whereas maintaining a high level of sensitivity, accuracy, acquisition speed, and tolerance for heterogeneous analytes (21). We previously reported the application of electrospray ionization MS for rapid analysis of carbohydrate compositions of Listeria WTAs (22). However, this method is unable to discriminate between diastereoisomeric monosaccharides (e.g. galactose versus glucose substitution), which commonly exist in type II WTAs. Toward this end, we have developed a novel hydrophilic interaction ultra-performance liquid chromatography-coupled electrospray ionization tandem mass spectrometry (UPLC-MS/MS) method, for detailed analysis of WTA structures. Given that certain serovars are responsible for most cases of human listeriosis, the structures we elucidate here are likely to represent secondary pathogenicity factors, rendering them not only interesting subjects for biochemical studies, but also exposing them as possible targets for vaccines, diagnostics, and virulence-related antimicrobial therapy (23). In this light, we also investigate the binding specificity of cell wall-binding domains (CBDs) from Listeria bacterio-phage endolysins. As these molecules can recognize and bind to the Listeria cell wall in a serovar-dependent manner (24,25), and have been proposed as novel tools for diagnostics and biocontrol (26,27), we evaluated the specificity and binding kinetics of certain CBDs for Listeria WTAs.

Validating UPLC-MS/MS for structural determination of type I Listeria WTAs
First, purified (supplemental Fig. S1, A and B) and HF-depolymerized (supplemental Fig. S1C) type I WTAs (including SV 1/2a, 3, and 7) were analyzed to validate our methodology, as their structures are already known from previous NMR studies (18). We identified repeating units with their individual carbohydrate constituents based on retention time and mass detection of each peak ( Fig. 2A)  Diversity in Listeria cell wall teichoic acids significant peak of m/z 415 (8.01 min) indicates a species that consists of glucose-glucose-Gro, in agreement with a previous report (20). Fragmentation (MS/MS) of m/z 415 identifies the Glc(133)Glc connectivity due to the lack of cross-ring fragments (Fig. 2B). Interestingly, we found this linkage also exists in the infrequent serovars 3a and 7, representing a unique marker for type I WTAs (Fig. 2C). The other portion of the linkage unit, ManNAc-GlcNAc, could not be detected in the chromatogram, likely due to the acid treatment during WTA extractions from the cell wall. Nevertheless, we do not anticipate any change as this disaccharide unit is known to be highly conserved within the Listeria genus (5). In SV 3a, a dominant peak m/z 354 (6.88 min) reveals GlcNAc-Rbo as the major component of the repeating unit ( Fig. 2A). This composition was confirmed by NMR spectroscopic analysis of the WTA polymer prior to depolymerization. Its 1 H spectrum is shown in supplemental Fig. S2A. Unexpectedly in SV 7, a small percentage of Rbo (about 8%) was found to be substituted with a hexose residue, corresponding to the peak m/z 313. The peak m/z 262 suggests a species of hexosamine with two acetyl groups. However, these two species could not be detected by NMR analysis of corresponding WTA polymers (supplemental Fig. S2B).

Structural analysis of type II WTAs from a Listeria SV 4b strain by UPLC-MS/MS
Next, we analyzed the WTAs from the laboratory serovar 4b strain WSLC 1042, the serovar representing the majority of listeriosis outbreaks. The chromatogram suggests that the WTA of 4b is comprised of 8 carbohydrate species with variable retention times that differ by mass (supplemental Fig. S3A) or structure (Fig. 3A). Because the NMR structure of 4b WTA has been previously determined (18), we were able to deduce the 8 individual species' compositions by mass comparison, as well as their monosaccharide connectivity by MS/MS (Fig. 3B). The first two species identified were m/z 354 (7.08 min) and m/z 678 (13.88 min), as their masses correspond to GlcNAc-Rbo and [Gal][Glc]-GlcNAc-Rbo, respectively. Two m/z 516 species were distinguished by their fragmentation patterns. The one eluting at 9.93 min suggests a hexose(133)GlcNAc linkage (diagnostic m/z 202 fragment) (28) in contrast to a (136)-linkage for the peak at 11.19 min (diagnostic m/z 281 and 221 fragments) (29). In agreement with previous reports, the above two species are denoted as Glc(133)GlcNAc-Rbo and Gal(136)GlcNAc-Rbo for the peaks at 9.93 and 11.19 min, respectively. In addition to the glycosylation, two O-acetylated species, m/z 396 (4.05 min) and m/z 558 (7.43 min) were detected. Further MS/MS fragmentation of m/z 396 revealed that the aminosugar bears two acetyl (Ac) groups, one on the nitrogen (N-acetyl) and one on an oxygen (O-acetyl). We observed a fragment with m/z 262 corresponding to Ac-GlcNAc, indicating the Ac group is located on the GlcNAc, and not on the Rbo. This is supported by the fragment with m/z 151 (Rbo), and lack of a signal with m/z 193 (Ac-Rbo). Hence, we infer m/z 396 to be Ac-GlcNAc-Rbo. MS/MS of m/z 558 shows a strong presence of the deacetylated fragment m/z 516 and its corresponding cross-ring fragments in line with the Gal(136)-GlcNAc-Rbo linkage, and is thus denoted as [Gal(136)][3-OAc]-GlcNAc-Rbo. We propose that the O-acetyl group is at C3 as it is the only open position on GlcNAc (C4 is occupied by the phosphodiester bond linking together the WTA polymer). Assuming a conserved O-acetyl position, the mentioned m/z 396 fragment is therefore proposed to be O-acetylated at C3 as well. By comparing the area under the peaks denoting the species with and without O-acetylation (supplemental Fig. S3B), the relative degree of secondary O-acetylation in 4b WTAs was determined to be about 26% (see "Experimental procedures" for Equation 1). In summary, the updated WTA structure of the linkage and repeating units of 4b is shown in Fig. 3C.
Interestingly, a pair of glycerol (Gro)-related species, m/z 294 (5.32 min) and m/z 456 (7.94 min) were detected and identified as the linkage unit (Fig. 3A). MS/MS (Fig. 3B) shows that m/z 294 reflects a GlcNAc-Gro composition, whereas the fragmentation pattern of m/z 456 indicates a Glc(133)GlcNAc-Gro connectivity, representing a different type of linkage unit compared with type I WTAs.

Confirmation of connectivity and O-acetylation by NMR spectroscopy
In a parallel approach, a series of NMR experiments including DQF-COSY, 13 C-HSQC, 13 C-HMBC, and 1 H-31 P-COSY ( Fig. 4) were employed to determine the composition and connectivity of intact WTA polymers of SV 4b, and to rule out any possible confounding by-products resulting from HF digestion. The HP-COSY spectrum of the polymer is consistent with the existence of a phosphodiester linkage from C1Ј of Rbo to C4Ј of GlcNAc as determined previously for the monomeric units. Cross-peaks in the HSQC spectrum could be assigned to three differently decorated repeating units of the WTA sample (Table 1). H3Ј in the variants with the O-acetylated GlcNAc shows a distinct downfield shift to 5.10 ppm. Integration of the respective peaks confirmed that the degree of O-acetylation is similar to what UPLC-MS demonstrated earlier. NMR experiments corroborate that WTA O-acetylation and glucosylation occur at the same C3-OH of GlcNAc and are mutually exclusive. The configuration of the anomeric protons was confirmed by analysis of 3 J H1Ј-H2Ј coupling constants (Table 2). For the ␤-configuration, a value around 8 Hz is expected, whereas for the ␣-configuration it is about 3 Hz. In agreement with previous reports (17,18), the configuration of the anomeric center is ␤ for GlcNAc and Glc and ␣ for Gal. Due to the low abundance within the overall polymer it was not possible to identify signals of any Gro-related subunits.

Structural diversity and distinct modifications of type II Listeria WTAs
Various type I and II WTA polymers were analyzed by native PAGE (separates by charge, size, and shape), followed by Alcian blue staining (supplemental Fig. S4). Our results demonstrate that there are clear differences in mass of the WTA polymers among different SVs, likely due to differences in decoration, but also overall polymer chain length (or number of repeating units n), for which there is some variation among different strains (18). Using the chromatogram of 4b WTAs as a reference, we next sought to investigate the correlation between the WTA structure and serovar (Fig. 5A). The chromatogram of 4a

Diversity in Listeria cell wall teichoic acids
reveals a peak with m/z 354 (6.87 min) that elutes differently from 4b (7.08 min), suggesting two diastereoisomers. Despite the presence of two stereogenic centers, ribitol itself is an achiral meso compound due to the internal plane of symmetry through C3. However, substitution (at C2 or C4) breaks this symmetry, resulting in a diastereomorphic relationship between the m/z 354 species in 4a and 4b. In UPLC-MS analy-sis, the different arrangement of the two disaccharides results in a minor shift (ϳ12 s) in retention time. In agreement with previous work, NMR analysis (supplemental Fig. S5A) confirmed that the difference lies in the connectivity between GlcNAc and Rbo: GlcNAc(␤132)-Rbo in 4a and GlcNAc(␤134)-Rbo in 4b, whereas the linkage between GlcNAc and the phosphate stays the same for both serovars. The other two dominant peaks m/z

Diversity in Listeria cell wall teichoic acids
Ac-GlcNAc-Rbo and GlcNAc-Gro, respectively. Hence, the comprehensive WTA structure of the 4a repeating unit is denoted as: the linkage unit as GlcNAc-(133)-Gro-P.
The complete WTA structures of SV 5 and 6b were determined for the first time. Their chromatograms (Fig. 5A) and NMR spectra (supplemental Fig. S5, B and C) are almost identical and are indicative of a composition similar to 4d, but with a C2-connectivity like in 4a, suggesting that other determinants differentiate SV 5 and 6b, 4d and 4e. SV 6a was found to possess three new peaks with m/z 336 (9.  Fig. 5B and Table 3.

The structure of Listeria WTAs determines the recognition specificity of bacteriophage endolysins
The structure of Listeria WTAs are known to be recognized as binding ligands by bacteriophage tail fiber proteins and the encoded endolysins (11) (30). Endolysins are peptidoglycan hydrolases that function to degrade the bacterial cell wall from within, to release progeny virions at the end of the phage lytic cycle. As bacteriolytic enzymes, they can also lyse susceptible Gram-positive bacteria from the outside. These enzymes are typically composed of an N-terminal catalytic domain responsible for proteolytic cleavage of the PG, and a C-terminal CBD that confers target specificity by recognizing and binding to diverse carbohydrate epitopes on the bacterial cell wall, such as WTAs (31). To elucidate the structural specificity of CBDs for WTA, the binding interactions of three fluorescently-labeled Listeria-specific endolysin CBDs (CBDP35, CBD500, and CBD025) were investigated due to the correlation of their binding patterns with serovar (Fig. 6A). Quantitative surface plasmon resonance analysis demonstrated that CBDs indeed possess discriminative binding specificity for purified WTAs (Fig.  6B). CBDP35 strongly binds to WTA units substituted with an undecorated GlcNAc residue (SVs 1/2a, 3a, and 4a); and only weakly to the other subtypes of SV 4 and SV 5. In contrast, CBD500 recognizes WTAs equipped with O-acetylated GlcNAc (SVs 4 and 5) even when they are glycosylated by different monosaccharides such as glucose and/or galactose. CBD025 exclusively recognizes GlcNAc bound via the C2 position of the ribitol backbone (SV 4a, 4c, 5, and 6b), but not C4-bound GlcNAc, such as in SVs 4b, 4d, and 6a (data not shown). Furthermore, we demonstrated that CBDs interact with the WTA partner in a dose-dependent manner (supplemental Fig. S7), and found that the binding affinity generally lies in the submicromolar range (Table 4). In summary, we show that CBDs recognize the Listeria cell surface based on the specific constitution of their WTA ligands. Together, this demonstrates both the extreme sensitivity and specificity with which endolysins recognize and bind to their ligands, and promotes their use for rapidly probing select WTA structures.

Discussion
The cell wall of most Gram-positive bacteria contains two major complex glycopolymers: peptidoglycan and teichoic  13

C, and 31 P chemical shifts in Lmo WSLC 1042 WTA in D 2 O (in ppm)
Assignment of the two minor sets of GlcNAc signals to B2 and B3, respectively, is based on the position of the 5Ј cross-peak in the HSQC spectrum, which is expected to be significantly shifted in the absence of Gal. The abbreviations used are: GlcNAc, N-acetylglucosamine; OAc, O-acetyl group; Glc, glucose.     GlcNAc-glycerol a a, the incorporated GlcNAc refers to a GlcNAc residue that is integrated into polyribitol-phosphate while forming the repeating unit and b, pendant GlcNAc refers to a GlcNAc residue without any substitution.

Diversity in Listeria cell wall teichoic acids acids (including both wall teichoic acids and lipoteichoic acids).
A recent report has demonstrated the usefulness of ultra-performance liquid chromatography to uncover the significant complexity and variability in the composition and structure of peptidoglycan (32). To better understand the intricacies of the Gram-positive cell wall, we focus on the WTAs of Listeria, which are structurally heterogeneous, polydisperse, and highly negatively charged mixtures of glycan polymers. Similar to peptidoglycan, WTAs play important roles in maintaining the integrity of the bacterial cell, and contribute to various processes during growth and division. It has been recently suggested that the absence of WTA or loss of its compositional identity or integrity leads to attenuated virulence of Lmo (16,17), demonstrating that virulence is also dependent upon maintenance of a specific WTA structure. This hypothesis is in congruence with the understanding that WTAs are the major determinants of the antigenic pattern known as serovar, a phenotypic identifier for strain diversity and an indicator of virulence. Given that subtle constitutional and configurational differences are known to produce molecules with very different biological activities, and that WTA repeating units can be glycosylated by different isomeric monosaccharides, full structural elucidation is warranted. MS/MS has previously been used to discriminate between constitutional variants based on patterns of diagnostic fragment ions (33). Similarly, identification of isomeric monosaccharides and their mode of attachment has been traditionally determined by GC-MS, following methylation, hydrolysis, and derivatization of purified target molecules (34). However, this does not permit clarification of the sequence order, nor the linkage configuration of the monosaccharides within the oligosaccharide. To elucidate the WTA diversity within the genus Listeria, we describe a novel bioanalytical approach that allows for analysis of both compositional and structural features of WTA polymers. We show that UPLC-MS/MS can be employed with an accuracy on par with NMR, requiring lower sample amount and less time, whereas maintaining excellent reproducibility. Additionally, we have compiled a database (supplemental Table S1) with the masses of [M-H] Ϫ for all identified WTA fragments. This database can be further developed as a diagnostic toolbox for probing the surface chemistry of unknown Listeria strains, providing valuable information on serovar and associated pathogenicity or virulence of a given strain or isolate. This could be especially relevant, given that the most pathogenic serovars 1/2 and 4b harbor unique and characteristic WTA structures and glycosidic substitutions.
Previous efforts primarily focused on identification of the WTA repeating unit (18,22). However, our findings described here also allow for elucidation of the linkage unit structure. For the first time, we demonstrate that two types of linkage units exist in Listeria WTA: Glc-Glc-Gro in type I (SVs 1/2, 3, and 7), and [Glc] x GlcNAc-Gro (x ϭ 0 or 1) in type II (SVs 4, 5, and 6). In theory, these linkage units should be denoted as [Hex] x Hex-NAc-Gro (x ϭ 0 or 1), because our UPLC-MS/MS could not discriminate between monosaccharide isomers. However, we are confident in our assumption because Glc-Gro has been reported to be the linkage unit of type I WTA (20), and GlcNAc is the sole constituent utilized within all type II WTAs. It would be unlikely (and inefficient) to utilize a different substrate (e.g. GalNAc or ManNAc) for WTA synthesis. Nonetheless, the presence of HexNAc (or GlcNAc as we speculate) represents a novel linkage type that significantly differs from the other type previously known. In addition, structural modification of the linkage unit (through glycosylation and acetylation) can further diversify the overall composition of the WTA molecule.
Our methodology also revealed that O-acetylation of the incorporated GlcNAc in the repeating unit is a unique modification of all type II WTAs (SVs 4, 5, and 6). Interestingly, O-acetylation, along with N-acetylation have been found to be involved in numerous biological functions in Gram-positive bacteria, including the ability to confer intrinsic resistance to lysozyme through O-acetylation of MurNAc in the peptidoglycan backbone (35). Additionally, O-acetylation of GlcNAc in PG of Lmo may confer resistance to antimicrobial compounds and limit innate immune responses (36). Although O-acetylation on MurNAc of PG is a widely known modification in Gram-positive bacteria, O-acetylation of WTAs has only recently been reported (17). Here, we were able to demonstrate that (i) this modification only occurs in type II WTAs, and (ii) that the modification is not ubiquitous, but only affects a fraction (20% to 40%) of the WTA repeating units. The two classes of Listeria WTAs also fundamentally differ in the anomeric configuration of their repeating GlcNAc unit. The UPLC-MS/MS method is unable to discern the configuration of the GlcNAc-Rbo linkage, as both m/z 354 in 3a (shown as ␣-GlcNAc: Fig. 2A) and m/z 354 in 4a elute at the same retention time (ϳ6.88 min: Fig. 5A). However, NMR analysis clearly revealed that the configuration of GlcNAc differs between two types, e.g. pendant ␣-GlcNAc in type I and backbone-integrated ␤-GlcNAc in type II.
Despite existing knowledge on the genetic background of WTA biosynthesis in Listeria (14,17,37,38), very little is known about the mechanism of the biochemical synthesis pathway. Previous studies on Staphylococcus and Bacillus may be relevant, as these genera also utilize polyribitolphosphate as the WTA repeating unit. However, their modification patterns are

Diversity in Listeria cell wall teichoic acids
different, with Staphylococcus featuring ␣-GlcNAc, ␤-GlcNAc, and D-alanine (8), and Bacillus containing ␣-glucose and D-alanine (39). Based on the aforementioned data, Listeria appears to possess specific enzymes to recognize (␣-or ␤-linked) GlcNAc to produce the corresponding type of WTAs, which might be functionally similar to the glycosyltransferase TarM of Staphylococus (40). Given the extreme structural diversity of WTAs, even within a single bacterial species, understanding the intricacies of the biochemical synthesis will certainly aid in understanding differences in structure, and their biological roles. On this note, we have here determined the WTA structures of SV 4c, 5, 6a, and 6b, whose WTA compositions and constitutions have not been previously described. Interestingly, we find that 4c shares the same repeating unit structure as a previously reported 4-"non-b" strain (17), suggesting that they probably represent the same serovar. We also reveal that SV 7 appears to possess a unique hexose substitution, unlike the GlcNAc residue in all type I and II WTAs. Overall, this new profile of WTA structures expands our knowledge about the diversity of Listeria WTAs, and further strengthens the already established theory that WTA structure is the basis for O-antigen diversity. Further research will focus on elucidating the biochemical pathways involved in synthesis of Listeria WTAs, to better understand the source of this structural diversity. For the first time, we report the binding specificity and affinity of Listeria endolysins to purified WTA polymers, which have previously been shown to have a nanomolar affinity only to whole cells (25). We found that the interaction between CBD and WTA is purely dependent upon electrostatic forces, as 1 M NaCl alone was able to regenerate the functionalized SPR chip surface after each measurement. Taken together, these data provide key information for further understanding the molecular mechanisms underlying the WTA-endolysin interactions, supporting the potential application of these CBDs for detection and characterization of Listeria.

Bacterial strains and growth conditions
Listeria strains were routinely grown in brain heart infusion medium, with constant shaking at 30°C. All strains used in this study are listed in Table 3.

Extraction and purification of Listeria cell walls and WTAs
Cell walls from Listeria strains were prepared for extraction and purification of WTAs as previously described (24). Purified WTA polymers were depolymerized into monomeric repeating units by hydrolysis of the phosphodiester bonds using 48% hydrofluoric acid for 20 h at 0°C (7). The degraded products were subject to a Superdex 200 size exclusion column (GE Healthcare, Glattbrugg, Switzerland) in distilled water at 25°C (flow rate, 0.4 ml/min). Corresponding fractions (smaller than 1 kDa) were identified by a UV detector at 205 nm, collected, and pooled for dialysis (MWCO 100 -500 Da, Spectra laboratories, Inc.) in distilled water. The WTA monomers were then lyophilized and subjected to UPLC-MS/MS for compositional and structural analysis.

UPLC-MS/MS
A Waters Acquity UPLC system equipped with an Acquity UPLC BEH Amide column (2.1 ϫ 100 mm, 1.7 m) was used, coupled to a Synapt G2 MS system composed of an electrospray ionization (ESI) source and a quadrupole time-of-flight (qTOF) analyzer (Waters Corp., Milford, MA). The MS was used in the negative mode exclusively, and calibrated with a sodium formate solution with leucine-enkephalin (m/z 554.2615) as the lock mass, which was acquired every 60 s during UPLC-MS measurements (correction applied with 5 scans averaged).
Chromatographic conditions-ACN and H 2 O were used as eluents with a flow rate of 0.17 ml/min, both with 0.1% NH 3 additive. To avoid strong solvent effects during injection, mixtures of ACN/H 2 O, 8:2 and 2:8 (v/v), were used as weak and strong needle wash solutions, respectively. The dialyzed WTA monomer materials (see above) were dissolved in 50 l of ACN/ H 2 O, 1:1 (v/v), and 0.1-1 l were injected for the UPLC-MS analysis (partial loop mode; column temperature 35°C). The gradient began with 20% H 2 O and 80% ACN, and was linearly increased to 40% H 2 O over 18 min, followed by a 2-min reequilibration at the initial conditions.
MS conditions-The spectra were acquired in resolution mode. The voltages of capillary, sample cone, and extraction cone were set at 2000, 25, and 4 V, respectively. The desolvation gas flow rate was 850 liters/h at 350°C. The cone gas flow was 20 liters/h and the source temperature 120°C. Full scan mass spectra were acquired from m/z 50 to 1200 with a 0.3-s scan time in centroid mode. Besides the full scan, exact masses were selected to perform MS/MS through collision-induced dissociation. Argon was used as the collision gas, the low mass resolution was set to 15 for a very narrow m/z isolation width of the selected ions, and the transfer collision energies were ramps ranging from 5-15 to 10 -30 V for molecules with m/z around 300 (lower end) and 700 (higher end). All data were collected and processed using MassLynx software, version 4.1 (Waters Corp., Milford, MA) and MS spectra were background-corrected by subtracting the signals between 0 and 1 min of their respective chromatograms. Retention time shifts due to column aging were corrected in the base peak ion chromatograms on the basis of the major peaks from a WTA 1042 (4b) monomer-sample, which was always included in each sequence batch as a positive control (stored at Ϫ20°C between batches; samples analyzed over a time course of several months).
Integration-For extracted ion chromatograms of specific analytes, the corresponding exact masses were selected with a 0.05 Da window. Integration was conducted with enabled smoothing (window size (scans): Ϯ 4; number of smooths: 2; Savitzky Golay smoothing method) and automatic peak detection parameters. The relative degree of O-acetylation was determined by the ratio in Equation 1.

Diversity in Listeria cell wall teichoic acids
tains peaks m/z 396, 558, 354, 516 (two peaks), and 678, which correspond to all identified repeating units in the polymer, both with and without O-acetylation. Potential differences in ionization efficiency of the involved species were ignored for this rough estimation. A dilution series of sample WTA 1042 (4b) confirmed that the relative signal ratios remained constant in the range of relevant signal abundances that were observed for the major species in the various samples (ratios stable up to a signal height of ϳ3e 5 for the major species, or an area of ϳ60,000 (arbitrary units); injection volumes lowered if value exceeded).

NMR spectroscopy
The WTA polymers of WSLC1034, 1485, 1042, 1020, 3009, and 2012 were analyzed by NMR spectroscopy for detailed structural elucidation (41). A series of experiments, including DQF-COSY, 1 H-13 C-HSQC, 1 H-13 C-HMBC, and a 1 H-31 P-COSY (42) were acquired in D 2 O at 25°C. NMR spectra were recorded on a Bruker Avance III HD 600 MHz spectrometer equipped with a Prodigy triple-resonance probe and a Bruker Avance III HD 500MHz spectrometer equipped with a BBFO broadband probe.

Protein purification
Expression and purification of His-tagged CBDP35, CBD500, and CBD025 of respective phage endolysins fused to a green fluorescence protein (GFP) were carried out as previously described (25).

Fluorescence binding assay
The abilities of GFP-tagged CBDs to bind to Listeria cell surface were tested using fluorescence binding assay as previously reported (24). Briefly, Listeria cells from log phase cultures (A 600 nm ϭ ϳ0.5) were harvested by centrifugation (10,000 ϫ g, 1 min), and resuspended in 1/5 volume of PBS (pH 7.4). 100 l of cells were incubated with 5 l of 1 mg/ml of GFP-CBDs and incubated for 5 min at room temperature. The cells were spun down, washed three times, and finally resuspended in PBS buffer. The samples were then subjected to confocal laser scanning microscopy.

Surface plasmon resonance analysis
Sensorgrams of binding of purified Listeria WTA polymers to immobilized CBDs were obtained using surface plasmon resonance (Biacore X, GE Healthcare, Glattbrugg, Switzerland) as previously described (43,44), with slight modifications. First, the carboxymethylated surface of a CMD500L chip (Xantec bioanalytics GmbH, Duesseldorf, Germany) was coated with 0.2 mg/ml of CBDP35 (or CBD500 or CBD025) at a flow rate of 5 l/min; 10 mM sodium acetate, pH 5.0), using the amine coupling procedure according to the manufacturer's manual. To control nonspecific binding, a second flow cell was treated in the same manner, but without immobilization of CBD proteins. For specificity studies, the WTA samples (analyte) isolated from different Listeria strains were flowed through both cells in running buffer (10 mM BisTris, 100 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20, pH 6) at 10 l/min at 25°C. For each strain, association was measured for 180 s and dissociation was mea-sured for 720 s. After each injection, the surface was regenerated with a 45-s injection of regeneration buffer (1 M NaCl, 50 mM Tris, pH 9), at a flow rate of 10 l/min. This cycle was repeated after each measurement. For kinetics studies, the interaction between CBDs and various WTA concentrations (ranging from 0 to 50000 nM of the respective WTAs) were performed at 10 l/min at 25°C. For each concentration, association was measured for 180 s, and dissociation was monitored for 720 s. The surface was then regenerated by regeneration buffer prior to the next measurement. For all curves, the "twostate confirmation change" model gave the best fit and was therefore used for calculation of K A and K D .
Author contributions-Conception and design, acquisition of data, analysis and interpretation of data, drafting or revising the article was performed by Y. S., S. B., E. S., and M. O. E., acquisition of data, analysis, and interpretation of data was performed by B. G., A. J. R., M. R. E., and conception and design, drafting, or revising the article was performed by L. F., L. N., and M. J. L.