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PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.


Introduction
In Gram-positive bacteria, a cell wall outside the plasma membrane determines shape, ensures integrity, and protects the cell from environmental stresses and host immune defense mechanisms. In many streptococci, lactococci, and enterococci, the cell wall consists of multiple layers of peptidoglycan covalently modified with rhamnose (Rha)-containing polysaccharides 1 .
These essential polysaccharides are functional homologs of the canonical poly(glycerolphosphate) and poly(ribitol-phosphate) wall teichoic acids (WTAs) expressed by Bacillus subtilis and Staphylococcus aureus 2 , and non-canonical choline-containing WTAs produced by the Streptococcus mitis group 3 .
Streptococcus pyogenes (Group A Streptococcus, GAS) is a human pathogen of global significance for which a vaccine is not currently available 4 . The key antigenic surface polymer of GAS, the Lancefield group A carbohydrate (GAC), is composed of a repeating  5 . GAC has attracted significant interest from the scientific and medical communities as a vaccine candidate due to its uniform and invariant expression in all GAS serotypes and the absence of the constitutive component of GAC, Rha, in humans [6][7][8][9][10][11] . Furthermore, in proof-of-principle mouse immunization studies, protein-conjugated GAC vaccines demonstrated efficacy against GAS strains 6,7 . To provide well-defined GAC for immunological studies and vaccine design, knowledge of the structure and biogenesis of GAC is essential 9 . The molecular mechanism of GAC biosynthesis was proposed based on genetic and biochemical studies of two genomic loci encoded by gacABCDEFGHIJKL and gacO 8,[12][13][14][15][16] . GAC biosynthesis is initiated on the inside of the plasma residue GlcNAc for the polysaccharide biosynthesis ( Supplementary Fig. 1) 2,8,13,17 . In the final steps of peptidoglycan decoration with WTAs, the LCP phosphotransferases attach GlcNAc 1phosphate to peptidoglycan MurNAc residues forming a GlcNAc phosphodiester bond to peptidoglycan 18 . The WTA-peptidoglycan phosphodiester bond is acid labile 19 , and this aspect of the S. aureus and B. subtilis cell walls has been used to prepare undegraded, highly-enriched WTAs, freed of peptidoglycan remnants 20,21 . Accordingly, the GAC-peptidoglycan linkage should also be sensitive to these conditions. We subjected purified GAS cell wall and intact sacculi to mild acid conditions (0.02 N HCl, 100ºC, 20 min), known to hydrolyze GlcNAc 1phosphate, and measured the release of water-soluble GAC. Surprisingly, these wellestablished conditions released less than 25 % of GAC from GAS ( Fig. 1 b, c). The resistance of GAC to release by mild acid hydrolysis suggested the presence of an un-acetylated amino sugar in the linkage region as has been reported for Lactobacillus plantarum, which contains a GlcN 1-phosphate-linked WTA. A phosphodiester bond connecting the L. plantarum WTA to peptidoglycan is resistant to mild acid hydrolysis unless first chemically N-acetylated 20 . The chemical basis for the extraordinary resistance of GlcN 1-phosphate to acid hydrolysis is illustrated in Supplementary Fig. 2 a, b. Hydrolysis of the glycosidic bond of GlcNAc 1phosphate is facilitated by protonation of the O1-oxygen and subsequent formation of a resonance stabilized oxonium ion intermediate ( Supplementary Fig. 2 a). However, this reaction is dramatically slowed during incubation of GlcN 1-phosphate under these conditions, because the positively charged GlcN-ammonium group destabilizes the adjacent oxonium ion intermediate ( Supplementary Fig. 2 b). To test the possibility that a hexosamine residue, devoid of an N-acetyl group, is present in the GAC-peptidoglycan linkage, purified GAS cell wall or sacculi were first chemically N-acetylated and then subjected to mild acid hydrolysis. GAC was nearly quantitatively released from peptidoglycan under these conditions, supporting our hypothesis ( Fig. 1 b, c). 6 Sugars with primary amino groups, lacking N-acetyl groups, undergo deamination by reaction with nitrous acid (HONO). Deamination of polysaccharides containing GlcN residues results in the formation of 2,5-anhydromannose and hydrolysis of the glycosidic bond 22 , as illustrated in Supplementary Fig. 2 c. This selective cleavage has been utilized successfully to release the L. plantarum WTA from peptidoglycan 20 . Nitrite is rapidly protonated to form HONO in acidic conditions 23 . We observed that incubation of isolated cell wall material and intact sacculi with acidified nitrite released 70-80% of GAC in a water-soluble form ( Fig. 1 b, d). In contrast, chemically N-acetylated GAS cell wall or sacculi were resistant to this treatment ( Fig. 1 b, d). Taken together, these results strongly suggest that GAC is attached to peptidoglycan via a hexosamine 1-phosphate linkage.

The GAC-peptidoglycan linkage region contains GlcN and GlcNAc. Our observation that
HONO deamination cleaves only 70-80% of GAC from peptidoglycan, and that treatment with mild acid released ~20-25 % of GAC suggests that GAS produces two classes of rhamnopolysaccharide composed of major and minor fractions containing either hexosamine or N-acetylated hexosamine, respectively, in the linkage region. Importantly, mild acid hydrolysis of chemically N-acetylated cell wall released GAC nearly quantitatively ( Fig. 1 b), indicating that failure to achieve complete release of GAC by these treatments was not due to incomplete hydrolysis of GAC from peptidoglycan or poor recovery of released material. To verify this conclusion, we sequentially subjected previously deaminated cell walls to mild acid hydrolysis and measured the release of soluble GAC ( Supplementary Fig. 3). We observed that the HONO-resistant rhamnopolysaccharide fraction was fully released by the subsequent mild acid treatment. Furthermore, deamination of previously mild acid treated cell walls cleaves the remaining ~80 % of the GAC which is resistant to mild acid hydrolysis ( Supplementary Fig. 3).
To identify the hexosamine at the reducing end of the acetylated and un-acetylated forms of GAC, the polysaccharide was released from GAS cell wall by either deamination with HONO (nitrous acid sensitive GAC, or GAC NA ) or mild acid hydrolysis (mild acid sensitive GAC, or 7 GAC MA ), and reduced chemically with sodium borohydride. Sodium borohydride reduces the aldehyde of 2,5-anhydromannose to form 2,5-anhydromannitol and the hemiacetal of GlcNAc to form N-acetyl-D-glucosaminitol (GlcNAcitol). The reduced GAC NA and GAC MA preparations were then fractionated by size-exclusion chromatography (SEC) on BioGel P150. As confirmed by the composition analysis, both treatments released similar, uniformly-sized polysaccharides eluting near the middle of the column elution volume (Fig. 2 a and b). When column fractions were assayed by GC-MS as TMS-methyl glycosides, the GAC NA and GAC MA preparations were found to contain Rha and GlcNAc as the only component sugars detected (Fig. 2 a and b, upper panels). The reducing-end sugars were detected as alditol acetates by GC-MS analysis. This method provides better detection of these minor terminal sugars in the presence of high concentrations of the polymer sugars, Rha and GlcNAc. We identified 2,5-anhydromannitol in the GAC NA preparations, and GlcNAcitol in the GAC MA preparations (Fig. 2 a and b, bottom panels), co-eluting with the peak of carbohydrate, as the only sugar alcohols in these fractions, strongly suggesting that the extracted GAC material contains GlcN and GlcNAc residues as the reducing moiety. Notably, we observed a lower apparent recovery of 2,5-anhydromannitol in the HONO-released fraction (Fig. 2 b), compared to the recovery of GlcNAcitol in the mild acid released material (Fig. 2 a). This result is expected because 2,5-anhydromannitol is not the sole product of deamination of hexosamines 22,24 . In agreement, when we treated commercially available GlcN 1-phosphate with HONO, only ~ 25% of the expected 2,5-anhydromannitol was recovered (Supplementary Figure 4).
We have previously shown that GAS and Streptococcus mutans produce rhamnopolysaccharides with variable GroP content, and these forms can be separated by ionexchange chromatography 15,25 . Further experiments were performed to examine whether the GlcN and GlcNAc linker residues are restricted to a specific ionic isoform of GAC. Fractionation of GAC NA and GAC MA on DEAE Toyopearl revealed that both polysaccharides contained a similar array of ionic forms: a neutral glycan, not bound to the ion-exchange column, and a 8 strongly bound component (Fig. 2 c). Similar heterogeneity of GAC was previously observed when the polysaccharide was cleaved from peptidoglycan by the peptidoglycan hydrolases 15 .
As expected, the neutral glycan contains very low phosphate content indicating that it has a low degree of GroP modification (Fig. 2 c and d). The glycosyl composition of the pooled GAC fractions revealed that the column-bound and unbound fractions from both polysaccharide preparations contain similar Rha/GlcNAc ratio (Fig. 2 d). Furthermore, 2,5-anhydromannitol was detected in both the column-bound and unbound fractions of the GAC NA preparations, and GlcNAcitol in both the column-bound and unbound fractions of the GAC MA preparations (Fig. 2   d). These observations provide evidence that the GlcN and GlcNAc linker isoforms are not specifically linked to the presence or absence of GroP in GAC and that the only apparent difference in the two populations of GAC is the acetylation status of the linkage GlcN unit.
Chemical structure of the GAC-peptidoglycan linkage region. To elucidate the exact chemical structure of the linkage region, GAC liberated from peptidoglycan by the peptidoglycan hydrolases, PlyC and mutanolysin 15 , was subjected to NMR analysis. As expected from our previous study 15 , when GAC material was analyzed by 1 H, 13 C-HSQC NMR, the GAC trisaccharide repeating units, observed at highest level of intensity. The polysaccharide decorated by sn-Gro-1-phosphate groups as substituents at O6 of the side-chain D-GlcpNAc residues were identified at the second intensity level of NMR spectra 15 . To unravel how GAC is linked to peptidoglycan, the analysis was performed at the third level of intensity in the NMR spectra. In the 31   This cell wall-binding domain consists of six LysM repeats (Pfam PF01476) that recognize peptidoglycan 41 . The protein fusion was added exogenously to the GAS sacculi subjected to either no treatment, mild acid hydrolysis, or deamination. Fluorescence microscopy imaging revealed that the protein fusion did not associate with the majority of untreated sacculi ( Fig. 4 and Supplementary Table 2). In contrast, the sacculi treated with HONO demonstrated uniform distribution of GFP-AtlA Efs over the cell surface (Fig. 4). Interestingly, the majority of sacculi PgdA is a homolog of the peptidoglycan GlcNAc N-deacetylase from Streptococcus pneumoniae 42 . Pdi was originally identified in the fish pathogen Streptococcus iniae 43 . While the function of this protein has not been described, it was named Pdi for polysaccharide deacetylase of S. iniae. We renamed this protein to PplD (polysaccharide-peptidoglycan linkage deacetylase) for the reasons provided below. To examine the functions of PplD and PgdA in deacetylation of the GAC linker region, we generated pplD and pgdA single deletion mutants and the double deletion mutant in a GAS NZ131 strain, creating GASΔpplD, GASΔpgdA and GASΔpplDΔpgdA, respectively. Since the chemical treatments produced similar results with the GAS cell wall and sacculi ( Fig. 1 b, c and d), for convenience we used the sacculi of the constructed strains to test the sensitivity of the phosphodiester linkage to mild acid and HONO treatment. Mild acid hydrolysis liberated the polysaccharide almost quantitatively from the GASΔpplD and GASΔpplDΔpgdA sacculi ( Fig. 1 c). Conversely, the sacculi of these mutants were almost completely resistant to deaminative cleavage with HONO ( Fig. 1 d). Moreover, chemical N-acetylation of the GASΔpplD and GASΔpplDΔpgdA sacculi prior to the treatments with either mild acid or HONO did not significantly affect the release of GAC ( Fig. 1 c and d). In contrast, the GASΔpgdA sacculi behaved in these assays like the WT control ( Fig. 1 c and d).
These data indicate that PplD is required for deacetylation of the GAC-peptidoglycan linker region.
PplD structure. PplD is predicted to contain an N-terminal transmembrane helix and an extracellular catalytic domain (ePplD) connected by a linker region. To facilitate the functional analysis of PplD, we solved the crystal structure of ePpLD (residues 94-320) to 1.78 Å resolution (Supplementary Table 3). There were two molecules in the asymmetric unit that are very similar to each other with a r.m.s.d. of 0.3 Å over 226 aligned Cα atoms. Analysis of the crystal contacts revealed that the dimer interface is the largest with 632 Å 2 buried surface area that scored below the stable complex formation criteria 44 . In addition, ePplD behaves as a monomer in solution according to SEC. The structure of ePplD revealed a (β/α) 7 -barrel fold that is characteristic of carbohydrate esterases of the CE4 family (Fig. 5 a). The analysis of the putative active site revealed the presence of a metal ion, which we identified as a Zn 2+ based on analysis of anomalous difference maps and temperature factors of the ion and coordinating atoms. The Zn 2+ ion is coordinated by the side chains of residues D168, H223 and H227 (Fig. 5 b). This coordination pattern of the D-H-H residues is common for the metal coordination in CE4 family members. An additional electron density observed next to the Zn 2+ ion was assigned as acetate, one of the products of the deacetylation reaction. The compound makes a bi-dentate contact with the Zn 2+ ion. Altogether, the side chains of D168, H223 and H227, acetate and a water molecule complete an octahedral coordination of the Zn 2+ ion (Fig. 5 b). One of the oxygen atoms of acetate makes a hydrogen bond with the backbone amide of S264, an oxyanion hole, while another oxygen atom of acetate forms a hydrogen bond with catalytic residue H105. A semi-buried D167, which is likely involved in coordination of a catalytic water molecule, makes a bi-dentate salt bridge with R302 and an additional hydrogen bond with N191.
Altogether, the structural features of ePplD are consistent with the deacetylase function of this enzyme.
The structural comparison of PplD with the available protein structures shows that this enzyme is more distantly related to S. pneumoniae PgdA 45 , but similar to the exopolysaccharide poly-N-acetylglucosamine (PNAG) deacetylases PgaB and IcaB produced by Escherichia coli and Ammonifex degensii, respectively, (Supplementary Table 4 51 . In addition to N-acetylmuramidase activity, lysozyme kills bacteria by a non-enzymatic mechanism 52 . This mechanism is similar to killing by small cationic peptides 53 , and is attributed to lysozyme crossing the cell wall and binding to negatively charged bacterial membranes thus leading to membrane permeabilization. Deacetylation of GlcNAc in peptidoglycan by PgdA directly alters lysozyme binding to its substrate negatively affecting lysozyme-mediated degradation of peptidoglycan. Interestingly, in S. iniae, the PplD homolog also confers resistance to lysozyme 43 . Hence, to understand the role of cell wall deacetylation in protection of GAS against lysozyme, first, we assessed muramidase-dependent lysis of the WT GAS, GASΔpplD, GASΔpgdA and GASΔpplDΔpgdA cells treated with lysozyme. When GAS strains were incubated with 1 mg/ml lysozyme for 18 hours, no lysozyme-dependent decrease in turbidity was observed consistent with the established high resistance of GAS to lysozyme. In contrast, incubation of GAS strains with an alternative highly efficient Nacetylmuramidase, mutanolysin, produced a substantial drop in turbidity compared to control ( Supplementary Fig. 7). Since the sensitivity of GAS to lysozyme appeared to be below the limits of detection detected by turbidity analysis, cell wall degradation was estimated with a more sensitive assay using dot-blot analysis for the water-soluble form of GAC released from the bacteria during digestion ( Supplementary Fig. 8 a). Lysozyme produced a very weak solubilization of GAC from WT GAS and GASΔpplD cells, and more pronounced release of GAC from GASΔpgdA and GASΔpplDΔpgdA cells ( Supplementary Fig. 8 b). Furthermore, lysozyme released GAC from GASΔpplDΔpgdA more efficiently, than from GASΔpgdA ( Supplementary Fig. 8 b). Altogether, these data indicate that lytic degradation of the GAS cell wall by lysozyme is minor due to the presence of GlcN in peptidoglycan as the result of PgdA catalytic activity. However, when PgdA is absent, deacetylation of the GAC linker region by PplD can be detected as a factor conferring protection against lysozyme-mediated lysis.
We previously reported that in comparison to the parental strain, the GroP-deficient mutant of GAS, GASΔgacH, is significantly more resistant to a highly cationic enzyme human group IIA secreted phospholipase A 2 (hGIIA sPLA 2 hereafter hGIIA) 15 . This phenotype is likely due to either decreased binding of hGIIA to the cell surface or reduced permeability of the cell wall to hGIIA. Since deacetylation of GlcNAc residues increases the cell wall net positive charge, it might lead to neutralization of negative charges conferred by GroP moieties and as a result, affect the translocation of cationic AMPs across the cell wall. To understand how modulation of the cell wall net charge by deacetylation of GlcNAc residues alters susceptibility of GAS to cationic AMPs, we analyzed the resistance of WT GAS, GASΔpplD, GASΔpgdA, GASΔpplDΔpgdA to the antimicrobial action of hGIIA, lysozyme, or histone mixture (Fig. 6 a, b, c and Supplementary Fig. 8 c). We observed that deletion of either pplD or pgdA, or both genes, yielded strains with enhanced susceptibility to hGIIA (Fig. 6 a). In contrast, deletion of pplD did not affect susceptibility of bacteria to lysozyme (Fig. 6 b and Supplementary Fig. 8 c) or a histone mixture (Fig. 6 c). However, sensitivity to killing by these AMPs was significantly increased in GASΔpgdA and GASΔpplDΔpgdA, with a more pronounced effect observed in GASΔpplDΔpgdA. Moreover, the expression of WT PplD in GASΔpplD and GASΔpplDΔpgdA significantly increased the resistance of the parental strains to the AMPs, especially for hGIIA ( Fig. 6 a, b, c and Supplementary Fig. 8 c). This observation correlates with the conclusion that the level of deacetylation of the GAC-peptidoglycan linkage region in the complemented strains is higher than in the WT strain, and indicates that both PgdA and PplD enhance resistance of GAS to the tested AMPs.
Finally, we examined susceptibility of the GroP-deficient mutant, GASΔgacH, and its complemented strain, GASΔgacH:pgacH, to the antimicrobial action of lysozyme or histones to confirm that the mechanisms of lysozyme and histone killing of GAS are charge-dependent.
Deletion of gacH rendered GAS more resistant to lysozyme and histones, and the phenotype was reversed by complementation of GASΔgacH with WT gacH (Fig. 7 a, b). Collectively, our results are consistent with a model in which the addition of positive residues by deacetylation of GlcNAc in peptidoglycan and the GAC linkage region counteracts the cell wall net negative charge provided by GroP moieties resulting in reduced translocation of cationic AMPs across the cell wall (Fig. 7 c).

PplD homologs deacetylate respective rhamnopolysaccharides in streptococci.
To understand whether deacetylation of the polysaccharide-peptidoglycan linkage region is widespread in bacteria, we examined the distribution of the PplD family of proteins throughout the bacterial kingdom. PplD homologs were predominantly identified in the genomes of streptococcal species (Supplementary Fig. 9). Interestingly, members of the S. mitis group, that express choline-containing WTAs, and not Rha-containing polysaccharides, possess only the PgdA homolog, supporting the conclusion that PplD is only active on rhamnopolysaccharides.
We also observed that genes encoding pplD homologs in a number of streptococcal strains contain a frame shift mutation and thus are likely non-functional. Hence, to investigate the function of PplD in streptococcal species, we selected for analysis the following strains: Streptococcus agalactiae (Group B Streptococcus, GBS) A909, S. mutans Xc, and

Streptococcus equi subsp. equi CF32 in which pplD is an intact gene, and GBS COH1 and
Streptococcus thermophilus LMG 18311 in which the pplD homologs have a frame shift mutation ( Supplementary Fig. 10). Additionally, we deleted the pplD homologs in S. mutans Xc and GBS A909 strains creating the SMUΔpplD and GBSΔpplD strains. In agreement with the function of PplD in deacetylation of the polysaccharide-peptidoglycan linker, the rhamnopolysaccharides were liberated from the cell walls by HONO in the strains with the intact pplD (Fig. 8 a, b). In contrast, this treatment did not cleave the polysaccharides from the strains carrying the inactivated pplD. However, these polysaccharides were released by mild acid hydrolysis (Fig. 8 a, b). Furthermore, expression of the GAS WT PplD but not the catalytically inactive versions of PplD, PplD-D167N and PplD-H105A/D167N, restored the phenotype of SMUΔpplD (Fig. 8 a). Taken together, the results support the conclusion that in the large group of streptococci expressing the Rha-containing polysaccharides, the polysaccharidepeptidoglycan linkage region is deacetylated by PplD homologs suggesting that PplD recognizes for catalysis the polyrhamnose linker region sugars that are conserved in many streptococci 1,16 .

Discussion
Currently, no licensed vaccine exists for GAS which is a leading cause of morbidity and We previously demonstrated that modification of GAC and the S. mutans rhamnopolysaccharide with negatively charged GroP moieties renders GAS and S. mutans more sensitive to hGIIA activity 15 . In this study, we reveal that the GroP modification confers sensitivity of GAS to lysozyme and histone mixture also. Thus, these data support the conclusion that PplD and PgdA contribute to protection of bacteria against AMPs by neutralizing a net negative charge of the cell wall polysaccharides (Fig. 7 c).
In sum, the results of this study form a foundation for the design of significantly improved GAS glycoconjugate vaccine based on GAC and provide insight into the function of cell wall modifications in protection of bacteria against cationic AMPs.

Bacterial strains, growth conditions and media
All plasmids, strains and primers used in this study are listed in Supplementary Tables 5 and 6.
Streptococcal strains (GAS, GBS, S. equi and S. thermophiles) were grown in BD Bacto Todd-Hewitt broth supplemented with 1% yeast extract (THY) without aeration at 37°C. S. mutans strains were grown on THY agar at 37°C with 5% CO 2 . E. coli strains were grown in Lysogeny Broth (LB) medium or on LB agar plates at 37°C. When required, antibiotics were included at the following concentrations: ampicillin and streptomycin at 100 µ g mL -1 for E. coli; erythromycin at 500 µ g mL -1 for E. coli and 5 µ g mL -1 for streptococci; chloramphenicol at 10 µ g mL -1 for E. coli and 2 µ g mL -1 for streptococci; spectinomycin at 200 µ g mL -1 for E. coli and 200 µ g mL -1 for streptococci; kanamycin at 50 µ g mL -1 for E. coli and 300 µ g mL -1 for streptococci.

Construction of mutant strains
For construction of the GAS, GBS and S. mutans deletion mutants, GAS NZ131, GBS A909 and S. mutans Xc chromosomal DNA, respectively, were used as the templates for amplification of DNA fragments encompassing the upstream and downstream regions flanking the gene of interest. The primers used to generate the deletion mutants are listed in Supplementary Table 6.
To construct GASΔpplD, the DNA fragments were amplified using two primers pairs JC480/JC481 and JC482/JC483, digested with the restriction enzymes and cloned into pFED760 76 . The antibiotic resistance cassette for kanamycin (aph3A), amplified from pOskar 77 using two primers pairs JC292/JC304, was cloned between the upstream and downstream fragments to generate the plasmid pJC344-kan for selective allelic replacement. The plasmid was electroporated into the GAS NZ131 cells.
To construct GASΔpplDΔpgdA, two DNA fragments were amplified using two primers pairs To construct GASΔgacH, GBSΔpplD and SMUΔpplD, we used a PCR overlapping mutagenesis approach, as previously described 15   The assembled DNA fragments for mutagenesis of GAS gacH and GBS pplD were cloned into pHY304 by the use of restriction enzymes to generate the plasmids pHY304ΔgacH-NZ131 and pHY304ΔpplD-GBS, respectively. To plasmids were electroporated into the GAS NZ131
A two-step temperature-dependent selection process was used to isolate the desired GAS and GBS mutants 15 . The transformants were selected on THY agar containing the corresponding antibiotic and screened for sensitivity to erythromycin. Single colonies sensitive to erythromycin were picked. Double-crossover recombination was confirmed by PCR and Sanger sequencing using the primers listed in Supplementary Table 6.
For complementation of GASΔpplD, GASΔpplDΔpgdA, and SMUΔpplD, a promoterless pplD gene was amplified from GAS NZ131 using the primers listed in Supplementary Table 6. The

24
To create a vector for expression of the extracellular domain of PplD (ePplD), the gene was amplified from GAS 5005 chromosomal DNA using the primer pair pplD-NcoI-f and pplD-XhoI-r.
The PCR product was digested with NcoI and XhoI, and ligated into NcoI/XhoI-digested pCDF- The structure was solved by single-wavelength anomalous diffraction phasing using an automated Phaser SAD pipeline 80 . The initial zinc positions were found using SHELXD 81 .
Following density modification with Parrot, the initial model was built using Buccaneer 82,83 . The model was improved using manual rebuilding in Coot alternating with refinement by phenix.refine 84,85 . The final model had 96% of residues in the favorable region of the Ramachandran plot and 4% in additionally allowed region.

Lysozyme, hGIIA and histone susceptibility assay
Frozen stocks of GAS variants were used for microbial susceptibility tests with hGIIA, histone mixture from calf thymus (Millipore Sigma, 10223565001) and lysozyme from egg white (Millipore Sigma, L6876). Recombinant hGIIA was produced as described previously 86 . To make frozen aliquots, GAS strains, cultured overnight in THY medium, were diluted 1:100 into fresh THY medium and allowed to grow to mid-logarithmic phase (OD 600 =0.

Muramidase activity of lysozyme on GAS
Overnight cultures of the GAS strains were diluted (1:100) into fresh THY broth and grown to an OD 600 of 0.5. Cell suspension (10 mL) was centrifuged (3,200 g, 10 min) and resuspended with either 1 mg mL -1 lysozyme or 62.5 U/ml mutanolysin (Sigma, M9901) or no enzyme (negative control) in sterile PBS (10 mL). After 0 and 18 hours of incubation at 37°C, 0.2 mL was centrifuged (16,000 g, 3 min). The supernatant was transferred to a fresh centrifuge tube and concentrated to 30 µ l final volume using SpeedVac vacuum concentrator. Supernatant (5 µ l) was spotted on a nitrocellulose membrane. The membrane was blocked for 1 hour with 7% skim milk in PBS/0.1% Tween 20 and incubated with an anti-GAC antibody diluted 1:5000 (Abcam, ab9191). Bound antibodies were detected with a peroxidase-conjugated goat anti-rabbit IG antibody and the Amersham Bioscience ECL (enhanced chemiluminescence) western blotting system. ImageJ software was used to quantify the signal of polysaccharides released.

Isolation of cell wall and sacculi
Cell wall was isolated from exponential phase cultures by the SDS-boiling procedure as described for S. pneumoniae 87 . Purified cell wall samples were lyophilized and stored at -20°C before the analysis. S. mutans and GAS sacculi were obtained by the SDS-boiling procedure followed by 2 washes with 1 M NaCl and 5 washes with water as outlined 25 . To analyze sensitivity of GAC cleavage to chemical treatments, the GAS sacculi were additionally treated with 5 mg mL -1 trypsin (Sigma, T-8253) at 37°C overnight, sedimented (32,000 g for 15 min), and washed 4 times by resuspension in water. This treatment removes binding of the GAS sacculi to the microcentrifuge plastic tubes. Sacculi were resuspended in water and stored at 4 C before the analysis.

HONO deamination
Polysaccharide was released from sacculi or highly enriched cell wall preparations by HONO deamination essentially as described 22 with minor modification. Briefly, reactions contained sacculi (OD 600 = 3.4) or isolated cell walls (2-5 mg mL -1 ), in 0.2 N Na Acetate pH 4.5 and 1.5 M NaNO 2 (added in three equal portions over a period of 90 minutes) at room temperature.
Following deamination, the reaction was stopped by the addition of one equivalent of ethanolamine and centrifuged (60,000 g, 10 min). The supernatant was removed and transferred to an Amicon Ultra-0.5 mL (3,000 MWCO) spin filter. The pellet was washed two times by resuspension in water (1/2 of the original reaction volume), re-sedimented, and the washings were combined with the initial supernatant. The washed pellet was re-suspended in water and reserved for analysis, or discarded. The released material was desalted by several sequential cycles of dilution with water and re-concentration on the spin filter, recovered in a convenient volume of water and either reserved for analysis or purified further by chromatography on Bio Gel P150 and ion exchange on DEAE-Toyopearl.

Mild acid hydrolysis
Mild acid hydrolysis was performed, either with or without prior N-acetylation, as previously described for WTA of L. plantarum 20 with some modifications. Chemical N-acetylation reactions contained sacculi (OD 600 = 3.4) or isolated cell walls (2-5 mg/ml), resuspended in 1 mL of saturated NaHCO 3 , and 2 % acetic anhydride. After 1 h on ice, the reactions were allowed to warm to room temperature overnight. After incubation at room temperature overnight, the reactions were diluted with 2 volumes of water and sedimented (50,000 x g, 10 min). The insoluble residue was washed three times by resuspension with 3 mL water followed by sedimentation (50,000 x g, 10 min). For mild acid hydrolysis, sacculi or cell wall material was resuspended in 0.02 N HCl and heated to 100°C for 20 min. The reactions were cooled on ice, neutralized by the addition of 1 N NaOH (1 equivalent) and sedimented (50,000 x g, 10 min).
The supernatant fraction was removed and reserved, and the pellet was re-suspended in water and re-sedimented. The supernatant fractions were combined and either analyzed or purified further. The pellet fractions were resuspended in an equal volume of water and reserved for further analysis for rhamnose content or used for fluorescent microscopy.

Fluorescent and differential interference contrast (DIC) microscopy
GAS sacculi obtained by the SDS-boiling procedure were subjected to mild acid hydrolysis or deamination with HONO as outlined above. Untreated or treated sacculi were resuspended in PBS to an OD 600 =0.3, and incubated with 19.8 µ

Phosphate assays
Total phosphate content of SCCs was determined by the malachite green method following digestion with perchloric acid, as previously described 15 .

Modified anthrone assay
Total Rha content was estimated using a minor modification of the anthrone procedure as previously described 15 . Rha concentrations were estimated using L-Rha standard curves.

Analysis of glycosyl composition by gas chromatography-mass spectrometry (GC-MS)
Saccharide compositions were determined as either trimethylsilyl (TMS) derivatives of O-methyl glycosides, following methanolysis or as alditol acetates following hydrolysis with 2 N TFA and peracetylation, as described below 88  which, A x is GC area of analyte, A IS is GC area of internal standard, C x is amount of analyte and C is is amount of internal standard. Amounts of sugars were calculated using the formula: C x = C is ×(A x /A IS )/Rf. In this analysis, an exponential increase in response factor associated with increasing amounts of analyte relative to internal standard was observed. To correct for this change in response factor, standard curves of increasing amounts of analytes to a constant quantity of internal standard were prepared and used to estimate the appropriate response factor.

NMR spectroscopy
Cell wall isolated from GAS 5005 was used for purification of GAC for NMR analysis 15

Statistical analysis
Unless otherwise indicated, statistical analysis was carried out on pooled data from at least three independent biological repeats. Quantitative data was analyzed using either one-way or two-way ANOVA with either Tukey's or Bonferroni multiple comparison test. Symbols and error bars represent the mean and S.D., respectively. A p-value equal to or less that 0.05 was considered statistically significant.

Data availability
Atomic coordinates and structure factors of PplD crystal structure have been deposited to the Protein Data Bank with accession code 6DQ3. All data generated during this study are included in the article and Supplementary information files or will be available from the corresponding author upon reasonable request.  Fractions unbound (flow-through) and bound (eluted with a NaCl gradient) to the DEAE column were pooled, concentrated, desalted by spin column and analyzed by GC-MS to determine the Rha, GlcNAc, 2,5-anhydromannitol and GlcNAcitol concentrations as described above.

Figure legends
Malachite green assay was used to assay the phosphate concentration. The concentrations of GlcNAc, 2,5-anhydromannitol, GlcNAcitol and phosphate are expressed as moles per 30 moles of Rha. Columns and error bars represent the mean and S.D., respectively (n = 3 biologically independent replicates).      respectively (n = 3 biologically independent replicates). P values were calculated by two-way ANOVA with Tukey's multiple comparisons test.