sign in sign up
<<
Home , Polysaccharide

capsular polysaccharide is linked to peptidoglycan via a direct to β-D-N-acetylglucosamine

Thomas R. Larsona and Janet Yothera,1

aDepartment of , University of Alabama at Birmingham, Birmingham, AL 35294-2170

Edited by Emil C. Gotschlich, The Rockefeller University, New York, NY, and approved April 14, 2017 (received for review December 20, 2016) For many , including those important in pathogenesis, (Und-P). In S. pneumoniae serotype 2 CPS, Glcp-1-P is trans- expression of a surface-localized capsular polysaccharide (CPS) can ferred from UDP-Glcp (11), and this is followed by addition of be critical for survival in host environments. In Gram-positive the remaining (12, 13) to form the complete repeat unit bacteria, CPS linkage is to either the cytoplasmic membrane or the (Fig. 1). Und-P-P- repeat units are translocated wall. Despite the frequent occurrence and essentiality of these to the outer face of the cytoplasmic membrane by Wzx and po- , the exact nature of the linkage has not been lymerized into high molecular weight (MW) polysaccharide by described in any bacterial species. Using the Streptococcus pneu- Wzy. Growth occurs at the reducing end, with single or multiple moniae serotype 2 CPS, which is synthesized by the widespread repeat units being transferred en bloc from Und-P-P to an ac- Wzy mechanism, we found that linkage occurs via the reducing ceptor Und-P-P-oligosaccharide repeat unit. of the β N- end of CPS and the -D- acetylglucosamine (GlcNAc) res- donor Und-P-P that remains after transfer yields Und-P, which is idues of peptidoglycan (PG). Hydrofluoric resistance, 31P-NMR, 32 recycled back into the cell. In Gram-negative bacteria, CPS is and P labeling demonstrated the lack of phosphodiester bonds, ultimately transported across, and assembled on, the outer mem- which typically occur in PG–polysaccharide linkages. Component – brane (9, 14). For Gram-positive bacteria, CPS is covalently linked analysis of purified CPS PG identified only CPS and PG sug- to the cell wall and cytoplasmic membrane (15–17); however, the

ars in the appropriate ratios, suggesting the absence of an oligo- mechanisms involved in ligation are unknown. Failure to transfer MICROBIOLOGY saccharide linker. Time of flight mass spectrometry confirmed a CPS from Und-P may result in lethality due to lipid intermediate direct glycosidic linkage between CPS and PG and showed that a accumulation and/or reduced turnover of Und-P for essential single CPS repeat unit can be transferred to PG. The linkage was pathways such as peptidoglycan (PG) (12, 18). acetolysis susceptible, indicative of a 1,6 glycosidic bond between A partial description of a CPS cell wall linkage in a Gram- CPS and the GlcNAc C-6. The acetylation state of GlcNAc did not affect linkage. A direct glycosidic linkage to PG was also demon- positive bacterium has been reported for the Wzy-dependent strated for serotypes 8 and 31, whose reducing end sugars are type III CPS, in which an oligosaccha- glucose and , respectively. These results provide the most ride linker and phosphodiester bond are proposed to mediate detailed descriptions of CPS–PG linkages for any microorganism. linkage to the N-acetylglucosamine (GlcNAc) of PG (16). Ex- Identification of the linkage is a first step toward identifying the actly how these constituents are linked together or to GlcNAc linking and potential inhibitors of its activity. has not been described. Other PG-linked polysaccharides include wall teichoic and teichuronic and the mycobacterial arabino- capsule | Wzy | Gram positive | pneumococcus | glycobiology . The linkage for each of these structures is to the N-acetylmuramic acid (MurNAc) C-6 via a phosphodiester bond. For teichoic acids and arabinogalactans, an oligosaccharide linker is apsular polysaccharide (CPS) assembly and localization in – Cbacteria is a complex, multienzyme process leading to an- also present (19 24). In this study, we provide a detailed description choring of the CPS on the outer surface of the cell. For of a CPS linkage to PG, demonstrating that the S. pneumoniae type pathogens, the protective layer of the CPS can be important for adhesion, formation, and resistance to complement- Significance mediated opsonophagocytosis and lysis (1, 2). Although substantial information regarding the syntheses of these polymers has accumu- Bacterial surface polysaccharides form the interface between lated, less is known about the critical steps involved in their attach- the bacterium and its environment. In Gram-positive bacteria, ment to the bacterial surface. the intricate architecture of the peptidoglycan (PG) serves A large number of diverse CPS structures can be elaborated, as the scaffolding to which other surface polysaccharides, in- as exemplified in the Gram-positive pathogen Streptococcus cluding capsules, can be anchored. For , pneumoniae, where 98 CPS serotypes differing in sugar compo- proper localization of capsular polysaccharide (CPS) is essential Streptococcus pneumoniae sition and linkages are known (3, 4). S. pneumoniae , to cause disease. In , CPS is also the occurring primarily as pneumonia and sepsis in young children primary target for . Here, we identify a structure for polysaccharide attachment to PG that involves a direct glyco- and the elderly, are a significant global disease burden (5). sidic linkage. Knowledge of this linkage lays the groundwork Elaboration of a CPS is essential for these bacteria to cause for identifying the enzyme(s) involved in its synthesis and for disease and to colonize the nasopharyngeal cavity (6–8). Despite developing new targets for therapeutic interventions. their structural diversity, all but two of the 98 S. pneumoniae CPS

serotypes are synthesized by the Wzy polymerase-dependent Author contributions: T.R.L. and J.Y. designed research; T.R.L. performed research; T.R.L. mechanism, which is the most common mechanism of CPS syn- and J.Y. analyzed data; and T.R.L. and J.Y. wrote the . thesis in Gram-positive and Gram-negative bacteria, and is also The authors declare no conflict of interest. used in synthesis of many O- in Gram- This article is a PNAS Direct Submission. negative bacteria (9, 10). Polymer synthesis in the Wzy pathway 1To whom correspondence should be addressed. Email: [email protected]. begins with transfer of a sugar-1-phosphate from a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. diphosphosugar donor to the C55 lipid undecaprenyl-phosphate 1073/pnas.1620431114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1620431114 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Fig. 1. Proposed structure of serotype 2 CPS linked to PG, as determined in this study. The CPS structure is from refs. 11 and 25. PG residues are indicated in blue. Asterisk indicates 80% of the GlcNAc residues are deacetylated in S. pneumoniae (28). GlcUA, glucuronic acid; Rha, .

2 CPS is linked via its reducing end Glc to the GlcNAc of PG by a the teichoic acid-specific ribitol and N-acetylgalactosamine from the direct 1,6 glycosidic bond. high MW fraction (Fig. 5A, a) and their appearance in the low MW fraction, where neither CPS nor PG sugars were detected (Fig. 5A, Results d). The CPS–PG linkage is thus resistant to HF and likely not CPS Is Linked to PG. A cell wall fraction containing GlcNAc– mediated by a phosphodiester bond. MurNAc and any linked components was gener- To further test for phosphate-mediated linkages, CPS–PG was ated by treatment of S. pneumoniae with mutanolysin, a β-1,4-N- analyzed using 31P-NMR. Several distinct chemical shifts indicating acetylmuramidase that cleaves the PG backbone, and LytA, the phosphate and phosphate-mediated bonds were observed (Fig. 5B, pneumococcal N-acetylmuramoyl-L-alanine amidase that removes a). After treatment with HF, phosphate was removed from the CPS- the side chain from MurNAc. The negatively charged CPS containing fraction (Fig. 5B, b) and identified in only the lower MW was purified from the digest using anion exchange chromatog- fraction (Fig. 5B, c), consistent with its association with teichoic acid. raphy. CPS eluted in broad fractions, consistent with a range of The complete removal of phosphate without hydrolyzing the CPS– polysaccharide sizes of varying numbers of repeat units. Gas PG bond demonstrates the lack of phosphodiester bond involvement. and mass spectrometry (GC/MS) analysis identi- To test for any phosphate in the CPS–PG structure, cells were fied the sugars present in CPS (Rha, Glc, and GlcUA) and PG grown in the presence of [32P]-phosphate and then separated (GlcNAc and MurNAc) (Fig. 2). Ribitol was detected in only small into cell wall- and membrane-containing fractions. Following amounts, possibly indicative of teichoic acid and CPS on the same SDS/PAGE, 32P incorporation was observed in low MW products PG or incomplete PG hydrolysis resulting in copur- only, consistent with the small number of repeat units present in ification of distantly linked CPS and teichoic acid. The molar ratios teichoic acid (26) and the detection of teichoic acid-specific bands of Glc, Rha, and GlcUA were those expected from the published by immunoblotting (Fig. 5C, a and b). The greater 32P incorporation serotype 2 structure (11, 25). The GlcNAc and MurNAc ratio observed in the membrane fraction reflects the higher amount of was consistent with that of PG. The results show no evidence for membrane-linked lipoteichoic acid, which also contains phospho- additional sugars, suggesting that no oligosaccharide linker is diester bonds (19). No 32P was detected in higher MW bands, in- – present in the CPS PG structure. dicative of the absence of phosphate in the CPS–PG linkage (Fig. 5C, a and c). The lack of 32P signal and CPS overlap also indicates CPS Is Linked to GlcNAc. Purified CPS linked to PG disaccharides that single repeat units of PG disaccharides rarely contain both CPS (which also contains low amounts of teichoic acid, as noted and teichoic acid (an outcome previously suggested by failure of above, but henceforth referred to as CPS–PG) was subjected to antisera to teichoic acid to react with higher MW bands) (15, 27). β-N-acetylglucosaminidase to hydrolyze β-D-GlcNAc-1,4-β-D- MurNAc bonds. After ultrafiltration (3,000 Da cutoff), MurNAc Linkage of CPS to PG Involves a Direct Glycosidic Bond. Component but not GlcNAc was identified in the low MW fraction, indicating sugar analysis of CPS–PG with or without HF treatment identified that the latter remained linked to high MW CPS (Fig. 3). Sodium the CPS and PG sugars in their expected ratios, showing no evidence borodeuteride (NaBD4) treatment of the high MW fraction resul- of a linker (see above). To further test for a linkage unit, low MW ted in reduction of GlcNAc to deuterated N-acetylglucosaminitol (GlcNAc-ol), indicating that MurNAc was released by the di- gestion and GlcNAc had remained at the reducing end of the high MW CPS (Fig. 4 A, C, and E). Similar treatment of a non– β-N-acetylglucosaminidase-digested control sample demonstrated no reduction of GlcNAc (Fig. 4 B and D). Sodium borodeuteride- reduced products were not detected for Glc, Rha, or GlcUA, con- sistent with linkage of their anomeric C-1 carbons to other sugars. Taken together, these data indicate that CPS and PG are covalently linked, and GlcNAc is located at the reducing end of CPS.

CPS–PG Linkage Does Not Involve Phosphate Bonds. To test for phosphodiester bonds, CPS–PG was subjected to by 48% hydrofluoric acid (HF). After ultrafiltration (3,000 Da cutoff), all CPS sugars were identified in the high MW fraction in a ratio consistent with the serotype 2 CPS structure (Fig. 5A, a). GlcNAc – Fig. 2. CPS is linked to PG. GC/MS total ion chromatogram of cell wall-linked and MurNAc were also present in this fraction (Fig. 5A, a c). CPS. The molar ratios of Glc, Rha, and GlcUA are consistent with those of serotype 2 Consistent with the known phosphodiester bonds in S. pneumoniae CPS. The ratios of the GlcNAc and MurNAc peaks reflect equimolar concentrations, teichoic acid repeat units (19, 22), HF treatment resulted in loss of as determined using purified sugar standards. Rib-ol, ribitol. MCounts, megacounts.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1620431114 Larson and Yother Downloaded by guest on September 30, 2021 are generally resistant (30, 31). The CPS backbone contains one 1,4 linkage and no 1,6 linkages; a single 1,6 linkage occurs between Glc and the terminal GlcUA in the side chain (Fig. 1). Following acetolysis of CPS–PG, the majority of the polymer was degraded but Glc and Rha were detected in the high MW fraction, indicative of incomplete acetolysis of 1,4 linkages (Fig. 8A). No GlcUA was identified in this fraction (Fig. 8A), demonstrating that all 1,6 bonds had been cleaved. Likewise, no GlcNAc was present in the high MW fraction, demonstrating that it was linked to CPS by a 1,6 bond (Fig. 8B). The lower MW fraction contained the majority of the Glc and Rha (Fig. 8C), and all of the GlcUA (Fig. 8C)andGlcNAc(Fig. 8D). The acetolysis labile nature of the CPS–PG linkage is indicative Fig. 3. CPS is linked to the GlcNAc of PG. GC/MS analysis of low MW of a 1,6 glycosidic bond. As detailed in Discussion,thecollective products following β-N-acetylglucosaminidase digestion of CPS–PG. (A)Se- results provide strong evidence for linkage occurring between the lective ion monitoring for MurNAc at m/z 187. (B) Selective ion monitoring C-6 of GlcNAc in the PG and C-1 of Glc in the CPS backbone. for GlcNAc at m/z 173. Direct Glycosidic Bonds Mediate CPS–PG Linkages of Other S. pneumoniae Serotypes. More than 75% of the 96 known CPS was purified by isolation and ultrafiltration of early elutions of S. pneumoniae Wzy serotypes, including serotype 2, initiate CPS – CPS PG from anion exchange chromatography. Triple Q-time-of- synthesis with Glcp (3, 4). To examine the CPS–PG linkages in flight mass spectrometry (Q-TOF MS) demonstrated two major serotype 8, which also initiates with Glcp (Fig. S2), and serotype peaks (Fig. 6). The larger peak at m/z 1,361.99 corresponded to the 31, which initiates with galactose (Galf)(Fig. S3A), isolated mass expected for a single repeat unit of CPS linked by a glycosidic CPS–PG was treated with HF and purified by ultrafiltration. bond to a PG disaccharide with the loss of m/z 73, which is likely that GC/MS analysis of the high MW fractions demonstrated only the of the lactate group on MurNAc. The smaller peak at 1,435.02 with a shift of m/z 73 represents the intact MurNAc on the PG. Taken together, these results show that CPSisdirectlylinkedtoPGviaa MICROBIOLOGY glycosidic bond, and a single repeat unit of CPS can be transferred to PG.

Linkage of CPS to PG Does Not Require Deacetylation of GlcNAc. Approximately 80% of the S. pneumoniae PG GlcNAc is deace- tylated at the 2-acetylamino position, resulting in resistance to ly- sozyme (28). Inactivation of the deacetylase-encoding gene (pgdA) did not alter CPS localization to the cell wall (Fig. S1). The acetylation state is thus not relevant, and there is likely no role for PgdA or the 2-acetylamino position of GlcNAc in the attachment of CPS to PG.

CPS Linkage Does Not Occur Through the GlcNAc C-2 or C-3. Smith degradation (29) was used to determine whether the GlcNAc C-2 and C-3 are protected from periodate oxidation, as would be expected if CPS linkage occurred at either of these sites. GC/MS of the reaction products demonstrated degradation of Glc but not Rha (Fig. 7A), consistent with the presence and absence, respectively, of vicinal hydroxyls in these sugars in CPS (Fig. 1). For the PG sugars, MurNAc was not degraded but GlcNAc was reduced to 16.7% of the amount contained in a nonperiodate- treated sample (Fig. 7 A and B). This value is consistent with the observation that more than 80% of S. pneumoniae PG GlcNAc is deacetylated and exists as (28 and see above). In the absence of a linkage to the C-3, glucosamines are periodate sus- ceptible. This result precludes the possibility of linkage occurring through the C-2 or C-3 of either the PG GlcNAc or the CPS backbone Glc, as well as the C-2, C-3, or C-4 of the side chain Glc.

Linkage of CPS to PG Occurs Through a Direct 1,6 Glycosidic Bond to GlcNAc. Based on the known PG structure and the results de- Fig. 4. GlcNAc is at the reducing end of CPS. CPS–PG was either treated with

scribed thus far, the glycosidic linkage between CPS and GlcNAc NaBD4 or first digested with β-N-acetylglucosaminidase followed by isolation is unlikely to occur at the GlcNAc C-1 or C-4 (due to linkage to of high MW product and subsequent NaBD4 treatment. (A) GC/MS total ion – MurNAc), or C-2 or C-3 (due to susceptibility to Smith degra- chromatogram of enzyme plus NaBD4-treated CPS PG. (B) Total ion chro- – dation and lack of effect of N-acetylation on CPS linkage). The matogram of NaBD4-treated CPS PG. (C) Selective ion monitoring at m/z 173 GlcNAc C-6 is the least sterically hindered residue available for (GlcNAc) and 174 (deuterated GlcNAc-ol) of enzyme plus NaBD4-treated CPS–PG. The arrow is the reduced GlcNAc-ol. (D) Selective ion monitoring at glycosidic linkages, and the analogous MurNAc C-6 is the at- m/z 173 and 174 of NaBD4-treated CPS–PG. (E) Electron impact mass spectrum of tachment site for other PG-linked polysaccharides. Acetolysis the GlcNAc-ol peak in C demonstrates the mass spectral fragmentation pattern preferentially cleaves 1,6 glycosidic bonds, with 1,4 linkages of deuterated GlcNAc-ol, which resulted from reduction of GlcNAc after release of showing lesser susceptibility; linkages between 1, 2 and 1,3 residues MurNAc by β-N-acetylglucosaminidase. kCounts, kilocounts; MCounts, megacounts.

Larson and Yother PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 positives) (9, 10). Cell surface attachment is the final step in each of these processes. In Gram-negative bacteria, linkage is to the outer membrane, either via a linker and a phosphatidylgly- cerol anchor (ABC transporter pathway) (32) or as a result of ionic interactions with an outer membrane that serves as a nucleation point (Wzy pathway) (14). In Gram-positive bacteria, cytoplasmic membrane linkage via phosphatidylglycerol occurs in the synthase pathway (33). In the Wzy pathway, CPS synthesized on Und-P is transferred to PG, and there is also evidence for transfer to an alternate membrane acceptor (18). The most detailed descriptions of PG–polysaccharide linkages are for the teichoic acids, teichuronic acids, and mycobacterial arabinogalactans. The reducing ends of these polymers are linked to the MurNAc C-6 through a phosphodiester bond and, for teichoic acids and arabinogalactan, an oligosaccharide linker (polysaccharide– oligosaccharide–phosphodiester–MurNAc) (19–24). Similar compo- nents may comprise the S. agalactiae type III CPS–PG structure, except that this linkage is proposed to be CPS–phosphodiester– oligosaccharide–GlcNAc (16). However, complete analyses to con- firm such a structure have not been reported. It was suggested that, like teichoic acid, the S. aureus CPSmaybelinkedtotheMurNAc C-6 (34); however, no experimental evidence has been presented for this possibility. Additional information regarding any aspect of CPS linkages to the cell wall is sparse. The present study provides a detailed description of a CPS–PG linkage and demonstrates that, unlike other surface poly- saccharide–PG linkages, it is mediated by a direct glycosidic bond. The results provide a clear demonstration that the phospho- diester bonds and oligosaccharide linkers common to other polysaccharide–PG linkages are missing here. Evidence for the lack of a phosphodiester bond came from resistance of the linkage to HF and absence of detectable phosphorous by either 31PNMRor[32P]-phosphate metabolic labeling. Likewise, there is no evidence for an oligosaccharide linker as: (i) GC/MS analyses demonstrated only CPS- and PG-specific sugars in the expected ratios, and (ii) the molecular mass of a single CPS re- peat unit linked to a PG disaccharide was consistent with the presence of only these sugars. Susceptibility of the CPS–GlcNAc linkage to acetolysis is in- dicative of a 1,6 glycosidic bond, and is consistent with observations Fig. 5. The CPS–PG linkage does not contain phosphate bonds. (A) Re- making linkage to carbons other than the GlcNAc C-6 unlikely, as sistance to HF. GC/MS analysis of HF-treated CPS–PG. (a) Total ion chro- discussed above. During CPS synthesis, Glcp is the matogram of high MW fraction. (b) Selective ion monitoring of high following addition of Glc-1-P to Und-P to form β-D-Glc-1-P-P-Und MW fraction for GlcNAc at m/z 173. (c) Selective ion monitoring of high MW (11). As demonstrated here, the Glc anomeric C-1 remains bonded fraction for MurNAc at m/z 187. (d) Total ion chromatogram of low MW fraction demonstrating teichoic acid sugars ribitol (Rib-ol) and N-acetylga- lactosamine (GalNAc). (B) Absence of phosphodiester bonds. 31P-NMR spec- tra of CPS–PG and HF-treated products. (a) CPS–PG before HF treatment. (b)HighMWCPS–PG fraction obtained after HF treatment. (c)LowMWteichoic acid-containing fraction obtained after HF treatment. (C) Absence of phos- phate. Cell wall (CW) and membrane (M) fractions from bacteria grown with 32P and separated by SDS-polyacrylamide . (a) Phosphor- image identifies incorporating 32P. (b) Immunoblot using antiserum to teichoic acid. (c) Immunoblot using antiserum to CPS serotype 2.

CPS and PG sugars in the expected ratios (Figs. S2 and S3A). The absence of ribitol in this fraction was indicative of HF cleavage of the phosphodiester bonds of teichoic acid repeat units. Q-TOF MS of CPS–PG from serotype 31 revealed a peak corresponding to the mass expected for a single repeat unit of CPS linked by a direct glycosidic bond to a PG disaccharide (Fig. S3B). These results demonstrate the lack of phosphodiester bonds and oligosaccharide linkers in these CPS structures. Discussion Fig. 6. The CPS–PG linkage is a direct glycosidic bond. Mass spectrum of intact Bacterial CPS synthesis occurs by three mechanisms: ABC trans- CPS–PG analyzed by triple Q-TOF MS. Peaks are as follows: 1,435.02 m/z,intactsingle porter dependent (Gram negatives), synthase dependent (Gram repeat unit of CPS linked to a PG disaccharide; 1,361.99, intact single repeat unit of positives), and Wzy dependent (Gram negatives and Gram CPS linked to a PG disaccharide wherein the MurNAc has lost its lactate group.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1620431114 Larson and Yother Downloaded by guest on September 30, 2021 enzyme. WchA use nucleotide sugar donors (Leloir enzymes) and their known active sites are in the cytoplasm. Similar to the proposed CPS–PG transfer mechanism, Wzy recognizes the reducing end of Und-P-P–linked polymers and generates a glyco- sidic linkage to a single Und-P-P–linked repeat unit. A bifunctional Wzy, capable of recognizing both the PG and CPS acceptor sub- strates, could potentially form the CPS–PG linkage. The 96 Wzy CPS serotypes comprise 44 serogroups, wherein the serogroups contain immunologically and chemically related serotypes (3, 4). Wzy exhibit high diversity, with 40 homology groups that are largely serogroup specific (3). These enzymes could potentially provide the specificity necessary for the recognition of such diverse polymers, and the generation of a similar linkage to PG across serotypes. CPS transfer to PG appears to occur essentially at random, as the polymer sizes on the membrane and cell wall are similar and range from very small to very large (15) (Fig. S1). Indeed, the results of the present study demonstrate that a single CPS repeat unit can be transferred to PG. Simultaneous modification of adjacent MurNAc and GlcNAc residues with teichoic acid and CPS, respectively, appears rare, as neither 32P nor immunore- active teichoic acid colocalizes with CPS on PG disaccharides, and only low levels of teichoic acid sugars copurify with CPS–PG. The structure of the S. pneumoniae CPS linkage is distinct among PG-linked polysaccharides. Its identification opens the – Fig. 7. The GlcNAc C-2 and C-3 are not involved in CPS linkage. GC/MS way for comparison with other CPS PG linkages and provides analysis following Smith degradation of CPS–PG. (A) Total ion chromato- the foundation for identifying the linking enzyme and potential gram. The degradation product (blue) is overlaid with a nontreated control inhibitors of its activity. (red). Glc, but not Rha, is largely degraded. (B) Selective ion monitoring for MICROBIOLOGY PG sugars at m/z 173 and 187 following degradation (blue) overlaid with the Materials and Methods untreated control (red). (Inset) MurNAc at higher magnification. kCounts, S. pneumoniae serotype 2 strain D39 was used for CPS isolation. Other kilocounts. strains, growth conditions, procedures for 32P incorporation, and immuno- blotting are described in SI Materials and Methods. analyses – by GC/MS were performed essentially as described previously (39) and are to another in CPS PG, as sodium borodeuteride re- detailed in SI Materials and Methods. duction did not produce a deuterated glucitol. The observation of a single, complete CPS repeat unit linked to GlcNAc is consistent CPS–PG Preparation and Treatments. and cell wall with a that recognizes the reducing end of CPS fractions were generated by incubation of bacteria with mutanolysin and and forms a Glc-1,6-GlcNAc linkage; any other mechanism would autolysin (LytA). CPS–PG was purified from the cell wall fractions using DEAE likely require two enzymes to cleave and transfer an oligosaccharide anion exchange chromatography. Details for enzyme digestions, internal to the polymer chain. Cleavage at the reducing end, be- HF treatment, sodium borodeuteride reduction, Smith degradation (29), and tween the Glc and terminal P of Und-P-P, would leave the latter acetolysis (as in ref. 40 with minor modifications) are provided in SI Materials intact. Similar to the polymerization reaction in reducing end and Methods. High and low MW products were separated using ultrafil- polysaccharide synthesis (35), this would allow for recycling of Und- tration (Amicon; 3,000 Da cutoff). P following hydrolysis of the pyrophosphate bond. Glcp is the most common initiating sugar in CPS synthesis in S. pneumoniae and other Gram-positive bacteria, but other sugars can be used (3). The observation that the CPS–PG linkages of serotypes 2, 8, and 31, which initiate with Glcp,Glcp, and Galf, respectively, are the same, suggests a common linking mechanism may occur across serotypes. The S. pneumoniae enzyme responsible for linking CPS to PG has not been identified, but proteins of the LytR–CpsA–Psr family are proposed to play a role in this and other bacteria (34, 36). The initial suggestion for a ligase activity of these proteins was based on copurification of a polyprenol phosphate, along with phosphotransferase activity associated with recombinant CpsA from S. pneumoniae (37). However, CPS attachment is not affected in S. pneumoniae mutants where cpsA alone is mutated (15, 36, 38) and, as shown here, linkage of CPS to PG does not involve phosphoryl transfer. Thus, proteins other than, or in ad- dition to, CpsA must be involved. The enzyme catalyzing the CPS–PG linkage is expected to be a glycosyltransferase that uses a polyprenol pyrophosphate sugar donor (i.e., a non-Leloir enzyme) and possesses an extrac- – ytoplasmic domain. Such domains are present in the S. pneumoniae Fig. 8. The CPS PG linkage is susceptible to acetolysis. GC/MS analysis of CPS–PG following acetolysis. (A) Total ion chromatogram of the high Wzy polymerase, Wzx transporter, CpsA, CpsC, and the initiating MW fraction. (B) Selective ion monitoring of the high MW fraction at m/z of the WchA family (including CpsE for sero- 173 revealed no GlcNAc. (C) Total ion chromatogram of the low MW fraction. type 2) (3). Of these, only the Wzy and WchA enzymes are known (D) Selective ion monitoring of the low MW fraction at m/z 173. kCounts, to catalyze glycosidic linkages, and only Wzy is a non-Leloir kilocounts.

Larson and Yother PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 Structural Analyses. Purified CPS–PG samples were analyzed using an AB ACKNOWLEDGMENTS. We thank Yuriy Knirel for advice on acetolysis and Sciex 5600 Triple-TOF mass spectrometer. Data were collected in the high Landon Wilson for expertise with Q-TOF MS instrumentation. This work was resolution TOF-MS positive ion mode over a mass range of 400–3,000 m/z. supported by NIH Awards R01 AI28457 (to J.Y.), T32 AI07041 (to T.R.L.), P30 Postacquisition analysis was carried out using PeakView Software version CA13148 [University of Alabama at Birmingham (UAB) Cancer Center High- 31 Field NMR Facility], and the Mizutani Foundation for Glycoscience (J.Y.). The 1.2 (AB Sciex). One-dimensional P-NMR was performed using a 500-MHz UAB Targeted Metabolomics and Proteomics Laboratory is funded in part by AVANCE III HD spectrometer with a triple-resonance broadband inverse NIH Awards S10 RR027822, P30 DK079337 (UAB O’Brien Acute Injury (TBI) probe; 1,024 scans were collected. Additional details of both procedures Center), P30 AR50948 (UAB Skin Disease Research Center), and the UAB Lung are provided in SI Materials and Methods. Health Center and UAB Center for Free Radical .

1. Hyams C, Camberlein E, Cohen JM, Bax K, Brown JS (2010) The Streptococcus pneu- 25. Jansson PE, Lindberg B, Anderson M, Lindquist U, Henrichsen J (1988) Structural moniae capsule inhibits complement activity and neutrophil phagocytosis by multiple studies of the capsular polysaccharide from Streptococcus pneumoniae type 2, a re- mechanisms. Infect Immun 78:704–715. investigation. Carbohydr Res 182:111–117. 2. Limoli DH, Jones CJ, Wozniak DJ (2015) Bacterial extracellular polysaccharides in bi- 26. Yother J, Leopold K, White J, Fischer W (1998) Generation and properties of a ofilm formation and . Microbiol Spectr 3. Streptococcus pneumoniae mutant which does not require choline or analogs for 3. Bentley SD, et al. (2006) Genetic analysis of the capsular biosynthetic locus from all growth. J Bacteriol 180:2093–2101. 90 pneumococcal serotypes. PLoS Genet 2:e31. 27. Yother J (2006) Integration of capsular polysaccharide biosynthesis with metabolic 4. Geno KA, Saad JS, Nahm MH (2017) Discovery of novel pneumococcal serotype, 35D: and pathways in Streptococcus pneumoniae. Virulence Mechanisms of A natural WciG-deficient variant of serotype 35B. J Clin Microbiol 55:1416–1425. Bacterial Pathogens, eds Brogden KA, et al. (ASM Press, Washington, DC), 4th Ed, 5. Pilishvili T, Noggle B, Moore MR (2012) Pneumococcal Disease. VPD Surveillance pp 51–65. th Manual (Centers for Disease Control, Atlanta), 5 Ed, Chap 11. Available at https:// 28. Vollmer W, Tomasz A (2000) The pgdA gene encodes for a peptidoglycan N-acetylglu- www.cdc.gov/vaccines/pubs/surv-manual/chpt11-pneumo.pdf. Accessed May 2, 2017. cosamine deacetylase in Streptococcus pneumoniae. J Biol Chem 275:20496–20501. 6. Hardy GG, Magee AD, Ventura CL, Caimano MJ, Yother J (2001) Essential role for 29. Goldstein IJ, Hay GW, Lewis BA, Smith F (1965) Controlled degradation of polysac- cellular phosphoglucomutase in virulence of type 3 Streptococcus pneumoniae. Infect charides by periodate oxidation, reduction, and hydrolysis. Methods in Carbohydrate – Immun 69:2309 2317. Chemistry, ed Whistler RL (Academic Press, New York), Vol 5, pp 361–370. 7. Magee AD, Yother J (2001) Requirement for capsule in colonization by Streptococcus 30. Aspinall GO (1977) The selective degradation of carbohydrate polymers. Pure Appl – pneumoniae. Infect Immun 69:3755 3761. Chem 49:1105–1134. 8. Nelson AL, et al. (2007) Capsule enhances pneumococcal colonization by limiting 31. Rosenfeld L, Ballou CE (1974) Acetolysis of disaccharides: Comparative kinetics and – mucus-mediated clearance. Infect Immun 75:83 90. mechanism. Carbohydr Res 32:287–298. 9. Whitfield C (2006) Biosynthesis and assembly of capsular polysaccharides in Escher- 32. Willis LM, et al. (2013) Conserved glycolipid termini in capsular polysaccharides syn- – ichia coli. Annu Rev Biochem 75:39 68. thesized by ATP-binding cassette transporter-dependent pathways in Gram-negative 10. Yother J (2011) Capsules of Streptococcus pneumoniae and other bacteria: Paradigms pathogens. Proc Natl Acad Sci USA 110:7868–7873. – for polysaccharide biosynthesis and regulation. Annu Rev Microbiol 65:563 581. 33. Cartee RT, Forsee WT, Yother J (2005) Initiation and synthesis of the Streptococcus 11. Cartee RT, Forsee WT, Bender MH, Ambrose KD, Yother J (2005) CpsE from type 2 pneumoniae type 3 capsule on a phosphatidylglycerol membrane anchor. J Bacteriol Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate 187:4470–4479. to a polyprenyl phosphate acceptor, initiating type 2 capsule repeat unit formation. 34. Chan YG, Kim HK, Schneewind O, Missiakas D (2014) The capsular polysaccharide of J Bacteriol 187:7425–7433. is attached to peptidoglycan by the LytR-CpsA-Psr (LCP) family 12. James DB, Gupta K, Hauser JR, Yother J (2013) Biochemical activities of Streptococcus of enzymes. J Biol Chem 289:15680–15690. pneumoniae serotype 2 capsular glycosyltransferases and significance of suppressor 35. Robbins PW, Bray D, Dankert BM, Wright A (1967) Direction of chain growth in mutations affecting the initiating glycosyltransferase Cps2E. J Bacteriol 195: polysaccharide synthesis. Science 158:1536–1542. 5469–5478. 36. Eberhardt A, et al. (2012) Attachment of capsular polysaccharide to the cell wall in 13. James DB, Yother J (2012) Genetic and biochemical characterizations of enzymes Streptococcus pneumoniae. Microb Drug Resist 18:240–255. involved in Streptococcus pneumoniae serotype 2 capsule synthesis demonstrate that 37. Kawai Y, et al. (2011) A widespread family of bacterial cell wall assembly proteins. Cps2T (WchF) catalyzes the committed step by addition of β1-4 rhamnose, the second EMBO J 30:4931–4941. sugar residue in the repeat unit. J Bacteriol 194:6479–6489. 38. Morona JK, Morona R, Paton JC (2006) Attachment of capsular polysaccharide to the 14. Bushell SR, et al. (2013) Wzi is an outer membrane that underpins group cell wall of Streptococcus pneumoniae type 2 is required for invasive disease. Proc 1 capsule assembly in . Structure 21:844–853. Natl Acad Sci USA 103:8505–8510. 15. Bender MH, Cartee RT, Yother J (2003) Positive correlation between tyrosine phos- 39. Calix JJ, et al. (2012) Biochemical, genetic, and serological characterization of two phorylation of CpsD and capsular polysaccharide production in Streptococcus pneu- capsule subtypes among Streptococcus pneumoniae Serotype 20 strains: Discovery of moniae. J Bacteriol 185:6057–6066. a new pneumococcal serotype. J Biol Chem 287:27885–27894. 16. Deng L, Kasper DL, Krick TP, Wessels MR (2000) Characterization of the linkage be- tween the type III capsular polysaccharide and the bacterial cell wall of group B 40. Oyamada H, et al. (2008) Structural analysis of cell wall mannan of Candida sojae,a – Streptococcus. J Biol Chem 275:7497–7504. new species isolated from defatted soybean flakes. Arch Microbiol 189:483 490. 17. Sørensen UB, Henrichsen J, Chen HC, Szu SC (1990) Covalent linkage between the 41. Avery OT, Macleod CM, McCarty M (1944) Studies on the chemical nature of the capsular polysaccharide and the cell wall peptidoglycan of Streptococcus pneumoniae substance inducing transformation of pneumococcal types: Induction of trans- revealed by immunochemical methods. Microb Pathog 8:325–334. formation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. – 18. Xayarath B, Yother J (2007) Mutations blocking side chain assembly, polymerization, J Exp Med 79:137 158. or transport of a Wzy-dependent Streptococcus pneumoniae capsule are lethal in the 42. Sanz JM, Lopez R, Garcia JL (1988) Structural requirements of choline derivatives for ‘ ’ absence of suppressor mutations and can affect polymer transfer to the cell wall. conversion of pneumococcal amidase. A new single-step procedure for purification – J Bacteriol 189:3369–3381. of this autolysin. FEBS Lett 232:308 312. 19. Fischer W, Behr T, Hartmann R, Peter-Katalinic J, Egge H (1993) Teichoic acid and 43. Berry AM, Lock RA, Hansman D, Paton JC (1989) Contribution of autolysin to virulence lipoteichoic acid of Streptococcus pneumoniae possess identical chain structures. A of Streptococcus pneumoniae. Infect Immun 57:2324–2330. reinvestigation of teichoid acid (C polysaccharide). Eur J Biochem 215:851–857. 44. Yother J, Handsome GL, Briles DE (1992) Truncated forms of PspA that are secreted 20. Kojima N, Araki Y, Ito E (1985) Structure of the linkage units between ribitol teichoic from Streptococcus pneumoniae and their use in functional studies and cloning of the acids and peptidoglycan. J Bacteriol 161:299–306. pspA gene. J Bacteriol 174:610–618. 21. McNeil M, Daffe M, Brennan PJ (1990) Evidence for the nature of the link between 45. Hardy GG, Caimano MJ, Yother J (2000) Capsule biosynthesis and basic in the arabinogalactan and peptidoglycan of mycobacterial cell walls. J Biol Chem 265: Streptococcus pneumoniae are linked through the cellular phosphoglucomutase. 18200–18206. J Bacteriol 182:1854–1863. 22. Mosser JL, Tomasz A (1970) Choline-containing teichoic acid as a structural compo- 46. Dische Z, Shettles LB (1951) A new spectrophotometric test for the detection of nent of pneumococcal cell wall and its role in sensitivity to lysis by an autolytic en- methylpentose. J Biol Chem 192:579–582. zyme. J Biol Chem 245:287–298. 47. Slomiany BL, Meyer K (1972) Isolation and structural studies of sulfated glycoproteins 23. Neuhaus FC, Baddiley J (2003) A continuum of anionic charge: Structures and func- of hog gastric mucosa. J Biol Chem 247:5062–5070. tions of D-alanyl-teichoic acids in gram-positive bacteria. Microbiol Mol Biol Rev 67: 48. Jones JKN, Perry MB (1957) The structure of the type VIII pneumococcus specific 686–723. polysaccharide. J Am Chem Soc 79:2787–2793. 24. Ward JB (1981) Teichoic and teichuronic acids: Biosynthesis, assembly, and location. 49. Batavyal L, Roy N (1983) Structure of the capsular polysaccharide of Diplococcus Microbiol Rev 45:211–243. pneumoniae type 31. Carbohydr Res 119:300–302.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1620431114 Larson and Yother Downloaded by guest on September 30, 2021