O-acetylation controls the glycosylation of bacterial serine-rich repeat glycoproteins

Abstract

The serine-rich repeat (SRR) glycoproteins of gram-positive bacteria are a family of adhesins that bind to a wide range of host ligands, and expression of SRR glycoproteins is linked with enhanced bacterial virulence. The biogenesis of these surface glycoproteins involves their intracellular glycosylation and export via the accessory Sec system. Although all accessory Sec components are required for SRR glycoprotein export, Asp2 of Streptococcus gordonii also functions as an O-acetyltransferase that modifies GlcNAc residues on the SRR adhesin gordonii surface protein B (GspB). Because these GlcNAc residues can also be modified by the glycosyltransferases Nss and Gly, it has been unclear whether the post-translational modification of GspB is coordinated. We now report that acetylation modulates the glycosylation of exported GspB. Loss of O-acetylation due to aps2 mutagenesis led to the export of GspB glycoforms with increased glucosylation of the GlcNAc moieties. Linkage analysis of the GspB glycan revealed that both O-acetylation and glucosylation occurred at the same C6 position on GlcNAc residues and that O-acetylation prevented Glc deposition. Whereas streptococci expressing nonacetylated GspB with increased glucosylation were significantly reduced in their ability to bind human platelets in vitro, deletion of the glycosyltransferases nss and gly in the asp2 mutant restored platelet binding to WT levels. These findings demonstrate that GlcNAc O-acetylation controls GspB glycosylation, such that binding via this adhesin is optimized. Moreover, because O-acetylation has comparable effects on the glycosylation of other SRR adhesins, acetylation may represent a conserved regulatory mechanism for the post-translational modification of the SRR glycoprotein family.

Keywords: O-acetylation; Streptococcus gordonii; accessory; glycoprotein:glucosylation.

Figure 1

Gordonii surface protein (GspB) and accessory Sec operon.A, the schematic of WT GspB and the recombinant constructs, GspB2061 and GspB1060FLAG. B, the gspB-aSec operon. Components of the accessory aSec (aSec) system are shown in green, and glycosyltransferases are in blue. C, sequential steps in the modification of GspB by GtfAB, Nss, and Gly. GspB glycan structures were determined previously by MALDI-TOF mass spectrometry analysis (27). SP is the signal peptide; SRR1 and SRR2 are the serine-rich repeat regions that undergo post-translational modification; Asp, accessory Sec protein; BR, ligand-binding region; GtfAB, glycosyltransferase complex; LPxTG, cell wall anchoring domain.

Figure 2

Loss of GlcNAc O-acetylation leads to an increase in glucosylation of exported GspB by Nss and Gly.A and B, western blot analysis of GspB1060FLAG and GspB2061 exported by isogenic variants of M99 containing a deletion in gtfA (ΔgtfA), gly (Δg), gly and nss (Δgn), and/or an asp2^S362A mutation (asp2∗). Samples of culture media were separated by SDS-PAGE and probed with anti-FLAG antibodies and biotinylated sWGA, to detect levels of GspB and core GlcNAc, respectively. Exported GspB2061 was simultaneously probed with biotinylated sWGA (700-nm channel red) and anti-GspB polyclonal antisera raised against the WT GspB glycan (800-nm channel-green in overlay). C, saponification of GspB glycoforms. Exported GspB2061 glycoforms were treated with 100-mM NaOH at 37 °C for 1 h, to remove O-acetyl moieties. Changes to the GspB glycan were assessed by GspB2061 binding to both sWGA (700-nm channel red) and anti-GspB (800-nm channel green in overlay) glycan probes. GspB, gordonii surface protein.

Figure 3

Structures of O-acetylated and Nss and Gly modified glycans of GspB.A, The structure of O-acetylated GlcNAc was obtained from published glycopeptide CID fragmentation analysis of GspB and the SRR adhesin Srr1 of S. agalactiae (27). B, Structure of GlcNAc modified by both Nss and Gly was obtained by GC-MS profiling of partially methylated alditol acetates (PMAA) derived from glycans released from a recombinant GST-SRR protein co-expressed with GtfAB, Nss, and Gly. GlcNAc carbon positions are numbered. C, PMAA derivatives of the O-glycans released from GST-SRR1. Terminally linked glucose (t-Glc), terminally linked GlcNAc (t-GlcNAc), 2-linked glucose, and 6-linked GlcNAc were identified based on retention times in reference to carbohydrate standards. GspB, gordonii surface protein; GtfAB, glycosyltransferase complex; SRR, serine-rich repeat.

Figure 4

Effect of O-acetylation on the subsequent glycosylation of GspB1060FLAG.A, SDS-PAGE analysis of purified NssH6 after expression in E. coli BL21 and purification on Ni-NTA resin. B, glucosylation of GspB1060FLAG before and after saponification. Acetylated (Δgn) or nonacetylated (Δgn asp2∗) GspB1060FLAG was combined with Nss_H6 and UDP-Glc as indicated. Glucosylation reactions were carried out at 37 °C for 1 h and samples analyzed by SDS-PAGE and Western blot analysis with anti-FLAG antibodies. Samples were saponified by incubation with 100-mM NaOH for 1 h, to release O-acetyl moieties.

Figure 5

Effect of O-acetylation on the binding of Streptococcus gordonii to human platelets. WT strain M99 and mutants PS3536 (M99 asp2^S362A), PS3319 (M99 Δgn), PS3880 (M99 Δgn asp2^S362A), and PS666 (M99 ΔgtfA) expressing glycovariants of GspB were assessed for their binding to immobilized human platelets. Binding is expressed as the percent of input bacteria that remained bound to platelets after repeated washing of the wells (n = 5 + S.D. of triplicate results from a representative experiment). ∗∗p < 0.01, compared with WT M99 or PS3880 as indicated. Of note, no significant differences in platelet binding were seen between M99 and PS388.

Figure 6

Asp2 mediates the O-acetylation of SRR adhesins from other gram-positive pathogens.A, organization of the srr-aSec loci of strain M99 and the homologous regions of S. agalactiae strain A909, Staphylococcus aureus strain ISP479C, and S. mitis strain SF100. The genes encoding the SRR proteins are shown in gray, the aSec system components are in green, and the glycosyltransferases in blue. B, amino acid alignment of the conserved GxSxG motif in Asp2 homologues. The catalytic Ser residues are highlighted in pink. C, western blot analysis of full-length SRR proteins exported by S. aureus and S. agalactiae and truncated SF100-SRRFLAG expressed by S. mitis SF100. Cell wall (CW), culture medium (CM), and protoplast (P) fractions were prepared from strains in the exponential phase of growth. Proteins were separated by SDS-PAGE (3–8%) and analyzed by Western blotting using either biotinylated sWGA or anti-FLAG antibodies. White arrows indicate the position of the WT SRR glycoform. Asp2, aSec protein 2; SRR, serine-rich repeat.

Figure 7

Determined glycan linkages from known SRR protein glycan structures. Glycan structures of Fap1 and SRRP₅₃₆₀₈ as reviewed by Latousakis et al. (6). Monosaccharide symbols follow the Symbol Nomenclature for Glycans system (50). GlcNAc (blue square), glucose (Glc: blue circle), rhamnose (Rha; green triangle).

Figure 8

Model for GspB glycoprotein biogenesis. GspB is sequentially glycosylated by the GtfAB complex that deposits GlcNAc along the SRR region. GlcNAc-modified GspB engages with the aSec system where upon GlcNAc residues are O-acetylated. Before full engagement with SecA2 at the cell membrane, GspB is further modified by Nss and Gly. GtfAB, glycosyltransferase AB complex; GspB, gordonii surface protein B; SRR, serine-rich repeat.