Saponification analysis of exported GspB736flag and GspB1060flag.

<p>A) GspB736flag and GspB1060flag secreted from parental strains PS1225 (lane 1) and PS921 (lane 3) and the corresponding derivative strains harboring the S362A mutation within <i>asp2</i>, PS3539 (lane 2), PS3540 (lane 4) were separated by SDS-PAGE and subjected to Western blot analysis using anti-FLAG antibodies to detect GspB levels. Saponified reaction products were obtained through incubation with 100 mM NaOH to release ester-linked acetate (lanes 5–8). B) Blot A was simultaneously probed for GlcNAc reactivity of GspB736flag and GspB1060flag before and after saponfication. GlcNAc reactivity was assessed by lectin blot analysis using biotinylated sWGA as a GlcNAc probe. Western and lectin blot analysis of secreted GspB variants are representative of 3 different genetic transformants analyzed from each strain.</p

List of GspB736flag glycopeptides identified by Q-TOF LC/MS.

<p>List of GspB736flag glycopeptides identified by Q-TOF LC/MS.</p

O-acetylation of the serine-rich repeat glycoprotein GspB is coordinated with accessory Sec transport

<div><p>The serine-rich repeat (SRR) glycoproteins are a family of adhesins found in many Gram-positive bacteria. Expression of the SRR adhesins has been linked to virulence for a variety of infections, including streptococcal endocarditis. The SRR preproteins undergo intracellular glycosylation, followed by export via the accessory Sec (aSec) system. This specialized transporter is comprised of SecA2, SecY2 and three to five accessory Sec proteins (Asps) that are required for export. Although the post-translational modification and transport of the SRR adhesins have been viewed as distinct processes, we found that Asp2 of <i>Streptococcus gordonii</i> also has an important role in modifying the SRR adhesin GspB. Biochemical analysis and mass spectrometry indicate that Asp2 is an acetyltransferase that modifies <i>N</i>-acetylglucosamine (GlcNAc) moieties on the SRR domains of GspB. Targeted mutations of the predicted Asp2 catalytic domain had no effect on transport, but abolished acetylation. Acetylated forms of GspB were only detected when the protein was exported via the aSec system, but not when transport was abolished by <i>secA2</i> deletion. In addition, GspB variants rerouted to export via the canonical Sec pathway also lacked <i>O</i>-acetylation, demonstrating that this modification is specific to export via the aSec system. Streptococci expressing GspB lacking O-acetylated GlcNAc were significantly reduced in their ability bind to human platelets <i>in vitro</i>, an interaction that has been strongly linked to virulence in the setting of endocarditis. These results demonstrate that Asp2 is a bifunctional protein involved in both the post-translational modification and transport of SRR glycoproteins. In addition, these findings indicate that these processes are coordinated during the biogenesis of SRR glycoproteins, such that the adhesin is optimally modified for binding. This requirement for the coupling of modification and export may explain the co-evolution of the SRR glycoproteins with their specialized glycan modifying and export systems.</p></div

Proposed model for transport-mediated O-acetylation of GspB.

<p>GspB is glycosylated by the GtfAB complex to deposit GlcNAc along the SRR regions [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006558#ppat.1006558.ref025" target="_blank">25</a>], [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006558#ppat.1006558.ref013" target="_blank">13</a>]. Glycosylated GspB maybe delivered to SecA2 via an interaction with one or more Asps [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006558#ppat.1006558.ref018" target="_blank">18</a>], [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006558#ppat.1006558.ref019" target="_blank">19</a>]. Asp3 binds both Asp1 and Asp2 forming a protein complex [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006558#ppat.1006558.ref017" target="_blank">17</a>] localizing at the membrane [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006558#ppat.1006558.ref039" target="_blank">39</a>]. Collectively, the Asps enhance the interaction between the GspB preprotein and SecA2 enabling full substrate engagement with the translocation (SecA2/SecY2) machinery [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006558#ppat.1006558.ref016" target="_blank">16</a>], [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006558#ppat.1006558.ref019" target="_blank">19</a>]. Following full engagement of aSec translocation, GspB is O-acetylated by Asp2 (GspB glycosylation by Nss and Gly have been omitted for clarity).</p

Determination of acetylesterase activity of Asp2.

<p>A) MalE-Asp2-H6 and the MalE-Asp2^S362A-H6 catalytic mutant following expression in <i>E</i>. <i>coli</i> BL21 were purified on amylose resin, followed by Ni-NTA purification, and were analyzed by SDS-PAGE and Coomassie staining of 5 μg of the final purified recombinant protein. B) Time course of <i>p</i>-nitrophenol release from <i>p</i>NP-Ac in the presence of glycosylated SRR1 (red circle), MalE-Asp2-H6 (black open square), MalE-Asp2^S362A-H6 (grey triangle), SRR1 with MalE-Asp2-H6 (black square) or SRR1 with MalE-Asp2^S362A-H6 (grey open triangle). Reactions were incubated at 25°C in 50 mM sodium phosphate buffer (pH 7) and rates of <i>p</i>-nitrophenol release were measured spectrophotometrically at 405 nm. Assays were performed in triplicate and data expressed as mean ± S.D nmol <i>p</i>NP released. Results shown are representative of at least two independent experiments.</p

Assessment of anti-FLAG and sWGA reactivity of GspB736flag when exported through the general secretory pathway.

<p>A) Western blot analysis of GlcNAc-modified GspB736flag exported through the aSec system from the Δ<i>gly-nss</i> parental strain PS3309 (lane 1) and derivative strain harboring the Asp2 S362A mutation, PS3540 (lane 2), or when rerouted to the Sec pathway (via the *G3 signal sequence mutation [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006558#ppat.1006558.ref026" target="_blank">26</a>]) by the Δ<i>gly-nss</i> accessory Sec deficient strains PS3316 (lane 3), PS3317 (lane 4). B) sWGA reactivity of GspB736flag exported via the aSec or Sec system in defined glycosylation backgrounds. C) GlcNAc reactivity of secreted GspB736flag following saponification. GlcNAc reactivity was assessed through lectin blot analysis using biotinylated sWGA as a GlcNAc probe. D) Densitometry analysis of GspB736flag levels (green bars) and GlcNAc reactivity before (red bars) and after saponification treatment (open red bars). The y-axis represents GspB736flag levels and GlcNAc reactivity based on band intensity analysis via LI-COR imaging. Western and lectin blot analysis of secreted GspB variants together with corresponding densitometry analysis are representative of at least three different genetic transformants of each strain analyzed.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Effects of transport upon sWGA reactivity of GspB736flag.

<p>A) Upper panel shows Western blot analysis of GspB736flag exported from a <i>nss</i> and <i>gly</i> deletion (Δ<i>gn</i>) variant derivative strain PS3309 (lane 1) and intracellular GspB736flag expressed in the aSec deficient <i>nss</i> and <i>gly</i> deletion variant strains PS3310 (lane 2) and PS3584 (lane 3). Anti-FLAG antibodies were used to measure GspB736flag protein levels, while GlcNAc reactivity was assessed by lectin blot analysis using biotinylated sWGA as a GlcNAc probe. Lower panel shows densitometry analysis of GspB736flag levels and GlcNAc reactivity was determined by band intensity analysis via LI-COR imaging. B) Western blot analysis of GspB736flag exported from strains PS3309 (Δ<i>gn</i>)(lane 1), PS3310 (Δ<i>gn</i>Δ<i>secA2</i>)(lane 2) and PS3605 (Δ<i>gn</i>Δ<i>secA2</i>::<i>asp2</i>^S362A)(lane 3). (M) media fraction, (P) protoplast fraction. GspB736flag was detected with anti-FLAG antibodies. C) GlcNAc reactivity of GspB736flag before and after saponfication of GspB736flag obtained from protoplasts in the defined aSec background. Non-exported GspB736flag was expressed in the following accessory Sec/Δ<i>gn</i> deletion strains, PS3310 (lane 1), PS3605 (lane 2). Saponified intracellular GspB736flag was achieved through incubation with 100 mM NaOH to release ester-linked acetate. Western and lectin blot analysis of secreted GspB variants, together with corresponding densitometry analysis, are representative of at least three different genetic transformants analyzed for each strain.</p

Effects of mutating the Asp2 catalytic triad upon the export of GspB736flag.

<p>Western blot analysis of Asp2-dependent export of GspB736flag by <i>S</i>. <i>gordonii</i> Δ<i>asp2</i> strain PS1244 (lane 1), parental strain PS1225 (lane 2) and derivative strains harboring the designated alanine substitution within the catalytic triad of Asp2, PS3539 (lane 3), PS3551 (lanes 4), PS3552 (lane 5), PS3553 (lane 6), PS3554 (lane 7). Culture media (M) and protoplasts (P) were collected from exponentially growing strains. Proteins were separated by SDS-PAGE and analyzed by Western blotting, using anti-FLAG antibody to detect GspB736flag. The data shown is representative of at least three different genetic transformants for each strain.</p

Molecular Basis for the Attachment of S‑Layer Proteins to the Cell Wall of <i>Bacillus anthracis</i>

Bacterial surface (S) layers are paracrystalline arrays of protein assembled on the bacterial cell wall that serve as protective barriers and scaffolds for housekeeping enzymes and virulence factors. The attachment of S-layer proteins to the cell walls of the <i>Bacillus cereus sensu lato</i>, which includes the pathogen <i>Bacillus anthracis</i>, occurs through noncovalent interactions between their S-layer homology domains and secondary cell wall polysaccharides. To promote these interactions, it is presumed that the terminal <i>N</i>-acetylmannosamine (ManNAc) residues of the secondary cell wall polysaccharides must be ketal-pyruvylated. For a few specific S-layer proteins, the O-acetylation of the penultimate <i>N</i>-acetylglucosamine (GlcNAc) is also required. Herein, we present the X-ray crystal structure of the SLH domain of the major surface array protein Sap from <i>B. anthracis</i> in complex with 4,6-<i>O</i>-ketal-pyruvyl-β-ManNAc-(1,4)-β-GlcNAc-(1,6)-α-GlcN. This structure reveals for the first time that the conserved terminal SCWP unit is the direct ligand for the SLH domain. Furthermore, we identify key binding interactions that account for the requirement of 4,6-<i>O</i>-ketal-pyruvyl-ManNAc while revealing the insignificance of the O-acetylation on the GlcNAc residue for recognition by Sap