HMW1C-like proteins in two categories: Those encoded by loci that contain obvious substrate genes and those encoded by isolated genes without adjacent substrate genes.

<p>Numbers below <i>hmw1C</i>-like genes represent translated protein sequence percent identity/similarity when compared to <i>H. influenzae</i> HMW1C. (<b>A</b>) HMW1C-like enzymes encoded in apparent TPS systems. (<b>B</b>) HMW1C-like enzymes encoded in loci without obvious surface protein targets for glycosylation. Abbreviations: <i>Hi</i>, <i>H. influenzae</i> 86-028NP; <i>Bsp</i>, <i>Burkholderia</i> species GCE1003; <i>Ec</i>, Enterotoxigenic <i>E. coli</i> H10407; <i>Yp</i>, <i>Y. pseudotuberculosis</i> YPIII; <i>Ap</i>, <i>Actinobacillus pleuropneumoniae</i> L20; <i>Hd</i>, <i>H. ducreyi</i> HD35000; <i>Kk</i>, <i>K. kingae</i> 269–492; <i>hyp</i>, hypothetical with no conserved domains; <i>hyp</i>¹, predicted lipoprotein; <i>hyp</i>², predicted UDP-glcNAc carboxyvinyltransferase; <i>hyp</i>³, predicted 2 C-methyl-D erythritol-4-phosphate cytidyltransferase; <i>hyp</i>⁴, predicted deoxyguanosinetriphosphate triphosphohydrolase.</p

Glycosylation by HMW1C may play several roles in promoting HMW1 stability, export, folding, and function.

<p>HMW1C has the potential to contribute to several different processes that occur during HMW1 synthesis and transit across the inner and outer membranes. First of all, HMW1C glycosylates the HMW1 adhesin in the cytoplasm and is likely to be involved in the stability of the HMW1 adhesin during or after its synthesis. Glycosylation may contribute to stability of HMW1 in the (<b>A</b>) cytoplasm or (<b>C</b>) periplasm <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-Grass1" target="_blank">[5]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-StGeme1" target="_blank">[7]</a>. Alternatively, the HMW1C protein may improve stability of HMW1 by acting as a (<b>B</b>) chaperone prior to secretion of the adhesin. It is unlikely that the activity of HMW1C is required for export of the adhesin across either the inner or outer membrane, as fully processed HMW1 is found in the supernatant in the absence of HMW1C <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-Grass1" target="_blank">[5]</a>. It is unclear whether glycosylation influences interaction of HMW1 with the (<b>D</b>) HMW1B periplasmic domain prior to transit, (<b>E</b>) the HMW1B pore during transit, or (<b>F</b>) the docking region of HMW1B upon surface tethering <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-Grass1" target="_blank">[5]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-Buscher2" target="_blank">[11]</a>. It is also unclear whether glycosylation participates in (<b>G</b>) protein folding upon export. Evidence from the nonglycosylated <i>Bordetella</i> prototypic, two-partner, secreted adhesin FHA indicates that this adhesin remains unfolded in the cytoplasm and folds very rapidly upon export via its TpsB secretion pore <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-Hodak1" target="_blank">[30]</a>. One hypothesis is that the energy generated by this rapid folding is at least part of what drives export of TpsA proteins across the outer membrane <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-JacobDubuisson1" target="_blank">[2]</a>. Finally, glycosylation of HMW1 may be required for (<b>H</b>) adherence to host cells or host interaction in a particular niche.</p

Initial velocity of ApHMW1C.

<p>(<b>A</b>) Double reciprocal plots of the initial velocity of ApHMW1C as a function of UDP-glucose concentration at the fixed His-HMW1ct concentrations, as indicated (inset). (<b>B</b>) Double reciprocal plots of the initial velocity of ApHMW1C as a function of His-HMW1ct concentration at the fixed UDP-glucose concentrations, as indicated (inset). The true K_m values of UDP-glucose and His-HMW1ct corresponded to 54.5 µM and 2.3 µM, respectively, as obtained by eq. 2.</p

Ability of ApHMW1C to complement a deficiency in HMW1C.

<p>Panel <b>A</b> shows Western immunoblots of whole cell sonicates of <i>E. coli</i> BL21(DE3)/pACYC-HMW1ΔC (lane 1), <i>E. coli</i> BL21(DE3)/pACYC-HMW1ΔC + pET45b-HMW1C (lane 2), and <i>E. coli</i> BL21(DE3)/pACYC-HMW1ΔC + pET45b-ApHMW1C (lane 3). Lane 1 contains twice as much protein as loaded in lanes 2 and 3 to increase the visibility of the non-glycosylated HMW1 species. The blot in the upper panel was performed with a guinea pig antiserum reactive with HMW1, and the blot in the lower panel was performed with a guinea pig antiserum reactive with <i>H. influenzae</i> HMW1C. The asterisk indicates the glycosylated HMW1 pro-protein, and the plus sign indicates the non-glycosylated HMW1 pro-protein. The diamond indicates the glycosylated HMW1 mature protein, and the circle indicates the non-glycosylated HMW1 mature protein. Panel <b>B</b> shows <i>in vitro</i> adherence results comparing adherence by <i>E. coli</i> BL21(DE3)/pACYC-HMW1ΔC (<i>hmw1AB</i>), <i>E. coli</i> BL21(DE3)/pACYC-HMW1ΔC + pET45b-HMW1C (<i>hmw1AB</i> + <i>hmw1C</i>), and <i>E. coli</i> BL21(DE3)/pACYC-HMW1ΔC + pET45b-ApHMW1C (<i>hmw1AB</i> + <i>Aphmw1C</i>) to Chang epithelial cells. Bars and error bars represent mean and standard error measurements from a representative assay with measurements performed in triplicate.</p

Glycosylation of HMW1ct by ApHMW1C.

<p>To define the donor substrate specificity of ApHMW1C, glycosylation reactions were carried out in the reaction buffer with (R-lanes) or without (C-lanes) ApHMW1C using different UDP (or GDP) activated sugars. HMW1ct (without fusion tag) was used as the acceptor protein (lanes 1, and 3 to 6). As a control, His-tagged HMW1ct (His-HMW1ct) was also tested in a reaction with UDP-glucose as the donor sugar (lanes 2). (<b>A</b>) After the glycosylation reactions, samples were separated by SDS-PAGE, and the gel was stained with Coomassie Blue. (<b>B</b>) In parallel, a duplicate gel was transferred to a PVDF membrane and subjected to a detection reaction using the GlycoProfile III Fluorescent Glycoprotein Detection kit (Sigma). Glycosylated HMW1ct proteins are indicated by arrows: ‘a’ and ‘c’ are glycosylated HMW1ct reacted with UDP-glucose and UDP-galactose, respectively, and ‘b’ is glycosylated His-HMW1ct reacted with UDP-glucose. The lanes labeled “M1,” “M2,” and “HMW1ct only” indicate pre-staining protein markers (Precision Plus Protein Standards, Bio-Rad), glycosylated protein markers (ProteoProfile PTM Marker, Sigma), and HMW1ct only as a control, respectively.</p

Primers used for cloning, site-directed mutagenesis, and sequence analysis.

<p>Primers used for cloning, site-directed mutagenesis, and sequence analysis.</p

N-linked glycosylation of HMW1ct.

<p>The glycosylation reactions were carried out in standard conditions using single (N1348Q, N1352Q, N1366Q) mutants of His-HMW1ct as acceptor proteins and UDP-glucose as donor substrate. (<b>A</b>) After the glycosylation reaction, the samples were separated by SDS-PAGE, and the gel was stained with Coomassie Blue. (<b>B</b>) A duplicate gel was transferred to a PVDF membrane and subjected to a detection reaction using the GlycoProfile III Fluorescent Glycoprotein Detection kit (Sigma). The lanes labeled “Native” and “His-HMW1ct only” are control reaction samples with and without ApHMW1C, respectively. M1 is a pre-staining protein marker (Precision Plus Protein Standards, Bio-Rad), and M2 is a glycosylated protein marker (ProteoProfile PTM Marker, Sigma).</p

Kinetic parameters of ApHMWC and its derivative proteins.

a<p>These are apparent values, determined by varying the concentration of one substrate (sugar donor substrate) at a fixed concentration of the second (protein acceptor).</p

Specificity of HMW1ct glycosylation.

<p>The glycosylation reactions were carried out in standard conditions using His-HMW1ct as acceptor protein and UDP-glucose or UDP-galactose as donor substrate. (<b>A</b>) At each time point, an aliquot of the reaction was stopped by adding an equal volume of 2X SDS-PAGE sample buffer and followed by heating at 95°C for 4 min. (<b>B</b>) At each time point, SDS-PAGE samples were prepared as in A. However, two hrs after reaction with the first donor substrate, the second donor substrate was added to the reaction, as indicated. All samples were separated by 12% SDS-PAGE, and the gel was stained with Coomassie blue. The distinct shifts due to incorporated sugars are indicated by symbols (•, 0 hexose; ▪, 2 hexoses; ⋆, 4 or 5 hexoses; and ⋆’, 5 or 6 hexoses). (<b>C</b>) The glycosylation reactions were carried out in standard conditions using double mutants of His-HMW1ct (N1348Q/N1352Q, N1348Q/N1366Q, and N1352Q/N1366Q) by ApHMW1C using UDP-glucose or UDP-galactose as donor substrates. C1, C2, and C3 indicate control reactions without ApHMW1C. Samples were separated by 12% SDS-PAGE and were stained with Coomassie blue. (<b>D</b>) In parallel, a duplicated gel was transferred to a PVDF membrane and subjected to a detection reaction using the GlycoProfile III Fluorescent Glycoprotein Detection kit (Sigma). The glycosylated proteins by UDP-glucose or by UDP-galactose are indicated by arrows. (<b>E</b>) Model of hexose modifications at Asn-1348, Asn-1352, and Asn-1366.</p

Structure of capsule polysaccharide repeating unit (top) and galactan exopolysaccharide repeating unit (bottom).

<p>Structure of capsule polysaccharide repeating unit (top) and galactan exopolysaccharide repeating unit (bottom).</p