Effects of Glucosylation and O-Acetylation on the Conformation of Shigella flexneri Serogroup 2 O-Antigen Vaccine Targets

Abstract

Shigellosis is an enteric disease with high morbidity and mortality, particularly in developing countries. There is currently no licensed vaccine available. Most infection is caused by Shigella flexneri, of which 30 serotypes have been recognized based on O-antigen polysaccharide structure. Almost all S. flexneri serotypes share the same repeating unit backbone (serotype Y), with varying glucosylation, O-acetylation and phosphorylation. The O-antigen is the primary vaccine target; the vaccine valency (and hence cost) can be reduced by cross-protection. Our planned systematic conformational study of S. flexneri starts here with 2a, the dominant cause of infection globally. We employ microsecond molecular dynamics simulations to compare the conformation of the unsubstituted serotype Y backbone with the serogroup 2 O-antigens, to investigate the effect of glucosylation and O-acetylation (O-factor 9) on conformation. We find that serotype Y is highly flexible, whereas glucosylation in 2a restricts flexibility and induces C-curve conformations. Further, the glucose side-chains adopt two distinct conformations, corroborated by the antibody-bound crystal structure data. Additional substitution on O-3 of rhamnose A (whether O-acetylation in 2a or glucosylation in 2b) induces helical conformations. Our results suggest that the O-3-acetylated 2a antigen will elicit cross-protection against 2b, as well as other serotypes containing O-factor 9.

Molecular Modeling of the Shigella flexneri Serogroup 3 and 5 O-Antigens and Conformational Relationships for a Vaccine Containing Serotypes 2a and 3a

“…The S. flexneri O-Ags have glycosidic linkages described by two dihedral angles, ϕ and ψ, defined as ϕ = H1-C1-O1-C x ' and ψ = C1-O1-C x '-H x '. These definitions are analogous to ϕ H and ψ H in IUPAC nomenclature and are consistent with our previous carbohydrate modeling [26,38] This work follows our established methodology for the computational study of carbohydrate antigens. Potential of mean force (PMF) calculations for the disaccharide fragments of the O-Ag repeating unit indicate the global minima for each linkage, which are then used to build short 3 RU chains for initial 300 ns MD simulations in solution.…”

“…Figure 3 compares the conformational families for serotype Y (Figure 3a) and the serogroup 2 O-Ags (Figure 3b-d) with the serogroup 3 (Figure 3e,f) and serogroup 5 (Figure 3g,h) O-Ags. As discussed in prior work [26], the flexible Y backbone transitions between extended conformations (Figure 3a Y-1, Y-4, and Y-6) to more curved arrangements of the chain (Figure 3a Y-2, Y-3, and Y-5). Further, we showed that O-factor II (O-4 glucosylation at rhamnose C, serogroup 2) has a dramatic effect on the chain conformations, removing the extended conformations and restricting the O-Ag to a wide range of "C-curves" (Figure 3b).…”

“…However, 3a remains more flexible than 2b and can adopt a wide range of helical conformations (3a-3, 3a-5), as well as partially extended chains (3a-2), fully extended chains (3a-4), and S-bends (3a-6). Therefore, the serogroup 3 O-Ags follow a similar trend to serogroup 2 [26]: substitution on rhamnose C restricts the chains to curved conformations for both serotypes 2a and 3b (Figure 3b,f) and additional substitution on O-3 of rhamnose A shifts the conformations towards helices for serotypes 2a-3Ac, 2b, and 3a (Figure 3c-e). Further, the axially orientated O-acetyl groups of serogroup 3 are readily accessible for antibody binding in both serogroup 3 O-Ags, which supports the reported immunodominance of O-factor 6 [31].…”

“…VMD's internal cluster command was employed to calculate the conformational clusters in the production runs with an RMSD fit of the non-hydrogen atoms in the central 4 RUs with a cut-off of 5.5 Å. Clusters comprising less than 5% of the simulation were excluded. The conformations of the previously published serotype Y and serogroup 2 simulations [26] were recalculated under these criteria for a fair comparison between the serogroups.…”

“…Here we define r as the distance between C-2 of rhamnose B in RU1 and C-1 of rhamnose C in RU6 (Figure 2a). Figure 2 compares the r time series and corresponding histograms for the simulations of the serotype Y backbone and the serogroup 2 O-Ags previously published [26] with simulations of serogroup 3 (Figure 2f,g) and serogroup 5 (Figure 2h,i).…”