Impact of branching on the conformational heterogeneity of the lipopolysaccharide from Klebsiella pneumoniae: Implications for vaccine design

Highlights

• Antigenic D-Gal-I and D-Gal-III O polysaccharides of K. pneumoniae are studied computationally.

• An extended conformation dominates, though it occurs in both species in different populations.

• The OPS are hindered from interactions with antibodies due to the branched D-Gal-III OPS.

• D-Gal-II regions sample exposed conformations that may be targeted for vaccine development.

• Results suggest a strategy for vaccine development against K. pneumoniae.

Abstract

Resistance of Klebsiella pneumoniae (KP) to antibiotics has motivated the development of an efficacious KP human vaccine that would not be subject to antibiotic resistance. Klebsiella lipopolysaccharide (LPS) associated O polysaccharide (OPS) types have provoked broad interest as a vaccine antigen as there are only 4 that predominate worldwide (O1, O2a, O3, O5). Klebsiella O1 and O2 OPS are polygalactans that share a common D-Gal-I structure, for which a variant D-Gal-III was recently discovered. To understand the potential impact of this variability on antigenicity, a detailed molecular picture of the conformational differences associated with the addition of the D-Gal-III (1 → 4)-α-Galp branch is presented using enhanced-sampling molecular dynamics simulations. In D-Gal-I two major conformational states are observed while the presence of the 1 → 4 branch in D-Gal-III resulted in only a single dominant extended state. Stabilization of the more folded states in D-Gal-I is due to a O4-H⋯O2 hydrogen bond in the linear backbone that cannot occur in D-Gal-III as the O4 is in the Galp(1 → 4)Galp glycosidic linkage. The impact of branching in D-Gal-III also significantly decreases the accessibility of the monosaccharides in the linear backbone region of D-Gal-I, while the accessibility of the terminal D-Gal-II region of the OPS is not substantially altered. The present results suggest that a vaccine that targets both the D-Gal-I and D-Gal-III LPS can be developed by using D-Gal-III as the antigen combined with cross-reactivity experiments using the Gal-II polysaccharide to assure that this region of the LPS is the primary epitope of the antigen.

Introduction

Klebsiella pneumoniae is a gram negative encapsulated bacterial pathogen, common in the environment, and is a major cause of hospital acquired infections, for which the recent development of widespread antimicrobial resistance among clinical isolates has become an urgent threat [1]. Individuals with impaired host defenses due to chronic illness or immunosenescence are generally at highest risk [[2], [3], [4], [5], [6]]. With the limited pipeline of new and novel antibiotics, development of an efficacious Klebsiella vaccine is urgently needed. K. pneumoniae typically expresses both lipopolysaccharide (LPS), comprised of a conserved core polysaccharide (CP) linked to lipid A and a repeating polymer of O polysaccharide (OPS), and a capsular polysaccharide (CPS, K-antigen), both of which contribute to virulence. While there are greater than 80 different Klebsiella capsule serotypes for which no single type predominates, there are only 8 recognized OPS serotypes of which 4 (O1, O2a, O3, O5) account for most human disease globally [2,7,8]. Thus, the development of vaccines based on OPS is preferable due to the lower valency requirement to enable broad coverage. However, variability in the composition of OPS subtypes may impact the utility of OPS as a K. pneumoniae vaccine antigen [[2], [3], [4],[9], [10], [11]].

Klebsiella O1, a poly-galactan, is formed by a short stretch of a D-Galactan-I (D-Gal-I) comprised of repeats of →3)-β-Galf-(1 → 3)-α-Galp-(1→, that is linked at the non-reducing end to a longer stretch of D-galactan-II repeat units (D-Gal-II) that is generated by →3)-β-Galp-(1 → 3)-α-Galp-(1→ (Scheme 1 and Fig. 1). The D-Gal-I structure, when uncapped with D-Gal-II, forms the O2a serotype [4]. A recent report documented a variant of D-Gal-I, that contains a (1 → 4)-α-Galp branch, which has been designated as D-Gal-III [12]. In this work, we investigate the conformational properties associated with the addition of this branch to the K. pneumoniae O1/O2a OPS (Scheme 1) [4,12]. To investigate the conformational properties we apply computational molecular dynamics (MD) simulations with enhanced sampling via Hamiltonian replica exchange, a technique that has been successfully used to elucidate the conformational properties of other polysaccharides [3,4,[13], [14], [15], [16], [17], [18], [19], [20]]. Enhanced sampling was achieved through the use of the Solute Tempering 2 method (HREST) in conjunction with the use of biasing potentials in the context of the correction map (CMAP) [21,22] approach, termed HREST-bpCMAP [18,20,[23], [24], [25], [26]]. This approach is applied in the present study to elucidate the changes in the conformational properties associated with the addition of 1 → 4 branches to the D-Gal-I repeat units (RU) yielding D-Gal-III [27]. The mechanism by which branching on the K. pneumoniae serotypes O1 and O2a that may impact antigenicity is explored in terms of both the conformational properties and accessibility of the monosaccharide components of the LPS.