Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence.

Melissa Robb,Samantha J King,Sarah Shapiro-Ward,Shireen A Woodiga,Michael D L Suits,Joanne K Hobbs,Harry Brumer,Alisdair B Boraston,Hasan Yesilkaya,Nicholas Mcgregor

doi:10.1371/journal.ppat.1006090

Abstract

The carbohydrate-rich coating of human tissues and cells provide a first point of contact for colonizing and invading bacteria. Commensurate with N-glycosylation being an abundant form of protein glycosylation that has critical functional roles in the host, some host-adapted bacteria possess the machinery to process N-linked glycans. The human pathogen Streptococcus pneumoniae depolymerizes complex N-glycans with enzymes that sequentially trim a complex N-glycan down to the Man3GlcNAc2 core prior to the release of the glycan from the protein by endo-β-N-acetylglucosaminidase (EndoD), which cleaves between the two GlcNAc residues. Here we examine the capacity of S. pneumoniae to process high-mannose N-glycans and transport the products. Through biochemical and structural analyses we demonstrate that S. pneumoniae also possesses an α-(1,2)-mannosidase (SpGH92). This enzyme has the ability to trim the terminal α-(1,2)-linked mannose residues of high-mannose N-glycans to generate Man5GlcNAc2. Through this activity SpGH92 is able to produce a substrate for EndoD, which is not active on high-mannose glycans with α-(1,2)-linked mannose residues. Binding studies and X-ray crystallography show that NgtS, the solute binding protein of an ABC transporter (ABCNG), is able to bind Man5GlcNAc, a product of EndoD activity, with high affinity. Finally, we evaluated the contribution of EndoD and ABCNG to growth of S. pneumoniae on a model N-glycosylated glycoprotein, and the contribution of these enzymes and SpGH92 to virulence in a mouse model. We found that both EndoD and ABCNG contribute to growth of S. pneumoniae, but that only SpGH92 and EndoD contribute to virulence. Therefore, N-glycan processing, but not transport of the released glycan, is required for full virulence in S. pneumoniae. To conclude, we synthesize our findings into a model of N-glycan processing by S. pneumoniae in which both complex and high-mannose N-glycans are targeted, and in which the two arms of this degradation pathway converge at ABCNG.

Highlights

The covalent attachment of carbohydrates to macromolecules, such as proteins and lipids, to form glycoconjugates is one of the most abundant modifications of molecules in nature
S. pneumoniae relies on its ability to process the carbohydrates presented on the surface of host cells for full-virulence
We examine the capability of the bacterium to process high-mannose N-linked sugars, a heretofore unknown ability for S. pneumoniae

Summary

Introduction

The covalent attachment of carbohydrates to macromolecules, such as proteins and lipids, to form glycoconjugates is one of the most abundant modifications of molecules in nature. The glycans displayed on glycoproteins and glycolipids in and on the outer surface of cells help to form the complex carbohydrate-rich glycocalyx that surround cells and contribute to key functions such as cell-cell interactions, cell signaling, and physical protection [1]. N-glycans are essential to the proper function of glycoproteins by providing a range of biochemical properties important to the folding, stability and activity of their protein scaffold, enabling these glycoconjugates to assume their essential roles in a wide range of physiological processes in the human body [4]. Glycoproteins in the human body with this modification contribute to an abundance of critical biological roles including structural functions, protection of tissues, immune response, hormone signaling, cell and molecule attachment, blood coagulation, and many more

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