The Sodium Sialic Acid Symporter From Staphylococcus aureus Has Altered Substrate Specificity.

Rachel A North,Sarah A Kessans,Jane R Allison,Elin Dunevall,Elin Claesson,Renwick C J Dobson,Tatiana P Soares Da Costa,Mariafrancesca Scalise,Matthew A Perugini,Cesare Indiveri,Weixiao Y Wahlgren,S Ramaswamy,Rosmarie Friemann,Daniela M Remus

doi:10.3389/fchem.2018.00233

Abstract

Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from Staphylococcus aureus (SaSiaT). We demonstrate that SaSiaT rescues an Escherichia coli strain lacking its endogenous sialic acid transporter when grown on the sialic acids N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SaSiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SaSiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SaSiaT that may explain the altered specificity. SaSiaT is shown to be electrogenic, and transport is dependent upon more than one Na+ ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SaSiaT, and developing the in vivo and in vitro assay systems, our work underpins the design of inhibitors to this transporter.

Highlights

Mammalian cell surfaces are decorated with complex glycoconjugates, such as glycoproteins and glycolipids
To demonstrate sialic acid transport by SaSiaT, we first showed that SaSiaT rescues the growth of an E. coli strain lacking the endogenous NanT sialic acid transporter when grown on Neu5Ac or Neu5Gc (Figure 1)
We demonstrate that SaSiaT is a functional sialic acid transporter, with a considerably higher binding affinity for Neu5Gc over Neu5Ac

Summary

Introduction

Mammalian cell surfaces are decorated with complex glycoconjugates, such as glycoproteins and glycolipids. Found at the terminal non-reducing positions of these cell-surface glycoconjugates are negatively charged sialic acids, which mediate a diverse array of cellular interactions, recognition and adhesion. Sialic acids comprise a large family of nine-carbon acidic monosaccharides, the most common of which is Nacetylneuraminic acid (Neu5Ac). While Neu5Ac is ubiquitously synthesized, the closely related sialic acid, N-glycolylneuraminic acid (Neu5Gc) is not. Neu5Ac and Neu5Gc sialic acids are widely expressed on mammalian tissues, human cells do not synthesize Neu5Gc. Neu5Ac and Neu5Gc sialic acids are widely expressed on mammalian tissues, human cells do not synthesize Neu5Gc This is because humans have an inactivating mutation in the gene encoding CMP-N-acetylneuraminic acid hydroxylase, the rate-limiting enzyme for the generation of Neu5Gc in the cells of other mammals (Varki, 2001)

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