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

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.

Keywords: SiaT; Staphylococcus aureus; antibiotic resistance; sialic acids; sodium solute symporter.

Figure 1

Bacterial growth experiments demonstrate SaSiaT function. (A) The chemical structures of Neu5Ac and Neu5Gc. (B) Growth of E. coli wild type (orange), ΔnanT (green), and its complemented derivative Δnant_siaT (blue) on Neu5Ac and Neu5Gc. While ΔnanT is unable to utilize Neu5Ac and Neu5Gc, SaSiaT is able to rescue the growth of ΔnanT on both Neu5Ac and Neu5Gc sialic acids as the sole carbon source.

Figure 2

Recombinant SaSiaT can be stably purified and occupies a predominantly single oligomeric state. (A) Size-exclusion chromatography trace of SaSiaT at its final purification step. To the left of a main dominant peak there is a small shoulder, which may represent aggregate. (B) van Holde-Weischet sedimentation coefficient distributions show a dominant component at ~8 S. (C) 2DSA-Monte Carlo analysis of sedimentation velocity data of SaSiaT at 0.6 mg/mL, as implemented by UltraScan III, shows a main peak comprising 70% of the signal.

Figure 3

Orthogonal binding experiments demonstrate substrate ambiguity. (A) Microscale thermophoresis binding assay to measure the affinity of SaSiaT for Neu5Ac and Neu5Gc sialic acids. Raw data are shown with the fit for three independent experiments with Neu5Ac (left) and Neu5Gc (middle). The K_d values are reported as the mean ± uncertainty in the mean of the fit using the signal from Thermophoresis + T-jump, from triplicate experiments, where n = 1 (we define n as the number of different recombinant protein preparations, which we view as equivalent to biological replicates). The K_d and associated error of each fit is given in Supplementary Table 2. The shift in K_d between both sialic acids is shown (right). SaSiaT has a tighter affinity for Neu5Gc than Neu5Ac. (B) Representative isothermal titration calorimetry raw data (top panel) and binding isotherm (bottom panel) of one isothermal titration calorimetry experiment obtained by successive titration of Neu5Ac (left) or Neu5Gc (right) with purified SaSiaT. The fit of a single binding site is shown in the bottom panels (black line). K_d values are reported as the mean ± SEM of the fit from three experiments using different protein preparations (n = 3).

Figure 4

Sequence alignment and homology modeling probe substrate ambiguity. (A) Amino acid sequence alignment of SaSiaT with SiaT transporters from eight additional bacterial species (Wahlgren et al., 2018). SiaT transporters from S. aureus, P. mirabilis, Morganella morganii, S. enterica, Vibrio fischeri, Plesiomonas shigelloides, Photobacterium profundum, Clostridium perfringens, C. difficile, and Streptococcus pneumoniae are aligned. Important residues in the Neu5Ac binding site in PmSiaT (pdb entry 5nv9) are shown. Residues highlighted with black boxes are highly conserved, and important residues implicated in Neu5Ac binding in PmSiaT are numbered according to SaSiaT. (B) Superposition of the SaSiaT homology model (green) and PmSiaT (gray) with Neu5Ac bound (black). Residues are labeled according to SaSiaT, with PmSiaT in parentheses. A water molecule from PmSiaT is shown in yellow. PmSiaT coordinates are from pdb entry 5nv9. Black dashed lines depict hydrogen bonds, or a salt bridge with Arg136. On the right, the binding site has been rotated 90° and the substituted residues are shown. (C) The PmSiaT-Neu5Ac interaction network (Wahlgren et al., 2018) with Gln82, Phe78, and Phe243 is represented as a Ligplot⁺ diagram (Laskowski and Swindells, 2011) using PDB entry 5nv9. Hydrogen bonds (dashed lines), hydrophobic contacts (arcs with spokes), and an interacting water molecule (yellow) are shown.

Figure 5

Proteoliposome assays demonstrate the ability to transport sialic acid, which is dependent on Na+. (A) Proteoliposome transport was started by adding 50 μM [³H]-Neu5Ac together with 25 mM NaCl to proteoliposomes reconstituted with purified recombinant SaSiaT. In °, □, valinomycin was added to facilitate K⁺ movement prior to transport. In •, ethanol was added instead of valinomycin as a control. On the left Y-axis, specific transport activity is reported. In □, transport was measured in empty liposomes, with transport in empty liposomes reported on the right Y-axis. Transport was stopped at indicated times by passing proteoliposomes through Sephadex-G75 columns. Data were fitted to the first-order rate equation. (B) The transport of [³H]-Neu5Ac over a range of concentrations in the presence of 25 mM NaCl was measured in proteoliposomes reconstituted with purified recombinant SaSiaT, with an imposed K⁺ diffusion membrane potential, over 5 min. Data were fitted to the Michaelis-Menten equation. (C) The transport of 50 μM [³H]-Neu5Ac in the presence of NaCl over a range of concentrations was measured in proteoliposomes reconstituted with purified recombinant SaSiaT, with an imposed K⁺ diffusion membrane potential, over 5 min. Data were fitted to the Hill equation. All data are presented as mean ± SD from three independent experiments.