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

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

Structural and Functional Studies of Membrane Proteins For Future Development of Antimicrobial Drugs

Antibiotic resistance is a world-wide occurring problem which threatens human health. Without development of any new and effective antibiotics, the rapid growth of antibiotic-resistant bacterial infections could put society in a situation resembling the pre-antibiotic era when a simple lung infection could kill a human being. This thesis presents two venues for targeting antibiotic resistance. Pathogenic bacteria present in mucus rich environments are able to utilize host-derived sialic acid either as an alternative food source or by incorporating sialic acid to their surface glycoconjugates as a way to evade the host´s immune system. Hence, molecular mimicry enables bacteria to secure an ecological niche for survival. Transport of scavenged sialic acid into the cytoplasm of bacteria occurs through specific membrane bound sialic acid transporters. The cell wall is an essential protective barrier for bacteria. The membrane bound enzyme MraY catalyzes the synthesis of lipid I, an intermediate step in the biosynthesis of peptidoglycan, the cell wall of bacteria. This thesis presents work aimed to structurally and functionally characterize sialic acid transporters and MraY for future development of antibacterial drugs. Starting with a broad approach for expression and purification of sialic acid transporters resulted in low-resolution diffracting crystals of the Pasteurella multocida sialic acid TRAP transporter. In addition the X-ray crystallography structure of the sialic acid transporter SiaT from Proteus mirabilis was determined at 1.95 Å resolution in a substrate-bound outward-open conformation revealing a new sodium site. Furthermore, SiaT transporters have been characterized in vivo and the sialic acid specificity has been characterized for SiaT from Staphylococcus aureus. Structural comparison between MraY and the human homologue GPT have highlighted regions where to modify the natural product inhibitor tunicamycin to selectively target MraY. Further characterization of tunicamycin analogues identified potent inhibitors with reduced eukaryotic toxicity

Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature

Cytochrome c oxidase catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane. Although an extremely well-studied biological system, the molecular mechanism of proton pumping by cytochrome c oxidase is still not understood. Here we report a method to produce large quantities of highly diffracting microcrystals of ba3-type cytochrome c oxidase from Thermus thermophilus suitable for serial femtosecond crystallography. The room-temperature structure of cytochrome c oxidase is solved to 2.3 Å resolution from data collected at an X-ray Free Electron Laser. We find overall agreement with earlier X-ray structures solved from diffraction data collected at cryogenic temperature. Previous structures solved from synchrotron radiation data, however, have shown conflicting results regarding the identity of the active-site ligand. Our room-temperature structure, which is free from the effects of radiation damage, reveals that a single-oxygen species in the form of a water molecule or hydroxide ion is bound in the active site. Structural differences between the ba3-type and aa3-type cytochrome c oxidases around the proton-loading site are also described

Table_1_The Sodium Sialic Acid Symporter From Staphylococcus aureus Has Altered Substrate Specificity.PDF

<p>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.</p