%0 Journal Article %J Microb Biotechnol %D 2018 %T Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies. %A Bairy, Sneha %A Gopalan, Lakshmi Narayanan %A Setty, Thanuja Gangi %A Srinivasachari, Sathya %A Manjunath, Lavanyaa %A Kumar, Jay Prakash %A Guntupalli, Sai R %A Bose, Sucharita %A Nayak, Vinod %A Ghosh, Swagatha %A Sathyanarayanan, Nitish %A Caing-Carlsson, Rhawnie %A Wahlgren, Weixiao Yuan %A Friemann, Rosmarie %A Ramaswamy, S %A Neerathilingam, Muniasamy %X

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.

%B Microb Biotechnol %V 11 %P 420-428 %8 2018 Mar %G eng %N 2 %R 10.1111/1751-7915.13041 %0 Journal Article %J Biophys Rev %D 2018 %T "Just a spoonful of sugar...": import of sialic acid across bacterial cell membranes. %A North, Rachel A %A Horne, Christopher R %A Davies, James S %A Remus, Daniela M %A Muscroft-Taylor, Andrew C %A Goyal, Parveen %A Wahlgren, Weixiao Yuan %A Ramaswamy, S %A Friemann, Rosmarie %A Dobson, Renwick C J %X

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.

%B Biophys Rev %V 10 %P 219-227 %8 2018 Apr %G eng %N 2 %R 10.1007/s12551-017-0343-x %0 Journal Article %J Front Chem %D 2018 %T The Sodium Sialic Acid Symporter From Has Altered Substrate Specificity. %A North, Rachel A %A Wahlgren, Weixiao Y %A Remus, Daniela M %A Scalise, Mariafrancesca %A Kessans, Sarah A %A Dunevall, Elin %A Claesson, Elin %A Soares da Costa, Tatiana P %A Perugini, Matthew A %A Ramaswamy, S %A Allison, Jane R %A Indiveri, Cesare %A Friemann, Rosmarie %A Dobson, Renwick C J %X

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 (SiaT). We demonstrate that SiaT rescues an strain lacking its endogenous sialic acid transporter when grown on the sialic acids -acetylneuraminic acid (Neu5Ac) or -glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SiaT 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 SiaT that may explain the altered specificity. SiaT 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 SiaT, and developing the and assay systems, our work underpins the design of inhibitors to this transporter.

%B Front Chem %V 6 %P 233 %8 2018 %G eng %R 10.3389/fchem.2018.00233 %0 Journal Article %J Nat Commun %D 2018 %T Substrate-bound outward-open structure of a Na-coupled sialic acid symporter reveals a new Na site. %A Wahlgren, Weixiao Y %A Dunevall, Elin %A North, Rachel A %A Paz, Aviv %A Scalise, Mariafrancesca %A Bisignano, Paola %A Bengtsson-Palme, Johan %A Goyal, Parveen %A Claesson, Elin %A Caing-Carlsson, Rhawnie %A Andersson, Rebecka %A Beis, Konstantinos %A Nilsson, Ulf J %A Farewell, Anne %A Pochini, Lorena %A Indiveri, Cesare %A Grabe, Michael %A Dobson, Renwick C J %A Abramson, Jeff %A Ramaswamy, S %A Friemann, Rosmarie %X

Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na ions. One Na binds to the conserved Na2 site, while the second Na binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na sites regulate N-acetylneuraminic acid transport.

%B Nat Commun %V 9 %P 1753 %8 2018 May 01 %G eng %N 1 %R 10.1038/s41467-018-04045-7 %0 Journal Article %J Acta Crystallogr F Struct Biol Commun %D 2017 %T Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum. %A Caing-Carlsson, Rhawnie %A Goyal, Parveen %A Sharma, Amit %A Ghosh, Swagatha %A Setty, Thanuja Gangi %A North, Rachel A %A Friemann, Rosmarie %A Ramaswamy, S %K Adenosine Triphosphate %K Amino Acid Sequence %K Bacterial Proteins %K Binding Sites %K Cloning, Molecular %K Crystallography, X-Ray %K Escherichia coli %K Fusobacterium nucleatum %K Gene Expression %K Genetic Vectors %K Hexosamines %K Models, Molecular %K Phosphotransferases (Alcohol Group Acceptor) %K Protein Binding %K Protein Conformation, alpha-Helical %K Protein Conformation, beta-Strand %K Protein Interaction Domains and Motifs %K Protein Multimerization %K Recombinant Proteins %K Sequence Alignment %K Sequence Homology, Amino Acid %K Substrate Specificity %X

Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5'-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.

%B Acta Crystallogr F Struct Biol Commun %V 73 %P 356-362 %8 2017 Jun 01 %G eng %N Pt 6 %R 10.1107/S2053230X17007439