Exploring and Exploiting Acceptor Preferences of the Human Polysialyltransferases as a Basis for an Inhibitor Screen.

α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

A biosynthetic route for polysialylating proteins in Escherichia coli.

Polysialic acid (polySia) is a posttranslational modification found on only a handful of proteins in the central nervous and immune systems. The addition of polySia to therapeutic proteins improves pharmacokinetics and reduces immunogenicity. To date, polysialylation of therapeutic proteins has only been achieved in vitro by chemical or chemoenzymatic strategies. In this work, we develop a biosynthetic pathway for site-specific polysialylation of recombinant proteins in the cytoplasm of Escherichia coli. The pathway takes advantage of a bacterial cytoplasmic polypeptide-glycosyltransferase to establish a site-specific primer on the target protein. The glucose primer is extended by glycosyltransferases derived from lipooligosaccharide, lipopolysaccharide and capsular polysaccharide biosynthesis from different bacterial species to synthesize long chain polySia. We demonstrate the new biosynthetic route by modifying green fluorescent proteins and a therapeutic DARPin (designed ankyrin repeat protein).

Engineering the product profile of a polysialyltransferase.

Oligo- and polysaccharides have myriad applications as therapeutic reagents from glycoconjugate vaccines to matrices for tissue engineering. Polysaccharide length may vary over several orders of magnitude and is a critical determinant of both their physical properties and biological activities. Therefore, the tailored synthesis of oligo- and polysaccharides of defined size is a major goal for glycoengineering. By mutagenesis and screening of a bacterial polysialyltransferase (polyST), we identified a single-residue switch that controls the size distribution of polymeric products. Specific substitutions at this site yielded distributive enzymes that synthesize polysaccharides with narrow size distribution ideal for glycoengineering applications. Mechanistic investigation revealed that the wild-type enzyme has an extended binding site that accommodates at least 20 residues of the growing polymer; changes in affinity along this binding site allow fine-tuning of the enzyme's product distribution.

Dissection of hexosyl- and sialyltransferase domains in the bifunctional capsule polymerases from Neisseria meningitidis W and Y defines a new sialyltransferase family.

Crucial virulence determinants of disease causing Neisseria meningitidis species are their extracellular polysaccharide capsules. In the serogroups W and Y, these are heteropolymers of the repeating units (→6)-α-d-Gal-(1→4)-α-Neu5Ac-(2→)n in NmW and (→6)-α-d-Glc-(1→4)-α-Neu5Ac-(2→)n in NmY. The capsule polymerases, SiaDW and SiaDY, which synthesize these highly unusual polymers, are composed of two predicted GT-B fold domains separated by a large stretch of amino acids (aa 399-762). We recently showed that residues critical to the hexosyl- and sialyltransferase activity are found in the predicted N-terminal (aa 1-398) and C-terminal (aa 763-1037) GT-B fold domains, respectively. Here we use a mutational approach and synthetic fluorescent substrates to define the boundaries of the hexosyl- and sialyltransferase domains. Our results reveal that the active sialyltransferase domain extends well beyond the predicted C-terminal GT-B domain and defines a new glycosyltransferase family, GT97, in CAZy (Carbohydrate-Active enZYmes Database).

Surface Cross-Linking by Macromolecular Tethers Enhances Virus-like Particles' Resilience to Mucosal Stress Factors.

Virus-like particles (VLPs) are emerging as nanoscaffolds in a variety of biomedical applications including delivery of vaccine antigens and cargo such as mRNA to mucosal surfaces. These soft, colloidal, and proteinaceous structures (capsids) are nevertheless susceptible to mucosal environmental stress factors. We cross-linked multiple capsid surface amino acid residues using homobifunctional polyethylene glycol tethers to improve the persistence and survival of the capsid to model mucosal stressors. Surface cross-linking enhanced the stability of VLPs assembled from Acinetobacter phage AP205 coat proteins in low pH (down to pH 4.0) and high protease concentration conditions (namely, in pig and mouse gastric fluids). Additionally, it increased the stiffness of VLPs under local mechanical indentation applied using an atomic force microscopy cantilever tip. Small angle X-ray scattering revealed an increase in capsid diameter after cross-linking and an increase in capsid shell thickness with the length of the PEG cross-linkers. Moreover, surface cross-linking had no effect on the VLPs' mucus translocation and accumulation on the epithelium of in vitro 3D human nasal epithelial tissues with mucociliary clearance. Finally, it did not compromise VLPs' function as vaccines in mouse subcutaneous vaccination models. Compared to PEGylation without cross-linking, the stiffness of surface cross-linked VLPs were higher for the same length of the PEG molecule, and also the lifetimes of surface cross-linked VLPs were longer in the gastric fluids. Surface cross-linking using macromolecular tethers, but not simple conjugation of these molecules, thus offers a viable means to enhance the resilience and survival of VLPs for mucosal applications.

A single amino acid toggles Escherichia coli polysialyltransferases between mono- and bifunctionality.

The enteropathogenic Escherichia coli K92 synthesizes a unique capsule consisting of polysialic acid (polySia) with alternating α2,8- and α2,9-linkages. The fact that a single enzyme is responsible for the synthesis of these alternating regioisomeric linkages raises questions as to how this controlled bifunctionality is achieved mechanistically. Aiming to identify the sequence elements responsible for dual regiospecificity, we have utilized a high-throughput polysialyltransferase (polyST) activity screen to explore the relevant sequence space between this enzyme and its close monofunctional homolog from E. coli K1. The linkage specificity of selected mutants was subsequently confirmed using a polySia permethylation linkage analysis technique. We have identified a single amino acid exchange at residue 52 that toggles these enzymes between mono and dual regiospecificity. The results have implications for the mechanism by which the E. coli K92 polyST achieves bifunctional elongation.

Bacterial genome-wide association study substantiates papGII of Escherichia coli as a major risk factor for urosepsis.

BACKGROUND: Urinary tract infections (UTIs) are among the most common bacterial infections worldwide, often caused by uropathogenic Escherichia coli. Multiple bacterial virulence factors or patient characteristics have been linked separately to progressive, more invasive infections. In this study, we aim to identify pathogen- and patient-specific factors that drive the progression to urosepsis by jointly analysing bacterial and host characteristics. METHODS: We analysed 1076 E. coli strains isolated from 825 clinical cases with UTI and/or bacteraemia by whole-genome sequencing (Illumina). Sequence types (STs) were determined via srst2 and capsule loci via fastKaptive. We compared the isolates from urine and blood to confirm clonality. Furthermore, we performed a bacterial genome-wide association study (bGWAS) (pyseer) using bacteraemia as the primary clinical outcome. Clinical data were collected by an electronic patient chart review. We concurrently analysed the association of the most significant bGWAS hit and important patient characteristics with the clinical endpoint bacteraemia using a generalised linear model (GLM). Finally, we designed qPCR primers and probes to detect papGII-positive E. coli strains and prospectively screened E. coli from urine samples (n = 1657) at two healthcare centres. RESULTS: Our patient cohort had a median age of 75.3 years (range: 18.00-103.1) and was predominantly female (574/825, 69.6%). The bacterial phylogroups B2 (60.6%; 500/825) and D (16.6%; 137/825), which are associated with extraintestinal infections, represent the majority of the strains in our collection, many of which encode a polysaccharide capsule (63.4%; 525/825). The most frequently observed STs were ST131 (12.7%; 105/825), ST69 (11.0%; 91/825), and ST73 (10.2%; 84/825). Of interest, in 12.3% (13/106) of cases, the E. coli pairs in urine and blood were only distantly related. In line with previous bGWAS studies, we identified the gene papGII (p-value < 0.001), which encodes the adhesin subunit of the E. coli P-pilus, to be associated with 'bacteraemia' in our bGWAS. In our GLM, correcting for patient characteristics, papGII remained highly significant (odds ratio = 5.27, 95% confidence interval = [3.48, 7.97], p-value < 0.001). An independent cohort of cases which we screened for papGII-carrying E. coli at two healthcare centres further confirmed the increased relative frequency of papGII-positive strains causing invasive infection, compared to papGII-negative strains (p-value = 0.033, chi-squared test). CONCLUSIONS: This study builds on previous work linking papGII with invasive infection by showing that it is a major risk factor for progression from UTI to bacteraemia that has diagnostic potential.

A high-throughput screen for polysialyltransferase activity.

Polysialic acid is common to humans and a few bacterial pathogens and it holds great potential for the development of new therapeutic reagents. Currently, the bacterial polysialyltransferases (polySTs) are the only source of polysialic acid for research and biotechnological purposes either directly, by enzymatic polysialylation of therapeutic proteins, or indirectly, by harvest of polysialic acid from bacterial fermentation. Further engineering and optimization of these enzymes is hindered by the lack of high-throughput screening methodologies for polysialyltransferase activity. Here we report the development of an efficient in vivo activity screen for bacterial polySTs. The screen exploits complementation of a dormant capsule export complex in the expression strain, Escherichia coli BL21-Gold(DE3). This strain was metabolically engineered to synthesize CMP-Neu5Ac, the donor sugar for the polysialylation reaction. Using the new strain, a colony blotting procedure that enables the routine testing of more than 10(4) polyST genes was developed. To test the usefulness of the methodology, we screened a library of N-terminally truncated polySTs derived from the Neisseria meningitidis serogroup B (NmB)-polyST. We identified truncations that remove a putative membrane interaction domain, resulting in soluble and active enzymes.

A universal fluorescent acceptor for high-performance liquid chromatography analysis of pro- and eukaryotic polysialyltransferases.

Polysialyltransferases (polySTs) play critical roles in diverse biological processes, including neural development, tumorigenesis, and bacterial pathogenesis. Although the bacterial enzymes are presumed to have evolved to provide molecular mimics of the host-specific polysialic acid, no analytical technique is currently available to facilitate a direct comparison of the bacterial and vertebrate enzymes. Here we describe a new fluorescent acceptor, a 1,2-diamino-4,5-methylenedioxybenzene (DMB)-labeled trimer of α2,8-linked sialic acid (DMB-DP3), which primes both pro- and eukaryotic polySTs. High-performance liquid chromatography separation and fluorescence detection (HPLC-FD) of reaction products enabled the sensitive and quantitative detection of polyST activity, even using cell lysates as enzyme source, and revealed product profiles characteristic of each enzyme. Single product resolution afforded by this assay system revealed mechanistic insights into a kinetic lag phase exhibited by the polyST from Neisseria meningitidis serogroup B during chain elongation. DMB-DP3 is the first fluorescent acceptor shown to prime the mammalian polySTs. Moreover, product profiles obtained for the two murine polySTs provided direct biochemical evidence for enzymatic properties that had, until now, only been inferred from the analysis of biological samples. With DMB-DP3, we introduce a universal acceptor that provides an easy, fast, and reliable system for the comprehensive mechanistic and comparative analysis of polySTs.

Biosynthesis of Glycoconjugate Virus-like Particles (VLPs).

The outermost surface of bacterial pathogens consists primarily of complex carbohydrate structures-polysaccharides, glycolipids, and glycoproteins. To raise a long-lasting and effective immune response against carbohydrate antigens, they generally require covalent attachment to an immunogenic carrier protein-a so-called glycoconjugate vaccine. One hurdle to the development of glycoconjugate vaccines is that carbohydrate antigens remain inaccessible to recombinant production. Thus, the carbohydrate antigen is typically purified from the pathogen and then chemically conjugated to an immunogenic protein. Recent developments in the field of bacterial glycoengineering have opened the opportunity for total recombinant production of glycoconjugate vaccines. In this method, we describe the production of proteinaceous, virus-like particles (VLPs) bearing the conserved N-glycan of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumoniae.

Cytoplasmic glycoengineering enables biosynthesis of nanoscale glycoprotein assemblies.

Glycosylation of proteins profoundly impacts their physical and biological properties. Yet our ability to engineer novel glycoprotein structures remains limited. Established bacterial glycoengineering platforms require secretion of the acceptor protein to the periplasmic space and preassembly of the oligosaccharide substrate as a lipid-linked precursor, limiting access to protein and glycan substrates respectively. Here, we circumvent these bottlenecks by developing a facile glycoengineering platform that operates in the bacterial cytoplasm. The Glycoli platform leverages a recently discovered site-specific polypeptide glycosyltransferase together with variable glycosyltransferase modules to synthesize defined glycans, of bacterial or mammalian origin, directly onto recombinant proteins in the E. coli cytoplasm. We exploit the cytoplasmic localization of this glycoengineering platform to generate a variety of multivalent glycostructures, including self-assembling nanomaterials bearing hundreds of copies of the glycan epitope. This work establishes cytoplasmic glycoengineering as a powerful platform for producing glycoprotein structures with diverse future biomedical applications.

Screening a natural product-based combinatorial library using FTICR mass spectrometry.

This manuscript reports the use of Fourier transform ion cyclotron resonance mass spectrometry to screen a combinatorially generated natural product-based library for binding affinity to bovine carbonic anhydrase II (bCAII). The fungal natural product 3-chloro-4-hydroxyphenylacetamide was the library template, with 11 secondary amide analogues of this template constituting the combinatorial library. 2-(3-Chloro-4-hydroxyphenyl)-N-(4-sulfamoylphenethyl)acetamide (compound 11) of this library was identified as a tight binding inhibitor of bCAII, by detection of a noncovalent complex corresponding to [bCAII+11] in the mass spectrum. A competitive bCAII enzyme binding assay validated the mass spectrometry screening result. The equilibrium dissociation constant (K(i)) for 11 was measured as 77.4 nM. Preliminary structure-activity investigations of the bioactive natural product analogue are also reported.