The development and characterization of an E. coli O25B bioconjugate vaccine.

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.

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.

Characterization and immunogenicity of a Shigella flexneri 2a O-antigen bioconjugate vaccine candidate.

Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Vaccination represents a promising preventive measure to fight the burden of the disease, but despite enormous efforts, an efficacious vaccine is not available to date. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of a multivalent conjugate vaccine to prevent shigellosis. This bioconjugation approach has been used to produce the Shigella dysenteriae type O1 conjugate that has been successfully tested in a phase I clinical study in humans. In this report, we describe a similar approach for the production of an additional serotype required for a broadly protective shigellosis vaccine candidate. The Shigella flexneri 2a O-polysaccharide is conjugated to introduced asparagine residues of the carrier protein exotoxin A (EPA) from Pseudomonas aeruginosa by co-expression with the PglB oligosaccharyltransferase. The bioconjugate was purified, characterized using physicochemical methods and subjected to preclinical evaluation in rats. The bioconjugate elicited functional antibodies as shown by a bactericidal assay for S. flexneri 2a. This study confirms the applicability of bioconjugation for the S. flexneri 2a O-antigen, which provides an intrinsic advantage over chemical conjugates due to the simplicity of a single production step and ease of characterization of the homogenous monomeric conjugate formed. In addition, it shows that bioconjugates are able to raise functional antibodies against the polysaccharide antigen.

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).

Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli.

Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Glycoconjugate vaccines consisting of bacterial surface polysaccharides conjugated to carrier proteins are the most effective vaccines for controlling invasive bacterial infections. Nevertheless, the development of a multivalent conjugate vaccine to prevent Shigellosis has been hampered by the complex manufacturing process as the surface polysaccharide for each strain requires extraction, hydrolysis, chemical activation and conjugation to a carrier protein. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of glycoconjugates. Herein, the Shigella dysenteriae type 1 (Sd1) O-polysaccharide is expressed and its functional assembly on an E. coli glycosyl carrier lipid is demonstrated by HPLC analysis and mass spectrometry. The polysaccharide is enzymatically conjugated to specific asparagine residues of the carrier protein by co-expression of the PglB oligosaccharyltransferase and the carrier protein exotoxin A (EPA) from Pseudomonas aeruginosa. The extraction and purification of the Shigella glycoconjugate (Sd1-EPA) and its detailed characterization by the use of physicochemical methods including NMR and mass spectrometry is described. The report shows for the first time that bioconjugation provides a newly developed and improved approach to produce an Sd1 glycoconjugate that can be characterized using state-of-the-art techniques. In addition, this generic process together with the analytical methods is ideally suited for the production of additional Shigella serotypes, allowing the development of a multivalent Shigella vaccine.

A genomewide mutagenesis screen identifies multiple genes contributing to Vi capsular expression in Salmonella enterica serovar Typhi.

A transposon-based, genomewide mutagenesis screen exploiting the killing activity of a lytic ViII bacteriophage was used to identify Salmonella enterica serovar Typhi genes that contribute to Vi polysaccharide capsule expression. Genes enriched in the screen included those within the viaB locus (tviABCDE and vexABCDE) as well as oxyR, barA/sirA, and yrfF, which have not previously been associated with Vi expression. The role of these genes in Vi expression was confirmed by constructing defined null mutant derivatives of S. Typhi, and these were negative for Vi expression as determined by agglutination assays with Vi-specific sera or susceptibility to Vi-targeting bacteriophages. Transcriptome analysis confirmed a reduction in expression from the viaB locus in these S. Typhi mutant derivatives and defined regulatory networks associated with Vi expression.

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component.

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.

Molecular characterization of the viaB locus encoding the biosynthetic machinery for Vi capsule formation in Salmonella Typhi.

The Vi capsular polysaccharide (CPS) of Salmonella enterica serovar Typhi, the cause of human typhoid, is important for infectivity and virulence. The Vi biosynthetic machinery is encoded within the viaB locus composed of 10 genes involved in regulation of expression (tviA), polymer synthesis (tviB-tviE), and cell surface localization of the CPS (vexA-vexE). We cloned the viaB locus from S. Typhi and transposon insertion mutants of individual viaB genes were characterized in Escherichia coli DH5α. Phenotype analysis of viaB mutants revealed that tviB, tviC, tviD and tviE are involved in Vi polymer synthesis. Furthermore, expression of tviB-tviE in E. coli DH5α directed the synthesis of cytoplasmic Vi antigen. Mutants of the ABC transporter genes vexBC and the polysaccharide copolymerase gene vexD accumulated the Vi polymer within the cytoplasm and productivity in these mutants was greatly reduced. In contrast, de novo synthesis of Vi polymer in the export deficient vexA mutant was comparable to wild-type cells, with drastic effects on cell stability. VexE mutant cells exported the Vi, but the CPS was not retained at the cell surface. The secreted polymer of a vexE mutant had different physical characteristics compared to the wild-type Vi.

A conserved acetyl esterase domain targets diverse bacteriophages to the Vi capsular receptor of Salmonella enterica serovar Typhi.

A number of bacteriophages have been identified that target the Vi capsular antigen of Salmonella enterica serovar Typhi. Here we show that these Vi phages represent a remarkably diverse set of phages belonging to three phage families, including Podoviridae and Myoviridae. Genome analysis facilitated the further classification of these phages and highlighted aspects of their independent evolution. Significantly, a conserved protein domain carrying an acetyl esterase was found to be associated with at least one tail fiber gene for all Vi phages, and the presence of this domain was confirmed in representative phage particles by mass spectrometric analysis. Thus, we provide a simple explanation and paradigm of how a diverse group of phages target a single key virulence antigen associated with this important human-restricted pathogen.

Two newly identified SipA domains (F1, F2) steer effector protein localization and contribute to Salmonella host cell manipulation.

Salmonella Typhimurium causes bacterial enterocolitis. The type III secretion system (TTSS)-1 is a key virulence determinant of S. Typhimurium mediating host cell invasion and acute enterocolitis. The TTSS-1 effector protein SipA is transported into host cells, accumulates in characteristic foci at the bacteria-host cell interface, manipulates signalling and affects virulence. Two functional domains of SipA have previously been characterized: The N-terminal SipA region (amino acids 1-105) mediates TTSS-1 transport and the C-terminal SipA 'actin-binding' domain (ABD; amino acids 446-685) manipulates F-actin assembly. Little is known about the central region of SipA. In a deletion analysis we found that the central SipA region harbours two distinct functional domains, F1 and F2. They are involved in SipA focus formation and host manipulation. The F1 domain (amino acids 170-271) drives SipA focus formation and domain F2 (amino acids 280-394) enhances this process by mediating SipA-SipA interactions. SipA variants lacking the F1-, the F2- or the actin binding domain were attenuated in virulence assays, namely host cell invasion and/or virulence in a mouse model for enterocolitis. Our results show that the newly identified SipA domains have distinct functions. Nevertheless, cooperation between the SipA domains F1, F2 and ABD is required to promote Salmonella virulence.