Virulence of the emerging pathogen, Burkholderia pseudomallei, depends upon the O-linked oligosaccharyltransferase, PglL.

Aim: We sought to characterize the contribution of the O-OTase, PglL, to virulence in two Burkholderia spp. by comparing isogenic mutants in Burkholderia pseudomallei with the related species, Burkholderia thailandensis. Materials & methods: We utilized an array of in vitro assays in addition to Galleria mellonella and murine in vivo models to assess virulence of the mutant and wild-type strains in each Burkholderia species. Results: We found that pglL contributes to biofilm and twitching motility in both species. PglL uniquely affected morphology; cell invasion; intracellular motility; plaque formation and intergenus competition in B. pseudomallei. This mutant was attenuated in the murine model, and extended survival in a vaccine-challenge experiment. Conclusion: Our data support a broad role for pglL in bacterial fitness and virulence, particularly in B. pseudomallei

Production and efficacy of a low-cost recombinant pneumococcal protein polysaccharide conjugate vaccine.

Streptococcus pneumoniae is the leading cause of bacterial pneumonia. Although this is a vaccine preventable disease, S. pneumoniae still causes over 1 million deaths per year, mainly in children under the age of five. The biggest disease burden is in the developing world, which is mainly due to unavailability of vaccines due to their high costs. Protein polysaccharide conjugate vaccines are given routinely in the developed world to children to induce a protective antibody response against S. pneumoniae. One of these vaccines is Prevnar13, which targets 13 of the 95 known capsular types. Current vaccine production requires growth of large amounts of the 13 serotypes, and isolation of the capsular polysaccharide that is then chemically coupled to a protein, such as the diphtheria toxoid CRM197, in a multistep expensive procedure. In this study, we design, purify and produce novel recombinant pneumococcal protein polysaccharide conjugate vaccines in Escherichia coli, which act as mini factories for the low-cost production of conjugate vaccines. Recombinant vaccine efficacy was tested in a murine model of pneumococcal pneumonia; ability to protect against invasive disease was compared to that of Prevnar13. This study provides the first proof of principle that protein polysaccharide conjugate vaccines produced in E. coli can be used to prevent pneumococcal infection. Vaccines produced in this manner may provide a low-cost alternative to the current vaccine production methodology

Cytoplasmic glycoengineering of Apx toxin fragments in the development of Actinobacillus pleuropneumoniae glycoconjugate vaccines.

BACKGROUND: Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and represents a major burden to the livestock industry. Virulence can largely be attributed to the secretion of a series of haemolytic toxins, which are highly immunogenic. A. pleuropneumoniae also encodes a cytoplasmic N-glycosylation system, which involves the modification of high molecular weight adhesins with glucose residues. Central to this process is the soluble N-glycosyl transferase, ngt, which is encoded in an operon with a subsequent glycosyl transferase, agt. Plasmid-borne recombinant expression of these genes in E. coli results in the production of a glucose polymer on peptides containing the appropriate acceptor sequon, NX(S/T). However to date, there is little evidence to suggest that such a glucose polymer is formed on its target peptides in A. pleuropneumoniae. Both the toxins and glycosylation system represent potential targets for the basis of a vaccine against A. pleuropneumoniae infection. RESULTS: In this study, we developed cytoplasmic glycoengineering to construct glycoconjugate vaccine candidates composed of soluble toxin fragments modified by glucose. We transferred ngt and agt to the chromosome of Escherichia coli in order to generate a native-like operon for glycoengineering. A single chromosomal copy of ngt and agt resulted in the glucosylation of toxin fragments by a short glycan, rather than a polymer. CONCLUSIONS: A vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression

Genome-wide saturation mutagenesis of Burkholderia pseudomallei K96243 predicts essential genes and novel targets for antimicrobial development

This is the final version of the article. Available from the publisher via the DOI in this record.Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 10(6) transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. IMPORTANCE: Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.This work was partially funded by the Defence Science and Technology Laboratories (DSTL)

Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines.

BACKGROUND: Glycoengineering, in the biotechnology workhorse bacterium, Escherichia coli, is a rapidly evolving field, particularly for the production of glycoconjugate vaccine candidates (bioconjugation). Efficient production of glycoconjugates requires the coordinated expression within the bacterial cell of three components: a carrier protein, a glycan antigen and a coupling enzyme, in a timely fashion. Thus, the choice of a suitable E. coli host cell is of paramount importance. Microbial chassis engineering has long been used to improve yields of chemicals and biopolymers, but its application to vaccine production is sparse. RESULTS: In this study we have engineered a family of 11 E. coli strains by the removal and/or addition of components rationally selected for enhanced expression of Streptococcus pneumoniae capsular polysaccharides with the scope of increasing yield of pneumococcal conjugate vaccines. Importantly, all strains express a detoxified version of endotoxin, a concerning contaminant of therapeutics produced in bacterial cells. The genomic background of each strain was altered using CRISPR in an iterative fashion to generate strains without antibiotic markers or scar sequences. CONCLUSIONS: Amongst the 11 modified strains generated in this study, E. coli Falcon, Peregrine and Sparrowhawk all showed increased production of S. pneumoniae serotype 4 capsule. Eagle (a strain without enterobacterial common antigen, containing a GalNAc epimerase and PglB expressed from the chromosome) and Sparrowhawk (a strain without enterobacterial common antigen, O-antigen ligase and chain length determinant, containing a GalNAc epimerase and chain length regulators from Streptococcus pneumoniae) respectively produced an AcrA-SP4 conjugate with 4 × and 14 × more glycan than that produced in the base strain, W3110. Beyond their application to the production of pneumococcal vaccine candidates, the bank of 11 new strains will be an invaluable resource for the glycoengineering community

Ferric Citrate Regulator FecR Is Translocated across the Bacterial Inner Membrane via a Unique Twin-Arginine Transport-Dependent Mechanism.

In Escherichia coli, citrate-mediated iron transport is a key nonheme pathway for the acquisition of iron. Binding of ferric citrate to the outer membrane protein FecA induces a signal cascade that ultimately activates the cytoplasmic sigma factor FecI, resulting in transcription of the fecABCDE ferric citrate transport genes. Central to this process is signal transduction mediated by the inner membrane protein FecR. FecR spans the inner membrane through a single transmembrane helix, which is flanked by cytoplasm- and periplasm-orientated moieties at the N and C termini. The transmembrane helix of FecR resembles a twin-arginine signal sequence, and the substitution of the paired arginine residues of the consensus motif decouples the FecR-FecI signal cascade, rendering the cells unable to activate transcription of the fec operon when grown on ferric citrate. Furthermore, the fusion of beta-lactamase C-terminal to the FecR transmembrane helix results in translocation of the C-terminal domain that is dependent on the twin-arginine translocation (Tat) system. Our findings demonstrate that FecR belongs to a select group of bitopic inner membrane proteins that contain an internal twin-arginine signal sequence.IMPORTANCE Iron is essential for nearly all living organisms due to its role in metabolic processes and as a cofactor for many enzymes. The FecRI signal transduction pathway regulates citrate-mediated iron import in many Gram-negative bacteria, including Escherichia coli The interactions of FecR with the outer membrane protein FecA and cytoplasmic anti-sigma factor FecI have been extensively studied. However, the mechanism by which FecR inserts into the membrane has not previously been reported. In this study, we demonstrate that the targeting of FecR to the cytoplasmic membrane is dependent on the Tat system. As such, FecR represents a new class of bitopic Tat-dependent membrane proteins with an internal twin-arginine signal sequence

PglB function and glycosylation efficiency is temperature dependent when the pgl locus is integrated in the Escherichia coli chromosome.

BACKGROUND: Campylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens. To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni. RESULTS: We generated a glycoengineering E. coli strain containing the conserved C. jejuni heptasaccharide coding region integrated in its chromosome as a model glycan. This methodology confers three advantages: (i) reduction of plasmids and antibiotic markers used for PGCT, (ii) swift generation of many glycan-protein combinations and consequent rapid identification of the most antigenic proteins or peptides, and (iii) increased genetic stability of the polysaccharide coding-region. In this study, by using the model glycan expressing strain, we were able to test proteins from C. jejuni, Pseudomonas aeruginosa (both Gram-negative), and Clostridium perfringens (Gram-positive) as acceptors. Using this pgl integrant E. coli strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from C. jejuni (double-hit vaccines), and two glycoconjugates, where the glycan antigen is conjugated to a detoxified toxin from a different pathogen (single-hit vaccines). Because the downstream application of Live Attenuated Vaccine Strains (LAVS) against C. jejuni is to be used in poultry, which have a higher body temperature of 42 °C, we investigated the effect of temperature on protein expression and glycosylation in the E. coli pgl integrant strain. CONCLUSIONS: We determined that glycosylation is temperature dependent and that for the combination of heptasaccharide and carriers used in this study, the level of PglB available for glycosylation is a step limiting factor in the glycosylation reaction. We also demonstrated that temperature affects the ability of PglB to glycosylate its substrates in an in vitro glycosylation assay independent of its transcriptional level

Inactivation of bpsl1039-1040 ATP-binding cassette transporter reduces intracellular survival in macrophages, biofilm formation and virulence in the murine model of Burkholderia pseudomallei infection.

Burkholderia pseudomallei, a gram-negative intracellular bacillus, is the causative agent of a tropical infectious disease called melioidosis. Bacterial ATP-binding cassette (ABC) transporters import and export a variety of molecules across bacterial cell membranes. At present, their significance in B. pseudomallei pathogenesis is poorly understood. We report here characterization of the BPSL1039-1040 ABC transporter. B. pseudomallei cultured in M9 medium supplemented with nitrate, demonstrated that BPSL1039-1040 is involved in nitrate transport for B. pseudomallei growth under anaerobic, but not aerobic conditions, suggesting that BPSL1039-1040 is functional under reduced oxygen tension. In addition, a nitrate reduction assay supported the function of BPSL1039-1040 as nitrate importer. A bpsl1039-1040 deficient mutant showed reduced biofilm formation as compared with the wild-type strain (P = 0.027) when cultured in LB medium supplemented with nitrate under anaerobic growth conditions. This reduction was not noticeable under aerobic conditions. This suggests that a gradient in oxygen levels could regulate the function of BPSL1039-1040 in B. pseudomallei nitrate metabolism. Furthermore, the B. pseudomallei bpsl1039-1040 mutant had a pronounced effect on plaque formation (P < 0.001), and was defective in intracellular survival in both non-phagocytic (HeLa) and phagocytic (J774A.1 macrophage) cells, suggesting reduced virulence in the mutant strain. The bpsl1039-1040 mutant was found to be attenuated in a BALB/c mouse intranasal infection model. Complementation of the bpsl1039-1040 deficient mutant with the plasmid-borne bpsl1039 gene could restore the phenotypes observed. We propose that the ability to acquire nitrate for survival under anaerobic conditions may, at least in part, be important for intracellular survival and has a contributory role in the pathogenesis of B. pseudomallei

Actinobacillus pleuropneumoniae serovar 8 predominates in England and Wales

This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (BBSRC grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G018553/1) and Zoetis (formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) Consortium

Pathotyping the Zoonotic Pathogen Streptococcus suis: Novel Genetic Markers To Differentiate Invasive Disease-Associated Isolates from Non-Disease-Associated Isolates from England and Wales.

Streptococcus suis is one of the most important zoonotic bacterial pathogens of pigs, causing significant economic losses to the global swine industry. S. suis is also a very successful colonizer of mucosal surfaces, and commensal strains can be found in almost all pig populations worldwide, making detection of the S. suis species in asymptomatic carrier herds of little practical value in predicting the likelihood of future clinical relevance. The value of future molecular tools for surveillance and preventative health management lies in the detection of strains that genetically have increased potential to cause disease in presently healthy animals. Here we describe the use of genome-wide association studies to identify genetic markers associated with the observed clinical phenotypes (i) invasive disease and (ii) asymptomatic carriage on the palatine tonsils of pigs on UK farms. Subsequently, we designed a multiplex PCR to target three genetic markers that differentiated 115 S. suis isolates into disease-associated and non-disease-associated groups, that performed with a sensitivity of 0.91, a specificity of 0.79, a negative predictive value of 0.91, and a positive predictive value of 0.79 in comparison to observed clinical phenotypes. We describe evaluation of our pathotyping tool, using an out-of-sample collection of 50 previously uncharacterized S. suis isolates, in comparison to existing methods used to characterize and subtype S. suis isolates. In doing so, we show our pathotyping approach to be a competitive method to characterize S. suis isolates recovered from pigs on UK farms and one that can easily be updated to incorporate global strain collections.This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) Knowledge Transfer Network CASE studentship co-funded by Zoetis (previously Pfizer Animal Health UK) and with significant contribution from BQP Ltd (Award Reference: BB/L502479/1). Funding bodies provided scholarship support but had no part in study design, data collection, analysis and interpretation of data or in writing the manuscript. AWT is supported by a BBSRC Longer and Larger (LoLa) grant (Award Reference: BB/G019274/1). LAW is supported by a Dorothy Hodgkin Fellowship funded by the Royal Society (Grant Number: DH140195) and a Sir Henry Dale Fellowship co-funded by the Royal Society and Wellcome Trust (Grant Number: 109385/Z/15/Z)