Engineering Escherichia coli for increased Und-P availability leads to material improvements in glycan expression technology.

BACKGROUND: Bacterial surface glycans are assembled by glycosyltransferases (GTs) that transfer sugar monomers to long-chained lipid carriers. Most bacteria employ the 55-carbon chain undecaprenyl phosphate (Und-P) to scaffold glycan assembly. The amount of Und-P available for glycan synthesis is thought to be limited by the rate of Und-P synthesis and by competition for Und-P between phosphoglycosyl transferases (PGTs) and GTs that prime glycan assembly (which we collectively refer to as PGT/GTs). While decreasing Und-P availability disrupts glycan synthesis and promotes cell death, less is known about the effects of increased Und-P availability. RESULTS: To determine if cells can maintain higher Und-P levels, we first reduced intracellular competition for Und-P by deleting all known non-essential PGT/GTs in the Gram-negative bacterium Escherichia coli (hereafter called ΔPGT/GT cells). We then increased the rate of Und-P synthesis in ΔPGT/GT cells by overexpressing the Und-P(P) synthase uppS from a plasmid (puppS). Und-P quantitation revealed that ΔPGT/GT/puppS cells can be induced to maintain 3-fold more Und-P than wild type cells. Next, we determined how increasing Und-P availability affects glycan expression. Interestingly, increasing Und-P availability increased endogenous and recombinant glycan expression. In particular, ΔPGT/GT/puppS cells could be induced to express 7-fold more capsule from Streptococcus pneumoniae serotype 4 than traditional E. coli cells used to express recombinant glycans. CONCLUSIONS: We demonstrate that the biotechnology standard bacterium E. coli can be engineered to maintain higher levels of Und-P. The results also strongly suggest that Und-P pathways can be engineered to increase the expression of potentially any Und-P-dependent polymer. Given that many bacterial glycans are central to the production of vaccines, diagnostics, and therapeutics, increasing Und-P availability should be a foremost consideration when designing bacterial glycan expression systems.

Revised Model for the Type A Glycan Biosynthetic Pathway in Clostridioides difficile Strain 630Δerm Based on Quantitative Proteomics of cd0241-cd0244 Mutant Strains.

The bacterial flagellum is involved in a variety of processes including motility, adherence, and immunomodulation. In the Clostridioides difficile strain 630Δerm, the main filamentous component, FliC, is post-translationally modified with an O-linked Type A glycan structure. This modification is essential for flagellar function, since motility is seriously impaired in gene mutants with improper biosynthesis of the Type A glycan. The cd0240-cd0244 gene cluster encodes the Type A biosynthetic proteins, but the role of each gene, and the corresponding enzymatic activity, have not been fully elucidated. Using quantitative mass spectrometry-based proteomics analyses, we determined the relative abundance of the observed glycan variations of the Type A structure in cd0241, cd0242, cd0243, and cd0244 mutant strains. Our data not only confirm the importance of CD0241, CD0242, and CD0243 but, in contrast to previous data, also show that CD0244 is essential for the biosynthesis of the Type A modification. Combined with additional bioinformatic analyses, we propose a revised model for Type A glycan biosynthesis.

Flagellin O-linked glycans are required for the interactions between Campylobacter jejuni and Acanthamoebae castellanii.

The predation and engulfment of bacteria by Acanthamoebae facilitates intimate interactions between host and prey. This process plays an important and underestimated role in the physiology, ecology and evolution of pathogenic bacteria. Acanthamoebae species can be reservoirs for many important human pathogens including Campylobacter jejuni. C. jejuni is the leading cause of bacterial foodborne enteritis worldwide, despite being a microaerophile that is incapable of withstanding atmospheric levels of oxygen long-term. The persistence and transmission of this major pathogen in the natural environment outside its avian and mammalian hosts is not fully understood. Recent evidence has provided insight into the relationship of C. jejuni and Acanthamoebae spp. where Acanthamoebae are a transient host for this pathogen. Mutations to the flagella components were shown to affect C. jejuni-A. castellanii interactions. Here, we show that the motility function of flagella is not a prerequisite for C. jejuni-A. castellanii interactions and that specific O-linked glycan modifications of the C. jejuni major flagellin, FlaA, are important for the recognition, interaction and phagocytosis by A. castellanii. Substitution of the O-linked glycosylated serine 415 and threonine 477 with alanine within FlaA abolished C. jejuni interactions with A. castellanii and these mutants were indistinguishable from a ΔflaA mutant. By contrast, mutation to serine 405 did not affect C. jejuni 11168H and A. castellanii interactions. Given the abundance of flagella glycosylation among clinically important pathogens, our observations may have a wider implication for understanding host-pathogen interactions.

Development of a novel glycoengineering platform for the rapid production of conjugate vaccines.

Conjugate vaccines produced either by chemical or biologically conjugation have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccine assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel biotechnological platform that overcomes the limitations of the current conjugate vaccine design method(s). As a model, we focused our design on a leading bioconjugation method using N-oligosaccharyltransferase (OTase), PglB. The installation of MAGIC led to at least twofold increase in glycoconjugate yield via MAGIC when compared to conventional N-OTase based bioconjugation method(s). Then, we improved MAGIC to (a) allow rapid installation of glycoengineering component(s), (b) omit the usage of antibiotics, (c) reduce the dependence on protein induction agents. Furthermore, we show the modularity of the MAGIC platform in performing glycoengineering in bacterial species that are less genetically tractable than the commonly used Escherichia coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against serious bacterial pathogens. We anticipate the utility of the MAGIC platform could enhance vaccines production due to its compatibility with virtually any bioconjugation method, thus expanding vaccine biopreparedness toolbox.

Identification of novel p-cresol inhibitors that reduce Clostridioides difficile's ability to compete with species of the gut microbiome.

Treatment of Clostridioides difficile infection (CDI) is expensive and complex, with a high proportion of patients suffering infection relapse (20-35%), and some having multiple relapses. A healthy, unperturbed gut microbiome provides colonisation resistance against CDI through competition for nutrients and space. However, antibiotic consumption can disturb the gut microbiota (dysbiosis) resulting in the loss of colonisation resistance allowing C. difficile to colonise and establish infection. A unique feature of C. difficile is the production of high concentrations of the antimicrobial compound para-cresol, which provides the bacterium with a competitive advantage over other bacteria found in the gut. p-cresol is produced by the conversion of para-Hydroxyphenylacetic acid (p-HPA) by the HpdBCA enzyme complex. In this study, we have identified several promising inhibitors of HpdBCA decarboxylase, which reduce p-cresol production and render C. difficile less able to compete with a gut dwelling Escherichia coli strain. We demonstrate that the lead compound, 4-Hydroxyphenylacetonitrile, reduced p-cresol production by 99.0 ± 0.4%, whereas 4-Hydroxyphenylacetamide, a previously identified inhibitor of HpdBCA decarboxylase, only reduced p-cresol production by 54.9 ± 13.5%. To interpret efficacy of these first-generation inhibitors, we undertook molecular docking studies that predict the binding mode for these compounds. Notably, the predicted binding energy correlated well with the experimentally determined level of inhibition, providing a molecular basis for the differences in efficacy between the compounds. This study has identified promising p-cresol production inhibitors whose development could lead to beneficial therapeutics that help to restore colonisation resistance and therefore reduce the likelihood of CDI relapse.

Progress towards a glycoconjugate vaccine against Group A Streptococcus.

The Group A Carbohydrate (GAC) is a defining feature of Group A Streptococcus (Strep A) or Streptococcus pyogenes. It is a conserved and simple polysaccharide, comprising a rhamnose backbone and GlcNAc side chains, further decorated with glycerol phosphate on approximately 40% GlcNAc residues. Its conservation, surface exposure and antigenicity have made it an interesting focus on Strep A vaccine design. Glycoconjugates containing this conserved carbohydrate should be a key approach towards the successful mission to build a universal Strep A vaccine candidate. In this review, a brief introduction to GAC, the main carbohydrate component of Strep A bacteria, and a variety of published carrier proteins and conjugation technologies are discussed. Components and technologies should be chosen carefully for building affordable Strep A vaccine candidates, particularly for low- and middle-income countries (LMICs). Towards this, novel technologies are discussed, such as the prospective use of bioconjugation with PglB for rhamnose polymer conjugation and generalised modules for membrane antigens (GMMA), particularly as low-cost solutions to vaccine production. Rational design of "double-hit" conjugates encompassing species specific glycan and protein components would be beneficial and production of a conserved vaccine to target Strep A colonisation without invoking an autoimmune response would be ideal.

A combinatorial DNA assembly approach to biosynthesis of N-linked glycans in E. coli.

Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the Campylobacter jejuni  N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli.

Survival of Campylobacter jejuni 11168H in Acanthamoebae castellanii Provides Mechanistic Insight into Host Pathogen Interactions.

Campylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis worldwide but is rarely transferred between human hosts. Although a recognized microaerophile, the majority of C. jejuni are incapable of growing in an aerobic environment. The persistence and transmission of this pathogen outside its warm-blooded avian and mammalian hosts is poorly understood. Acanthamoebae species are predatory protists and form an important ecological niche with several bacterial species. Here, we investigate the interaction of C. jejuni 11168H and Acanthamoebae castellanii at the single-cell level. We observe that a subpopulation of C. jejuni cells can resist killing by A. castellanii, and non-digested bacteria are exocytosed into the environment where they can persist. In addition, we observe that A. castellanii can harbor C. jejuni 11168H even upon encystment. Transcriptome analyses of C. jejuni interactions revealed similar survival mechanisms when infecting both A. castellanii and warm-blooded hosts. In particular, nitrosative stress defense mechanisms and flagellum function are important as confirmed by mutational analyses of C. jejuni 11168H. This study describes a new host-pathogen interaction for C. jejuni and confirms that amoebae are transient hosts for the persistence, adaptability, and potential transmission of C. jejuni.

Acinetobacter nosocomialis Causes as Severe Disease as Acinetobacter baumannii in Northeast Thailand: Underestimated Role of A. nosocomialis in Infection.

Infections by Acinetobacter species are recognized as a serious global threat due to causing severe disease and their high levels of antibiotic resistance. Acinetobacter baumannii is the most prevalent pathogen in the genus, but infection by Acinetobacter nosocomialis has been reported widely. Diagnosis of patients with A. baumannii infection is often misdiagnosed with other Acinetobacter species, especially A. nosocomialis. This study investigated whether there were significant differences in clinical outcomes between patients infected with A. baumannii versus A. nosocomialis in Northeast Thailand, and to characterize serological responses to infection with these pathogens. The results show that A. baumannii had higher levels of multidrug resistance. Despite this, clinical outcomes for infection with A. baumannii or A. nosocomialis were similar with mortalities of 33% and 36%, respectively. Both pathogens caused community-acquired infections (A. baumannii 35% and A. nosocomialis 29% of cases). Plasma from uninfected healthy controls contained IgG antibody that recognized both organisms, and infected patients did not show a significantly enhanced antibody response from the first week versus 2 weeks later. Finally, the patterns of antigen recognition for plasma IgG were similar for patients infected with A. baumannii or A. nosocomialis infection, and distinct to the pattern for patients infected with non-Acinetobacter. In conclusion, our data revealed that infection with A. nosocomialis was associated with a similarly high level of mortality as infection with A. baumannii, the high rate of community-acquired infection and antibodies in uninfected individuals suggesting that there is significant community exposure to both pathogens. IMPORTANCE Bacterial infections by Acinetobacter species are global threats due to their severity and high levels of antibiotic resistance. A. baumannii is the most common pathogen in the genus; however, infection by A. nosocomialis has also been widely reported but is thought to be less severe. In this study, we have prospectively investigated 48 reported cases of A. baumannii infection in Northeast Thailand, and characterized the serological responses to infection. We found that 14 (29%) of these infections were actually caused by A. nosocomialis. Furthermore, the incidence of antibiotic resistance among A. nosocomialis strains, APACHE II scores, and mortality for patients infected with A. nosocomialis were much higher than published data. Both A. baumannii and A. nosocomialis had unexpectedly mortality rates of over 30%, and both pathogens caused a high rate of community-acquired infections. Importantly, background antibodies in uninfected individuals suggest significant community exposure to both pathogens in the environment.

Defining the Genes Required for Survival of Mycobacterium bovis in the Bovine Host Offers Novel Insights into the Genetic Basis of Survival of Pathogenic Mycobacteria.

Tuberculosis has severe impacts on both humans and animals. Understanding the genetic basis of survival of both Mycobacterium tuberculosis, the human-adapted species, and Mycobacterium bovis, the animal-adapted species, is crucial to deciphering the biology of both pathogens. There are several studies that identify the genes required for survival of M. tuberculosis in vivo using mouse models; however, there are currently no studies probing the genetic basis of survival of M. bovis in vivo. In this study, we utilize transposon insertion sequencing in M. bovis AF2122/97 to determine the genes required for survival in cattle. We identify genes encoding established mycobacterial virulence functions such as the ESX-1 secretion system, phthiocerol dimycocerosate (PDIM) synthesis, mycobactin synthesis, and cholesterol catabolism that are required in vivo. We show that, as in M. tuberculosis H37Rv, phoPR is required by M. bovis AF2122/97 in vivo despite the known defect in signaling through this system. Comparison to studies performed in species that are able to use carbohydrates as an energy source, such as M. bovis BCG and M. tuberculosis, suggests that there are differences in the requirement for genes involved in cholesterol import (mce4 operon) and oxidation (hsd). We report a good correlation with existing mycobacterial virulence functions but also find several novel virulence factors, including genes involved in protein mannosylation, aspartate metabolism, and glycerol-phosphate metabolism. These findings further extend our knowledge of the genetic basis of survival in vivo in bacteria that cause tuberculosis and provide insight for the development of novel diagnostics and therapeutics. IMPORTANCE This is the first report of the genetic requirements of an animal-adapted member of the Mycobacterium tuberculosis complex (MTBC) in a natural host. M. bovis has devastating impacts on cattle, and bovine tuberculosis is a considerable economic, animal welfare, and public health concern. The data highlight the importance of mycobacterial cholesterol catabolism and identify several new virulence factors. Additionally, the work informs the development of novel differential diagnostics and therapeutics for TB in both human and animal populations.

Strain Specific Variations in Acinetobacter baumannii Complement Sensitivity.

The complement system is required for innate immunity against Acinetobacter baumannii, an important cause of antibiotic resistant systemic infections. A. baumannii strains differ in their susceptibility to the membrane attack complex (MAC) formed from terminal complement pathway proteins, but the reasons for this variation remain poorly understood. We have characterized in detail the complement sensitivity phenotypes of nine A. baumannii clinical strains and some of the factors that might influence differences between strains. Using A. baumannii laboratory strains and flow cytometry assays, we first reconfirmed that both opsonization with the complement proteins C3b/iC3b and MAC formation were inhibited by the capsule. There were marked differences in C3b/iC3b and MAC binding between the nine clinical A. baumannii strains, but this variation was partially independent of capsule composition or size. Opsonization with C3b/iC3b improved neutrophil phagocytosis of most strains. Importantly, although C3b/iC3b binding and MAC formation on the bacterial surface correlated closely, MAC formation did not correlate with variations between A. baumannii strains in their levels of serum resistance. Genomic analysis identified only limited differences between strains in the distribution of genes required for serum resistance, but RNAseq data identified three complement-resistance genes that were differentially regulated between a MAC resistant and two MAC intermediate resistant strains when cultured in serum. These data demonstrate that clinical A. baumannii strains vary in their sensitivity to different aspects of the complement system, and that the serum resistance phenotype was influenced by factors in addition to the amount of MAC forming on the bacterial surface.

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.

Transient internalization of Campylobacter jejuni in Amoebae enhances subsequent invasion of human cells.

The ubiquitous unicellular eukaryote, Acanthamoeba, is known to play a role in the survival and dissemination of Campylobacter jejuni. C. jejuni is the leading cause of bacterial foodborne gastroenteritis world-wide and is a major public health problem. The ability of C. jejuni to interact and potentially invade epithelial cells is thought to be key for disease development in humans. We examined C. jejuni grown under standard laboratory conditions, 11168HCBA with that harvested from within Acanthamoeba castellanii (11168HAC/CBA) or Acanthamoeba polyphaga (11168HAP/CBA), and compared their ability to invade different cell lines. C. jejuni harvested from within amoebae had a ~3.7-fold increase in invasiveness into T84 human epithelial cells and a striking ~11-fold increase for re-entry into A. castellanii cells. We also investigated the invasiveness and survivability of six diverse representative C. jejuni strains within Acanthamoeba spp., our results confirm that invasion and survivability is likely host-cell-dependent. Our survival assay data led us to conclude that Acanthamoeba spp. are a transient host for C. jejuni and that survival within amoebae pre-adapts C. jejuni and enhances subsequent cell invasion. This study provides new insight into C. jejuni interactions with amoebae and its increased invasiveness potential in mammalian hosts.

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.

MdaB and NfrA, Two Novel Reductases Important in the Survival and Persistence of the Major Enteropathogen Campylobacter jejuni.

The paralogues RrpA and RrpB, which are members of the MarR family of DNA binding proteins, are important for the survival of the global bacterial foodborne pathogen Campylobacter jejuni under redox stress. We report that RrpA is a positive regulator of mdaB, encoding a flavin-dependent quinone reductase that contributes to the protection from redox stress mediated by structurally diverse quinones, while RrpB negatively regulates the expression of cj1555c (renamed nfrA for NADPH-flavin reductase A), encoding a flavin reductase. NfrA reduces riboflavin at a greater rate than its derivatives, suggesting that exogenous free flavins are the natural substrate. MdaB and NfrA both prefer NADPH as an electron donor. Cysteine substitution and posttranslational modification analyses indicated that RrpA and RrpB employ a cysteine-based redox switch. Complete genome sequence analyses revealed that mdaB is frequently found in Campylobacter and related Helicobacter spp., while nfrA is predominant in C. jejuni strains. Quinones and flavins are redox cycling agents secreted by a wide range of cell types that can form damaging superoxide by one-electron reactions. We propose a model for stress adaptation where MdaB and NfrA facilitate a two-electron reduction mechanism to the less toxic hydroquinones, thus aiding survival and persistence of this major pathogen. IMPORTANCE Changes in cellular redox potential result in alteration in the oxidation state of intracellular metabolites and enzymes; consequently, cells make adjustments that favor growth and survival. The work we present here answers some of the many questions that have remained elusive over the years of investigation into the enigmatic microaerophile bacterium Campylobacter jejuni. We employed molecular approaches to understand the regulation mechanisms and functional analyses to reveal the roles of two novel quinone and flavin reductases; both serve as major pools of cellular redox-active molecules. This work extends our knowledge on bacterial redox sensing mechanisms and the significance of hemostasis.

Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS).

Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines.

Complete genome for Actinobacillus pleuropneumoniae serovar 8 reference strain 405: comparative analysis with draft genomes for different laboratory stock cultures indicates little genetic variation.

We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain.

Production of p-cresol by Decarboxylation of p-HPA by All Five Lineages of Clostridioides difficile Provides a Growth Advantage.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and is capable of causing severe symptoms, such as pseudomembranous colitis and toxic megacolon. An unusual feature of C. difficile is the distinctive production of high levels of the antimicrobial compound para-cresol. p-Cresol production provides C. difficile with a competitive colonization advantage over gut commensal species, in particular, Gram-negative species. p-Cresol is produced by the conversion of para-hydroxyphenylacetic acid (p-HPA) via the actions of HpdBCA decarboxylase coded by the hpdBCA operon. Host cells and certain bacterial species produce p-HPA; however, the effects of p-HPA on the viability of C. difficile and other gut microbiota are unknown. Here we show that representative strains from all five C. difficile clades are able to produce p-cresol by two distinct mechanisms: (i) via fermentation of p-tyrosine and (ii) via uptake and turnover of exogenous p-HPA. We observed strain-specific differences in p-cresol production, resulting from differential efficiency of p-tyrosine fermentation; representatives of clade 3 (CD305) and clade 5 (M120) produced the highest levels of p-cresol via tyrosine metabolism, whereas the toxin A-/B+ isolate from clade 4 (M68) produced the lowest level of p-cresol. All five lineages share at least 97.3% homology across the hpdBCA operon, responsible for decarboxylation of p-HPA to p-cresol, suggesting that the limiting step in p-cresol production may result from tyrosine to p-HPA conversion. We identified that elevated intracellular p-HPA, modulated indirectly via CodY, controls p-cresol production via inducing the expression of HpdBCA decarboxylase ubiquitously in C. difficile populations. Efficient turnover of p-HPA is advantageous to C. difficile as p-HPA has a deleterious effect on the growth of C. difficile and other representative Gram-negative gut bacteria, transduced potentially by the disruption of membrane permeability and release of intracellular phosphate. This study provides insights into the importance of HpdBCA decarboxylase in C. difficile pathogenesis, both in terms of p-cresol production and detoxification of p-HPA, highlighting its importance to cell survival and as a highly specific therapeutic target for the inhibition of p-cresol production across C. difficile species.

Evaluation of a Campylobacter jejuni N-glycan-ExoA glycoconjugate vaccine to reduce C. jejuni colonisation in chickens.

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide and handling or consumption of contaminated poultry meat is the key source of infection. Glycoconjugate vaccines containing the C. jejuni N-glycan have been reported to be partially protective in chickens. However, our previous studies with subunit vaccines comprising the C. jejuni FlpA or SodB proteins with up to two or three C. jejuni N-glycans, respectively, failed to elicit significant protection. In this study, protein glycan coupling technology was used to add up to ten C. jejuni N-glycans onto a detoxified form of Pseudomonas aeruginosa exotoxin A (ExoA). The glycoprotein, G-ExoA, was evaluated for efficacy against intestinal colonisation of White Leghorn chickens by C. jejuni strains M1 and 11168H relative to unglycosylated ExoA. Chickens were challenged with the minimum dose required for reliable colonisation, which was 102 colony-forming units (CFU) for strain M1 and and 104 CFU for strain 11168H. Vaccine-specific serum IgY was detected in chickens vaccinated with both ExoA and G-ExoA. However, no reduction in caecal colonisation by C. jejuni was observed. While the glycan dose achieved with G-ExoA was higher than FlpA- or SodB-based glycoconjugates that were previously evaluated, it was lower than that of glycoconjugates where protection against C. jejuni has been reported, indicating that protection may be highly sensitive to the amount of glycan presented and/or study-specific variables.

Multivalent poultry vaccine development using Protein Glycan Coupling Technology.

BACKGROUND: Poultry is the world's most popular animal-based food and global production has tripled in the past 20 years alone. Low-cost vaccines that can be combined to protect poultry against multiple infections are a current global imperative. Glycoconjugate vaccines, which consist of an immunogenic protein covalently coupled to glycan antigens of the targeted pathogen, have a proven track record in human vaccinology, but have yet to be used for livestock due to prohibitively high manufacturing costs. To overcome this, we use Protein Glycan Coupling Technology (PGCT), which enables the production of glycoconjugates in bacterial cells at considerably reduced costs, to generate a candidate glycan-based live vaccine intended to simultaneously protect against Campylobacter jejuni, avian pathogenic Escherichia coli (APEC) and Clostridium perfringens. Campylobacter is the most common cause of food poisoning, whereas colibacillosis and necrotic enteritis are widespread and devastating infectious diseases in poultry. RESULTS: We demonstrate the functional transfer of C. jejuni protein glycosylation (pgl) locus into the genome of APEC χ7122 serotype O78:H9. The integration caused mild attenuation of the χ7122 strain following oral inoculation of chickens without impairing its ability to colonise the respiratory tract. We exploit the χ7122 pgl integrant as bacterial vectors delivering a glycoprotein decorated with the C. jejuni heptasaccharide glycan antigen. To this end we engineered χ7122 pgl to express glycosylated NetB toxoid from C. perfringens and tested its ability to reduce caecal colonisation of chickens by C. jejuni and protect against intra-air sac challenge with the homologous APEC strain. CONCLUSIONS: We generated a candidate glycan-based multivalent live vaccine with the potential to induce protection against key avian and zoonotic pathogens (C. jejuni, APEC, C. perfringens). The live vaccine failed to significantly reduce Campylobacter colonisation under the conditions tested but was protective against homologous APEC challenge. Nevertheless, we present a strategy towards the production of low-cost "live-attenuated multivalent vaccine factories" with the ability to express glycoconjugates in poultry.