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.

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.

Biotechnological and Ecological Potential of Micromonospora provocatoris sp. nov., a Gifted Strain Isolated from the Challenger Deep of the Mariana Trench.

A Micromonospora strain, isolate MT25T, was recovered from a sediment collected from the Challenger Deep of the Mariana Trench using a selective isolation procedure. The isolate produced two major metabolites, n-acetylglutaminyl glutamine amide and desferrioxamine B, the chemical structures of which were determined using 1D and 2D-NMR, including 1H-15N HSQC and 1H-15N HMBC 2D-NMR, as well as high resolution MS. A whole genome sequence of the strain showed the presence of ten natural product-biosynthetic gene clusters, including one responsible for the biosynthesis of desferrioxamine B. Whilst 16S rRNA gene sequence analyses showed that the isolate was most closely related to the type strain of Micromonospora chalcea, a whole genome sequence analysis revealed it to be most closely related to Micromonospora tulbaghiae 45142T. The two strains were distinguished using a combination of genomic and phenotypic features. Based on these data, it is proposed that strain MT25T (NCIMB 15245T, TISTR 2834T) be classified as Micromonospora provocatoris sp. nov. Analysis of the genome sequence of strain MT25T (genome size 6.1 Mbp) revealed genes predicted to responsible for its adaptation to extreme environmental conditions that prevail in deep-sea sediments.

Whole Genome Sequence of Dermacoccus abyssi MT1.1 Isolated from the Challenger Deep of the Mariana Trench Reveals Phenazine Biosynthesis Locus and Environmental Adaptation Factors.

Dermacoccus abyssi strain MT1.1T is a piezotolerant actinobacterium that was isolated from Mariana Trench sediment collected at a depth of 10898 m. The organism was found to produce ten dermacozines (A‒J) that belonged to a new phenazine family and which displayed various biological activities such as radical scavenging and cytotoxicity. Here, we report on the isolation and identification of a new dermacozine compound, dermacozine M, the chemical structure of which was determined using 1D and 2D-NMR, and high resolution MS. A whole genome sequence of the strain contained six secondary metabolite-biosynthetic gene clusters (BGCs), including one responsible for the biosynthesis of a family of phenazine compounds. A pathway leading to the biosynthesis of dermacozines is proposed. Bioinformatic analyses of key stress-related genes provide an insight into how the organism adapted to the environmental conditions that prevail in the deep-sea.

Investigation into the efficiency of diverse N-linking oligosaccharyltransferases for glycoengineering using a standardised cell-free assay.

The application of bacterial oligosaccharyltransferases (OSTs) such as the Campylobacter jejuni PglB for glycoengineering has attracted considerable interest in glycoengineering and glycoconjugate vaccine development. However, PglB has limited specificity for glycans that can be transferred to candidate proteins, which along with other factors is dependent on the reducing end sugar of glycans. In this study, we developed a cell-free glycosylation assay that offers the speed and simplicity of a 'yes' or 'no' determination. Using the assay, we tested the activity of eleven PglBs from Campylobacter species and more distantly related bacteria. The following assorted glycans with diverse reducing end sugars were tested for transfer, including Streptococcus pneumoniae capsule serotype 4, Salmonella enterica serovar Typhimurium O antigen (B1), Francisella tularensis O antigen, Escherichia coli O9 antigen and Campylobacter jejuni heptasaccharide. Interestingly, while PglBs from the same genus showed high activity, whereas divergent PglBs differed in their transfer of glycans to an acceptor protein. Notably for glycoengineering purposes, Campylobacter hepaticus and Campylobacter subantarcticus PglBs showed high glycosylation efficiency, with C. hepaticus PglB potentially being useful for glycoconjugate vaccine production. This study demonstrates the versatility of the cell-free assay in rapidly assessing an OST to couple glycan/carrier protein combinations and lays the foundation for future screening of PglBs by linking amino acid similarity to glycosyltransferase activity.

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.

Whole genome sequencing of four bacterial strains from South Shetland Trench revealing biosynthetic and environmental adaptation gene clusters.

Whole genome sequences of four bacterial strains Dietzia maris SST1, Pseudomonas zhaodongensis SST2, Pseudomonas sp. SST3 and Halomonas sulfidaeris SST4, recovered from the South Shetland Trench sediment in Antarctica were analyzed using Ion Torrent sequencing technology. The respective sizes of their genomes (3.88, 4.99, 5.60 and 4.25 Mb) and GC contents (70.0, 60.3, 59.9 and 53.8%) are in agreement with these values of other strains of the species. The bacterial strains displayed promising antimicrobial activity against a number of pathogenic bacterial and fungal species. Whole genomes have been assembled and biosynthetic gene clusters (BGCs) have been identified using the antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) web platform. Comparative analysis of the genome sequences revealed that the strains host abundant BGCs encoding for terpenes, siderophores, arylpolyene, bacteriocins, and lassopeptides. Furthermore, the key stress-related genes were identified and their distribution provided an insight into how these isolates adapt to key marine environmental conditions. This comprehensive study is a contribution to understanding the nature of life on the deep-sea environments.

Potential probiotic-associated traits revealed from completed high quality genome sequence of Lactobacillus fermentum 3872.

The article provides an overview of the genomic features of Lactobacillus fermentum strain 3872. The genomic sequence reported here is one of three L. fermentum genome sequences completed to date. Comparative genomic analysis allowed the identification of genes that may be contributing to enhanced probiotic properties of this strain. In particular, the genes encoding putative mucus binding proteins, collagen-binding proteins, class III bacteriocin, as well as exopolysaccharide and prophage-related genes were identified. Genes related to bacterial aggregation and survival under harsh conditions in the gastrointestinal tract, along with the genes required for vitamin production were also found.

Specific genetic features of Campylobacter jejuni strain G1 revealed by genome sequencing.

This report describes a draft genome sequence of Campylobacter jejuni strain G1. The strain contains no plasmids, despite being resistant to tetracycline (Tet). Comparison of the genome of this strain with that of a reference strain NCTC 11168 (Tet sensitive) revealed the presence of a candidate gene that may be responsible for antibiotic resistance, as well as the genes involved in the synthesis of ganglioside-mimicking molecules responsible for the development of a neurodegenerative disorder, Guillain-Barré syndrome.