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Vezina, B.

Publications and source records attributed to Vezina, B..

6 recordsLinked to original sources
bioRxiv2021

A curated collection of Klebsiella metabolic models reveals variable substrate usage and gene essentiality

The Klebsiella pneumoniae species complex (KpSC) is a set of seven Klebsiella taxa which are found in a variety of niches, and are an important cause of opportunistic healthcare-associated infections in humans. Due to increasing rates of multi-drug resistance within the KpSC, there is a growing interest in better understanding the biology and metabolism of these organisms to inform novel control strategies. We collated 37 sequenced KpSC isolates isolated from a variety of niches, representing all seven taxa. We generated strain-specific genome scale metabolic models (GEMs) for all 37 isolates and simulated growth phenotypes on 511 distinct carbon, nitrogen, sulphur and phosphorus substrates. Models were curated and their accuracy assessed using matched phenotypic growth data for 94 substrates (median accuracy of 96%). We explored species-specific growth capabilities and examined the impact of all possible single gene deletions on growth in 145 core carbon substrates. These analyses revealed multiple strain-specific differences, within and between species and highlight the importance of selecting a diverse range of strains when exploring KpSC metabolism. This diverse set of highly accurate GEMs could be used to inform novel drug design, enhance genomic analyses, and identify novel virulence and resistance determinants. We envisage that these 37 curated strain-specific GEMs, covering all seven taxa of the KpSC, provide a valuable resource to the Klebsiella research community.

microbiology↗
bioRxiv2021

Trycycler: consensus long-read assemblies for bacterial genomes

Assembly of bacterial genomes from long-read data (generated by Oxford Nanopore or Pacific Biosciences platforms) can often be complete: a single contig for each chromosome or plasmid in the genome. However, even complete bacterial genome assemblies constructed solely from long reads still contain a variety of errors, and different assemblies of the same genome often contain different errors. Here, we present Trycycler, a tool which produces a consensus assembly from multiple input assemblies of the same genome. Benchmarking using both simulated and real sequencing reads showed that Trycycler consensus assemblies contained fewer errors than any of those constructed with a single long-read assembler. Post-assembly polishing with Medaka and Pilon further reduced errors and yielded the most accurate genome assemblies in our study. As Trycycler can require human judgement and manual intervention, its output is not deterministic, and different users can produce different Trycycler assemblies from the same input data. However, we demonstrated that multiple users with minimal training converge on similar assemblies that are consistently more accurate than those produced by automated assembly tools. We therefore recommend Trycycler+Medaka+Pilon as an ideal approach for generating high-quality bacterial reference genomes. Data availabilitySupplementary figures, tables and code can be found at: github.com/rrwick/Trycycler-paper Reads, assemblies and reference sequences can be found at: bridges.monash.edu/articles/dataset/Trycycler_paper_dataset/14890734

bioinformatics↗
bioRxiv2020

Prediction of Burkholderia pseudomallei DsbA substrates identifies potential virulence factors and vaccine targets

Identification of bacterial virulence factors is critical for understanding disease pathogenesis, drug discovery and vaccine development. In this study we used two approaches to predict virulence factors of Burkholderia pseudomallei, the Gram-negative bacterium that causes melioidosis. B. pseudomallei is naturally antibiotic resistant and there are no melioidosis vaccines. To identify B. pseudomallei protein targets for drug discovery and vaccine development, we chose to search for substrates of the B. pseudomallei periplasmic disulfide bond forming protein A (DsbA). DsbA introduces disulfide bonds into extra-cytoplasmic proteins and is essential for virulence in many Gram-negative organism, including B. pseudomallei. The first approach to identify B. pseudomallei DsbA virulence factor substrates was a large-scale genomic analysis of 511 unique B. pseudomallei disease-associated strains. This yielded 4,496 core gene products, of which we hypothesise 263 are DsbA substrates. Manual curation of the 263 mature proteins yielded 73 associated with disease pathogenesis or virulence. These were screened for structural homologues to predict potential B-cell epitopes. In the second approach, we searched the B. pseudomallei genome for homologues of the more than 90 known DsbA substrates in other bacteria. Using this approach, we identified 15 potential B. pseudomallei DsbA virulence factor substrates. Two putative B. pseudomallei virulence factors were identified by both methods: homologues of PenI family β-lactamase and of succinate dehydrogenase flavoprotein subunit. These two proteins could serve as high priority targets for future B. pseudomallei virulence factor characterization.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioinformatics↗
bioRxiv2020

Crystal structure and site-directed mutagenesis of circular bacteriocin plantacyclin B21AG reveals cationic and aromatic residues important for antimicrobial activity

Plantacyclin B21AG is a circular bacteriocin produced by Lactobacillus plantarum B21 which displays antimicrobial activity against various Gram-positive bacteria including foodborne pathogens, Listeria monocytogenes and Clostridium perfringens. It is a 58-amino acid cyclised antimicrobial peptide, with the N and C termini covalently linked together. The circular peptide backbone contributes to remarkable stability, conferring partial proteolytic resistance and structural integrity under a wide temperature and pH range. Here, we report the first crystal structure of a circular bacteriocin from food grade Lactobacillus. The protein was crystallised using the hanging drop vapour diffusion method and the structure solved to a resolution of 1.8 Å. Sequence alignment against 17 previously characterised circular bacteriocins revealed the presence of conserved charged and aromatic residues. Alanine substitution mutagenesis validated the importance of these residues. Minimum inhibitory concentration analysis of these Ala mutants showed Phe8Ala and Trp45Ala mutants displayed a 48- and 32-fold reduction in activity, compared to wild type. Lys19Ala mutant displayed the weakest activity, with a 128-fold reduction. These experiments demonstrate the importance of aromatic and cationic residues for the antimicrobial activity of plantacyclin B21AG and by extension, other circular bacteriocins sharing these evolutionarily conserved residues.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗
bioRxiv2020

Discovery and characterisation of novel circular bacteriocin plantacyclin B21AG from Lactobacillus plantarum B21

Lactobacillus plantarum B21 isolated from Vietnamese sausage (nem chua) has previously displayed broad antimicrobial activity against gram positive bacteria including foodborne pathogens Listeria monocytogenes and Clostridium perfringens. This study successfully identified the antimicrobial agent as plantacyclin B21AG, a 5668 Da circular bacteriocin demonstrating high thermostability, resistance to a wide range of pH, proteolytic resistance and temporal stability. We report a reverse genetics approach used to identify and characterise plantacyclin B21AG. The bacteriocin was purified from culture supernatant by a short process consisting of concentration, n-butanol extraction and cation exchange chromatography. A de novo peptide sequencing using LC-MS/MS techniques identified two putative peptide fragments which were mapped to the genome of Lactobacillus plantarum B21. This revealed an ORF corresponding to a putative circular bacteriocin with a 33-amino acid leader peptide and 58-amino acid mature peptide found on native plasmid pB21AG01. The corresponding gene cluster, consisted of seven genes associated with post-translational circularisation, immunity and secretion. The robust nature of plantacyclin B21AG, its antimicrobial activity and associated machinery for cyclisation make it an interesting biotechnological target for further development, and application as a food-safe antimicrobial.

biochemistry↗