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Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

ABSTRACT: Whole genome sequencing (WGS) is useful for determining clusters of human cases, investigating outbreaks, and defining the population genetics of bacteria. It also provides information about other aspects of bacterial biology, including classical typing results, virulence, and adaptive strategies of the organism. Cell culture invasion and protein expression patterns of four related multilocus sequence type 21 (ST21) C. jejuni isolates from a significant Canadian water-borne outbreak were previously associated with the presence of a CJIE1 prophage. Whole genome sequencing was used to examine the genetic diversity among these isolates and confirm that previous observations could be attributed to differential prophage carriage. Moreover, we sought to determine the presence of genome sequences that could be used as surrogate markers to delineate outbreak-associated isolates.Differential carriage of the CJIE1 prophage was identified as the major genetic difference among the four outbreak isolates. High quality single-nucleotide variant (hqSNV) and core genome multilocus sequence typing (cgMLST) clustered these isolates within expanded datasets consisting of additional C. jejuni strains. The number and location of homopolymeric tract regions was identical in all four outbreak isolates but differed from all other C. jejuni examined. Comparative genomics and PCR amplification enabled the identification of large chromosomal inversions of approximately 93 kb and 388 kb within the outbreak isolates associated with transducer-like proteins containing long nucleotide repeat sequences. The 93-kb inversion was characteristic of the outbreak-associated isolates, and the gene content of this inverted region displayed high synteny with the reference strain.The four outbreak isolates were clonally derived and differed mainly in the presence of the CJIE1 prophage, validating earlier findings linking the prophage to phenotypic differences in virulence assays and protein expression. The identification of large, genetically syntenous chromosomal inversions in the genomes of outbreak-associated isolates provided a unique method for discriminating outbreak isolates from the background population. Transducer-like proteins appear to be associated with the chromosomal inversions. CgMLST and hqSNV analysis also effectively delineated the outbreak isolates within the larger C. jejuni population structure.

SUBMITTER: Clark CG

PROVIDER: S-EPMC5135748 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

Clark Clifford G CG Berry Chrystal C Walker Matthew M Petkau Aaron A Barker Dillon O R DO Guan Cai C Reimer Aleisha A Taboada Eduardo N EN

BMC genomics 20161203 1

<h4>Background</h4>Whole genome sequencing (WGS) is useful for determining clusters of human cases, investigating outbreaks, and defining the population genetics of bacteria. It also provides information about other aspects of bacterial biology, including classical typing results, virulence, and adaptive strategies of the organism. Cell culture invasion and protein expression patterns of four related multilocus sequence type 21 (ST21) C. jejuni isolates from a significant Canadian water-borne ou ...[more]

PMID: 27912729

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Project description:Whole genome sequencing (WGS) has been used to assess the phylogenetic relationships, virulence and metabolic differences, and the relationship between gene carriage and host or niche differentiation among populations of C. jejuni isolates. We previously characterized the presence and expression of CJIE4 prophage proteins in four C. jejuni isolates using WGS and comparative proteomics analysis, but the isolates were not assessed further. In this study we compare the closed, finished genome sequences of these isolates to the total proteome. Genomes of the four isolates differ in phage content and location, plasmid content, capsular polysaccharide biosynthesis loci, a type VI secretion system, orientation of the ~92 kb invertible element, and allelic differences. Proteins with 99% sequence identity can be differentiated using isobaric tags for relative and absolute quantification (iTRAQ) comparative proteomic methods. GO enrichment analysis and the type of artefacts produced in comparative proteomic analysis depend on whether proteins are encoded in only one isolate or common to all isolates, whether different isolates have different alleles of the proteins analyzed, whether conserved and variable regions are both present in the protein group analyzed, and on how the analysis is done. Several proteins encoded by genes with very high levels of sequence identity in all four isolates exhibited preferentially higher protein expression in only one of the four isolates, suggesting differential regulation among the isolates. It is possible to analyze comparative protein expression in more distantly related isolates in the context of WGS data, though the results are more complex to interpret than when isolates are clonal or very closely related. Comparative proteomic analysis produced log2 fold expression data suggestive of regulatory differences among isolates, indicating that it may be useful as a hypothesis generation exercise to identify regulated proteins and regulatory pathways for more detailed analysis.

Project description:Campylobacter jejuni is the leading cause of bacterial gastroenteritis, which has motivated the monitoring of genetic profiles circulating in Luxembourg since 13 years. From our integrated surveillance using a genotyping strategy based on an extended MLST scheme including gyrA and porA markers, an unexpected endemic pattern was discovered in the temporal distribution of genotypes. We aimed to test the hypothesis of stable lineages occurrence by implementing whole genome sequencing (WGS) associated with comprehensive and internationally validated schemes. This pilot study assessed four WGS-based typing schemes to classify a panel of 108 strains previously identified as recurrent or sporadic profiles using this in-house typing system. The strain collection included four common lineages in human infection (N = 67) initially identified from recurrent combination of ST-gyrA-porA alleles also detected in non-human samples: veterinary (N = 19), food (N = 20), and environmental (N = 2) sources. An additional set of 19 strains belonging to sporadic profiles completed the tested panel. All the strains were processed by WGS by using Illumina technologies and by applying stringent criteria for filtering sequencing data; we ensure robustness in our genomic comparison. Four typing schemes were applied to classify the strains: (i) the cgMLST SeqSphere+ scheme of 637 loci, (ii) the cgMLST Oxford scheme of 1,343 loci, (iii) the cgMLST INNUENDO scheme of 678 loci, and (iv) the wgMLST INNUENDO scheme of 2,795 loci. A high concordance between the typing schemes was determined by comparing the calculated adjusted Wallace coefficients. After quality control and analyses with these four typing schemes, 60 strains were confirmed as members of the four recurrent lineages regardless of the method used (N = 32, 12, 7, and 9, respectively). Our results indicate that, regardless of the typing scheme used, epidemic or endemic signals were detected as reflected by lineage B (ST2254-gyrA9-porA1) in 2014 or lineage A (ST19-gyrA8-porA7), respectively. These findings support the clonal expansion of stable genomes in Campylobacter population exhibiting a multi-host profile and accounting for the majority of clinical strains isolated over a decade. Such recurring genotypes suggest persistence in reservoirs, sources or environment, emphasizing the need to investigate their survival strategy in greater depth.

Project description:BACKGROUND: Protein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins. RESULTS: In this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5) were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA). We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were subjected to cassette saturation mutagenesis. With the exception of I571C only hydrophobic residues were found in active variants. Variant I571V performed equally well as the wild type, cysteine at the same position reduced glycoprotein yield slightly, while a change to phenylalanine reduced activity by a factor of three. CONCLUSIONS: This study provides novel structure-function relationships for the periplasmic domain of the Campylobacter jejuni N-oligosaccharyltransferase PglB and describes procedures for generating and screening oligosaccharyltransferase mutant libraries in an engineered E. coli system.

Dataset Information

Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

Publications

Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

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