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Assessment of the Genome Sequencing Paper for Bordetella Pertussis

All of the content in this section discusses the following genome sequencing paper:

• Parkhill, J., Sebaihia, M., Preston, A., Murphy, L. D., et al. (2003). Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Nature genetics, 35(1), 32-40. doi:10.1038/ng1227
• PubMed Abstract: http://www.ncbi.nlm.nih.gov/pubmed/12910271
• PubMed Central: Not available on PubMed Central.
• Publisher Full Text (HTML): http://www.nature.com/ng/journal/v35/n1/full/ng1227.html
• Publisher Full Text (PDF): http://www.nature.com/ng/journal/v35/n1/pdf/ng1227.pdf
• Copyright: ©2003 Nature Publishing Group (information found on PDF version of article). This article is not Open Access, but it is freely available 6 months after publication.
• Publisher: Nature Publishing Group (for-profit).
• Availability: In print and online.
• Did LMU pay a fee for this article: Yes, LMU pays a subscription fee for access to the journal Nature Genetics.

The Parkhill et al. (2003) paper was accessed using the link listed above as "Publisher Full Text (PDF)".

Defining Unknown Biological Terms from Parkhill et al.

Unknown terms from the Parkhill et al. (2003) paper were entered into search engines, and results were vetted until definitions for each were found in quality sources. Links to the sites from which the definitions were attained are included below.

1. Pseudogenes
   • Pseudogenes are genomic DNA sequences similar to normal genes but non-functional; they are regarded as defunct relatives of functional genes.
   • Citation: http://pseudogene.org/background.php
2. Fimbriae
   • Modern term for short, hair-like projections or appendages (organelles) on the outer surface of certain bacteria composed of protein subunits (pilin) extending outward from the surface that act as a virulence factor by promoting adherence; formerly known as pili.
   • Citation: http://www.life.umd.edu/classroom/bsci424/Definitions.htm
3. Auxotrophy
   • The inability of a organism to synthesize a particular organic compound required for its growth.
   • Citation: http://goldbook.iupac.org/A00537.html
4. Ortholog
   • Orthologs are genes that have evolved from a common ancestor gene. These genes can be in different species or within the same species.
   • Citation: http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2010/Piper/Orthologs.html
5. Insertion Sequence Elements (ISEs)
   • Insertion sequences, or insertion-sequence (IS) elements, are now known to be segments of bacterial DNA that can move from one position on a chromosome to a different position on the same chromosome or on a different chromosome. When IS elements appear in the middle of genes, they interrupt the coding sequence and inactivate the expression of that gene.
   • Citation: http://www.ncbi.nlm.nih.gov/books/NBK21779/
6. Horizontal Gene Transfer
   • The movement of genetic material between bacteria other than by descent in which information travels through the generations as the cell divides.
   • Citation: https://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/genetic-exchange/exchange/exchange.html
7. Prophage
   • A phage chromosome inserted as part of the linear structure of the DNA chromosome of a bacterium. A temperate phage integrated into the host chromosome.
   • Citation: http://groups.molbiosci.northwestern.edu/holmgren/Glossary/Definitions/Def-P/prophage.html
8. Autotransporter
   • The key feature of an autotransporter is that it contains all the information for secretion in the precursor of the secreted protein itself. Autotransporters comprise three functional domains: 1) an N-terminal targeting domain (amino-terminal leader sequence) that functions as a signal peptide to mediate targeting to and translocation across the inner membrane 2) a C-terminal translocation domain (carboxy-terminal) that forms a beta-barrel pore to allow the secretion of 3) the passenger domain, the secreted mature protein.
   • Citation: https://www.ebi.ac.uk/interpro/entry/IPR006315
9. Type-III Secretion System
   • The protein Type III Secretion System (T3SS) is a supramolecular, organic nanomachine that injects bacterial virulence proteins into eukaryotic cells to modulate their physiology for the benefit of the pathogen.
   • Citation: http://lab.rockefeller.edu/stebbins/research/T3SS
10. Constitutive Expression
    • Expression of a gene that is transcribed at a constant level.
    • Citation: http://www.biology-online.org/dictionary/Constitutive_expression

Comparative Analysis of the Genome Sequences of Bordetella Pertussis, Bordetella Parapertussis and Bordetella Bronchiseptica Outline

The following outline was adapted from the original paper published by Parkhill et al. (2003) in Nature Genetics.

Introduction

Establishing the Importance of B. pertussis, B. parapertussis, and B. bronchiseptica

• All three of these bacteria are pathogens that colonize the respiratory tracts of mammals.
  • B. bronchiseptica- infects a wide range of mammals.
  • B. parapertussis- infects both humans and sheep.
  • B. pertussis- specific to humans vectors.
    • B. pertussis is the causative agent of whooping cough.
    • Despite vaccination programs, whooping cough is still endemic in some countries, causing hundreds of thousands of deaths every year.
• Evidence suggests that B. pertussis and B. parapertussis may have evolved in the recent past from a common ancestor, possibly the more genetically diverse species B. bronchiseptica.
  • The species exhibit similar virulence factors.
  • Gene expression in these species is regulated by the two-component BvgA/S regulatory system.
    • Bvg-plus phase: vector detected. Virulence-activated genes (vags) up-regulated and virulence-repressed genes (vrgs) down-regulated.
    • Bvg-minus phase: in the environment. Standard gene expression occurs.

Experimental Design: Sequencing B. pertussis, B. parapertussis, and B. bronchiseptica

• Specific strains of each Bordetella species were sequenced.
• Genome sequences were compared to:
  1. Compare genetic background.
  2. Assess factors influencing variable disease severity.
  3. Assess factors influencing variable host range.

Results

Structure of the Genomes

• Figure 1 compares the general properties of the three sequenced genomes using circular representations. Genes are labelled to show similarities & differences between genomes and color-coded to show association with several key gene ontology (GO) terms.
  • Suggests that B. pertussis and B. parapertussis evolved from an ancestor similar to B. bronchiseptica.
  • Calculations of time to most recent common ancestor (MRCA) suggest a recent bottleneck as opposed to recent descent from B. bronchiseptica
• Table 1 quantifies the general features of the sequenced genomes presented in Figure 1. Counts for categories such as "pseudogenes" and insertion sequence elements (ISEs) are included for use in later analyses.
  • Initially used to support the conclusions drawn from Figure 1.
• Figure 2 linear genomic comparison between the sequenced genomes. Red lines indicate similarities and black triangles indicate ISEs.
  • B. parapertussis & B. bronchiseptica are more similar than B. pertussis & B. bronchiseptica.
  • Suggest that frequent recombination and deletion has occurred in the genomes of B. bronchiseptica and B. pertussis.
  • Losses in the B. pertussis genome are due to expansion in ISEs in this species, resulting in ISE-mediated deletion events.

Gene Complements

• Figure 3 presents a venn diagram comparing the gene complements of the three Bordetella spp..
  • Support the hypothesis that B. pertussis and B. parapertussis are derivatives of B. bronchiseptica.
    • Very few genes are unique to B. pertussis and B. parapertussis (114 and 50, respectively).
    • B. bronchiseptica contains over 600 genes that are not present in the genomes of the other two species.
• Figure 4 categories genes lost by B. pertussis and B. parapertussis based on their associated GO terms.
  • Demonstrates that genes lost in the derivative Bordetella species are involved in the following processes: membrane transport, small-molecule metabolism, regulation of gene expresion, and synthesis of surface structures.
• B. pertussis and B. parapertussis appear to have lost the function of many genes present in B. bronchiseptica through the formation of pseudogenes (in addition to deletion losses).
  • Figure 4 demonstrates that the genes lost in this manner are, once again, associated in the same processes as above (e.g. membrane transport).

Metabolism

• Genomic analysis demonstrates that all three isolates predominantly share a common central and intermediary metabolism.
• Bordetella all share glutamate as their main carbon source, as they do not synthesize or break down glucose.
• Bordetella all do not have a complete pathway for the biosynthesis of cysteine.
  • Suggests Bordetella have lost an ancestral ability to use external sulfur sources for cysteine synthesis.
• The fact that B. pertussis and B. parapertussis cannot survive in the environment without a host while B. bronchisepticacan, despite these similarities, suggests the loss of many accessory pathways for use of alternative nutrient sources.

Host Range and Pathogenicity

• FhaB (protein involved in attachment to host cells) variation.
  • Ortholog present in all three species, but there are internal variations in each.
  • Two extra genes encoding FhaB-like proteins are contained in the B. pertussis genome.
  • Variation Present in other fimbrial systems as well.
  • Suggests that the Bordetella variable host specificity may be influenced by changes in this receptor-ligand interaction.
• Autotransporter protein variation.
  • Table 2 demonstrate differing autotransporter complements amongst the three species.
  • B. pertussis and B. parapertussis have fewer genes and more pseudogenes.
  • May also impact host specificity.
• Siderophore similarities and variation.
  • All three species contain operons for the siderophore alcaligin, suggesting they scavenge iron from their hosts.
  • B. pertussis and B. parapertussis have fewer genes and more pseudogenes that code for siderophores.
  • Lacking necessary siderophores may influence host specificity.
• Variation is present in the following virulence structures: type-III secretion systems, O-antigens, and flagella.
  • Variations in the O-antigen that were identified supported claims made in previous literature.
  • The full flagellar operon is intact in B. bronchiseptica but inactivated in B. parapertussis and B. pertussis.
    • Suggests this lack of motility confers host-restrictions.
• Discovered a locus that codes for a polysaccharide capsule.
  • Typical type II capsule arrangement.
    • The presence of this type of capsule in Bordetella spp. was argued for in previous literature.
  • Expression of this capsule is variable among the three species:
    • B. bronchiseptica has the intact capsule locus.
    • B. parapertussis has an introduced stop codon, prevent expression of this capsule.
    • B. pertussis has mutations at the beginning and end of the locus, suggesting that little to no capsule expression occurs.
  • These findings suggest that the capsule does not influence pathogenesis in humans, and that it instead aids in survival in the environment.
• Overall conclusion: The absence of surface structures such as flagella, fimbriae, and a capusle, increase the virulence of B. pertussis (and B. parapertussis) by reducing immune system targets.
• Variations in toxin production.
  • The pertussis toxin operon is present in all three species but expressed differently:
    • B. parapertussis and B. bronchiseptica contain the operon but do not express it due to changes in the ptxA promoter region.
      • This supports previous claims made in the literature.
    • B. pertussis produces the toxin.
  • Figure 5 compares the ptxA promoter region present in the pertussis toxin operon among all three Bordetella species.
    • Demonstrates that the majority of base changes (62%) are present only in B. pertussis.
    • Suggests recent mutations allowed for pertussis toxin expression.

Discussion

Comparing the Genomes of B. pertussis, B. parapertussis, and B. bronchiseptica elucidates differences in host range and pathogenesis.

• Host interaction factors and virulence determinants were not recently acquired by the more virulent strains (B. pertussis and B. parapertussis). This was contrary to expectation.
• Individual traits in the derivative species (B. pertussis and B. parapertussis) that have enabled virulence were generated by independent gene deletions and inactivations by creation of pseudogenes.
• Increased virulence can be explained by:
  1. Overexpression of virulence traits in human vectors.
  2. Loss of structures that allowed for simpler immune recognition.
  • In the process of explaining increased virulence, several findings supported pre-existing literature:
    • The pertussis toxin operon is present in B. parapertussis and B. bronchiseptica but not expressed.
    • B. bronchiseptica and possibly B. pertussis have polysaccharide capsules.
    • O-antigen variations are present between the three species.
• Why were these changes selected for?
  • Coevolution with the expansion of Homo sapiens.
  • Higher transmission rates no longer necessitate environmental survival (e.g. loss of capsule) or limited damage to the host (e.g. pertussis toxin).

Overall Impact This paper sequenced the genomes of B. pertussis, B. parapertussis, and B. bronchiseptica; provided evidence for the common ancestry of these organisms; identified new information regarding their capsules; and proposed the genetic roots of increased virulence in the two strains that are human pathogens. These findings can be applied to genomic analyses of future pathogens to assess virulence.

Methods

DNA Preparation and Sequencing

• Strains were gifted to the researchers from the following sources:
  • Food and Drug Administration (B. pertussis Tomaha I).
  • UCLA (B. bronchiseptica RB50).
  • Universitat Erlangen (B. parapertussis strain 12822).
• Bacteria were grown on Bordet Gengou agar supplemented with 15% defibrinated horse blood at 37 degrees Celcius for three days.
• Initial genome assemblies were obtained from thousands of paired-end sequences derived from genomic shotgun libraries using dye terminator chemistry on automated sequencers.
• Thousands of paired-end sequences from a pBACe3.6 library with sizes ranging from 15-48k kb were used as scaffolding.
• Thousands of further sequence reads were generated during sequencing.

Annotation and Analysis

• Artemis was used for data collection and annotation.
• Best-match FASTA comparisons were used to identify orthologous genes.
• Pseudogenes were identified by direct comparison.
• Calculations were made using the genome data produced:
  • Synonymous substitution frequency values were calculated from orthologous gene pairs.
  • Ages of divergence were estimated.

Bordetella Pertussis Model Organism Database

Gene Database: http://www.genedb.org/Homepage/Bpertussis Example Link: http://www.genedb.org/gene/BP3783?actionName=%2FQuery%2FquickSearch&resultsSize=1&taxonNodeName=Root

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