Anuvarsh Week 11

Burkholderia cenocepacia Genome Paper

Holden, M. T., Seth-Smith, H. M., Crossman, L. C., Sebaihia, M., Bentley, S. D., Cerdeño-Tárraga, A. M., ... & Parkhill, J. (2009). The genome of Burkholderia cenocepacia J2315, an epidemic pathogen of cystic fibrosis patients. Journal of bacteriology, 191(1), 261-277.

Biological Terms and Definitions

1. Pseudogenes
   • Pseudogenes are genomic DNA sequences similar to normal genes but non-functional; they are regarded as defunct relatives of functional genes.
   • Citation: [1]
2. Melioidosis
   • Melioidosis, also called Whitmore's disease, is an infectious disease that can infect humans or animals. The disease is caused by the bacterium Burkholderia pseudomallei.
   • Citation: [2]
3. Glanders
   • Glanders is an infectious disease that is caused by the bacterium Burkholderia mallei.
   • Citation: [3]
4. Orthologous
   • Orthologs are genes in different species that evolved from a common ancestral gene by speciation.
   • Citation: [4]
5. Genomic islands
   • Genomic islands (GIs) are commonly defined as clusters of genes of probable horizontal origin >8 kb in size in bacterial and archaeal genomes (Langille et al., 2010).
   • Citation: [5]
6. Mobile genetic elements
   • Mobile genetic elements (MGEs) are segments of DNA that encode enzymes and other proteins that mediate the movement of DNA within genomes (intracellular mobility) or between bacterial cells (intercellular mobility).
   • Citation: [6]
7. Horizontal transfer of genes
   • Horizontal gene transfer is defined to be the movement of genetic material between bacteria other than by descent in which information travels through the generations as the cell divides. It is most often thought of as a sexual process that requires a mechanism for the mobilization of chromosomal DNA among bacterial cells.
   • Citation: [7]
8. Exoenzymes
   • An enzyme that acts outside the cell that secretes it.
   • Citation: http://medical-dictionary.thefreedictionary.com/exoenzyme
9. Metalloproteases
   • Metalloproteases are a group of enzymes comprising a section of the protease family that contain a catalytic metal ion at their active site which aids in the hydrolysis of peptide bonds leading to protein degradation.
   • Citation: [8]
10. Actinobacterium
    • The Actinobacteria are a group of Gram-positive bacteria. Most are found in the soil, and they include some of the most common soil life, playing important roles in decomposition and humus formation.
    • Citation: [9]
11. Nonmucoid
    • (noun)Any of various glycoproteins, especially a mucoprotein, similar to the mucins. (adj) Of, relating to, or resembling mucus.
    • Citation: [10]

Outline of Paper

• Introduction
  • Burkholderia cenocepacia is a known cystic fibrosis pathogen.
    • It appeared in 1980 when CF patients experienced several symptoms that resulted in early death.
    • The resulting decline in health was then on referred to as “cepacia syndrome”.
    • The specific strain associated with the “cepacia syndrome” is the ET12 strain.
  • B. cenocepecia J2315 is part the ET12 ancestral linage.
  • The researchers sequenced the J2315 strain in order to find information regarding the how the ET12 linage adapted to result in the previously mentioned clinical symptoms.
    • The sequence of the J2315 strain would also give insight on the performance of the J2315 strain in patients with CF.
• Materials and Methods
  • Bacterial strains
    • B. cenocepacia was first isolated in 1989 in the UK.
    • J2315 is resistant to several different drugs
    • In addition to J2315, 7 other previously sequenced strains were also used in this study.
  • Genome sequencing
    • Whole genome sequencing
      • DNA was extracted from J2315 and sequence data was obtained.
      • Sequence was annotated using the Artemis software.
      • Initial coding sequences were identified using Orpheus.
      • The CDSs were verified using protein databases in conjunction with BLAST and the DNA sequence was verified through all 6.
    • Comparative genomics
      • Comparisons of genome sequences were run using Artemis Comparison Tool
    • B. cenocepecia strain J2315 was compared to several other burkholderia species
  • PCR screening
    • CRP amplification was done using Platinum Pfx DNA polymerase with suppliers protocols.
• Results
  • Fig. 1: Genome contained 3 circular chromosomes and a plasmid. The figure features diagrams of each of the chromosome and plasmid that are color coded according to CDSs.
  • Table 1: This table contained general features of the plasmid and each of the chromosomes in the B. cenocapacia J2315 genome. Chromosome 1 has the greatest number of base pairs, CDSs, and pseudogenes and partial genes while the plasmid has the fewest of each of those categories
  • Fig. 2: The general distributions of CDSs among chromosomes and the functional classes they are associated with.
    • Chromosome 1 has more CDSs that are involved with core functions of the cell (cell division, metabolism, etc.). Chromosome 2 and 3 have more CDSs that are involved with accessory functions (protective responses, gene transfer), and other unknown functions.
  • Fig 3: The orthologs associated with B. cenocepacia J2315 include B. cenocepacia HI2424, B. cenocepacia AU1054, B. contaminans 383, B. ambifaria AMMD, B. vietnamiensis G4, B. xenovorans LB400, B. pseudomallei K96243, B. thailandensis H264 and Ralstonia solanacearum GM11000.
    • B. cenocepacia was compared to 5 other Burkholderia cenocepacia complex (BCC) genomes in the public database as well as 4 other strains of B. cenocepacia known to cause melioidosis and glanders. The largest number of orthologs (found using FASTA) were found in the BCCs and Ralstonia solanacearum.
  • Table 2: RODs exist in the B. cenocepacia J21315 genome.
    • Comparison between B. cenocepacia strains found regions of difference (RODs) indicating that 21% of J2315 contains RODs.
      • These include genomic islands that appeared most likely due to horizontal gene transfer.
      • Genomic islands were defined as regions displaying irregular %G+C content or other indications of recent horizontal transfer of genes. Genes associated with mobile genetic elements (MGEs) were also identified (bacteriophages, transposons, and plasmids).
      • Other RODs in J2315: indel regions representing lineage specific DNA insertions or deletions ad allelic variants that have divergent sequences at the same locus.
        • RODs not including at least one complete CDS were not considered in this analysis
    • ET12 linage of B. cenocepacia emerged recently and spread between patients readily, thereby causing disease. This may be attributed to 14 genomic islands in J2315 strain.
  • Table 3: 9 special functions encoded in the J2315 genome were identified that may have added effects to the virulence of J2315. These functions include exoproteins, secretion, LPS and capsule, adhesins, fimbriae and pili, quorum sensing, siderophores, intracellular stress, and motility.
    • Each of these functions are specific to J2315, and do not appear in other strains in the BCC.
  • Table 4: J2315 exhibits incredible drug resistance, which has been attributed to specific functions and products of each chromosome.
    • Chromosome 2 has the largest number of products that result in drug resistance.

Some drugs J2315 is known to be resistant to include: Fosmidomycin, aminoglycosides, trimethoprim, tetracycline, and others.

• Discussion
  • Horizontal gene transfer has played a large role in genomic plasticity and allowed for strains to develop specific niches – such as CF patients lungs.
  • J2315 contains 14 genetic islands absent from other B. cenocepacia strains.
  • Some genetic islands are similar to species of Burkholderia indicating that the genome spreads further than the species, while others are specific to the ET12 lineage and result in pathogenesis in CF patients.
  • The ET12 lineage can be seen to be pathogenic in the RODs which lack MGEs.
  • J2315 has a higher level of antibiotic resistance compared to other strains.
    • Extensive antibiotic resistance has come through the form of point mutations
  • Table 5: Several pseudogenes exist in the genome of strain J2315 that are usually active and attributed with virulence activity in other strains.
    • These pseudogenes came about IS interruptions and frameshift mutations. The level of mutation found in J2315 is comparable to the mutations found in other bacteria, so J2315 does not have an elevated level of mutation.
  • Table 6: Virulence factors exist in other B. cenocepacia IIIA strains including J2315, BCC0011, BCC0179, BCC0162, BCC0313, BCC0016, K56-2, BCC1261, and BCC0222.
    • Analyzing the appearance of different virulence factors among these strains provides information regarding the timeline of mutations.
  • In Toronto and Cleveland, a study was performed that indicated that pyochelin-positive strains of CF existed in patients with severe pulmonary disease. Lack of expression of pyochelin in ET12 and its related strains may result in resistance in the CF lungs, thereby helping the bacteria spread from patient to patient.
  • Another study in Vancouver found that 50% of the 506 B. cenocepacia strains collected from CF patients were nonmucoid. The loss of a specific cluster of genes (EPS) may have resulted in increased virulence in a CF lung.
  • A recent study by Smith et al. found that virulence factors are the most mutated class of genes throughout the infection time frame. This study found that the O antigen in P. aeruginosa received mutations, and the same antigen was found to have mutations in J2315.

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