Determining the repertoire of immunodominant proteins via whole-genome amplification of intracellular pathogens

PLoS One. 2012;7(4):e36456. doi: 10.1371/journal.pone.0036456. Epub 2012 Apr 30.

Abstract

Culturing many obligate intracellular bacteria is difficult or impossible. However, these organisms have numerous adaptations allowing for infection persistence and immune system evasion, making them some of the most interesting to study. Recent advancements in genome sequencing, pyrosequencing and Phi29 amplification, have allowed for examination of whole-genome sequences of intracellular bacteria without culture. We have applied both techniques to the model obligate intracellular pathogen Anaplasma marginale and the human pathogen Anaplasma phagocytophilum, in order to examine the ability of phi29 amplification to determine the sequence of genes allowing for immune system evasion and long-term persistence in the host. When compared to traditional pyrosequencing, phi29-mediated genome amplification had similar genome coverage, with no additional gaps in coverage. Additionally, all msp2 functional pseudogenes from two strains of A. marginale were detected and extracted from the phi29-amplified genomes, highlighting its utility in determining the full complement of genes involved in immune evasion.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anaplasma marginale / genetics*
  • Anaplasma marginale / immunology
  • Anaplasma phagocytophilum / genetics*
  • Anaplasma phagocytophilum / immunology
  • Animals
  • Antigens, Bacterial / chemistry
  • Antigens, Bacterial / genetics
  • Antigens, Bacterial / immunology
  • Bacillus Phages / genetics
  • Bacterial Outer Membrane Proteins / chemistry
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Outer Membrane Proteins / immunology
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / immunology*
  • Base Sequence
  • Cattle
  • Genomics / methods*
  • HL-60 Cells
  • Humans
  • Intracellular Space / microbiology*
  • Molecular Sequence Data
  • Nucleic Acid Amplification Techniques / methods*
  • Polymorphism, Single Nucleotide
  • Pseudogenes / genetics
  • Sequence Analysis, DNA

Substances

  • Antigens, Bacterial
  • Bacterial Outer Membrane Proteins
  • Bacterial Proteins
  • msp2 protein, Anaplasma marginale