title: Determining the repertoire of immunodominant proteins via whole-genome amplification of intracellular pathogens.
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.
authors.affiliation: Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA. darkmich@ufl.edu
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Dark
authors.forename: Anna M
authors.initials: AM
authors.lastname: Lundgren
authors.forename: Anthony F
authors.initials: AF
authors.lastname: Barbet
mesh_terms: D000595:Amino Acid Sequence; D042322:Anaplasma marginale / Q000235:genetics* / Q000276:immunology; D041081:Anaplasma phagocytophilum / Q000235:genetics* / Q000276:immunology; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017103:Bacillus Phages / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D001483:Base Sequence; D002417:Cattle; D023281:Genomics / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D042541:Intracellular Space / Q000382:microbiology*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011544:Pseudogenes / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C103348:msp2 protein, Anaplasma marginale