title: The application of proteomics in defining the T cell antigens of Mycobacterium tuberculosis.
abstract: The complete sequencing of the Mycobacterium tuberculosis genome offers a unique opportunity to fully elucidate the biology of this human pathogen. One aspect of significant importance is the definition of T cell antigens. This report describes the development and implementation of a proteomic approach to defining such antigens. Large quantities of subcellular protein fractions of M. tuberculosis were resolved by two-dimensional liquid phase electrophoresis (2-D LPE), resulting in 355 and 299 fractions of culture filtrate and cytosolic proteins, respectively. Analysis of these fractions against splenocytes of C57Bl/6 mice infected with M. tuberculosis resulted in the identification of 37 fractions that stimulated a dominant T cell response, as measured by the production of interferon-gamma. Additionally, when the 2-D LPE fractions were assayed against splenocytes harvested at 10 and 40 days post infection significant changes in the T cell response were observed. Molecular characterization of the proteins contained in each of the 38 immunodominant fractions by liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry resulted in the identification of 30 individual proteins. Many of these represented previously defined antigens. However 17 of these proteins were novel T cell antigens. The data presented demonstrate that proteomics offers a rapid and facile approach for elucidation of immunodominant T cell antigens of pathogenic bacteria.
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: B A
authors.initials: BA
authors.lastname: Covert
authors.forename: J S
authors.initials: JS
authors.lastname: Spencer
authors.forename: I M
authors.initials: IM
authors.lastname: Orme
authors.forename: J T
authors.initials: JT
authors.lastname: Belisle
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional; D016680:Genome, Bacterial; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D051379:Mice; D008810:Mice, Inbred C57BL; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D020543:Proteome / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D020543:Proteome; D007371:Interferon-gamma