RESUMEN
Culture filtrates of Mycobacterium tuberculosis H37Rv highly enriched with secreted proteins were used to identify antigens recognized by a serum pool from tuberculosis patients. Two different approaches were used to separate the culture filtrate protein mixture: (i) proteins were fractionated according to their hydrophobicity using an HPLC-C18 chromatography column followed by separation based on their molecular mass by SDS-PAGE and subsequent immunoblotting or (ii) proteins were separated by two-dimensional gel electrophoresis, based on their isoelectric point and their molecular mass. Twenty serologically reactive proteins were ultimately identified by both methods, including four novel antigens. Further, to estimate the immunogenicity of the identified culture filtrate proteins, the relative antibody quantities were measured using Image master software. Our results show that the antibodies against proteins belonging to the antigen 85 complex were the most abundant in the serum of patients with active tuberculosis. The most immunogenic proteins in terms of high antibody-to-protein-ratio were Rv3881c and three lipoproteins Rv0934 (the 38 kDa antigen), Rv0932c (pstS2), and Rv3006 (LppZ). Rv 3881c [corrected] is located close to the RD1 region and is also present in BCG. [corrected] The proteins from the M. tuberculosis H37Rv culture filtrate are strong candidates to be evaluated for improvement of the serodiagnostic tests of tuberculosis.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/análisis , Mycobacterium tuberculosis/inmunología , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Células Cultivadas , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Filtración , Humanos , Procesamiento de Imagen Asistido por Computador , Immunoblotting , Tuberculosis Pulmonar/sangre , Tuberculosis Pulmonar/inmunologíaRESUMEN
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is one of the main killers among infectious pathogens in the world and represents an important factor that sustain poverty in developing countries. Failure of the BCG vaccine to protect in endemic regions, and increasing problems with multi-drug-resistant TB calls for development of better vaccines to prevent reactivation of tuberculosis. It has been estimated that an effective post-exposure vaccine will prevent 30-40% of the TB cases. New vaccines should also prevent development of TB in HIV-infected individuals. Recent characterization of M. tuberculosis H37Rv by proteomic methods has revealed a large number of novel secreted proteins that should be investigated in mouse models for latent and slowly progressive TB. There is an important balance between control of infection and tissue destruction in TB, and M. tuberculosis has developed strategies to prevent immune-mediated sterilization. Central to this strategy is inhibition of apoptosis of macrophages. Development of novel vaccines should therefore take into consideration the effects on central markers to obtain a better picture of regulation of immunity, including FasL and Bcl-2 which are essential in regulation of apoptosis.