Natural resistance to intracellular parasites: a study by two-dimensional gel electrophoresis coupled with multivariate analysis.
Natural resistance to Mycobacterium bovis bacillus Calmette-Guérin (BCG) is determined by the Bcg gene (Nramp1), which is exclusively expressed by mature macrophages. The Nramp1 gene is a dominant autosomal gene that has two allelic forms; r confers resistance and s confers susceptibility to infection with intracellular pathogen. Although the wide range of pleiotropic immunological effects of the Nramp1 gene has been described, the exact mechanism of its action remains elusive. In this study we searched for differentially expressed proteins that might provide clues in the studies on Nramp1 gene function. We performed two-dimensional gel electrophoresis of cellular proteins prepared from a B10R macrophage line derived from mice carrying the r allele of the Nramp1 gene, B10S macrophages carrying the s allele, and B10R-Rb macrophages transfected with Nramp1-ribozyme. The classification of protein patterns and selection of distinct proteins characteristic of r or s allele-carrying macrophages was performed using the principal component analysis. We found differential expression of four proteins with the following isoelectric point/molecular weight (pI/Mr) in B10R macrophages compared to B10S and B10R-Rb macrophages: 6.6/25, 7.0/22, 9.1/31.5, and 5.3/8.5. The protein 7.0/22 has been identified as Mn-superoxide dismutase and the best candidate for protein p6.6/25 seems to be Bcl-2 according to the immunoblot analysis. When the splenic macrophages carrying the r or s allele were analyzed, the changes in relative abundance for proteins 6.6/25 and p7.0/22 were satisfactorily reproduced. Overall, the two identified proteins are important in the regulation of intracellular redox balance and the regulation of apoptosis in macrophages, respectively. Our findings may suggest their possible biological role in the innate immunity against intracellular pathogens.
Kovárová
H
H
Purkyne Military Medical Academy, Hradec Králové, Czech Republic. kovarova@pmfhk.cz
Radzioch
D
D
Hajdúch
M
M
Sirová
M
M
Bláha
V
V
Macela
A
A
Stulík
J
J
Hernychová
L
L
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D027682:Cation Transport Proteins; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D007113:Immunity, Innate; D008264:Macrophages / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D015999:Multivariate Analysis; D009163:Mycobacterium bovis / Q000276:immunology*; D014376:Tuberculosis; D000099173:Natural Resistance-associated Macrophage Protein 1
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D008565:Membrane Proteins; D000099173:Natural Resistance-associated Macrophage Protein 1