NATURAL-RESISTANCE TO INTRACELLULAR PARASITES - A STUDY BY 2-DIMENSIONAL GEL-ELECTROPHORESIS COUPLED WITH MULTIVARIATE-ANALYSIS

Natural resistance to Mycobacterium bovis bacillus Calmette-Guerin (BC G) is determined by the Beg gene (Nramp1), which is exclusively expres sed by mature macrophages. The Nramp1 gene is a dominant autosomal gen e that has two allelic forms; r confers resistance and s confers susce ptibility 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 pr ovide clues in the studies on Nramp1 gene function. We performed two-d imensional gel electrophoresis of cellular proteins prepared from a B1 0R macrophage line derived from mice carrying the r allele of the Nram p1 gene, B10S macrophages carrying the s allele, and B10R-Rb macrophag es transfected with Nramp1-ribozyme. The classification of protein pat terns and selection of distinct proteins characteristic of r or s alle le-carrying macrophages was performed using the principal component an alysis. We found differential expression of four proteins with the fol lowing isoelectric point/molecular weight (pI/M-r) in B10R macrophages compared to B10S and B10R-Rb macrophages: 6.6/25, 7.0/22, 9.1/31.5, a nd 5.3/8.5. The protein 7.0/22 has been identified as Mn-superoxide di smutase and the best candidate for protein p6.6/25 seems to be Bcl-2 a ccording to the immunoblot analysis. When the splenic macrophages earr ing the I or s allele were analyzed, the changes in relative abundance for proteins 6.6/25 and p7.0/22 were satisfactorily reproduced. Overa ll, the two identified proteins are important in the regulation of int racellular redox balance and the regulation of apoptosis in macrophage s, respectively. Our findings may suggest their possible biological ro le in the innate immunity against intracellular pathogens.