A Comparative Functional Genomics Analysis of Gene Expression and Intra-Macrophage Adaptation of Strains of Francisella Tularensis

The molecular basis for the differences in virulence profiles of the strains of Francisella tularensis is not yet known. A series of studies comparing the expressed gene products, both proteomes and transcriptomes, of the strains of F.tularensis in both defined medium and in macrophages were undertaken to discover the most probable gene candidates for virulence. These studies identified candidate genes and proteins that are likely involved in virulence and intra-macrophage growth. The strains used in the transcriptome study were F.t.tularensis Schu4, F.t.novicida U112, F.t.holarctica OR960246, and F.t.holarctica LVS. All the strains were grown to an O.D. of 0.6 in synthetic media. The comparison of gene expression of virulent F.t.tularensis Schu4 to moderately virulent strain of F.t.holarctica OR960246 showed genes involved in polyamine synthesis, citrulline ureidase, isochorismatases, and many transporters to be highly expressed in F.t.tularensis Schu4. When F.t.tularensis Schu4 was compared to the avirulent F.t.novicida U112, a number of genes involved in general metabolism were highly expressed only in F.t.novicida, consistent with the phenomenon of genome decay. In contrast, genes involved in iron uptake, type I secretion system, and some hypothetical genes were highly expressed in Schu4. In addition, many of the genes which have low expression in OR960246 also have low expression in U112 making them likely candidates for high virulence. When OR960246 is compared with the vaccine strain LVS, there were only a few expression differences. Adaptation of F.tularensis strains to intra-macrophage growth was monitored in F.t.holarctica LVS and F.t.novicida U112. There were marked differences in the sets of modulated genes and operons between the two strains. However, many of the modulated pathways were similar. Iron response system was found to be one of the major pathways that were modulated inside the macrophage. From both baseline expression and intra-macrophage adaptation responses, it was shown that in spite of high genome identity the expressed gene products are markedly different. There is a large subset of genes which have high expression only in the virulent strains. Many of these genes are among the genes which are modulated inside the macrophage in the less decayed strains. Further characterization of these differences will lead to a better understanding of the virulence profiles and intra-macrophage adaptation factors of these strains.