IDENTIFICATION OF PROTEINS OF FRANCISELLA-TULARENSIS INDUCED DURING GROWTH IN MACROPHAGES AND CLONING OF THE GENE ENCODING A PROMINENTLY INDUCED 23-KILODALTON PROTEIN

The adaptation of facultative intracellular bacteria to host macrophag es involves regulation of the synthesis of bacterial proteins, We anal yzed the protein synthesis of Francisella tularensis LVS growing intra cellularly in the macrophage-like murine cell line J774 and extracellu larly in culture medium. After pulse-labeling with [S-35] methionine a nd separation by one- and two-dimensional polyacrylamide gel electroph oresis, induction of a few proteins during intracellular growth was de monstrated, One of them, a 23-kDa protein, was prominently induced in the macrophages and also when extracellularly growing F. tularensis wa s exposed to hydrogen peroxide. After isolation of the 23-kDa protein from a preparative two-dimensional gel, a 22-amino-acid N-terminal pep tide and two peptides obtained by trypsin digestion were sequenced, Ba sed on the sequences, degenerate oligonucleotides were constructed for use as primers in a PCR. Hybridization of amplified DNA to XbaI-diges ted LVS DNA identified the gene of the 23-kDa protein in a 1.3-kb DNA fragment, Nucleotide sequence analysis revealed an open reading frame encoding a putative protein of a calculated molecular mass of 22.2 kDa , The open reading frame was preceded by a sequence typical of ribosom e-binding sites in Escherichia coli, The amplified gene was successful ly expressed by the pTrc99A vector in E. coli under control of the trc promoter, The gene product showed the same mobility and immunoreactiv ity as the 23-kDa protein of F. tularensis, The deduced amino acid seq uence showed no significant homology with protein sequences in current data banks, Thus, intracellular growth of F. tularensis in macrophage s was associated with prominent upregulation of a novel 23-kDa protein .