An intracellularly inducible gene involved in virulence and polyphosphate production in Francisella

Abstract

Francisella tularensis is an intracellular pathogen capable of multiplying to high levels in macrophages. By protein analysis, only a few proteins have been shown previously to be expressed at high levels in macrophages relative to bacteria grown in culture media. To identify additional genes that show increased expression during intracellular growth, we developed a plasmid for use in Francisella based on the induction of expression of green fluorescent protein. Clones of F. tularensis subsp. novicida were identified that were fluorescent only intracellularly and not when grown in vitro. Sequencing identified a range of genes comprising some such as dnaK that are already known to be expressed intracellularly and some novel targets. One of these newly identified regulated genes, FTN1472/FTT1564, was selected for further study. Isogenic mutants were generated in F. tularensis subsp. novicida and subsp. tularensis by allelic replacement. Inactivation of the gene resulted in abolition of polyphosphate production by F. novicida, strongly supporting the bioinformatic analysis, which had suggested that the gene may encode a polyphosphate kinase. The mutants exhibited defects for intracellular growth in macrophages and were attenuated in mice, indicating a key role for the putative polyphosphate kinase in the virulence of Francisella.