Global Transcriptional Response to Spermine, a Component of the Intramacrophage Environment, Reveals Regulation of Francisella Gene Expression through Insertion Sequence Elements

Abstract

Tularemia is caused by the category A biodefense agent Francisella tularensis. This bacterium is associated with diverse environments and a plethora of arthropod and mammalian hosts. How F. tularensis adapts to these different conditions, particularly the eukaryotic intracellular environment in which it replicates, is poorly understood. Here, we demonstrate that the polyamines spermine and spermidine are environmental signals that alter bacterial stimulation of host cells. Genomewide analysis showed that F. tularensis LVS undergoes considerable changes in gene expression in response to spermine. Unexpectedly, analysis of gene expression showed that multiple members of two classes of Francisella insertion sequence (IS) elements, ISFtu1 and ISFtu2, and the genes adjacent to these elements were induced by spermine. Spermine was sufficient to activate transcription of these IS elements and of nearby genes in broth culture and in macrophages. Importantly, the virulent strain of F. tularensis, Schu S4, exhibited similar phenotypes of cytokine induction and gene regulation in response to spermine. Distinctions in gene expression changes between Schu S4 and LVS at one orthologous locus, however, correlated with differences in IS element location. Our results indicate that spermine and spermidine are novel triggers to alert F. tularensis of its eukaryotic host environment. The results reported here also identify an unexpected mechanism of gene regulation controlled by a spermine-responsive promoter contained within IS elements. Different arrangements of these mobile genetic elements among Francisella strains may contribute to virulence by conveying new expression patterns for genes from different strains.

Francisella tularensisΔpyrFMutants Show that Replication in Nonmacrophages Is Sufficient for PathogenesisIn Vivo

“…Buffy coats from blood donations (Central Blood Bank, Pittsburgh, PA) served as the source of human monocytes that were differentiated into macrophages by in vitro culture as has been detailed previously (9,10,27,29,50). For in vitro infections, primary human macrophages were washed and resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% human serum AB (complement replete; GemCell Gemini Bio-Products), 25 mM HEPES, and 1% GlutaMAX and then plated onto Primaria-coated 96-well culture dishes (Becton, Dickinson and Company) at a density of 5.0 ϫ 10 4 cells per well as we have described previously (27).…”

Subversion of Host Recognition and Defense Systems by Francisella spp

“…Furthermore, the importance of central metabolism (e.g., glycolysis) has also been shown to play a role during infection (149). Additionally, there is evidence that nutritional status may play a role in allowing Francisella to sense the host phagosome compared to being in the host cytosol (40) and to induce expression of virulence genes, including those required for phagosomal escape (40,67,191). Mutants that are unable to recognize particular metabolites, such as polyamines present in the host cytosol, are significantly attenuated and unable to alter temporal expression of virulence factors (191).…”

A Francisella tularensis Locus Required for Spermine Responsiveness Is Necessary for Virulence

“…Successful evasion of the host immune response may be partly due to an adaptation of F. tularensis to the intracellular environment (7,34). Loegering et al demonstrated that after replication within macrophages F. tularensis is less stimulatory in subsequent macrophage infections compared to F. tularensis cultured in bacterial growth media (34).…”

“…However, the mechanism(s) enabling F. tularensis to sense and adapt to the host environment must be delineated. Among possible signals, polyamines, temperature, and amino acid concentration are known to be important environmental cues that alter bacterial virulence (7,26,28).…”