AUTHOR=Woolard Matthew D., Barrigan Lydia M., Fuller James R., Buntzman Adam S., Bryan Joshua , Manoil Colin , Kawula Tom , Frelinger Jeffrey A.

TITLE=Identification of Francisella novicida mutants that fail to induce prostaglandin E2 synthesis by infected macrophages

JOURNAL=Frontiers in Microbiology

VOLUME=4

YEAR=2013

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2013.00016

DOI=10.3389/fmicb.2013.00016

ISSN=1664-302X

ABSTRACT=<p><italic>Francisella tularensis</italic> is the causative agent of tularemia. We have previously shown that infection with <italic>F. tularensis</italic> Live Vaccine Strain (LVS) induces macrophages to synthesize prostaglandin E<sub>2</sub> (PGE<sub>2</sub>). Synthesis of PGE<sub>2</sub> by <italic>F. tularensis</italic> infected macrophages results in decreased T cell proliferation <italic>in vitro</italic> and increased bacterial survival <italic>in vivo</italic>. Although we understand some of the biological consequences of <italic>F. tularensis</italic> induced PGE<sub>2</sub> synthesis by macrophages, we do not understand the cellular pathways (neither host nor bacterial) that result in up-regulation of the PGE<sub>2</sub> biosynthetic pathway in <italic>F. tularensis</italic> infected macrophages. We took a genetic approach to begin to understand the molecular mechanisms of bacterial induction of PGE<sub>2</sub> synthesis from infected macrophages. To identify <italic>F. tularensis</italic> genes necessary for the induction of PGE<sub>2</sub> in primary macrophages, we infected cells with individual mutants from the closely related strain <italic>F. tularensis</italic> subspecies <italic>novicida</italic> U112 (U112) two allele mutant library. Twenty genes were identified that when disrupted resulted in U112 mutant strains unable to induce the synthesis of PGE<sub>2</sub> by infected macrophages. Fourteen of the genes identified are located within the <italic>Francisella</italic> pathogenicity island (FPI). Genes in the FPI are required for <italic>F. tularensis</italic> to escape from the phagosome and replicate in the cytosol, which might account for the failure of U112 with transposon insertions within the FPI to induce PGE<sub>2</sub>. This implies that U112 mutant strains that do not grow intracellularly would also not induce PGE<sub>2</sub>. We found that U112 <italic>clpB</italic>::Tn grows within macrophages yet fails to induce PGE<sub>2</sub>, while U112 <italic>pdpA</italic>::Tn does not grow yet does induce PGE<sub>2</sub>. We also found that U112 <italic>iglC</italic>::Tn neither grows nor induces PGE<sub>2</sub>. These findings indicate that there is dissociation between intracellular growth and the ability of <italic>F. tularensis</italic> to induce PGE<sub>2</sub> synthesis. These mutants provide a critical entrée into the pathways used in the host for PGE<sub>2</sub> induction.</p>