Identification of Francisella novicida mutants that fail to induce prostaglandin E(2) synthesis by infected macrophages

Abstract

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

Keywords: Francisella; prostaglandin E.

FIGURE 1

U112 and Schu S4 induces the synthesis of PGE₂ from bone marrow-derived macrophages (BMDMs). BMDMs were either mock inoculated or inoculated with LVS, U112, or Schu S4 at an MOI of 200:1. Twenty-four hours after inoculation supernatants were collected and PGE₂ concentration was determined. Data represents three independent experiments and expressed as the mean ± SEM. Asterisk “*”denotes statistical difference (p ≤ 0.05) from uninfected BMDM (n = 3).

FIGURE 2

Identification of U112 genes necessary for the induction of PGE0₂ from bone marrow-derived macrophages (BMDMs). BMDMs were inoculated with F. novicida U112 or individual transposon insertion strains at an MOI of 200:1. Twenty-four hours after inoculation, supernatants were collected and PGE₂ concentration was determined. Each transposon insertion mutant strain was tested four times. Bars represents the mean of all independent transposon insertions mutants within the same gene ± SEM, Asterisk “*” denotes statistical difference (p ≤ 0.05) from U112 inoculated BMDM (n ≥ 4).

FIGURE 3

mglA, sspA, and dotU are necessary for LVS induction of PGE₂ synthesis. Bone marrow-derived macrophages were inoculated with LVS, LVS∆mglA, LVS∆mglA (pmglA), LVS∆sspA, LVS∆sspA (psspA), LVS∆dotU, LVS∆dotU (pdotU), or LVS at an MOI of 200:1. Twenty-four hours after inoculation the levels of PGE₂ were determined. Experiments were done in triplicate; error bars represent SEM (n = 3).

FIGURE 4

Induction of PGE₂ does not require full escape from the phagosome. (A) Bacterial association with LAMP-1 was scored using Volocity (n ≥ 20 imaged cells per strain with an average of one bacterium per macrophage). Co-localization was determined by the shared of red and green pixels at the same location. Data represents three independent experiments and expressed as the mean ± SEM. Asterisk “*” denotes statistical difference (p ≤ 0.05) from U112-infected cells. (B) BMDMs were inoculated with U112, U112 clpB::Tn, U112 iglC::Tn, or U112 pdpA::Tn at an MOI of 500:1. Association of the bacterium with the phagosomal membrane was determined 4 h post-inoculation using transmission electron microscopy. Open arrowheads denote bacteria no longer surrounded by an intact phagosomal membrane. Filled arrowheads denote bacteria surrounded by a phagosomal membrane.

FIGURE 5

Dissociation of intracellular growth and the induction of PGE₂ from bone marrow-derived macrophages (BMDMs). (A) BMDMs were inoculated with U112, U112 clpB::Tn, U112 iglC::Tn, or U112 pdpA::Tn at an MOI of 100:1. CFU were determined at 4 and 24 h post-inoculation. Data represents three independent experiments and expressed as the means ± SEM. Asterisk “*” denotes statistical difference (p ≤ 0.05) from corresponding 4 h sample. ^#BMDM denotes statistical difference (p ≤ 0.05) from 24 h U112-infected BMDM (n = 3). (B) BMDMs were inoculated with U112, U112 clpB::Tn, U112 iglC::Tn, or U112 pdpA::Tn at an MOI of 100:1. Twenty-four hours after inoculation supernatants were collected and PGE₂ concentration was determined. Data represents three independent experiments and expressed as the mean ± SEM. Asterisk “*” denotes statistical difference (p ≤ 0.05) from U112-infected BMDM.