Legionella pneumophila Is Directly Sensitive to 2-Deoxyglucose-Phosphate via Its UhpC Transporter but Is Indifferent to Shifts in Host Cell Glycolytic Metabolism

Abstract

Toll-like receptor (TLR) stimulation induces a pronounced shift to increased glycolytic metabolism in mammalian macrophages. We observed that bone marrow-derived macrophages (BMMs) increase glycolysis in response to infection with , but the role of host macrophage glycolysis in terms of intracellular replication is not currently understood. Treatment with 2-deoxyglucose (2DG) blocks replication in mammalian macrophages but has no effect on bacteria grown in broth. In addition, we found that 2DG had no effect on bacteria grown in amoebae. We used a serial enrichment strategy to reveal that the effect of 2DG on in macrophages requires the hexose-phosphate transporter UhpC. Experiments with UhpC-deficient revealed that mutant bacteria are also resistant to growth inhibition following treatment with phosphorylated 2DG in broth, suggesting that the inhibitory effect of 2DG on in mammalian cells requires 2DG phosphorylation. UhpC-deficient replicates without a growth defect in BMMs and protozoan host cells and also replicates without a growth defect in BMMs treated with 2DG. Our data indicate that neither TLR signaling-dependent increased macrophage glycolysis nor inhibition of macrophage glycolysis has a substantial effect on intracellular replication. These results are consistent with the view that can employ diverse metabolic strategies to exploit its host cells. We explored the relationship between macrophage glycolysis and replication of an intracellular bacterial pathogen, Previous studies demonstrated that a glycolysis inhibitor, 2-deoxyglucose (2DG), blocks replication of during infection of macrophages, leading to speculation that may exploit macrophage glycolysis. We isolated mutants resistant to the inhibitory effect of 2DG in macrophages, identifying a hexose-phosphate transporter, UhpC, that is required for bacterial sensitivity to 2DG during infection. Our results reveal how a bacterial transporter mediates the direct antimicrobial effect of a toxic metabolite. Moreover, our results indicate that neither induction nor impairment of host glycolysis inhibits intracellular replication of, which is consistent with a view of as a metabolic generalist.

IRG1 and iNOS act redundantly with other interferon gamma-induced factors to restrict intracellular replication ofLegionella pneumophila

“…In an attempt to identify specific factors that explain the ability of IFNγ to restrict L. pneumophila replication in macrophages to specific genetic factors associated with the immune response, we tested the ability of IFNγ to restrict L. pneumophila in BMMs derived from various knockout mice. Using an extensively validated strain of Δ flaA L. pneumophila that expresses luminescence (lux) genes from P. luminescens (20, 36–38), we confirmed that Δ flaA L. pneumophila replicates in unstimulated BMMs but does not replicate in BMMs stimulated with IFNγ ( Figure 1A ). As expected, BMMs lacking the IFNγ receptor ( Ifngr −/− ) do not restrict L. pneumophila replication in the presence of IFNγ ( Figure 1B ).…”

“…We next investigated the possibility that IFNγ may act to restrict L. pneumophila not through induction of any single antimicrobial factor but by changing the metabolic landscape of the host macrophage to be unsuitable for the metabolic needs of L. pneumophila . As macrophages infected with L. pneumophila and IFNγ-stimulated macrophages increase rates of glycolysis (1, 36, 42), we tested whether treatment with the glycolysis inhibitor 2-deoxyglucose (2DG) might interfere with IFNγ-dependent restriction observed in BMMs. 2DG is metabolized to 2DG-phosphate in cells, which is directly antimicrobial (36).…”

“…As macrophages infected with L. pneumophila and IFNγ-stimulated macrophages increase rates of glycolysis (1, 36, 42), we tested whether treatment with the glycolysis inhibitor 2-deoxyglucose (2DG) might interfere with IFNγ-dependent restriction observed in BMMs. 2DG is metabolized to 2DG-phosphate in cells, which is directly antimicrobial (36). However, by taking advantage of a newly identified strain of L. pneumophila resistant to the direct antimicrobial effect of 2DG(P) in BMMs (Δ flaA Δ uhpC L. pneumophila ) (36), we observed that addition of 2DG to BMMs partially restored L. pneumophila replication in IFNγ-treated macrophages ( Figure 2A ).…”

“…2DG is metabolized to 2DG-phosphate in cells, which is directly antimicrobial (36). However, by taking advantage of a newly identified strain of L. pneumophila resistant to the direct antimicrobial effect of 2DG(P) in BMMs (Δ flaA Δ uhpC L. pneumophila ) (36), we observed that addition of 2DG to BMMs partially restored L. pneumophila replication in IFNγ-treated macrophages ( Figure 2A ). We confirmed that 2DG disrupts the enhanced glycolysis observed in L. pneumophila -infected BMMs stimulated with IFNγ ( Figure 2B ).…”

“…Flagellin produced by wild-type L. pneumophila can trigger host cell pyroptosis via the NAIP/NLRC4 inflammasome; however, L. pneumophila that lack flagellin (Δ flaA ) are able to replicate to high levels in macrophages (29–35). We recently described a mutant strain of L. pneumophila (Δ flaA Δ uhpC ) that is able to replicate in macrophages treated with 2-deoxyglucose (2DG), an inhibitor of mammalian glycolysis (36). This strain allows us to probe the role that host cell metabolism plays in the immune response to L. pneumophila .…”