A specific gene expression program triggered by Gram-positive bacteria in the cytosol

Abstract

Innate and adaptive immunity depends critically on host recognition of pathogen-associated molecules. Toll-like receptors (TLRs) are key mediators of pathogen surveillance at the cell or phagocytic vacuole surface. However, mechanisms underlying recognition of pathogens in other cellular compartments remain unclear, and responses elicited by cytosolic challenge are poorly characterized. We therefore used mouse cDNA microarrays to investigate gene expression triggered by infection of bone marrow-derived macrophages with cytosol- and vacuole-localized Listeria monocytogenes (Lm), a model cytosolic pathogen. The resulting gene expression program included two basic categories of induced genes: an "early/persistent" cluster consistent with NF-kappaB-dependent responses downstream of TLRs, and a subsequent "late response" cluster largely composed of IFN-responsive genes (IRGs). The early/persistent cluster was observed upon infection with WT, heat-killed, or mutant Lm lacking listeriolysin O, the pore-forming hemolysin that promotes escape from phagocytic vacuoles. However, the IRG cluster depended on entry of WT Lm into the cytosol. Infection with listeriolysin O-expressing, cytosolic Bacillus subtilis (Bs) strikingly recapitulated the expression profile associated with WT Lm, including IRG induction. IRG up-regulation was associated with MyD88-independent induction of IFN-beta transcription and activity. Whereas Staphylococcus aureus (Sa) lipoteichoic acid treatment confirmed that many late-response genes could also be stimulated through TLRs, our study identified a cytosol-specific transcriptional program independent of TLR signaling through MyD88. Further characterization of cytosolic surveillance pathway(s) and their points of convergence with TLR- and IFN-dependent pathways will enhance our understanding of the means by which mammals detect and respond to pathogens.

Fig. 1.

Macrophage activation program elicited by intracytosolic or vacuolar bacteria and bacterial components. Shown is an Eisen plot of 1,445 genes selected as described in Materials and Methods. Columns represent BMDM treatments at 0, 1, 2, 4 and 8 hpi; rows colorimetrically represent expression ratios of individual genes. Cy5-labeled treatments were compared with matched Cy3-labeled uninfected or untreated controls. Intensity of red or green shading is proportional to, respectively, gene induction or repression relative to the uninfected or control. Zoom boxes represent clusters described in the text. HK, heat-killed Lm, 0.5-h treatment; HK⁺, persistent 8-h treatment; Hly, Δhly Lm (moi = 1); WT, WT Lm; Bs, B. subtilis ± LLO; Hly⁺, Δhly Lm (moi = 50); LTA, S. aureus LTA; LLO, liposomes ± LLO.

Fig. 3.

Cytosolic but not vacuolar bacteria significantly induce IFN-β mRNA and protein expression. (A) Mouse BMDM treated with cytosolic bacteria [WT, WT Lm; Bs⁺, Bs plus LLO] or vacuolar bacteria [hly, Δhly Lm (moi = 6), hly⁺, Δhly Lm (moi = 300)]. Shown is total RNA from 6 hpi, Ifnb RNA levels estimated by quantitative PCR, mean of three experiments ± SD. (B) Type I IFN bioassay, supernatants from BMDM treated with bacteria or bacterial products, legend as in Fig. 1 except Bs = B. subtilis-expressing LLO. Shown are the means of two separate bioassays of samples pooled from three experiments ± SEM. Data shown for 4 and 8 hpi; all samples before 4 hpi had titers ≤4. (C) Total RNA from MyD88^–/– BMDM at 6 hpi. Ifnb levels were estimated by quantitative PCR. Values represent the means of three experiments ± SD. In all panels, Un = untreated control.

Fig. 2.

The late expression cluster is dominated by IFN-dependent genes. Both cytosolic and LTA-induced signaling induce IFN-regulated gene expression; shown are the 25 most highly expressed (WT, 8 h) group II genes previously shown to exhibit IFN dependence. Abbreviations are as in Fig. 1.

Fig. 4.

Intracytosolic Lm and Bs induce cytosol-specific genes. Abbreviations are as in Fig. 1. Twenty-six genes representing the cytosol-specific pathway were selected based on the following criteria: >3-fold induction for WT Lm at 8 hpi, <3-fold induction by Sa LTA at 4 and 8 hpi, and a >3-fold difference between 8 hpi WT Lm and 8 hpi LTA expression levels.