Bacterial Ligands Generated in a Phagosome Are Targets of the Cytosolic Innate Immune System

Macrophages are permissive hosts to intracellular pathogens, but upon activation become microbiocidal effectors of innate and cell-mediated immunity. How the fate of internalized microorganisms is monitored by macrophages, and how that information is integrated to stimulate specific immune responses is not understood. Activation of macrophages with interferon (IFN)–γ leads to rapid killing and degradation of Listeria monocytogenes in a phagosome, thus preventing escape of bacteria to the cytosol. Here, we show that activated macrophages induce a specific gene expression program to L. monocytogenes degraded in the phago-lysosome. In addition to activation of Toll-like receptor (TLR) signaling pathways, degraded bacteria also activated a TLR-independent transcriptional response that was similar to the response induced by cytosolic L. monocytogenes. More specifically, degraded bacteria induced a TLR-independent IFN-β response that was previously shown to be specific to cytosolic bacteria and not to intact bacteria localized to the phagosome. This response required the generation of bacterial ligands in the phago-lysosome and was largely dependent on nucleotide-binding oligomerization domain 2 (NOD2), a cytosolic receptor known to respond to bacterial peptidoglycan fragments. The NOD2-dependent response to degraded bacteria required the phagosomal membrane potential and the activity of lysosomal proteases. The NOD2-dependent IFN-β production resulted from synergism with other cytosolic microbial sensors. This study supports the hypothesis that in activated macrophages, cytosolic innate immune receptors are activated by bacterial ligands generated in the phagosome and transported to the cytosol

Systems Level Analyses Reveal Multiple Regulatory Activities of CodY Controlling Metabolism, Motility and Virulence in Listeria monocytogenes.

Bacteria sense and respond to many environmental cues, rewiring their regulatory network to facilitate adaptation to new conditions/niches. Global transcription factors that co-regulate multiple pathways simultaneously are essential to this regulatory rewiring. CodY is one such global regulator, controlling expression of both metabolic and virulence genes in Gram-positive bacteria. Branch chained amino acids (BCAAs) serve as a ligand for CodY and modulate its activity. Classically, CodY was considered to function primarily as a repressor under rich growth conditions. However, our previous studies of the bacterial pathogen Listeria monocytogenes revealed that CodY is active also when the bacteria are starved for BCAAs. Under these conditions, CodY loses the ability to repress genes (e.g., metabolic genes) and functions as a direct activator of the master virulence regulator gene, prfA. This observation raised the possibility that CodY possesses multiple functions that allow it to coordinate gene expression across a wide spectrum of metabolic growth conditions, and thus better adapt bacteria to the mammalian niche. To gain a deeper understanding of CodY's regulatory repertoire and identify direct target genes, we performed a genome wide analysis of the CodY regulon and DNA binding under both rich and minimal growth conditions, using RNA-Seq and ChIP-Seq techniques. We demonstrate here that CodY is indeed active (i.e., binds DNA) under both conditions, serving as a repressor and activator of different genes. Further, we identified new genes and pathways that are directly regulated by CodY (e.g., sigB, arg, his, actA, glpF, gadG, gdhA, poxB, glnR and fla genes), integrating metabolism, stress responses, motility and virulence in L. monocytogenes. This study establishes CodY as a multifaceted factor regulating L. monocytogenes physiology in a highly versatile manner

Bacterial ligands generated in a phagosome are targets of the cytosolic innate immune system.

Macrophages are permissive hosts to intracellular pathogens, but upon activation become microbiocidal effectors of innate and cell-mediated immunity. How the fate of internalized microorganisms is monitored by macrophages, and how that information is integrated to stimulate specific immune responses is not understood. Activation of macrophages with interferon (IFN)-gamma leads to rapid killing and degradation of Listeria monocytogenes in a phagosome, thus preventing escape of bacteria to the cytosol. Here, we show that activated macrophages induce a specific gene expression program to L. monocytogenes degraded in the phago-lysosome. In addition to activation of Toll-like receptor (TLR) signaling pathways, degraded bacteria also activated a TLR-independent transcriptional response that was similar to the response induced by cytosolic L. monocytogenes. More specifically, degraded bacteria induced a TLR-independent IFN-beta response that was previously shown to be specific to cytosolic bacteria and not to intact bacteria localized to the phagosome. This response required the generation of bacterial ligands in the phago-lysosome and was largely dependent on nucleotide-binding oligomerization domain 2 (NOD2), a cytosolic receptor known to respond to bacterial peptidoglycan fragments. The NOD2-dependent response to degraded bacteria required the phagosomal membrane potential and the activity of lysosomal proteases. The NOD2-dependent IFN-beta production resulted from synergism with other cytosolic microbial sensors. This study supports the hypothesis that in activated macrophages, cytosolic innate immune receptors are activated by bacterial ligands generated in the phagosome and transported to the cytosol

ChIP-seq analysis of CodY binding sites under growth in BHI and LBMM media.

<p><b>A.</b> Venn diagram representing CodY binding sites associated with differentially expressed genes in BHI and LBMM. <b>B.</b> Genomic view of CodY binding sites in <i>L</i>. <i>monocytogenes</i> 10403S– the two outer circles represent the + and—strands of <i>L</i>. <i>monocytogenes</i> genome, each line is a gene. Orange lines represent CodY regulated genes, while grey lines represent genes unaffected by CodY (based on the RNA-Seq analysis). The third circle represents CodY binding sites associated with differentially expressed genes in BHI medium, while the forth circle represents CodY binding sites associated with differentially expressed genes in LBMM. The fifth circle represents CodY motifs associated with differentially expressed genes, identified by the MAST algorithm [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005870#pgen.1005870.ref046" target="_blank">46</a>] using a CodY binding consensus sequence as an input based on previous experimental CodY-box analyses [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005870#pgen.1005870.ref036" target="_blank">36</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005870#pgen.1005870.ref037" target="_blank">37</a>]. The consensus sequence was generated using the MEME algorithm [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005870#pgen.1005870.ref047" target="_blank">47</a>]. Only sites with E value < 10 and P value < 0.05 are shown.</p

CodY serves as both a repressor and as an activator of genes under rich and minimal growth conditions.

<p>Unlike previous studies describing a function for CodY only under rich conditions, here we show that CodY exhibit a global regulatory role as a repressor and as an activator of genes under both rich and minimal conditions, the latter limiting for BCAAs. The coordinated regulation of metabolic, stress and virulence genes by CodY, most likely allows <i>L</i>. <i>monocytogenes</i> bacteria to swiftly switch from being a saprophyte to virulent bacteria. The color-coded oval represents CodY protein under the different conditions bound to regulatory regions of genes. R represents repressor and A activator.</p

A Model for CodY regulation of <i>L</i>. <i>monocytogenes</i> central metabolism.

<p>Illustration of central metabolic pathways that are regulated by CodY in <i>L</i>. <i>monocytogenes</i> in BHI <b>A</b>. and LBMM <b>B</b>. In light blue—pyruvate metabolism, light green—nitrogen metabolism, light orange—arginine metabolism, in the middle the TCA cycle. Green arrows represent enzymes that are induced by CodY, while red arrows represent enzymes repressed by CodY, based on the RNA-Seq analysis.</p

Functional enrichment analysis of CodY regulated genes.

<p>Categories functionally enriched in each cluster of CodY regulated genes, analyzed using the MIPS server [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005870#pgen.1005870.ref044" target="_blank">44</a>]. All categories are significantly enriched (P-value <0.005).</p