How the innate immune system tailors specific responses to diverse microbial infections is not well understood. Cells use a limited number of host receptors and signaling pathways to both discriminate among extracellular and intracellular microbes, and also to generate responses commensurate to each threat. Here, we have addressed these questions by using DNA microarrays to monitor the macrophage transcriptional response to the intracellular bacterial pathogen Listeria monocytogenes. By utilizing combinations of host and bacterial mutants, we have defined the host transcriptional responses to vacuolar and cytosolic bacteria. These compartment-specific host responses induced significantly different sets of target genes, despite activating similar transcription factors. Vacuolar signaling was entirely MyD88-dependent, and induced the transcription of pro-inflammatory cytokines. The IRF3-dependent cytosolic response induced a distinct set of target genes, including IFNβ. Many of these cytosolic response genes were induced by secreted cytokines, so we further identified those host genes induced independent of secondary signaling. The host response to cytosolic bacteria was reconstituted by the cytosolic delivery of L. monocytogenes genomic DNA, but we observed an amplification of this response by NOD2 signaling in response to MDP. Correspondingly, the induction of IFNβ was reduced in nod2 −/− macrophages during infection with either L. monocytogenes or Mycobacterium tuberculosis. Combinatorial control of IFNβ induction by recognition of both DNA and MDP may highlight a mechanism by which the innate immune system integrates the responses to multiple ligands presented in the cytosol by intracellular pathogens.

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A highly conserved virulence plasmid encoding a type III secretion system is shared by the three Yersinia species most pathogenic for mammals. Although factors encoded on this plasmid enhance the ability of Yersinia to thrive in their mammalian hosts, the loss of this virulence plasmid does not eliminate growth or survival in host organs. Most notably, yields of viable plasmid-deficient Yersinia pseudotuberculosis (Yptb) are indistinguishable from wild-type Yptb within mesenteric lymph nodes. To identify chromosomal virulence factors that allow for plasmid-independent survival during systemic infection of mice, we generated transposon insertions in plasmid-deficient Yptb, and screened a library having over 20,000 sequence-identified insertions. Among the previously uncharacterized loci, insertions in mrtAB, an operon encoding an ABC family transporter, had the most profound phenotype in a plasmid-deficient background. The absence of MrtAB, however, had no effect on growth in the liver and spleen of a wild type strain having an intact virulence plasmid, but caused a severe defect in colonization of the mesenteric lymph nodes. Although this result is consistent with lack of expression of the type III secretion system by Wt Yptb in the mesenteric lymph nodes, a reporter for YopE indicated that expression of the system was robust. We demonstrate that the ATPase activity of MrtB is required for growth in mice, indicating that transport activity is required for virulence. Indeed, MrtAB appears to function as an efflux pump, as the ATPase activity enhances resistance to ethidium bromide while increasing sensitivity to pyocyanin, consistent with export across the inner membrane.

All three pathogenic Yersinia species share a conserved virulence plasmid that encodes a Type 3 Secretion System (T3SS) and its associated effector proteins. During mammalian infection, these effectors are injected into innate immune cells, where they block many bactericidal functions, including the production of reactive oxygen species (ROS). However, Y. pseudotuberculosis (Yptb) lacking the T3SS retains the ability to colonize host organs, demonstrating that chromosome-encoded factors are sufficient for growth within mammalian tissue sites. Previously we uncovered more than 30 chromosomal factors that contribute to growth of T3SS-deficient Yptb in livers. Here, a deep sequencing-based approach was used to validate and characterize the phenotype of 18 of these genes during infection by both WT and plasmid-deficient Yptb. Additionally, the fitness of these mutants was evaluated in immunocompromised mice to determine whether any genes contributed to defense against phagocytic cell restriction. Mutants containing deletions of the dusB-fis operon, which encodes the nucleoid associated protein Fis, were markedly attenuated in immunocompetent mice, but were restored for growth in mice lacking neutrophils and inflammatory monocytes, two of the major cell types responsible for restricting Yersinia infection. We determined that Fis was dispensable for secretion of T3SS effectors, but was essential for resisting ROS and regulated the transcription of several ROS-responsive genes. Strikingly, this protection was critical for virulence, as growth of ΔdusB-fis was restored in mice unable to produce ROS. These data support a model in which ROS generated by neutrophils and inflammatory monocytes that have not been translocated with T3SS effectors enter bacterial cells during infection, where their bactericidal effects are resisted in a Fis-dependent manner. This is the first report of the requirement for Fis during Yersinia infection and also highlights a novel mechanism by which Yptb defends against ROS in mammalian tissues.

Infection with wild-type Listeria monocytogenes activates a host cytosolic surveillance response characterized by the expression of beta interferon (IFN-␤). We performed a genetic screen to identify L. monocytogenes transposon insertion mutants that induced altered levels of host IFN-␤ expression. One mutant from this screen induced elevated levels of IFN-␤ and harbored a Tn917 insertion upstream of lmo0558. This study identified lmo0558 as the 6-phosphogluconolactonase gene (pgl), which encodes the second enzyme in the pentose phosphate pathway. pgl mutant L. monocytogenes accumulated and secreted large amounts of gluconate, likely derived from labile 6-phosphogluconolactone, the substrate of Pgl. The pgl deletion mutant had decreased growth in glucose-limiting minimal medium but grew normally when excess glucose was added. Microarray analysis revealed that the pgl deletion mutant had increased expression of several ␤-glucosidases, consistent with known inhibition of ␤-glucosidases by 6-phosphogluconolactone. While growth in macrophages was indistinguishable from that of wild-type bacteria, pgl mutant L. monocytogenes exhibited a 15-to 30-fold defect in growth in vivo. In addition, L. monocytogenes harboring an in-frame deletion of pgl was more sensitive to oxidative stress. This study identified L. monocytogenes pgl and provided the first link between the bacterial pentose phosphate pathway and activation of host IFN-␤ expression.Listeria monocytogenes is a gram-positive, food-borne facultative intracellular pathogen that causes invasive, life-threatening infections, mainly in pregnant women, newborns, the elderly, and the immunosuppressed (39). In addition, L. monocytogenes has been studied for decades as a model pathogen, illuminating many aspects of host-pathogen interaction. The cell biology of its intracellular life cycle has been particularly well characterized. After phagocytosis by a macrophage, L. monocytogenes rapidly escapes from the phagosome into the cytosol, an event mediated by the pore-forming cytolysin listeriolysin O (29). L. monocytogenes is well adapted to its cytosolic niche, possessing virulence factors that allow utilization of cytosolic sugar sources and polymerization of host actin to move from cell to cell (4, 25). It is evident that L. monocytogenes has evolved specific mechanisms to grow in the host cytosol; however, the presence of cytosolic L. monocytogenes triggers a host innate immune response. Upon entry of L. monocytogenes into cells, a host cell cytosolic surveillance pathway is activated, including a transcriptional program that leads to the robust expression of beta interferon (IFN-␤) (19,22,23,34). However, the host and bacterial components responsible for the activation of the cytosolic surveillance pathway remain largely unknown.Our lab previously performed a genetic screen to identify L. monocytogenes transposon insertion mutants that induced enhanced or diminished activation of the host cytosolic surveillance system (6). We found that bacterial multidrug resistance tra...