Abstract. Listeria monocytogenes was used as a model intracellular parasite to study stages in the entry, growth, movement, and spread of bacteria in a macrophage cell line. The first step in infection is phagocytosis of the Listeria, followed by the dissolution of the membrane surrounding the phagosome presumably mediated by hemolysin secreted by Listeria as nonhemolytic mutants remain in intact vacuoles. Within 2 h after infection, each now cytoplasmic Listeria becomes encapsulated by actin filaments, identified as such by decoration of the actin filaments with subfragment 1 of myosin. These filaments are very short. The Listeria grow and divide and the actin filaments rearrange to form a long tail (often 5/~m in length) extending from only one end of the bacterium, a "comet's tail," in which the actin filaments appear randomly oriented. The ~'steria "comet" moves to the cell surface with its tail oriented towards the cell center and becomes encorporated into a cell extension with the Listeria at the tip of the process and its tail trailing into the cytoplasm behind it. This extension contacts a neighboring macrophage that phagocytoses the extension of the first macrophage. Thus, within the cytoplasm of the second macrophage is a Listeria with its actin tail surrounded by a membrane that in turn is surrounded by the phagosome membrane of the new host. Both these membranes are then solubilized by the Listeria and the cycle is repeated. Thus, once inside a host cell, the infecting Listeria and their progeny can spread from cell to cell by remaining intracellular and thus bypass the humoral immune system of the organism. To establish if actin filaments are essential for the spread of Listeria from cell to cell, we treated infected macrophages with cytochalasin D. The Listeria not only failed to spread, but most were found deep within the cytoplasm, rather than near the periphery of the cell. Thin sections revealed that the net of actin filaments is not formed nor is a "comet" tail produced.

Intracellular bacterial pathogens, such as Listeria monocytogenes, are detected in the cytosol of host immune cells. Induction of this host response is often dependent on microbial secretion systems, and in L. monocytogenes, is dependent on multi-drug efflux pumps (MDRs). Using L. monocytogenes mutants that over-expressed MDRs, we identified cyclic diadenosine monophosphate (c-di-AMP) as a secreted molecule able to trigger the cytosolic host response. Over-expression of the di-adenylate cyclase, dacA (lmo2120), resulted in elevated levels of the host response during infection. C-di-AMP thus represents a putative bacterial secondary signaling molecule that triggers a cytosolic pathway of innate immunity and is predicted to be present in a wide variety of bacteria and archea.

Here we have identified 'triple D' (3d), a recessive N-ethyl-N-nitrosourea-induced mutation and phenotype in which no signaling occurs via the intracellular Toll-like receptors 3, 7 and 9 (sensors for double-stranded RNA, single-stranded RNA and unmethylated DNA, respectively). The 3d mutation also prevented cross-presentation and diminished major histocompatibility complex class II presentation of exogenous antigen; it also caused hypersusceptibility to infection by mouse cytomegalovirus and other microbes. By positional identification, we found 3d to be a missense allele of Unc93b1, which encodes the 12-membrane-spanning protein UNC-93B, a highly conserved molecule found in the endoplasmic reticulum with multiple paralogs in mammals. Innate responses to nucleic acids and exogenous antigen presentation, which both initiate in endosomes, thus seem to depend on an endoplasmic reticulum-resident protein, which suggests communication between these organellar systems.

Stimulator of interferon genes (STING) is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides (CDNs), activating TBK1/IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce robust type I interferon and proinflammatory cytokine responses. CDN ligands were formulated with granulocyte-macrophage colony-stimulating factor (GM-CSF)–producing cellular cancer vaccines—termed STINGVAX—that demonstrated potent in vivo antitumor efficacy in multiple therapeutic models of established cancer. We found that rationally designed synthetic CDN derivative molecules, including one with an Rp,Rp dithio diastereomer and noncanonical c[A(2′,5′)pA(3′,5′)p] phosphate bridge structure, enhanced antitumor efficacy of STINGVAX in multiple aggressive therapeutic models of established cancer in mice. Antitumor activity was STING-dependent and correlated with increased activation of dendritic cells and tumor antigen–specific CD8+ T cells. Tumors from STINGVAX-treated mice demonstrated marked PD-L1 (programmed death ligand 1) up-regulation, which was associated with tumor-infiltrating CD8+IFNγ+ T cells. When combined with PD-1 (programmed death 1) blockade, STINGVAX induced regression of palpable, poorly immunogenic tumors that did not respond to PD-1 blockade alone.

Summary Cytosolic bacterial pathogens activate the cytosolic surveillance pathway (CSP) and induce innate immune responses, but how the host detects vacuolar pathogens like Mycobacterium tuberculosis is poorly understood. We show that M. tuberculosis also initiates the CSP upon macrophage infection via limited perforation of the phagosome membrane mediated by the ESX-1 secretion system. Although the bacterium remains within the phagosome, this permeabilization results in phagosomal and cytoplasmic mixing and allows extracellular mycobacterial DNA to access host cytosolic receptors, thus blurring the distinction between “vacuolar” and “cytosolic” pathogens. Activation of cytosolic receptors induces signaling through the STING/TBK1/IRF3 axis, resulting in IFN-β production. Surprisingly, IRF3−/− mice, which cannot respond to cytosolic DNA, are resistant to long-term M. tuberculosis infection, suggesting that the CSP promotes M. tuberculosis infection. Thus, cytosolic sensing of mycobacterial DNA plays a key role in M. tuberculosis pathogenesis and likely contributes to the high type I IFN signature in tuberculosis.