Daniel A. Portnoy

c-di-AMP Secreted by Intracellular Listeria monocytogenes Activates a Host Type I Interferon Response

Bacterial (Interferon)ce Intracellular bacterial pathogens, such as Listeria monocytogenes, are detected in the cytosol of host immune cells, where they induce a host response that is often dependent on microbial secretion systems. Woodward et al. (p. 1703, published online 27 May) now show that L. monocytogenes produce and release cyclic diadenosine monophosphate into the host cytosol, which induces the production of host type I interferon. Because a number of intracellular pathogens contain the protein machinery to generate this nucleotide and also activate this same innate immune pathway, a common molecular mechanism may exist for host detection of cytosolic bacterial pathogens. Within the host cell, a small secondary signaling molecule from a pathogen triggers a pathway of innate immunity. 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 multidrug efflux pumps (MDRs). Using L. monocytogenes mutants that overexpressed MDRs, we identified cyclic diadenosine monophosphate (c-di-AMP) as a secreted molecule able to trigger the cytosolic host response. Overexpression 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.

Dual roles of plcA in Listeria monocytogenes pathogenesis

The plcA gene of Listeria monocytogenes encodes a secreted phosphatidylinositol‐speciftc phospholi‐pase C (PI‐PLC). Recent studies have established that transposon mutations within plcA result in avirulence for mice and pleiotropic effects when examined in tissue‐culture models of infection. Genetic analysis reveals that many of the effects of the transposon insertions are due to loss of readthrough transcription from plcA into the downstream gene prfA, which encodes an essential transcription factor of numerous L. monocytogenes virulence genes. Construction of an in‐frame deletion within plcA had no effect on expression of prfA thus allowing direct assignment of a role of the PI‐PLC in pathogenesis. PI‐PLC was shown to play a significant role in mediating escape of L. monocytogenes from phagosomes of primary murine macrophages. Interestingly, this defect manifested itself in vivo in the liver but not in the spleen of infected mice.

Mice Lacking the Type I Interferon Receptor Are Resistant to Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that induces a cytosolic signaling cascade resulting in expression of interferon (IFN)-β. Although type I IFNs are critical in viral defense, their role in immunity to bacterial pathogens is much less clear. In this study, we addressed the role of type I IFNs by examining the infection of L. monocytogenes in BALB/c mice lacking the type I IFN receptor (IFN-α/βR−/−). During the first 24 h of infection in vivo, IFN-α/βR−/− and wild-type mice were similar in terms of L. monocytogenes survival. In addition, the intracellular fate of L. monocytogenes in macrophages cultured from IFN-α/βR−/− and wild-type mice was indistinguishable. However, by 72 h after inoculation in vivo, IFN-α/βR−/− mice were ∼1,000-fold more resistant to a high dose L. monocytogenes infection. Resistance was correlated with elevated levels of interleukin 12p70 in the blood and increased numbers of CD11b+ macrophages producing tumor necrosis factor α in the spleen of IFN-α/βR−/− mice. The results of this study suggest that L. monocytogenes might be exploiting an innate antiviral response to promote its pathogenesis.

The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity

Listeria monocytogenes has emerged as a remarkably tractable pathogen to dissect basic aspects of cell biology, intracellular pathogenesis, and innate and acquired immunity. In order to maintain its intracellular lifestyle, L. monocytogenes has evolved a number of mechanisms to exploit host processes to grow and spread cell to cell without damaging the host cell. The pore-forming protein listeriolysin O mediates escape from host vacuoles and utilizes multiple fail-safe mechanisms to avoid causing toxicity to infected cells. Once in the cytosol, the L. monocytogenes ActA protein recruits host cell Arp2/3 complexes and enabled/vasodilator-stimulated phosphoprotein family members to mediate efficient actin-based motility, thereby propelling the bacteria into neighboring cells. Alteration in any of these processes dramatically reduces the ability of the bacteria to establish a productive infection in vivo.

The N-Ethyl-N-Nitrosourea-Induced Goldenticket Mouse Mutant Reveals an Essential Function of Sting in the In Vivo Interferon Response to Listeria monocytogenes and Cyclic Dinucleotides

ABSTRACT Type I interferons (IFNs) are central regulators of the innate and adaptive immune responses to viral and bacterial infections. Type I IFNs are induced upon cytosolic detection of microbial nucleic acids, including DNA, RNA, and the bacterial second messenger cyclic-di-GMP (c-di-GMP). In addition, a recent study demonstrated that the intracellular bacterial pathogen Listeria monocytogenes stimulates a type I IFN response due to cytosolic detection of bacterially secreted c-di-AMP. The transmembrane signaling adaptor Sting (Tmem173, Mita, Mpys, Eris) has recently been implicated in the induction of type I IFNs in response to cytosolic DNA and/or RNA. However, the role of Sting in response to purified cyclic dinucleotides or during in vivo L. monocytogenes infection has not been addressed. In order to identify genes important in the innate immune response, we have been conducting a forward genetic mutagenesis screen in C57BL/6 mice using the mutagen N-ethyl-N-nitrosourea (ENU). Here we describe a novel mutant mouse strain, Goldenticket (Gt), that fails to produce type I IFNs upon L. monocytogenes infection. By genetic mapping and complementation experiments, we found that Gt mice harbor a single nucleotide variant (T596A) of Sting that functions as a null allele and fails to produce detectable protein. Analysis of macrophages isolated from Gt mice revealed that Sting is absolutely required for the type I interferon response to both c-di-GMP and c-di-AMP. Additionally, Sting is required for the response to c-di-GMP and L. monocytogenes in vivo. Our results provide new functions for Sting in the innate interferon response to pathogens.

Innate recognition of bacteria by a macrophage cytosolic surveillance pathway

Host recognition of bacterial pathogens is a critical component of the immune response. Intracellular bacterial pathogens are able to evade the humoral immune system by residing within the host cell. Here we show the existence of an innate host surveillance mechanism in macrophages that specifically distinguishes bacteria in the cytosol from bacteria in the vacuole. Recognition of Gram-positive and Gram-negative bacterial products by this surveillance system results in transcription of the ifnb gene. The activation of cytosol-specific signaling is associated with translocation of NF-κB into the nucleus and phosphorylation of the p38 mitogen-activated protein (MAP) kinase. Activation of the p38 kinase is required for the induction of gene expression by the cytosolic surveillance pathway. Our studies suggest that infection by intracellular bacterial pathogens results in an immune response distinct from that of infection by extracellular bacterial pathogens.

Involvement of profilin in the actin-based motility of L. monocytogenes in cells and in cell-free extracts

Within hours of Listeria monocytogenes infection, host cell actin filaments form a dense cloud around the intracytoplasmic bacteria and then rearrange to form a polarized comet tail that is associated with moving bacteria. We have devised a cell-free extract system capable of faithfully reconstituting L. monocytogenes motility, and we have used this system to demonstrate that profilin, a host actin monomer-binding protein, is necessary for bacterial actin-based motility. We find that extracts from which profilin has been depleted do not support comet tail formation or bacterial motility. In extracts and host cells, profilin is localized to the back half of the surface of motile L. monocytogenes, the site of actin filament assembly in the tail. This association is not observed with L. monocytogenes mutants that do not express the ActA protein, a bacterial gene product necessary for motility and virulence. Profilin also fails to bind L. monocytogenes grown outside of host cytoplasm, suggesting that at least one other host cell factor is required for this association.

SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis

Pathogenic bacteria secrete proteins that promote invasion of host tissues and resistance to immune responses. However, secretion mechanisms that contribute to the enormous morbidity and mortality of Gram-positive bacteria are largely undefined. An auxiliary protein secretion system (SecA2) has recently emerged in Listeria monocytogenes and eight other Gram-positive pathogens. Here, a proteomics approach identified seventeen SecA2-dependent secreted and surface proteins of L. monocytogenes, the two most abundant of which [the p60 and N-acetylmuramidase (NamA) autolysins] hydrolyze bacterial peptidoglycan (PGN) and contribute to host colonization. SecA2-deficient (ΔSecA2) bacteria were rapidly cleared after systemic infection of murine hosts, and in cultured cells showed reduced cell-cell spread. p60 or NamA deficiencies (Δp60 and ΔNamA) caused intermediate reductions in bacterial virulence in vivo, yet showed no defect for infection of cultured cells. Restoration of virulence in Δp60 bacteria required full-length p60 with an intact catalytic domain, suggesting that PGN hydrolysis by p60 is crucial for L. monocytogenes virulence. Coordinated PGN hydrolysis by p60 and NamA activities is predicted to generate a muramyl glycopeptide, glucosaminylmuramyl dipeptide (GMDP), which is known to modify host inflammatory responses. Thus, SecA2-dependent secretion may promote release of muramyl peptides that subvert host pattern recognition.

The Listeria monocytogenes hemolysin has an acidic pH optimum to compartmentalize activity and prevent damage to infected host cells

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is ∼10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.

Listeria monocytogenes triggers AIM2-mediated pyroptosis upon infrequent bacteriolysis in the macrophage cytosol.

A host defense strategy against pathogens is the induction of cell death, thereby eliminating the pathogens intracellular niche. Pyroptosis, one such form of cell death, is dependent on inflammasome activation. In a genetic screen to identify Listeria monocytogenes mutants that induced altered levels of host cell death, we identified a mutation in lmo2473 that caused hyperstimulation of IL-1beta secretion and pyroptosis following bacteriolysis in the macrophage cytosol. In addition, strains engineered to lyse in the cytosol by expression of both bacteriophage holin and lysin or induced to lyse by treatment with ampicillin stimulated pyroptosis. Pyroptosis was independent of the Nlrp3 and Nlrc4 inflammasome receptors but dependent on the inflammasome adaptor ASC and the cytosolic DNA sensor AIM2. Importantly, wild-type L. monocytogenes were also found to lyse, albeit at low levels, and trigger AIM2-dependent pyroptosis. These data suggested that pyroptosis is triggered by bacterial DNA released during cytosolic lysis.