John-Demian Sauer

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.

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.

Type I IFN Signaling Constrains IL-17A/F Secretion by γδ T Cells during Bacterial Infections

Recognition of intracellular bacteria by macrophages leads to secretion of type I IFNs. However, the role of type I IFN during bacterial infection is still poorly understood. Francisella tularensis, the causative agent of tularemia, is a pathogenic bacterium that replicates in the cytosol of macrophages leading to secretion of type I IFN. In this study, we investigated the role of type I IFNs in a mouse model of tularemia. Mice deficient for type I IFN receptor (IFNAR1−/−) are more resistant to intradermal infection with F. tularensis subspecies novicida (F. novicida). Increased resistance to infection was associated with a specific increase in IL-17A/F and a corresponding expansion of an IL-17A+ γδ T cell population, indicating that type I IFNs negatively regulate the number of IL-17A+ γδ T cells during infection. Furthermore, IL-17A–deficient mice contained fewer neutrophils compared with wild-type mice during infection, indicating that IL-17A contributes to neutrophil expansion during F. novicida infection. Accordingly, an increase in IL-17A in IFNAR1−/− mice correlated with an increase in splenic neutrophil numbers. Similar results were obtained in a mouse model of pneumonic tularemia using the highly virulent F. tularensis subspecies tularensis SchuS4 strain and in a mouse model of systemic Listeria monocytogenes infection. Our results indicate that the type I IFN-mediated negative regulation of IL-17A+ γδ T cell expansion is conserved during bacterial infections. We propose that this newly described activity of type I IFN signaling might participate in the resistance of the IFNAR1−/− mice to infection with F. novicida and other intracellular bacteria.

Differential Requirements for NAIP5 in Activation of the NLRC4 Inflammasome

ABSTRACT Inflammasomes are cytosolic multiprotein complexes that assemble in response to infectious or noxious stimuli and activate the CASPASE-1 protease. The inflammasome containing the nucleotide binding domain-leucine-rich repeat (NBD-LRR) protein NLRC4 (interleukin-converting enzyme protease-activating factor [IPAF]) responds to the cytosolic presence of bacterial proteins such as flagellin or the inner rod component of bacterial type III secretion systems (e.g., Salmonella PrgJ). In some instances, such as infection with Legionella pneumophila, the activation of the NLRC4 inflammasome requires the presence of a second NBD-LRR protein, NAIP5. NAIP5 also is required for NLRC4 activation by the minimal C-terminal flagellin peptide, which is sufficient to activate NLRC4. However, NLRC4 activation is not always dependent upon NAIP5. In this report, we define the molecular requirements for NAIP5 in the activation of the NLRC4 inflammasome. We demonstrate that the N terminus of flagellin can relieve the requirement for NAIP5 during the activation of the NLRC4 inflammasome. We also demonstrate that NLRC4 responds to the Salmonella protein PrgJ independently of NAIP5. Our results indicate that NAIP5 regulates the apparent specificity of the NLRC4 inflammasome for distinct bacterial ligands.

Cyclic di-AMP Is Critical for Listeria monocytogenes Growth, Cell Wall Homeostasis, and Establishment of Infection

ABSTRACT Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection. IMPORTANCE Listeria monocytogenes is a Gram-positive intracellular pathogen and the causative agent of the food-borne illness listeriosis. Upon infection, L. monocytogenes stimulates expression of IFN-β and coregulated genes dependent upon host detection of a secreted bacterial signaling nucleotide, c-di-AMP. Using a forward genetic screen for mutants that induced high levels of host IFN-β expression, we identified a c-di-AMP phosphodiesterase, PdeA, that degrades c-di-AMP. Here we characterize L. monocytogenes mutants that express enhanced or diminished levels of c-di-AMP. Decreased c-di-AMP levels by conditional depletion of the diadenylate cyclase (DacA) or overexpression of PdeA attenuated bacterial growth and led to bacteriolysis, suggesting that its production is essential for viability and may regulate cell wall metabolism. Mutants lacking PdeA had a distinct transcriptional profile, which may provide insight into additional roles for the molecule. This work demonstrates that c-di-AMP is a critical signaling molecule required for bacterial replication, cell wall stability, and pathogenicity. Listeria monocytogenes is a Gram-positive intracellular pathogen and the causative agent of the food-borne illness listeriosis. Upon infection, L. monocytogenes stimulates expression of IFN-β and coregulated genes dependent upon host detection of a secreted bacterial signaling nucleotide, c-di-AMP. Using a forward genetic screen for mutants that induced high levels of host IFN-β expression, we identified a c-di-AMP phosphodiesterase, PdeA, that degrades c-di-AMP. Here we characterize L. monocytogenes mutants that express enhanced or diminished levels of c-di-AMP. Decreased c-di-AMP levels by conditional depletion of the diadenylate cyclase (DacA) or overexpression of PdeA attenuated bacterial growth and led to bacteriolysis, suggesting that its production is essential for viability and may regulate cell wall metabolism. Mutants lacking PdeA had a distinct transcriptional profile, which may provide insight into additional roles for the molecule. This work demonstrates that c-di-AMP is a critical signaling molecule required for bacterial replication, cell wall stability, and pathogenicity.

Listeria monocytogenes engineered to activate the Nlrc4 inflammasome are severely attenuated and are poor inducers of protective immunity

Inflammasomes are intracellular multiprotein signaling complexes that activate Caspase-1, leading to the cleavage and secretion of IL-1β and IL-18, and ultimately host cell death. Inflammasome activation is a common cellular response to infection; however, the consequences of inflammasome activation during acute infection and in the development of long-term protective immunity is not well understood. To investigate the role of the inflammasome in vivo, we engineered a strain of Listeria monocytogenes that ectopically expresses Legionella pneumophila flagellin, a potent activator of the Nlrc4 inflammasome. Compared with wild-type L. monocytogenes, strains that ectopically secreted flagellin induced robust host cell death and IL-1β secretion. These strains were highly attenuated both in bone marrow-derived macrophages and in vivo compared with wild-type L. monocytogenes. Attenuation in vivo was dependent on Nlrc4, but independent of IL-1β/IL-18 or neutrophil activity. L. monocytogenes strains that activated the inflammasome generated significantly less protective immunity, a phenotype that correlated with decreased induction of antigen-specific T cells. Our data suggest that avoidance of inflammasome activation is a critical virulence strategy for intracellular pathogens, and that activation of the inflammasome leads to decreased long-term protective immunity and diminished T-cell responses.

An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence.

Significance The small nucleotide cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) recently emerged as a ubiquitous signaling molecule among bacteria, with essential roles in both bacterial physiology and host–pathogen interactions. Bacterial mutants with abnormal c-di-AMP levels exhibit growth and virulence defects, reflecting the importance of regulating c-di-AMP synthesis and degradation for normal signal transduction and adaptation to changing environments. Previously documented phosphodiesterases hydrolyze c-di-AMP via the DHH-DHHA1 domain, but they are not present in all c-di-AMP synthesizing species. We identified a previously unrecognized class of His-Asp -domain phosphodiesterases that are widespread across several taxonomic groups. Furthermore, for the bacterial pathogen Listeria monocytogenes, phosphodiesterase mutants exhibit enhanced host inflammation, growth defects inside host cells, and significantly attenuated virulence in a murine model of infection. The nucleotide cyclic di-3′,5′- adenosine monophosphate (c-di-AMP) was recently identified as an essential and widespread second messenger in bacterial signaling. Among c-di-AMP–producing bacteria, altered nucleotide levels result in several physiological defects and attenuated virulence. Thus, a detailed molecular understanding of c-di-AMP metabolism is of both fundamental and practical interest. Currently, c-di-AMP degradation is recognized solely among DHH-DHHA1 domain-containing phosphodiesterases. Using chemical proteomics, we identified the Listeria monocytogenes protein PgpH as a molecular target of c-di-AMP. Biochemical and structural studies revealed that the PgpH His-Asp (HD) domain bound c-di-AMP with high affinity and specifically hydrolyzed this nucleotide to 5′-pApA. PgpH hydrolysis activity was inhibited by ppGpp, indicating a cross-talk between c-di-AMP signaling and the stringent response. Genetic analyses supported coordinated regulation of c-di-AMP levels in and out of the host. Intriguingly, a L. monocytogenes mutant that lacks c-di-AMP phosphodiesterases exhibited elevated c-di-AMP levels, hyperinduced a host type-I IFN response, and was significantly attenuated for infection. Furthermore, PgpH homologs, which belong to the 7TMR-HD family, are widespread among hundreds of c-di-AMP synthesizing microorganisms. Thus, PgpH represents a broadly conserved class of c-di-AMP phosphodiesterase with possibly other physiological functions in this crucial signaling network.

Innate immune pathways triggered by listeria monocytogenes and their role in the induction of cell-mediated immunity

Acquired cell-mediated immunity to Listeria monocytogenes is induced by infection with live, replicating bacteria that grow in the host cell cytosol, whereas killed bacteria, or those trapped in a phagosome, fail to induce protective immunity. In this chapter, we focus on how L. monocytogenes is sensed by the innate immune system, with the presumption that innate immunity affects the development of acquired immunity. Infection by L. monocytogenes induces three innate immune pathways: an MyD88-dependent pathway emanating from a phagosome leading to expression of inflammatory cytokines; a STING/IRF3-dependent pathway emanating from the cytosol leading to the expression of IFN-β and coregulated genes; and very low levels of a Caspase-1-dependent, AIM2-dependent inflammasome pathway resulting in proteolytic activation and secretion of IL-1β and IL-18 and pyroptotic cell death. Using a combination of genetics and biochemistry, we identified the listerial ligand that activates the STING/IRF3 pathway as secreted cyclic diadenosine monophosphate, a newly discovered conserved bacterial signaling molecule. We also identified L. monocytogenes mutants that caused robust inflammasome activation due to bacteriolysis in the cytosol, release of DNA, and activation of the AIM2 inflammasome. A strain was constructed that ectopically expressed and secreted a fusion protein containing Legionella pneumophila flagellin that robustly activated the Nlrc4-dependent inflammasome and was highly attenuated in mice, also in an Nlrc4-dependent manner. Surprisingly, this strain was a poor inducer of adaptive immunity, suggesting that inflammasome activation is not necessary to induce cell-mediated immunity and may even be detrimental under some conditions. To the best of our knowledge, no single innate immune pathway is necessary to mount a robust acquired immune response to L. monocytogenes infection.

Specificity of Legionella pneumophila and Coxiella burnetii Vacuoles and Versatility of Legionella pneumophila Revealed by Coinfection

ABSTRACT Legionella pneumophila and Coxiella burnetii are phylogenetically related intracellular bacteria that cause aerosol-transmitted lung infections. In host cells both pathogens proliferate in vacuoles whose biogenesis displays some common features. To test the functional similarity of their respective intracellular niches, African green monkey kidney epithelial (Vero) cells, A/J mouse bone marrow-derived macrophages, human macrophages, and human dendritic cells (DC) containing mature C. burnetii replication vacuoles were superinfected with L. pneumophila, and then the acidity, lysosome-associated membrane protein (LAMP) content, and cohabitation of mature replication vacuoles was assessed. In all cell types, wild-type L. pneumophila occupied distinct vacuoles in close association with acidic, LAMP-positive C. burnetii replication vacuoles. In murine macrophages, but not primate macrophages, DC, or epithelial cells, L. pneumophila replication vacuoles were acidic and LAMP positive. Unlike wild-type L. pneumophila, type IV secretion-deficient dotA mutants trafficked to lysosome-like C. burnetii vacuoles in Vero cells where they survived but failed to replicate. In primate macrophages, DC, or epithelial cells, growth of L. pneumophila was as robust in superinfected cell cultures as in those singly infected. Thus, despite their noted similarities, L. pneumophila and C. burnetii are exquisitely adapted for replication in unique replication vacuoles, and factors that maintain the C. burnetii replication vacuole do not alter biogenesis of an adjacent L. pneumophila replication vacuole. Moreover, L. pneumophila can replicate efficiently in either lysosomal vacuoles of A/J mouse cells or in nonlysosomal vacuoles of primate cells.

The phagosomal nutrient transporter (Pht) family

Phagosomal transporters (Phts), required for intracellular growth of Legionella pneumophila, comprise a novel family of multispanning alpha-helical proteins within the major facilitator superfamily (MFS). The members of this family derive exclusively from bacteria. Multiple paralogues are present in a restricted group of Alpha- and Gammaproteobacteria, but single members were also found in Chlamydia and Cyanobacteria. Their protein sequences were aligned, yielding a phylogenetic tree showing the relations of the proteins to each other. Topological analyses revealed a probable 12 alpha-helical transmembrane segment (TMS) topology. Motif identification and statistical analyses provided convincing evidence that these proteins arose from a six TMS precursor by intragenic duplication. The phylogenetic tree revealed some potential orthologous relationships, suggestive of common function. However, several probable examples of lateral transfer of the encoding genetic material between bacteria were identified and analysed. The Pht family most closely resembles a smaller MFS family (the UMF9 family) with no functionally characterized members. However, the UMF9 family occurs in a broader range of prokaryotic organism types, including Archaea. These two families differ in that organisms bearing members of the Pht family often have numerous paralogues, whereas organisms bearing members of the UMF9 family never have more than two. This work serves to characterize two novel families within the MFS and provides compelling evidence for horizontal transfer of some of the family members.