Jörn Coers

Modulation of phagosome biogenesis by Legionella pneumophila creates an organelle permissive for intracellular growth.

Modulation of phagosome biogenesis by Legionella pneumophila creates an organelle permissive for intracellular growth

Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth

Legionella pneumophila is a bacterial pathogen that can enter the human lung and grow inside alveolar macrophages. To grow within phagocytic host cells, the bacteria must create a specialized organelle that restricts fusion with lysosomes. Biogenesis of this replicative organelle is controlled by 24 dot and icm genes, which encode a type IV‐related transport apparatus. To understand how this transporter functions, isogenic L. pneumophila dot and icm mutants were characterized, and three distinct phenotypic categories were identified. Our data show that, in addition to genes that encode the core Dot/Icm transport apparatus, subsets of genes are required for pore formation and modulation of phagosome trafficking. To understand activities required for virulence at a molecular level, we investigated protein–protein interactions. Specific interactions between different Icm proteins were detected by yeast two‐hybrid and gel overlay analysis. These data support a model in which the IcmQ–IcmR complex regulates the formation of a translocation channel that delivers proteins into host cells, and the IcmS–IcmW complex is required for export of virulence determinants that modulate phagosome trafficking.

Guanylate binding proteins promote caspase-11–dependent pyroptosis in response to cytoplasmic LPS

Significance A major component of the cell envelope of Gram-negative bacteria is LPS, also known as endotoxin. LPS produced during bacterial infections triggers inflammation, which can lead to septic shock and death. Our immune system can recognize LPS both outside and inside of cells. The recognition of extracellular and vacuolar LPS by LPS binding proteins is well described, but little is known about the recognition of cytoplasmic LPS. Here, we show that cytoplasmic LPS derived from the intracellular bacterial pathogen Legionella activated a proinflammatory immune response. We further identified host guanylate binding proteins as critical mediators of immunity triggered by cytoplasmic LPS. These findings are likely to advance our understanding of how cells can sense intracellular LPS. IFN receptor signaling induces cell-autonomous immunity to infections with intracellular bacterial pathogens. Here, we demonstrate that IFN-inducible guanylate binding protein (Gbp) proteins stimulate caspase-11–dependent, cell-autonomous immunity in response to cytoplasmic LPS. Caspase-11–dependent pyroptosis is triggered in IFN-activated macrophages infected with the Gram-negative bacterial pathogen Legionella pneumophila. The rapid induction of pyroptosis in IFN-activated macrophages required a cluster of IFN-inducible Gbp proteins encoded on mouse chromosome 3 (Gbpchr3). Induction of pyroptosis in naive macrophages by infections with the cytosol-invading ΔsdhA L. pneumophila mutant was similarly dependent on Gbpchr3, suggesting that these Gbp proteins play a role in the detection of bacteria accessing the cytosol. Cytoplasmic LPS derived from Salmonella ssp. or Escherichia coli has recently been shown to trigger caspase-11 activation and pyroptosis, but the cytoplasmic sensor for LPS and components of the caspase-11 inflammasome are not yet defined. We found that the induction of caspase-11–dependent pyroptosis by cytoplasmic L. pneumophila-derived LPS required Gbpchr3 proteins. Similarly, pyroptosis induced by cytoplasmic LPS isolated from Salmonella was diminished in Gbpchr3-deficient macrophages. These data suggest a role for Gbpchr3 proteins in the detection of cytoplasmic LPS and the activation of the noncanonical inflammasome.

Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole

The immunity‐related GTPases (IRGs) constitute an interferon‐induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion‐driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild‐type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A–D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

Restriction of Legionella pneumophila growth in macrophages requires the concerted action of cytokine and Naip5/Ipaf signalling pathways

Macrophages from the C57BL/6 (B6) mouse strain restrict intracellular growth of Legionella pneumophila, whereas A/J macrophages are highly permissive. The mechanism by which B6 macrophages restrict Legionella growth remains poorly understood, but is known to require the cytosolic microbe sensors Naip5 (Birc1e) and Ipaf. We hypothesized that Naip5 and Ipaf may act in partnership with other antimicrobial signalling pathways in macrophages. Indeed, we found that macrophages lacking either tumour necrosis factor (TNF)‐α or type I interferon (IFN) signalling are permissive for growth of L. pneumophila, even in the presence of functional Naip5 and Ipaf alleles. Similarly, macrophages lacking Naip5 and/or Ipaf signalling were permissive even though we found that Naip5 or Ipaf were not required for induction of TNF‐α and type I IFN. Therefore, our data suggest that the mechanism by which B6 macrophages restrict intracellular replication of L. pneumophila is more complex than previously appreciated, and involves the concerted action of cytokine and intracellular microbe sensor signalling pathways.

Chlamydia muridarum Evades Growth Restriction by the IFN-γ-Inducible Host Resistance Factor Irgb10

Chlamydiae are obligate intracellular bacterial pathogens that exhibit a broad range of host tropism. Differences in host tropism between Chlamydia species have been linked to host variations in IFN-γ-mediated immune responses. In mouse cells, IFN-γ can effectively restrict growth of the human pathogen Chlamydia trachomatis but fails to control growth of the closely related mouse pathogen Chlamydia muridarum. The ability of mouse cells to resist C. trachomatis replication is largely dependent on the induction of a family of IFN-γ-inducible GTPases called immunity-related GTPases or IRGs. In this study we demonstrate that C. muridarum can specifically evade IRG-mediated host resistance. It has previously been suggested that C. muridarum inactivates the IRG protein Irga6 (Iigp1) to dampen the murine immune response. However, we show that Irga6 is dispensable for the control of C. trachomatis replication. Instead, an effective IFN-γ response to C. trachomatis requires the IRG proteins Irgm1 (Lrg47), Irgm3 (Igtp), and Irgb10. Ectopic expression of Irgb10 in the absence of IFN-γ is sufficient to reduce intracellular growth of C. trachomatis but fails to restrict growth of C. muridarum, indicating that C. muridarum can specifically evade Irgb10-driven host responses. Importantly, we find that Irgb10 protein intimately associates with inclusions harboring C. trachomatis but is absent from inclusions formed by C. muridarum. These data suggest that C. muridarum has evolved a mechanism to escape the murine IFN-γ response by restricting access of Irgb10 and possibly other IRG proteins to the inclusion.

IRG and GBP Host Resistance Factors Target Aberrant, “Non-self” Vacuoles Characterized by the Missing of “Self” IRGM Proteins

Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with “non-self” PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on “self” organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of “self” IRGM proteins from these structures.

The p47 GTPases Igtp and Irgb10 map to the Chlamydia trachomatis susceptibility locus Ctrq-3 and mediate cellular resistance in mice

Infections caused by the bacteria Chlamydia trachomatis contribute to diverse pathologies in a variety of human populations. We previously used a systemic model of C. trachomatis infection in mice to map three quantitative trait loci that influence in vivo susceptibility differences between the C57BL/6J and C3H/HeJ inbred strains of mouse. One of these quantitative trait loci, Ctrq-3, influences an IFN-γ-dependent susceptibility difference in primary embryonic fibroblasts isolated from these strains. Here we use fine structure mapping in congenic fibroblasts carrying DNA from the susceptible parent to localize the effect of Ctrq-3 to a 1.2-megabase interval of genomic DNA that contains Irgb10 and Igtp, two members of the IFN-γ-inducible p47 family of GTPases. This class of proteins has been widely implicated in resistance to intracellular pathogens in mice. We analyzed expression of Irgb10 and Igtp in parental and congenic embryonic fibroblasts treated with IFN-γ and found that relatively resistant fibroblasts express more Irgb10 than relatively susceptible fibroblasts. However, we also found that abolishing the expression of either Irgb10 or Igtp increases susceptibility of embryonic fibroblasts to C. trachomatis. Thus, we conclude that, although a difference in Irgb10 expression is likely responsible for the effect of Ctrq-3 on susceptibility to C. trachomatis, both genes play a role in intracellular resistance to C. trachomatis.

Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins

Significance The innate immune system protects the host against infections with a diverse set of microbes that include intracellular bacterial and protozoan pathogens residing within pathogen-containing vacuoles (PVs). Because PVs provide an intracellular niche permissive for microbial growth, their destruction is critical for host defense. In mammals, PV destruction is dependent on immunity-related GTPases and guanylate binding proteins (GBPs). Although it has been shown that GBPs translocate to and eliminate PVs, the mechanisms by which GBPs specifically bind to PVs were unknown. Here, we describe an immune pathway that results in the decoration of PVs with a small protein called ubiquitin. Ubiquitin-decorated PVs are subsequently recognized by GBPs, resulting in the elimination of PVs and their microbial inhabitants. Many microbes create and maintain pathogen-containing vacuoles (PVs) as an intracellular niche permissive for microbial growth and survival. The destruction of PVs by IFNγ-inducible guanylate binding protein (GBP) and immunity-related GTPase (IRG) host proteins is central to a successful immune response directed against numerous PV-resident pathogens. However, the mechanism by which IRGs and GBPs cooperatively detect and destroy PVs is unclear. We find that host cell priming with IFNγ prompts IRG-dependent association of Toxoplasma- and Chlamydia-containing vacuoles with ubiquitin through regulated translocation of the E3 ubiquitin ligase tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). This initial ubiquitin labeling elicits p62-mediated escort and deposition of GBPs to PVs, thereby conferring cell-autonomous immunity. Hypervirulent strains of Toxoplasma gondii evade this process via specific rhoptry protein kinases that inhibit IRG function, resulting in blockage of downstream PV ubiquitination and GBP delivery. Our results define a ubiquitin-centered mechanism by which host cells deliver GBPs to PVs and explain how hypervirulent parasites evade GBP-mediated immunity.

Guanylate Binding Proteins Enable Rapid Activation of Canonical and Noncanonical Inflammasomes in Chlamydia-Infected Macrophages

ABSTRACT Interferon (IFN)-inducible guanylate binding proteins (GBPs) mediate cell-autonomous host resistance to bacterial pathogens and promote inflammasome activation. The prevailing model postulates that these two GBP-controlled activities are directly linked through GBP-dependent vacuolar lysis. It was proposed that the rupture of pathogen-containing vacuoles (PVs) by GBPs destroyed the microbial refuge and simultaneously contaminated the host cell cytosol with microbial activators of inflammasomes. Here, we demonstrate that GBP-mediated host resistance and GBP-mediated inflammatory responses can be uncoupled. We show that PVs formed by the rodent pathogen Chlamydia muridarum, so-called inclusions, remain free of GBPs and that C. muridarum is impervious to GBP-mediated restrictions on bacterial growth. Although GBPs neither bind to C. muridarum inclusions nor restrict C. muridarum growth, we find that GBPs promote inflammasome activation in C. muridarum-infected macrophages. We demonstrate that C. muridarum infections induce GBP-dependent pyroptosis through both caspase-11-dependent noncanonical and caspase-1-dependent canonical inflammasomes. Among canonical inflammasomes, we find that C. muridarum and the human pathogen Chlamydia trachomatis activate not only NLRP3 but also AIM2. Our data show that GBPs support fast-kinetics processing and secretion of interleukin-1β (IL-1β) and IL-18 by the NLRP3 inflammasome but are dispensable for the secretion of the same cytokines at later times postinfection. Because IFN-γ fails to induce IL-1β transcription, GBP-dependent fast-kinetics inflammasome activation can drive the preferential processing of constitutively expressed IL-18 in IFN-γ-primed macrophages in the absence of prior Toll-like receptor stimulation. Together, our results reveal that GBPs control the kinetics of inflammasome activation and thereby shape macrophage responses to Chlamydia infections.