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Caspases are either apoptotic or inflammatory. Among inflammatory caspases, caspase-1 and -11 trigger pyroptosis, a form of programmed cell death. Whereas both can be detrimental in inflammatory disease, only caspase-1 has an established protective role during infection. Here, we report that caspase-11 is required for innate immunity to cytosolic, but not vacuolar, bacteria. Although Salmonella typhimurium and Legionella pneumophila normally reside in the vacuole, specific mutants (sifA and sdhA, respectively) aberrantly enter the cytosol. These mutants triggered caspase-11, which enhanced clearance of S. typhimurium sifA in vivo. This response did not require NLRP3, NLRC4, or ASC inflammasome pathways. Burkholderia species that naturally invade the cytosol also triggered caspase-11, which protected mice from lethal challenge with B. thailandensis and B. pseudomallei. Thus, caspase-11 is critical for surviving exposure to ubiquitous environmental pathogens.

Key Points Legionella pneumophila and Coxiella burnetii are two evolutionarily related intracellular bacterial pathogens that reside in distinct compartments in host cells during infection. Successful infection by both pathogens requires a functionally exchangeable type IV secretion system called Dot/Icm, which translocates hundreds of virulence factors, termed effectors, into host cells. The majority of Legionella spp. and Coxiella spp. effectors are unique to these pathogens, and functional redundancy exists among many of them. Functional domains that are associated with most of these effectors are enigmatic and cannot be readily predicted by currently available bioinformatics tools. Legionella spp. and Coxiella spp. promote intracellular bacterial replication by interfering with host gene expression through effectors that impose epigenetic modifications on host chromatin by different mechanisms. L. pneumophila extensively manipulates the early phases of the secretory branch of the host vesicle trafficking pathway by hijacking the activity of key regulatory proteins such as RAB small GTPases via multiple effectors. L. pneumophila effectors function coordinately to alter the composition of lipids, such as phosphoinositides, on the vacuole that contains the bacterium and other organelles to facilitate its intracellular growth. L. pneumophila co-opts the ubiquitin network of host cells by effectors that function through diverse biochemical mechanisms, including the SidE family effectors, which catalyse ubiquitylation by an E1 enzyme and E2 enzyme-independent mechanism, which represents a paradigm shift in our understanding of this important post-translational modification. The intracellular pathogens Legionella pneumophila and Coxiella burnetii use the Dot/Icm type IV secretion system to translocate effectors into host cells. Qiu and Luo explore the biochemical and cell biological functions of these effectors and their roles in our understanding of bacterial virulence. Legionella pneumophila and Coxiella burnetii are two evolutionarily related intracellular pathogens that use the Dot/Icm type IV secretion system to translocate effectors into host cells. These effectors are essential for the establishment of membrane-bound compartments known as replication vacuoles, which enable the survival and replication of bacteria inside host cells. The effectors interfere with diverse signalling pathways to co-opt host processes, such as vesicle trafficking, ubiquitylation, gene expression and lipid metabolism, to promote pathogen survival. In this Review, we explore Dot/Icm effectors from L. pneumophila and C. burnetii as key virulence factors, and we examine the biochemical and cell biological functions of these effectors and their roles in our understanding of bacterial virulence.

Endomembrane breaching is a crucial strategy employed by intracellular pathogens enclosed within vacuoles to access the nutrient-rich cytosol for intracellular replication. While bacteria use various mechanisms to compromise host membranes, the specific processes and factors involved are often unknown. Shigella flexneri , a major human pathogen, accesses the cytosol relying on the Type Three Secretion System (T3SS) and secreted effectors. Using in-cell correlative light and electron microscopy, we tracked the sequential steps of Shigella host cell entry. Moreover, we captured the T3SS, which projects a needle from the bacterial surface, in the process of puncturing holes in the vacuolar membrane. This initial puncture ensures disruption of the vacuole. Together this introduces the concept of mechanoporation via a bacterial secretion system as a crucial process for bacterial pathogen-induced membrane damage.

Interferon-inducible GTPases are required for the release of vacuolar Gram-negative bacteria into the cytoplasm and subsequent inflammasome-mediated caspase-11 activation. Bacteria versus the innate immune system Inflammasomes are multiprotein complexes that act as activation platforms for caspase-11, a major mediator of inflammation. Lipopolysaccharide (LPS) from Gram-negative bacteria is sensed by a novel inflammasome pathway that targets caspase-11 and is activated by type-I interferons. This study in mice shows that the activation of caspase-11 by vacuolar bacterial pathogens requires the expression of small, interferon-inducible GTPases, so-called GBP proteins. These GBP proteins attack the membrane of pathogen-containing vacuoles and induce their lysis and subsequent caspase- 11- mediated inflammasome activation. These findings demonstrate that host-induced destruction of pathogen-containing vacuoles or phagosomes is an essential immune function and assures recognition of vacuolar bacteria by cytosolic innate immune sensors. Lipopolysaccharide from Gram-negative bacteria is sensed in the host cell cytoplasm by a non-canonical inflammasome pathway that ultimately results in caspase-11 activation and cell death 1 , 2 , 3 . In mouse macrophages, activation of this pathway requires the production of type-I interferons 4 , 5 , indicating that interferon-induced genes have a critical role in initiating this pathway. Here we report that a cluster of small interferon-inducible GTPases, the so-called guanylate-binding proteins, is required for the full activity of the non-canonical caspase-11 inflammasome during infections with vacuolar Gram-negative bacteria. We show that guanylate-binding proteins are recruited to intracellular bacterial pathogens and are necessary to induce the lysis of the pathogen-containing vacuole. Lysis of the vacuole releases bacteria into the cytosol, thus allowing the detection of their lipopolysaccharide by a yet unknown lipopolysaccharide sensor. Moreover, recognition of the lysed vacuole by the danger sensor galectin-8 initiates the uptake of bacteria into autophagosomes, which results in a reduction of caspase-11 activation. These results indicate that host-mediated lysis of pathogen-containing vacuoles is an essential immune function and is necessary for efficient recognition of pathogens by inflammasome complexes in the cytosol.

Key Points Legionella pneumophila is a Gram-negative intracellular pathogen of both amoebae and humans that grows in lung macrophages. The intracellular replication strategy of this bacterium, which involves growth in a membrane-bound compartment called a vacuole, seems to be similar in all cell types in which it grows. A protein translocation apparatus that encodes more than 20 proteins, called Dot/Icm, is required for formation of the replication vacuole and intracellular growth. Proteins move through the apparatus across membranes in contact with the bacterium, and are thought to manipulate host cell proteins that are involved in host cell secretory traffic. Eighty five translocated protein substrates of Dot/Icm have been identified, but there are probably many more. The targets of several have been identified, and they include proteins that modulate the activation state of Arf1 and Rab1 and are involved in vesicle trafficking in host cells, as well as proteins that antagonize host cell death pathways. Mutations in single translocated substrates rarely result in strong defects in intracellular growth. This has led to the model that there is considerable functional redundancy among the substrates. A model is provided to attempt to explain how L. pneumophila acquired such a diverse set of translocated substrates. In this Review, the authors evaluate the strategies that the intracellular pathogen Legionella pneumophila uses to establish growth inside cells and probe why this microorganism has accumulated an unprecedented number of translocated substrates that are targeted to host cells. The pathogenesis of Legionella pneumophila is derived from its growth within lung macrophages after aerosols are inhaled from contaminated water sources. Interest in this bacterium stems from its ability to manipulate host cell vesicular-trafficking pathways and establish a membrane-bound replication vacuole, making it a model for intravacuolar pathogens. Establishment of the replication compartment requires a specialized translocation system that transports a large cadre of protein substrates across the vacuolar membrane. These substrates regulate vesicle traffic and survival pathways in the host cell. This Review focuses on the strategies that L. pneumophila uses to establish intracellular growth and evaluates why this microorganism has accumulated an unprecedented number of translocated substrates that are targeted at host cells.

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.

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes an emerging infectious disease, human granulocytic anaplasmosis. It undergoes a biphasic developmental cycle inside membrane-bound vacuoles within the host human neutrophils, maturing from a proliferating reticulate cell form to an infectious dense core (DC) form that is subsequently spontaneously released from host cells to initiate a new infection cycle. However, how A. phagocytophilum coordinates growth and release is unknown. Here, we found localized cortical F-actin disruption occurs where Anaplasma-containing vacuoles abut on the plasma membrane to release bacteria. Disruption of actin filaments by cytochalasin D and latrunculin B induced unrestrained release of almost all intracellular A. phagocytophilum from host cells, which were significantly less infectious than spontaneously released bacteria. A. phagocytophilum AnkA, a type IV secretion system (T4SS) effector, was found to localize in the cell periphery with cortical F-actin. By immunoprecipitation followed by mass spectrometry, AnkA was found to interact with actin, α-actinin 4 (Actn4) involved in actin cross-linking, and gelsolin for actin filament remodeling. shRNA-knockdown of Actn4 or gelsolin, enhanced release of premature A. phagocytophilum. Glutathione S-transferase (GST)-tagged C-terminus of AnkA (AnkA-C) interacted with actin and gelsolin, whereas the N-terminus (AnkA-N) interacted with Actn4. In vitro pyrene-actin polymerization assay showed that GST-AnkA-C has stronger actin polymerizing activity than GST-AnkA or GST-AnkA-N. Ectopically expressed GFP-AnkA-N localized to the plasma membrane and induced membrane ruffling, whereas GFP-AnkA-C colocalized with and enhanced stress fiber formation. These results demonstrate that AnkA is the first example of bacterial molecules interacting with gelsolin and Actn4. The result suggests that by colocalizing with cortical F-actin and controlling F-actin dynamics and cross-linking, AnkA regulates spatiotemporal release of A. phagocytophilum. The current study unravels a new paradigm of retention/release mechanism of intracellular pathogen regulated by a T4SS effector.

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

Autophagy plays a crucial role in the host response to Mycobacterium tuberculosis (Mtb) infection, yet the dynamics and regulation of autophagy induction on Mtb-containing vacuoles (MCVs) remain only partially understood. We employed time-lapse confocal microscopy to investigate the recruitment of LC3B (LC3), a key autophagy marker, to MCVs at the single cell level with our newly developed workflow for single cell and single MCV tracking and fluorescence quantification. We show that approximately 70% of MCVs exhibited LC3 recruitment but that was lost in about 40% of those MCVs. The LC3 recruitment to MCVs displayed a high variability in timing that was independent of the size of the MCV or the bacterial burden. Most notably, the LC3-positive MCVs did not acidify, indicating that LC3 recruitment does not necessarily lead to the formation of mature autophagolysosomes. Interferon-gamma pre-treatment did not affect LC3 recruitment frequency or autophagosome acidification but increased the susceptibility of the macrophage to Mtb-induced cell death. LC3 recruitment and lysotracker staining were mutually exclusive events, alternating on some MCVs multiple times thus demonstrating a reversible aspect of the autophagy response. The LC3 recruitment was associated with galectin-3 and oxysterol-binding protein 1 staining, indicating a correlation with membrane damage and repair mechanisms. ATG7 knock-down did not impact membrane repair, suggesting that autophagy is not directly involved in this process but is coregulated by the membrane damage of MCVs. In summary, our findings provide novel insights into the dynamic and variable nature of LC3 recruitment to the MCVs over time during Mtb infection. Our data does not support a role for autophagy in either cell-autonomous defense against Mtb or membrane repair of the MCV in human macrophages. In addition, the combined dynamics of LC3 recruitment and Lysoview staining emerged as promising markers for investigating the damage and repair processes of phagosomal membranes.

Shigella flexneri is a human intracellular pathogen responsible for bacillary dysentery (bloody diarrhea). S. flexneri invades colonic epithelial cells and spreads from cell to cell, leading to massive epithelial cell fenestration, a critical determinant of pathogenesis. Cell-to-cell spread relies on actin-based motility, which leads to formation of membrane protrusions, as bacteria project into adjacent cells. Membrane protrusions resolve into intermediate structures termed vacuole-like protrusions (VLPs), which remain attached to the primary infected cell by a membranous tether. The resolution of the membranous tether leads to formation of double-membrane vacuoles (DMVs), from which S. flexneri escapes to gain access to the cytosol of adjacent cells. Here, we identify the class III PI3K family member PIK3C3 as a critical determinant of S. flexneri cell-to-cell spread. Inhibition of PIK3C3 decreased the size of infection foci formed by S. flexneri in HT-29 cells. Tracking experiments using live-fluorescence confocal microscopy showed that PIK3C3 is required for efficient resolution of VLPs into DMVs. PIK3C3-dependent accumulation of PtdIns(3)P at the VLP membrane in adjacent cells correlated with the transient recruitment of the membrane scission machinery component Dynamin 2 at the neck of VLPs at the time of DMV formation. By contrast, Listeria monocytogenes did not form VLPs and protrusions resolved directly into DMVs. However, PIK3C3 was also required for L. monocytogenes dissemination, but at the stage of vacuole escape. Finally, we showed that PIK3C3 inhibition decreased S. flexneri dissemination in the infant rabbit model of shigellosis. We propose a model of Shigella dissemination in which vacuole formation relies on the PIK3C3-dependent accumulation of PtdIns(3)P at the VLP stage of cell-to-cell spread, thereby supporting the resolution of VLPs into DMVs through recruitment of the membrane scission machinery component, DNM2.