Alveolar Macrophages and Neutrophils Are the Primary Reservoirs for Legionella pneumophila and Mediate Cytosolic Surveillance of Type IV Secretion

Copenhaver, Alan M.; Casson, Cierra N.; Nguyen, Hieu; Fung, Thomas C.; Duda, Matthew; Roy, Craig R.; Shin, Sunny

doi:10.1128/iai.01891-14

Cited by 64 publications

(69 citation statements)

References 91 publications

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“…Alternatively, cytokines may be produced by bystander cells that are uninfected or have taken up bacteria that failed to translocate effectors (22). To determine whether T4SS-injected cells or uninjected bystander cells produce cytokines, we used a fluorescence-based system that detects the translocated L. pneumophila effector (RalF) fused to β-lactamase (BlaM) (22,23). In the absence of BlaM activity, 409-nm excitation of the host cell-permeable BlaM fluorescent substrate CCF4-AM results in emission of green fluorescence at 518 nm.…”

Section: Uninjected Cells Produce Key Inflammatory Mediators During Imentioning

confidence: 99%

“…However, whether mechanisms that enable selective translation of IL-1 also apply to other key cytokines and immune effector proteins has not been determined. Alternatively, as a significant fraction of cells present during infection both in vitro and in vivo remain uninfected bystander cells, we considered the possibility that these uninfected bystander cells might respond to the presence of infection to produce cytokines instead (22). Here, by tracking immune responses in L. pneumophila-infected cells at the single cell level, we demonstrate that although infected cells receiving T4SS effectors synthesize IL-1α and IL-1β, they are poor producers of other key inflammatory proteins.…”

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confidence: 99%

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IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis

Copenhaver

Casson

Nguyen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The innate immune system is critical for host defense against microbial pathogens, yet many pathogens express virulence factors that impair immune function. Here, we used the bacterial pathogen Legionella pneumophila to understand how the immune system successfully overcomes pathogen subversion mechanisms. L. pneumophila replicates within macrophages by using a type IV secretion system to translocate bacterial effectors into the host cell cytosol. As a consequence of effector delivery, host protein synthesis is blocked at several steps, including translation initiation and elongation. Despite this translation block, infected cells robustly produce proinflammatory cytokines, but the basis for this is poorly understood. By using a reporter system that specifically discriminates between infected and uninfected cells within a population, we demonstrate here that infected macrophages produced IL-1α and IL-1β, but were poor producers of IL-6, TNF, and IL-12, which are critical mediators of host protection. Uninfected bystander cells robustly produced IL-6, TNF, and IL-12, and this bystander response required IL-1 receptor (IL-1R) signaling during early pulmonary infection. Our data demonstrate functional heterogeneity in production of critical protective cytokines and suggest that collaboration between infected and uninfected cells enables the immune system to bypass pathogen-mediated translation inhibition to generate an effective immune response.L. pneumophila | IL-1 | IL-1R | TNF | type IV secretion system

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Section: Uninjected Cells Produce Key Inflammatory Mediators During Imentioning

confidence: 99%

mentioning

confidence: 99%

IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis

Copenhaver

Casson

Nguyen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…We utilized a FRET-based reporter system in which C. burnetii NMII expresses TEM-1 ␤-lactamase (BlaM) translationally fused to the T4SS effector CBU_0077 (33). As a positive control for T4SS-mediated translocation, we used the L. pneumophila ⌬flaA mutant expressing BlaM translationally fused to the T4SS effector RalF and lacking flagellin, thereby bypassing NAIP5 inflammasome activation and cell death in B6 BMDMs (71,72,77). Furthermore, as C. burnetii NMII does not replicate within macrophages by 24 h postinfection whereas L. pneumophila normally replicates to large numbers by 24 h postinfection, we utilized L. pneumophila thymidine auxotroph (thyA) strains for comparison in order to avoid the confounding effects of bacterial replication.…”

Section: To E) Tlr2mentioning

confidence: 99%

Primary Role for Toll-Like Receptor-Driven Tumor Necrosis Factor Rather than Cytosolic Immune Detection in Restricting Coxiella burnetii Phase II Replication within Mouse Macrophages

et al. 2016

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dCoxiella burnetii replicates within permissive host cells by employing a Dot/Icm type IV secretion system (T4SS) to translocate effector proteins that direct the formation of a parasitophorous vacuole. C57BL/6 mouse macrophages restrict the intracellular replication of the C. burnetii Nine Mile phase II (NMII) strain. However, eliminating Toll-like receptor 2 (TLR2) permits bacterial replication, indicating that the restriction of bacterial replication is immune mediated. Here, we examined whether additional innate immune pathways are employed by C57BL/6 macrophages to sense and restrict NMII replication. In addition to the known role of TLR2 in detecting and restricting NMII infection, we found that TLR4 also contributes to cytokine responses but is not required to restrict bacterial replication. Furthermore, the TLR signaling adaptors MyD88 and Trif are required for cytokine responses and restricting bacterial replication. The C. burnetii NMII T4SS translocates bacterial products into C57BL/6 macrophages. However, there was little evidence of cytosolic immune sensing of NMII, as there was a lack of inflammasome activation, T4SS-dependent cytokine responses, and robust type I interferon (IFN) production, and these pathways were not required to restrict bacterial replication. Instead, endogenous tumor necrosis factor (TNF) produced upon TLR sensing of C. burnetii NMII was required to control bacterial replication. Therefore, our findings indicate a primary role for TNF produced upon immune detection of C. burnetii NMII by TLRs, rather than cytosolic PRRs, in enabling C57BL/6 macrophages to restrict bacterial replication.T o initiate innate immune defense against bacterial pathogens, infected host cells utilize pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs) (1-3). Toll-like receptors (TLRs) located at the cell surface and within endosomes detect extracellular PAMPs such as bacterial lipoproteins and lipopolysaccharide (LPS) (4). Downstream of TLRs, the adaptor proteins MyD88 and Trif activate several signaling pathways, including NF-B, mitogen-activated protein kinases (MAPKs), and interferon (IFN) regulatory factor 3 (IRF3), which direct the expression of proinflammatory cytokines and other antimicrobial effectors (4). For intracellular bacterial pathogens, cytosolic PRRs, such as those of the nucleotide binding domain/ leucine-rich repeat (NLR) and RIG-I-like receptor (RLR) families, often are critical for host defense as they respond to PAMPs introduced into the host cell cytosol by bacterial pore-forming toxins or specialized secretion systems (5-8). In addition, cytosolic sensing can lead to the assembly of a multiprotein complex termed the inflammasome, which activates the host proteases caspase-1 and caspase-11, resulting in the release of IL-1 family cytokines and a form of cell death known as pyroptosis (9-16). These innate immune pathways collaborate to restrict intracellular bacterial infection through both cell-intrinsic and -extrinsic mechanisms (17)(18...

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“…The bacterium is a pathogen of amoebae and humans. In humans, L. pneumophila's primary manifestation of virulence is Legionnaires' disease, a potentially lethal pneumonia resulting from growth of the bacteria within alveolar macrophages (17,18). Biogenesis of the microbial replication vacuole is evolutionarily conserved from amoebae to eukaryotic cells (16,19), with the bacterium growing in an ER-encompassed compartment called the Legionella-containing vacuole (LCV) (13,15,20).…”

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confidence: 99%

Endoplasmic Reticulum Tubule Protein Reticulon 4 Associates with the Legionella pneumophila Vacuole and with Translocated Substrate Ceg9

et al. 2015

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Intracellular growth of Legionella pneumophila occurs in a replication vacuole constructed by host proteins that regulate vesicular traffic from the host endoplasmic reticulum (ER). This process is promoted by a combination of approximately 300 Icm/Dot translocated substrates (IDTS). One of these proteins, Ceg9, was previously identified in a screen for L. pneumophila IDTS that manipulate secretory traffic when overexpressed in yeast. Using ectopic expression of Ceg9 in mammalian cells, we demonstrate that Ceg9 interacts with isoforms of host reticulon 4 (Rtn4), a protein that regulates ER tubule formation. Binding occurs under conditions that prevent association with other known reticulon binding proteins, arguing that Ceg9 binding is stable. A tripartite complex was demonstrated among Rtn4, Ceg9, and atlastin 1, a previously characterized reticulon interacting partner. The binding of Ceg9 to Rtn4 was not due to bridging by atlastin 1 but resulted from the two interacting partners binding independently to reticulon. When Ceg9 is ectopically expressed in mammalian cells, it shows a localization pattern that is indistinguishable from that of Rtn4, perhaps due to interactions between and similar structural features of the two proteins. Consistent with Rtn4 playing a role in the formation of the Legionella-containing vacuole, it was recruited to almost 50% of the vacuoles within 20 min postinfection. Our studies suggest that L. pneumophila proteins interact with ER tubules at an early stage of replication vacuole formation. I ntravacuolar growth of intracellular pathogens involves intimate interaction with specific subsets of host cell membrane compartments (1). In the case of Chlamydia trachomatis, the association of the replication vacuole with the trans-Golgi and the endoplasmic reticulum (ER) network is a key step during the establishment of an intracellular niche (2-5). Similarly, vacuoles harboring Brucella, Salmonella, and Mycobacterium species also interface with defined membrane compartments that aid the growth of these bacteria in host cells (6-10). Failure to establish association with the appropriate organelles can have dire consequences, including loss of vacuolar membrane integrity, activation of inflammatory host cell death, and trafficking of the microorganism into degradative compartments (3,6,11,12).Legionella pneumophila is one such intravacuolar pathogen that targets specific intracellular compartments during its replication cycle (13-16). The bacterium is a pathogen of amoebae and humans. In humans, L. pneumophila's primary manifestation of virulence is Legionnaires' disease, a potentially lethal pneumonia resulting from growth of the bacteria within alveolar macrophages (17, 18). Biogenesis of the microbial replication vacuole is evolutionarily conserved from amoebae to eukaryotic cells (16,19), with the bacterium growing in an ER-encompassed compartment called the Legionella-containing vacuole (LCV) (13,15,20). The connection between the LCV and ER dynamics is well established, since the prop...

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Alveolar Macrophages and Neutrophils Are the Primary Reservoirs for Legionella pneumophila and Mediate Cytosolic Surveillance of Type IV Secretion

Cited by 64 publications

References 91 publications

IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis

IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis

Primary Role for Toll-Like Receptor-Driven Tumor Necrosis Factor Rather than Cytosolic Immune Detection in Restricting Coxiella burnetii Phase II Replication within Mouse Macrophages

Endoplasmic Reticulum Tubule Protein Reticulon 4 Associates with the Legionella pneumophila Vacuole and with Translocated Substrate Ceg9

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