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IntroductionNovel therapeutic strategies are urgently needed for Mycobacterium aviumcomplex pulmonary disease (MAC-PD). Human mesenchymal stromal cells (MSCs) can directly inhibit MAC growth, but their effect on intracellular bacilli is unknown. We investigated the ability of human MSCs to reduce bacterial replication and inflammation in MAC-infected macrophages and in a murine model of MAC-PD.MethodsHuman monocyte-derived macrophages (MDMs) were infected with M. aviumChester strain and treated with human bone marrow-derived MSCs. Intracellular and extracellular colony-forming units (CFUs) were counted at 72 hours. Six-week-old female balb/c mice were infected by nebulisation of M. aviumChester. Mice were treated with 1×106intravenous human MSCs or saline control at 21 and 28 days post-infection. Lungs, liver and spleen were harvested 42 days post-infection for bacterial counts. Cytokines were quantified by ELISA.ResultsMSCs reduced intracellular bacteria in MDMs over 72 hours (median 35% reduction, p=0.027). MSC treatment increased extracellular concentrations of prostaglandin E2 (PGE2) (median 10.1-fold rise, p=0.002) and reduced tumour necrosis factor-α (median 28% reduction, p=0.025). Blocking MSC PGE2 production by cyclo-oxygenase-2 (COX-2) inhibition with celecoxib abrogated the antimicrobial effect, while this was restored by adding exogenous PGE2. MSC-treated mice had lower pulmonary CFUs (median 18% reduction, p=0.012), but no significant change in spleen or liver CFUs compared with controls.ConclusionMSCs can modulate inflammation and reduce intracellular M. aviumgrowth in human macrophages via COX-2/PGE2 signalling and inhibit pulmonary bacterial replication in a murine model of chronic MAC-PD.

ABSTRACTLegionella pneumophilais a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophiladepends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophilaΔlpdAmutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophilainfection in vivo.

ABSTRACTThe Dot/Icm type IV secretion system (T4SS) of Legionella pneumophilais crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of the Legionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD471-626bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P] in vitroand colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide-binding L. pneumophilaeffector that has a role in intracellular bacterial replication.

ABSTRACTLegionella pneumophilais an intracellular bacterium that resides within amoebae and macrophages in a specialized compartment termed the Legionella-containing vacuole (LCV). As well as providing an intracellular niche for replication, the LCV helps to prevent the release of bacterial components into the cytoplasm. Recognition of these components as danger signals by the host activates immune responses leading to clearance of the bacterium. Here, we examined the role of two important virulence factors of L. pneumophila, the potent danger signal flagellin and the translocated Dot/Icm type IVB secretion system effector SdhA, which is crucial to maintain LCV integrity, in the Galleria mellonellainfection model. We demonstrate that flagellin expression does not contribute to virulence, replication, or induction of clearance mechanisms. Conversely, SdhA expression is important for virulence. We found that in the absence of SdhA, the LCV in hemocytes showed signs of instability and leakage. Furthermore, in contrast to wild-type L. pneumophila, a ?sdhAmutant caused a transient depletion of hemocytes and reduced mortality. Analysis of the ?sdhAmutant in the A/J mouse model also showed a significant replication defect. Together, our data underline the crucial importance of SdhA in infection across different model organisms.

ABSTRACTLegionella pneumophilais a facultative intracellular human pathogen and the etiological agent of severe pneumonia known as Legionnaires' disease. Its virulence depends on protein secretion systems, in particular, the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication-permissive vacuole in macrophages. The analysis of the role of these systems and their substrates for pathogenesis requires easy-to-use models which approximate human infection. We examined the effectiveness of the larvae of the wax moth Galleria mellonellaas a new model for L. pneumophilainfection. We found that the L. pneumophilastrains 130b, Paris, and JR32 caused mortality of the G. mellonellalarvae that was strain, infectious dose, growth phase, and T4SS dependent. Wild-type L. pneumophilapersisted and replicated within the larvae, whereas T4SS mutants were rapidly cleared. L. pneumophilastrain Lp02, which is attenuated in the absence of thymidine but has a functional T4SS, resisted clearance in G. mellonellaup to 18 h postinfection without inducing mortality. Immunofluorescence and transmission electron microscopy revealed that L. pneumophilaresided within insect hemocytes in a vacuole that ultrastructurally resembled the Legionella-containing vacuole (LCV) observed in macrophages. The vacuole was decorated with the T4SS effector and LCV marker SidC. Infection caused severe damage to the insect organs and triggered immune responses, including activation of the phenoloxidase cascade leading to melanization, nodule formation, and upregulation of antimicrobial peptides. Taken together, these results suggest that G. mellonellaprovides an effective model to investigate the interaction between L. pneumophilaand the host.

SUMMARYShigellaspp. are gram-negative pathogenic bacteria that evolved from harmless enterobacterial relatives and may cause devastating diarrhea upon ingestion. Research performed over the last 25 years revealed that a type III secretion system (T3SS) encoded on a large plasmid is a key virulence factor of Shigella flexneri. The T3SS determines the interactions of S. flexneriwith intestinal cells by consecutively translocating two sets of effector proteins into the target cells. Thus, S. flexnericontrols invasion into EC, intra- and intercellular spread, macrophage cell death, as well as host inflammatory responses. Some of the translocated effector proteins show novel biochemical activities by which they intercept host cell signal transduction pathways. An understanding of the molecular mechanisms underlying Shigellapathogenesis will foster the development of a safe and efficient vaccine, which, in parallel with improved hygiene, should curb infections by this widespread pathogen.

ABSTRACTLegionella pneumophila, the causative agent of Legionnaires’ disease, uses the Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effectors into host cells, where they subvert host cell signaling. The function and host cell targets of most effectors remain unknown. PieE is a 69-kDa Dot/Icm effector containing three coiled-coil (CC) regions and 2 transmembrane (TM) helices followed by a fourth CC region. Here, we report that PieE dimerized by an interaction between CC3 and CC4. We found that ectopically expressed PieE localized to the endoplasmic reticulum (ER) and induced the formation of organized smooth ER, while following infection PieE localized to the Legionella-containing vacuole (LCV). To identify the physiological targets of PieE during infection, we established a new purification method for which we created an A549 cell line stably expressing the Escherichia colibiotin ligase BirA and infected the cells with L. pneumophilaexpressing PieE fused to a BirA-specific biotinylation site and a hexahistidine tag. Following tandem Ni2+nitrilotriacetic acid (NTA) and streptavidin affinity chromatography, the effector-target complexes were analyzed by mass spectrometry. This revealed interactions of PieE with multiple host cell proteins, including the Rab GTPases 1a, 1b, 2a, 5c, 6a, 7, and 10. Binding of the Rab GTPases, which was validated by yeast two-hybrid binding assays, was mediated by the PieE CC1 and CC2. In summary, using a novel, highly specific strategy to purify effector complexes from infected cells, which is widely applicable to other pathogens, we identified PieE as a multidomain LCV protein with promiscuous Rab GTPase-binding capacity.IMPORTANCEThe respiratory pathogen Legionella pneumophilauses the Dot/Icm type IV secretion system to translocate more than 300 effector proteins into host cells. The function of most effectors in infection remains unknown. One of the bottlenecks for their characterization is the identification of target proteins. Frequently used in vitroapproaches are not applicable to all effectors and suffer from high rates of false positives or missed interactions, as they are not performed in the context of an infection. Here, we determine key functional domains of the effector PieE and describe a new method to identify host cell targets under physiological infection conditions. Our approach, which is applicable to other pathogens, uncovered the interaction of PieE with several proteins involved in membrane trafficking, in particular Rab GTPases, revealing new details of the Legionellainfection strategy and demonstrating the potential of this method to greatly advance our understanding of the molecular basis of infection.