How does legionella pneumophila survive in a phagocyte

Previous data indicate that activation of Rac1 and Rho, members of Rho-family GTPases, requires PI3K activity, which in turn activates pactivated kinase-1 [43] , and Rho-kinases [44] to control cytoskeletal rearrangements. Thus, this pathway may well play an important role in phagocytic events. We have shown that contact between L.

One of the responses to Akt activation is prevention of programmed cell death or apoptosis [45]. Prolonging the survival of infected cells is clearly in the best interest of L. Thus, further investigation of the effects of Akt activation on phagocytosis as well as subsequent intracellular survival and replication of L.

Cell entry of T. Even though our data show that entry of L. Our findings support the hypothesis that PI3K activation is required for efficient entry of L. Activation of PI3K leads to the formation of 3-phosphoinositides and activation of Akt. Further studies to determine the isoform s of PI3K involved would be useful, since this information will provide insight into the mechanisms of PI3K activation.

The conclusion that PI3K is involved in uptake of L. Collectively these results indicate that L. Anti-phosphotyrosine mouse monoclonal antibody 4G10 was purchased from Upstate Biotechnology, Inc. The L. This strain has been shown to be virulent in both in vitro and in vivo models of infection and was passaged no more than twice in the laboratory before use to avoid loss of virulence.

The murine macrophage-like cell line JA. The effects of PI3K inhibitors on L. Briefly, macrophages were plated at a density of 2. The next day, cells were suspended in fresh medium and treated with different concentrations of PI3K inhibitors 0. Cells were then co-incubated with L. Cells were washed three times with PBS and lysed in 1 ml of sterile water for 10 min at room temperature.

The percent invasion of bacterium-infected cells in the presence of inhibitors was calculated by dividing the number of CFUs per milliliter of these samples by the number of CFUs per milliliter of the control, multiplied by To correct for variation in the levels of uptake between experiments, entry was reported relative to L.

Wettstein and M. Davis, Stanford University. At 3 h post-transfection cells were suspended in serum containing medium. Western blot analysis was performed as previously described [51]. Cells were then infected with L. Sample buffer containing Western analysis was performed on the nitrocellulose membranes by adding the appropriate primary antibody followed by the use of horseradish peroxidase-conjugated anti-mouse Ig as the secondary antibody.

ECL reagents Amersham were used for detection of peroxidase activity. Immunoprecipitation was performed as previously described [52]. Cellular debris were removed by centrifugation at 13, rpm for 15 min and detergent lysates were pre-cleared with pre-immune serum, and protein concentration determined using the Bradford method [53].

Measurement of PI3K activity was carried out in a similar manner to that previously described [9]. Briefly, cell lysates were immunoprecipitated with anti-phosphotyrosine 4G10 and incubated overnight with constant mixing.

The immunoprecipitate was then suspended in 0. The standards were visualized with iodine vapor. Radiolabeled bands were located by autoradiography or phosphor imaging, and the PIP band was scraped into scintillation vials and the associated radioactivity determined using a scintillation counter.

We are grateful to Dr. The technical assistance of Russell J. Francis is also appreciated. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.

Abstract Background Legionella pneumophila , is an intracellular pathogen that causes Legionnaires' disease in humans, a potentially lethal pneumonia. Introduction Legionnaire's disease is a severe bacterial infection of the respiratory tract caused by Legionella pneumophila. Results PI3K inhibitors reduce non-opsonic phagocytosis of L. Download: PPT. Figure 1. PI3K inhibitors block L. Figure 2. Overexpression of PI3K mutant protein ablates L.

Figure 3. Figure 4. Virulent but not avirulent L. Arai T. FEMS Microbiol. Saukkonen K. Cabellos C. Burroughs M. Prasad S. Tuomanen E. Pasula R. Wisniowski P. Martin W.

Bermudez L. Parker A. Goodman J. Isberg R. Falkow S. Nature , — Voorhis D. Cell 50 , — Byrd S. Gelber R. Byrne B. Swanson M. Michael E.

McClellan M. Huebner H. Hostetter M. Wilson J. Tedder T. Fearon D. Schneider U. Schwenk H. Bornkamm G. Cancer 19 , — Balsam L. Liang T. Parkos C.

Diamond M. Garcia-Aguilar J. Bickford J. Corbi A. Springer T. Cell Biol. Shin J. Abraham S. Immunology , 2 — 7. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search.

Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Most nosocomial infections and hospital outbreaks have been linked to contaminated air or water supplies. Within water systems, L. Biofilm develop into mushroom-like structures with water channels that allow access to nutrients and oxygen within these bacterial communities. Humans are accidental dead-end hosts for L. While providing a niche for L. Replication within different protozoa enhances bacterial multiplication in human alveolar macrophages.

Growth within the protozoa also induces resistance to chemical disinfectant, biocides and antibiotics and induces phenotypic changes in L. The majority of people exposed to L. Susceptible patients to LD disease are likely to exhibit a defect in cell-mediated immunity rendering them less capable of limiting the early multiplication of L.

Cigaret smoking, chronic lung disease, and immunosuppression especially that caused by corticosteroid therapy have been consistently implicated as risk factors Carratala et al. Surgery is a major predisposing factor in nosocomial infection, with transplant recipients at highest risk. Other factors associated with the development of LD include, old age, cancer, and alcohol intake Marston et al. Legionella pneumophila multiplies in human monocytes Horwitz and Silverstein, ; Horwitz, b. The intracellular fate of L.

Attachments and binding of L. Following phagocytosis, L. The bacteria maintain interactions with ER-derived vesicles and replicate in a vacuole surrounded by a membrane that resembles rough ER Horwitz and Silverstein, ; Horwitz, b ; Figure 1.

The L. In contrast, formalin-killed L. Figure 1. Diagram depicting survival mechanisms employed by L. Upon entry into a human phagocyte, L. This pathogen inhibits host cell apoptosis by up regulating anti-apoptotic genes 4.

Alveolar macrophages are the most abundant potential effector cells in the lung and play a role in host defense against L. Lung tissue specimens from LD patients frequently contain large numbers of intracellular L. Furthermore, alveolar macrophages are a major feature of the alveolar secretions in lung biopsies from patients with LD Winn Jr.

Beside human monocytes and alveolar macrophages, investigators have employed a variety of cell lines that not only have similar characteristics to human phagocytes, but are also capable of supporting growth of this intracellular pathogen. For example, L. In HL cells, L. Similar to human phagocytes, the LCV is surrounded by ER-derived vesicles studded with ribosomes and therefore, HL cells are utilized as a model to study the interaction of human macrophages with the LD bacterium Marra et al.

Similarly, L. These cells can be differentiated to adherent, non-replicative cells with characteristics of tissue macrophages. Additionally, they express surface markers specific for cells of monocyte lineage and are able to differentiate between virulent and avirulent strains of L.

Their activation state is controlled experimentally and consequently can be standardized Rovera et al. THP-1 is another human monocytic cell line that matures into macrophage-like adherent cells following stimulation with phorbol esters or 1,dihydroxyvitamin D3.

Cirillo et al. Mono Mac 6 MM6 is an additional example of human monocytic cell line. MM6 was generated from peripheral blood of patient with monoblastic leukemia. Phenotypically and functionally, MM6 represents a more mature macrophage-like cell line. MM6 has been successfully utilized to study the molecular pathogenesis of L. Furthermore, non-professional phagocytic cell lines have been utilized to study L.

They are non-professional phagocytes and have characteristics of type II alveolar epithelial cells Maruta et al. It has been shown that L. Therefore, many human-derived cell lines have been used to characterize the molecular mechanisms associated with L. While some features are unique to the interactions between L. Host cells express a range of receptors that act as microbial sensors. These receptors sense microorganisms and transduce signals that activate immune responses.

Table 1. Differences between human and mice cells that affects Legionella permissiveness. Other means of sensing the presence of microbes or their factors in the host cytosol is mediated by NOD-like receptors NLRs , which in turn, initiate signaling cascades that mediate the production of inflammatory cytokines, recruitment of phagocytic cells and direction of the innate and the acquired immune responses.

There are 23 NLRs in human and 34 in mice Abdelaziz et al. One of the major NLRs contributing to the restriction of L. Human NLRC4 is highly homologous to the mouse Nlrc4 and is expressed in human macrophages but not in human lung epithelial cells Vinzing et al. Like most NLRs, mouse Nlrc4 assembles in a large multiprotein complex called the inflammasome leading to the activation of caspase Caspases are family of cysteine proteases that play a distinct role in apoptosis and inflammation Fink and Cookson, Caspases can be divided into two groups; one category of caspases is involved in apoptosis such as caspase-3 and -7 Martinon and Tschopp, Another category is involved in inflammation and is required for cytokine processing Creagh et al.

Wild-type mouse macrophages restrict L. However, human monocytes do not activate caspase-1 and -7 upon L. Although THP-1 cells were not shown to activate caspase-1 in response to L. This data implies the existence of uncharacterized caspaseindependent restriction mechanism of L. These proteins are called the neuronal apoptosis-inhibitory proteins NAIPs.

The LRR domain recognizes microbial products, while the BIR domains are essential for their interactions with caspase-3 and -7 Liston et al. The importance of Naip5 during L. Furthermore, depletion of the human orthologs of Naip5 results in increased replication of L. Studies with the human macrophage-like U and human epithelial cells A showed that L.

It is terminated quickly to prevent the autotoxic overproduction of inflammatory mediators. This activation is beneficial for the host since it triggers the innate immune response. Therefore, this phase seems to be beneficial for the pathogen. The host can employ several mechanisms to overcome intracellular infection.

Among these, is the elimination of infected cells by caspase-mediated apoptosis. Arising reports demonstrate new distinct roles for executioner caspases independent of cell death Li and Yuan, ; Walsh et al. The earlier studies with macrophages and epithelial cells demonstrated that high numbers of L. Remarkably, other studies demonstrated that the early caspase-3 activation by L. Notably, at physiological levels of infection, L. Activation of caspase-3 by low numbers of L. Rab-5 GTPase is involved in the maturation of the early endosome.

Therefore, according to the number of L. Several studies showed that in human cells, the LCV is decorated with polyubiquitinated proteins upon L. Ubiquitination is a reversible post-translational modification that controls the abundance of many critical regulatory proteins Craig and Tyers, ; Lomma et al. Degradation of intracellular proteins is mediated via means of the ubiquitin system and its specificity is determined at the level of substrate recognition by the E3 ubiquitin ligases, that catalyzes binding of the activated ubiquitin to the target protein for degradation Craig and Tyers, Additionally, the L.

F-box proteins are known components of E3 ubiquitin ligases named SCF complexes. Whereas ubiquitin-decoration was consistently observed for LpCVs, LdCVs found in the same cell remained ubiquitin-negative.

This result further confirmed that LdCVs are largely devoid of ubiquitin. Salmonella enterica serovar Typhimurium S. These results suggest that formation of LC3-positive LdCVs occurs via ubiquitin-independent mechanisms.

Nuclear and bacterial DNAs are stained with Hoechst , and ubiquitin is stained using anti-ubiquitin antibody clone FK2. Arrows describe locations of bacteria. B Quantification of ubiquitin association with vacuoles containing either L. Ubiquitin staining was performed using anti-ubiquitin FK2 antibody.

The blue Hoescht but not red bacteria are L. Typhimurium E. Both nuclear and bacterial DNAs are shown in blue Hoescht Data is average of two independent experiments performed in triplicate. Data is average from two independent experiments, each performed in triplicate.

See Figure S2 for detailed experimental procedure. We next examined involvement of signaling molecules and adaptor proteins that are commonly found to target pathogens for LC3-decoration. Once again we used S. Thus, it appears that decoration of L.

It is emerging that Atg proteins play roles in processes beyond classical autophagy Because LAP is not associated with ubiquitinated proteins 47 and LdCVs lack ubiquitin and common autophagy adaptors, we decided to determine the pathway required for LC3-association.

One of major differences between autophagy and LAP is the formation of a double-membrane-bound autophagosome during autophagy versus a single-membrane-bound LAP-phagosome. This technique allows confocal and electron- micrographs to be taken of the same cell The vast majority of LC3-positive bacteria resided in spacious vacuoles with a single membrane Fig.

Small intracellular vesicles were also observed within the lumen of these enlarged LC3-positive compartments Fig.

These may be artifacts or perhaps intraluminal vesicles LC3-negative vacuoles had attached vesicles Fig. We also examined a rare LC3-positive L. These results are consistent with the hypothesis in which a subset of LdCVs is targeted to LAP rather than conventional autophagy. Cells were fixed and fluorescent images obtained using a confocal microscope A.

Specimens were then further examined by transmission electron microscopy TEM rest of all panels. TEM images are of the same field as the fluorescent micrographs B.

A large black arrow shows nearby mitochondria with internal cristae visible and a white arrow shows vesicles attached to the LdCV. To avoid possible complications due to murine-specific restriction, we created a mutant L.

Our L. After differentiation into bone-marrow derived macrophages, cells were infected with mCherry expressing L. LC3 was detected by indirect immunofluorescence A , B and localization scored in three independent experiments C.

Data are the average of three independent experiments, each performed with triplicate wells. Conversely, conventional autophagy is Ulk1-dependent.

Under non-inducing conditions this protein is functionally inhibited by the mammalian-target of rapamycin mTOR , but after relief from repression the ULK complex activates formation of the initial isolation membrane The result on the transfectable cell-line enabled us to analyze the role of Ulk1 using siRNA knockdown.

Levels of LC3-association with L. To validate the effect of Ulk1 knockdown, in uninfected cells we compared the effect of the autophagy activator rapamycin on LC3-puncta formation following scrambled or Ulk1 siRNA treatment.

As expected, the addition of rapamycin to cells treated with scrambled siRNA led to increased LC3 puncta, but in Ulk1-knockdown cells LC3-puncta were absent despite rapamycin treatment Figure S4B bottom panels. Treatment with rapamycin has also been described to enhance levels of LC3 associated with pathogens, including S. Typhimurium However, treatment with this drug did not enhance LC3-decoration of L.

Rather levels were slightly decreased Figure S4C. Recently Rubicon was reported to be involved in LAP induction by fungal infection Further, unlike other pathogens that gain LC3 through multiple mechanisms, such as Salmonella , LAP appears to be the major mechanism targeting L. Classical autophagy is often associated with membrane damage 25 , 79 , but whether membrane damage is important for LAP-activation is not clear.

Galectin-3 has been used a marker for damaged phagosomes Thus, we examined the localization of galectin-3 on LdCVs as a measure of membrane integrity. As a control we again utilized S.

Typhimurium, as galectin-3 has been found on SCVs To further examine the relationship between LAP-targeting of LdCVs and membrane damage, we next examined LC3-positive phagosomes for the presence of galectin Typhimurium right , both organisms harbour plasmids for constitutive expression of mCherry red.

Typhimurium right or L. Bacteria are shown in blue Hoescht Galectin-3 was detected by indirect immunofluorescence using an anti-galectin-3 antibody Santa Cruz sc C Quantification of assay shown in panel B. Data are average of three independent experiments, each performed in triplicate. Typhimurium and L. Pharmacological inhibitors are shown in red. B Quantification of single phagosome analysis of LC3-recruitment to L.

In addition to other autophagic recognition mechanisms 38 , 39 , 40 , 41 , 42 , 43 , 82 , both S. Manipulation of DAG levels using chemical inhibitors was found to perturb LC3-association with both organisms. Similarly, we found that specific chemical interventions that perturb DAG production Fig. To elucidate whether this host response limits L.

Classically, when LC3-positive autophagosomes fuse with lysosomes, autophagolysosome will be decorated with both LC3 and LAMP-1 before degradation of phosphatidylethanolamine PE -conjugated LC3 found within the lumen of the membrane-bound compartment.

Hoescht blue was used to stain both nuclear and bacterial DNA. However, ubiquitin is not a common feature of the LC3-positive L. To examine the fate of bacteria within LC3-positive LdCVs we then performed live imaging experiments. Given that a number of pathogens interfere with host processes that acidify lysosomes to create a niche in which to replicate, we also confirmed that LdCVs become acidified by live imaging of RAW cells stably expressing GFP-LC3 infected with Hoescht-stained L.

Conversely, acidified L. Thus, acidification occurs post acquisition of LC3. This data further supports degradation of pathogenic L. Because LAP of L. We examined this hypothesis using a bacterial viability-based assay.

Taken together, these results demonstrated that LAP can limit intracellular survival of a virulent bacterial pathogen that resides within a membrane-bound compartment within host cells. Some L. This clearly indicates that the LAP avoidance of L. A THP-1 cells were infected with a L.

LC3 was stained with LC3 antibodies. Bacteria were visualized with DAPI-staining white arrows. C THP-1 cells were infected with a wild-type L.

On the other hand, L. When L. To lay a foundation for studies examining species-specific Legionella effector functions during intracellular infection, we set out to phenotypically compare L. However, we identified two points of difference between the organisms: L. Thus, the nature and kinetics of unproductive L.

Regarding the balance of LAP-targeting over autophagy, our data indicates that vacuolar integrity is a likely contributing factor. Based on the lack of galectin-3 or any other galectins we tested association with LdCVs Fig. Thus, vacuolar integrity of LdCVs may skews recognition away from membrane-damage responsive autophagy 25 , Alternatively, it remains possible that our membrane damage marker galectin-3 is somehow removed from LdCVs.

In this scenario, TLR- and DAG-signaling and delayed phagosome maturation would provide positive LAP-activation signals and ubiquitin-negative intact phagosomes would avoid activation of classical autophagy. This suggests that either L. Clarification of molecular mechanisms underlying L. A remaining question is the physiological importance of LAP for bacterial restriction in the context of other immune defense mechanisms.

Recently, LAP has been proposed to facilitate enhanced antigen presentation by delaying phagosome maturation It is tempting to speculate that in higher eukaryotes, LAP-mediated enhanced antigen presentation by macrophages infected with L. In contrast, LpCVs were reported to acquire PI3P right after infection in a model natural host Dictyostelium discoideum 87 , but soon PI4P becomes a dominant phosphoinositide species, which possibly involves the concerted actions of host PI 4 kinases, and host and bacterial phosphoinositide phosphatases 88 , 89 , 90 , Unless otherwise noted, all chemicals were purchased from Sigma.

Rapamycin was purchased from Santa Cruz sc Primary antibodies used include rabbit polyclonal antibodies to L. Primary antibodies used for immunofluorescence experiments are listed in figure legends. Bacterial strains used in this study are described in Table S1. Legionella strains were grown on charcoal-yeast extract plates or in aces-buffered yeast extract broth as already described Bacterial plasmids used in this study are listed in Table S2.

To induce constitutive expression of mCherry an internal region of the lacI repressor was disrupted. Strain Ld00 is a spontaneous streptomycin-resistant mutant of L. Chromosomal deletion mutants of L. Legionella strains constitutively expressing mCherry were created by introduction of pMMBmCherry by electroporation using the conditions described for L. Typhimurium expressing pMMBmCherry was also created by electroporation and selection on chloramphenicol resistance.

Wild-type MEF cells were provided by Dr. Miwa Sasai Osaka University, Japan. Masaaki Komatsu Niigata University, Japan. Murine BMDMs were obtained from mice as previously described , and used fresh or stored as described THP-1 cells were routinely passaged as non-adherent cells. The next day, infection experiments were performed. To create the RAW The sequence of the Ulk1 siRNA used was previously reported Infections with L.

The final OD of L. For infection of RAW For dual infection experiments of L. To propagate Salmonella for infections, overnight cultures were diluted to OD of 0. For growth curves in mammalian cells an MOI of 0. The primer pairs used to assess transcript levels are listed in Table S2.

Epifluorescence micrographs were taken using a TE Nikon inverted microscope and this microscope was used for all counting experiments.