Legionella pneumophila is the causative agent of human Legionnaires' disease. L. pneumophila has been shown to induce apoptosis of T-cells and this may be important pathologically and clinically. The present study has determined the molecular mechanisms underlying L. pneumophila-induced apoptosis, which were unclear. Wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T-cells. However, apoptosis was efficiently induced in T-cells only by wild-type L. pneumophila, and not flagellin-deficient or Dot/Icm-deficient Legionella. Induction of apoptosis involved activation of the initiator caspase 9 and effector caspase 3. Infection with L. pneumophila inhibited phosphorylation of Akt (also known as protein kinase B) and the Akt substrate GSK3β (glycogen synthase kinase 3β), and reduced the levels of β-catenin, a transcriptional activator regulated by GSK3β. It also caused the activation of the pro-apoptotic protein Bax and inhibited the expression of the anti-apoptotic protein XIAP (X-linked inhibitor of apoptosis) via inhibition of the Akt pathway. In conclusion, L. pneumophila induces mitochondria-mediated T-cell apoptosis through inhibition of the Akt/GSK3β signalling pathway.

INTRODUCTION

The Gram-negative bacterium Legionella pneumophila is the causative pathogen of the life-threatening pneumonia known as Legionnaires' disease, which affects both immunocompromised and immunocompetent subjects. This bacterium is a facultative intracellular pathogen of amoebae that live in natural and artificial aquatic environments. Human infections occur after inhalation of contaminated water aerosol droplets. Dependent on its type IV secretion system called Dot/Icm, L. pneumophila initiates the biogenesis of a specialized vacuole inside the host cell that is critical for Legionella replication [1]. This Legionella-containing vacuole avoids fusion with lysosomes and instead acquires membrane via vesicles from the endoplasmic reticulum [2]. The bacterial flagellum with its major component flagellin is also considered to be a virulence-associated factor [3].

Important findings on L. pneumophila pathogenesis were obtained by analysing infected protozoa or immune cells such as macrophages [4]. Previous studies have indicated that L. pneumophila also replicates in human alveolar epithelial cells [5,6]. Legionella replicates less efficiently within human T-cells than within macrophages [7], and little is known of the consequences of T-cell infection with Legionella.

A role for T-cells was proposed on the basis of two pieces of evidence. First, T-cells infiltrate infected lungs, and T-cell depletion renders mice more susceptible to infection with L. pneumophila [8]. Secondly, neutrophils seem to contribute by shaping the T-cell immune response whereby a Th1-response is initiated [9]. It therefore appears that a number of different human cell types are either directly infected or come into close contact with the bacteria during an infection with L. pneumophila.

Apoptosis levels increase during infections by a variety of pathogenic microbes, and are likely to affect the response of the host to these infectious agents. On a molecular level, apoptosis is triggered by widely varying stimuli, after which the cell activates a specialized intracellular pathway whose only known task is to kill and dispose of unwanted cells.

The induction of apoptosis by L. pneumophila has been demonstrated in the Jurkat T-cell line in vitro [10,11]. Induction of apoptosis by L. pneumophila requires contact between the bacteria and mammalian cells, but invasion does not appear to be necessary [1113]. The induction of apoptosis of Jurkat cells by L. pneumophila requires the mitochondrial pathway in the absence of death receptor signalling for caspase 3 activation [10,11].

The aim of the present study was to determine the molecular events involved in caspase-9-mediated apoptosis of T-cells induced by Legionella infection. The results strongly suggested that apoptosis is activated via inhibition of the Akt (also known as protein kinase B)/GSK3β (glycogen synthase kinase 3β) signalling pathway and is dependent on both the type IV secretion system and flagellin.

MATERIALS AND METHODS

Antibodies and reagents

Mouse monoclonal antibody against XIAP (X-linked inhibitor of apoptosis) was purchased from MBL. Mouse monoclonal antibody against caspase 9, rabbit monoclonal antibodies against cleaved caspase 3, survivin, Akt, phospho-Akt (Ser473), rictor [rapamycin-insensitive companion of mTOR (mammalian target of rapamycin)], PI3K (phosphoinositide 3-kinase) p85, PI3K p110β and phospho-TAK1 (transforming growth factor β-activated kinase 1) (Thr184 and Thr187), as well as rabbit polyclonal antibodies against cleaved PARP [poly(ADP-ribose) polymerase], Bcl-XL, Bax, phospho-Akt (Thr308), PDK1 (phosphoinositide-dependent kinase 1), phospho-PDK1 (Ser241), TAK1, MKK [MAPK (mitogen-activated protein kinase) kinase] 4, phospho-MKK4 (Thr261), JNK (c-Jun N-terminal kinase), phospho-JNK (Thr183 and Tyr185), phospho-c-Jun (Ser73) and phospho-GSK3β (Ser9) were purchased from Cell Signaling Technology. Mouse monoclonal antibodies against Bcl-2 and actin were purchased from NeoMarkers. Mouse monoclonal antibodies against active form-specific Bax, β-catenin and GSK3β were purchased from BD Transduction Laboratories. JNK inhibitor SP600125 was obtained from Sigma–Aldrich. Akt inhibitor IV and GSK3β inhibitor VIII were obtained from Calbiochem.

Cell culture

The Jurkat human T-cell line was maintained in RPMI 1640 medium containing 10% (v/v) fetal bovine serum, 50 units/ml penicillin G and 50 μg/ml streptomycin. PBMCs (peripheral blood mononuclear cells) were isolated from a healthy volunteer using Ficoll-Paque™ density gradient centrifugation (GE Healthcare Biosciences) and were stimulated with 10 μg/ml PHA (phytohaemagglutinin) for 72 h. On the day of the experiment, cells were re-fed with fresh antibiotic-free medium and co-cultured with L. pneumophila for various durations. Blood was obtained from a donor with informed consent and with ethical approval.

Bacterial strains

L. pneumophila serogroup 1 strain AA100jm [14] is a spontaneous streptomycin-resistant mutant of strain 130b, which is virulent in guinea-pigs, macrophages and amoebae. The avirulent dotO mutant was constructed by random transposon mutagenesis, as described previously [14]. This mutation causes severe defects in intracellular growth and evasion of the endocytic pathway [15]. The Corby flaA mutant derived from the wild-type Corby bacterium has defective flagellin [16]. L. pneumophila strains were grown at 35 °C in a humidified incubator either on buffered charcoal/yeast extract/agar medium supplemented with α-oxoglutarate or in buffered yeast extract broth supplemented with α-oxoglutarate. The flaA mutant was grown in an environment similar to those used for other strains, with the addition of 20 μg/ml kanamycin.

Infection conditions and intracellular growth kinetics experiments

Jurkat cells seeded in plates were inoculated with either AA100jm or dotO mutant and either Corby or flaA mutant at different MOIs (multiplicity of infections). At 2 h after infection, cells were centrifuged at 200 g for 5 min and the supernatant was discarded. Cells were washed three times with PBS and resuspended in fresh RPMI 1640 medium containing 100 μg/ml gentamycin for 2 h. The cells were again washed three times with PBS and then incubated further in fresh medium. The infected cells and supernatant in each well were harvested at the indicated times by washing the wells three times with sterilized distilled water. These bacterial suspensions were diluted in sterilized water and plated in known volumes on to buffered charcoal/yeast extract/agar medium supplemented with α-oxoglutarate agar. The numbers of CFUs (colony-forming units) in infected cells were counted at the indicated time points after infection.

Direct fluorescent antibody staining

Jurkat cells were infected with bacteria for 2 h, followed by washing three times with PBS, and 2 h of gentamycin treatment (100 μg/ml). The infected cells were cultured in fresh antibiotic-free RPMI 1640 medium for an additional 24 h. The cells were then harvested and fixed in 4% (w/v) paraformaldehyde for 15 min, washed with PBS and then permeabilized with PBS containing 0.1% saponin and 1% (w/v) BSA for 45 min at 37 °C. Permeabilized cells were washed and stained with fluorescein-conjugated mouse anti-L. pneumophila monoclonal antibody (PRO-LAB) for 45 min at 37 °C. Finally, the cells were washed and observed under a confocal laser-scanning microscope (Leica). Cells were stained with the nucleic acid dye DAPI (4′,6-diamidino-2-phenylindole).

RT (reverse transcription)–PCR

First-strand cDNA was synthesized using an RNA PCR kit (TaKaRa Bio). Thereafter, cDNA was amplified using 30 cycles for XIAP and β-catenin, and 28 cycles for β-actin. The specific primers used were as follows: XIAP sense, 5′-GTGCCACGCAGTCTACAAATTCTGG-3′, and antisense, 5′-CGTGCTTCATAATCTGCCATGGATGG-3′; β-catenin sense, 5′-TGATGGAGTTGGACATGGCCATGG-3′, and antisense, 5′-CAGACACCATCTGAGGAGAACGCA-3′; and β-actin sense, 5′-GTGGGGCGCCCCAGGCACCA-3′, and antisense, 5′-CTCCTTAATGTCACGCACGATTTC-3′. The product sizes were 502 bp for XIAP, 570 bp for β-catenin and 548 bp for β-actin. The thermocycling conditions for the targets were as follows: denaturing at 94 °C for 30 s for XIAP and β-actin, and for 60 s for β-catenin, annealing at 62 °C for 30 s for XIAP, at 60 °C for 40 s for β-catenin and for 30 s for β-actin, and extension at 72 °C for 90 s for XIAP and β-actin, and 50 s for β-catenin. The PCR products were fractionated on 2% agarose gels and visualized by ethidium bromide staining.

Western blot analysis

Cells lysates were prepared and equal amounts of protein (20 μg) were subjected to SDS/PAGE (8% gels), followed by transfer on to PVDF membranes and sequential probing with the specific antibodies. An ECL (enhanced chemiluminescence) kit (GE Healthcare) was used for detection of antibody reactions.

Cell viability and apoptosis assays

Cell viability was assessed using the cell proliferation reagent, WST-8 (water-soluble tetrazolium 8) (Wako Chemicals). Briefly, 105 cells/ml were incubated in a 96-well microculture plate in the absence or presence of various concentrations of Akt inhibitor IV. After 24 h of culture, WST-8 (5 μl) was added for the last 4 h of incubation, and absorbance was measured at 450 nm using an automated microplate reader. Measurement of mitochondrial dehydrogenase cleavage of WST-8 to formazan dye provided an indication of cell viability. Apoptotic events in cells were detected by staining with phycoerythrin-conjugated APO2.7 monoclonal antibody (Beckman Coulter) [17] and analysed by flow cytometry (Epics XL, Beckman Coulter).

In vitro measurement of caspase activity

Cell extracts were recovered using cell lysis buffer and assessed for caspase 3 and 9 activities using colorimetric probes (MBL). The colorimetric caspase assay kits are based on detection of the chromophore p-nitroanilide after cleavage from caspase-specific labelled substrates. Colorimetric readings were taken using an automated microplate reader at 400 nm.

RESULTS

Multiplication of L. pneumophila in human T-cells

To investigate the interaction between L. pneumophila and T-cells, we first examined intracellular growth of L. pneumophila strain AA100jm in Jurkat cells in 72-h continuous cultures. The CFU per well of AA100jm growing in Jurkat cells began to increase after 24 h and thereafter increased time-dependently (Figure 1A). However, the avirulent mutant strain lacking the expression of dotO, which encodes a protein essential for type IV secretion, showed no increase in CFU during the 72-h period (Figure 1A). In contrast, multiplication of the flaA mutant did not change in Jurkat cells compared with wild-type Corby-infected cells (Figure 1B). These observations suggest that L. pneumophila can replicate in human T-cells and that such replication involves the type IV secretion system.

Intracellular growth of L. pneumophila strains in Jurkat cells

Figure 1
Intracellular growth of L. pneumophila strains in Jurkat cells

Jurkat cells were infected with L. pneumophila strains, AA100jm and dotO mutant (MOI of 100) (A) or Corby and flaA mutant (MOI of 100) (B). The number of bacteria at 0 h was set as 1 and those at other points were presented as relative log10 CFU in cultures. Results are means±S.D. for triplicate cell cultures. (C) Representative confocal laser-scanning images of DAPI staining in L. pneumophila-infected Jurkat cells. Jurkat cells were infected with AA100jm or Corby (MOI of 100) for 24 h. Jurkat cells were labelled simultaneously with fluorescein-conjugated monoclonal antibody specific for L. pneumophila (green) or DAPI (blue). The arrows indicate apoptotic cells showing no intracellular bacterial replication. Magnification × 400.

Figure 1
Intracellular growth of L. pneumophila strains in Jurkat cells

Jurkat cells were infected with L. pneumophila strains, AA100jm and dotO mutant (MOI of 100) (A) or Corby and flaA mutant (MOI of 100) (B). The number of bacteria at 0 h was set as 1 and those at other points were presented as relative log10 CFU in cultures. Results are means±S.D. for triplicate cell cultures. (C) Representative confocal laser-scanning images of DAPI staining in L. pneumophila-infected Jurkat cells. Jurkat cells were infected with AA100jm or Corby (MOI of 100) for 24 h. Jurkat cells were labelled simultaneously with fluorescein-conjugated monoclonal antibody specific for L. pneumophila (green) or DAPI (blue). The arrows indicate apoptotic cells showing no intracellular bacterial replication. Magnification × 400.

Apoptosis of Jurkat cells induced by L. pneumophila

In these experiments, the infecting bacteria were labelled with an L. pneumophila-specific monoclonal antibody conjugated to fluorescein, and apoptosis was measured by DAPI staining (nuclear morphology). Staining of the infected Jurkat cells showed increased intracellular replication of AA100jm and Corby strains after 24 h in culture (Figure 1C). Whereas Jurkat cells showing detectable intracellular bacterial replication were apoptotic, some cells not showing obvious bacterial replication were also apoptotic (Figure 1C).

Infection of Jurkat cells with L. pneumophila strains AA100jm and Corby induced rapid apoptosis, as assessed by APO2.7 staining (Figures 2A and 2C), with the apoptosis rate correlating positively with the time and concentrations of L. pneumophila (Figure 2). In contrast with the strong induction of apoptosis seen in Jurkat cells, the avirulent dotO mutant and flaA-knockout mutant showed little apoptosis (Figures 2B and 2D). These results indicate that triggering of L. pneumophila-induced apoptosis in human T-cells is dependent on both type IV secretion and flagellin expression, and that intracellular replication is not required for L. pneumophila to induce apoptosis.

Induction of apoptosis by L. pneumophila in Jurkat cells

Figure 2
Induction of apoptosis by L. pneumophila in Jurkat cells

(A and C) Jurkat cells were either left untreated (control) or infected with the indicated strain of L. pneumophila at an MOI of 100 for various times. (B and D) Jurkat cells were infected with the indicated strain of L. pneumophila at different MOIs for 6 h. The cells were harvested, stained with the APO2.7 monoclonal antibody, and then analysed by flow cytometry. Results are means±S.D. for three independent experiments.

Figure 2
Induction of apoptosis by L. pneumophila in Jurkat cells

(A and C) Jurkat cells were either left untreated (control) or infected with the indicated strain of L. pneumophila at an MOI of 100 for various times. (B and D) Jurkat cells were infected with the indicated strain of L. pneumophila at different MOIs for 6 h. The cells were harvested, stained with the APO2.7 monoclonal antibody, and then analysed by flow cytometry. Results are means±S.D. for three independent experiments.

We next investigated the involvement of caspases in Legionella-associated apoptosis by measuring the cleavage of known caspase substrates by immunoblot analysis. As shown in Figure 3, the caspase-3-specific substrate PARP was cleaved into the characteristic 89 kDa fragments in Jurkat cells after infection with L. pneumophila strains Corby and AA100jm. In addition, the initiator caspase 9 and the executioner caspase 3 were processed in Jurkat cells after infection with Corby and AA100jm in a time- and concentration-dependent manner. In contrast, PARP, caspase 3 and caspase 9 were not processed in Jurkat cells after infection with flaA and dotO mutants.

Processing of caspase 3, caspase 9 and the caspase-3-specific substrate PARP assessed by immunoblot analysis

Figure 3
Processing of caspase 3, caspase 9 and the caspase-3-specific substrate PARP assessed by immunoblot analysis

Jurkat cells were infected with L. pneumophila strains Corby or flaA mutant (A) and either AA100jm or dotO mutant (B) at an MOI of 100 for various times. Jurkat cells were also infected with Corby (A) or AA100jm (B) at different MOIs for 6 h. Cellular proteins were resolved by SDS/PAGE, and caspase activity was detected by cleavage of PARP, caspase 3 and caspase 9 using immunoblot analysis. Arrows indicate the cleaved form of caspase 9. Actin was used as a protein-loading control. Blots are representative of three independent experiments with similar results.

Figure 3
Processing of caspase 3, caspase 9 and the caspase-3-specific substrate PARP assessed by immunoblot analysis

Jurkat cells were infected with L. pneumophila strains Corby or flaA mutant (A) and either AA100jm or dotO mutant (B) at an MOI of 100 for various times. Jurkat cells were also infected with Corby (A) or AA100jm (B) at different MOIs for 6 h. Cellular proteins were resolved by SDS/PAGE, and caspase activity was detected by cleavage of PARP, caspase 3 and caspase 9 using immunoblot analysis. Arrows indicate the cleaved form of caspase 9. Actin was used as a protein-loading control. Blots are representative of three independent experiments with similar results.

The immunoblotting allowed us to examine the processing of caspases, but did not indicate whether the cleavage products were enzymatically active. Therefore caspase 3 and 9 activities were determined by cleavage of DEVD (Asp-Glu-Val-Asp)-p-nitroanilide and LEHD (Leu-Glu-His-Asp)-p-nitroanilide respectively in colorimetric assays. Again, Jurkat cells infected with Corby and AA100jm strains for 4 h at an MOI of 100 showed markedly increased caspase 3 and 9 activation, but infection with flaA and dotO mutants had no such effect (Figure 4).

Determination of caspase activity

Figure 4
Determination of caspase activity

Jurkat cells were left untreated (control) or were infected with Corby or flaA mutant (A) and either AA100jm or dotO mutant (B) at an MOI of 100. After 4 h, cell lysates were prepared and incubated with the labelled caspase substrates, and caspase activity was measured using an automated microplate reader. Caspase activity is expressed relative to untreated cells, which were assigned a value of 1. Results are means±S.D. for three independent experiments.

Figure 4
Determination of caspase activity

Jurkat cells were left untreated (control) or were infected with Corby or flaA mutant (A) and either AA100jm or dotO mutant (B) at an MOI of 100. After 4 h, cell lysates were prepared and incubated with the labelled caspase substrates, and caspase activity was measured using an automated microplate reader. Caspase activity is expressed relative to untreated cells, which were assigned a value of 1. Results are means±S.D. for three independent experiments.

Expression of apoptosis-related proteins during L. pneumophila induced apoptosis

Next, we tested for an alteration in the spectrum of apoptosis-related proteins. A time course study showed that infection of Jurkat cells with Corby strain at an MOI of 100 for various intervals did not discernibly modify the expressions of survivin, Bcl-2 and Bcl-XL (Figure 5A). A dose-dependence study also showed that infection of Jurkat cells with various concentrations of Corby did not appreciably modify the expression of the same three proteins (Figure 5A). In contrast, immunoblot analysis revealed a time- and concentration-dependent decrease in expression of anti-apoptotic protein XIAP after Corby infection (Figure 5). To investigate whether Legionella infection affects the mRNA expression of XIAP, Jurkat cells infected with Corby strain were subjected to RT–PCR. The results showed no inhibition of XIAP mRNA expression (Figure 5B).

Expression of various apoptosis-related proteins with activated Bax protein and XIAP mRNA in Jurkat cells

Figure 5
Expression of various apoptosis-related proteins with activated Bax protein and XIAP mRNA in Jurkat cells

Jurkat cells were infected with Corby strain at an MOI of 100 for various times (A and B, left-hand panels). Jurkat cells were also infected with Corby at different MOIs for 6 h (A, righthand panels) or 4 h (B, right-hand panels). (A) L. pneumophila-infected Jurkat cells were lysed and the various apoptosis-related proteins were detected by immunoblotting using the antibodies indicated. Actin was used as a protein-loading control. (B) L. pneumophila-infected Jurkat cells were collected, and XIAP mRNA was detected by RT–PCR. Lane M, size markers. Blots are representative of three independent experiments with similar results.

Figure 5
Expression of various apoptosis-related proteins with activated Bax protein and XIAP mRNA in Jurkat cells

Jurkat cells were infected with Corby strain at an MOI of 100 for various times (A and B, left-hand panels). Jurkat cells were also infected with Corby at different MOIs for 6 h (A, righthand panels) or 4 h (B, right-hand panels). (A) L. pneumophila-infected Jurkat cells were lysed and the various apoptosis-related proteins were detected by immunoblotting using the antibodies indicated. Actin was used as a protein-loading control. (B) L. pneumophila-infected Jurkat cells were collected, and XIAP mRNA was detected by RT–PCR. Lane M, size markers. Blots are representative of three independent experiments with similar results.

The pro-apoptotic Bcl-2 family proteins, such as Bax, also play a central role in regulating apoptosis, thus the involvement of Bax activation in apoptosis was tested in our system. Bax-mediated cell death occurs via well-controlled steps, including a conformational change that facilitates the dimerization and translocation of Bax to the mitochondrial outer membrane [18,19]. A previous study distinguished the conformational states of Bax using conformation-specific anti-Bax antibodies [20]. In the present study, immunoblot analysis using an antibody specific for the active form of Bax (Clone 3) indicated that infection with the L. pneumophila strain Corby induced a change in Bax conformation in a time- and concentration-dependent manner. The experiments also confirmed that total Bax expression is not affected in Corby-infected cells (Figure 5A).

L. pneumophila inhibits the Akt/GSK3β signalling pathway

The serine/threonine kinase Akt is a primary mediator of PI3K downstream effects by co-ordinating a variety of intracellular signals and controlling cell responses to extrinsic stimuli to regulate cell proliferation and survival. Akt inhibits apoptosis through multiple pathways [21], which induce direct phosphorylation and inactivation of pro-apoptotic proteins, increased expression of anti-apoptotic proteins and down-regulation of pro-apoptotic proteins [21]. Thus L. pneumophila-induced Akt down-regulation could induce cell death. To test this possibility, we evaluated the activity of this kinase by detecting its phosphorylation forms. Jurkat cells were infected with either the parental strain Corby or flaA mutant strain and either the parental strain AA100jm or dotO mutant strain for various times. Akt activation was then determined in cell lysates by immunoblot analysis using phospho-specific antibodies. Interestingly, both parental strains, but not the flaA or dotO mutant, induced a rapid decrease in Akt activation after 1 h of infection before apoptosis induction (Figure 6A). In addition, the L. pneumophila parental strain Corby, but not the flaA mutant, inhibited Akt kinase activity in Jurkat cells by down-regulating the phosphorylation of GSK3β, one of the major downstream targets of Akt, and thus maintaining the levels of activated GSK3β. Similar results were seen in Jurkat cells following infection with the parental strain AA100jm or dotO mutant strain.

Time- and dose-dependent inhibition of the PI3K/Akt pathway by the infection of L. pneumophila

Figure 6
Time- and dose-dependent inhibition of the PI3K/Akt pathway by the infection of L. pneumophila

Jurkat cells were infected with either Corby or flaA mutant and either AA100jm or dotO mutant at an MOI of 100 for various times. Jurkat cells were also infected with Corby strain at different MOIs for 6 h (A, right-hand panels) or 4 h (B, right-hand panels). (A) Cell lysates were prepared and subjected to immunoblotting with the antibodies indicated. Actin was used as a protein-loading control. (B) L. pneumophila-infected Jurkat cells were collected, and β-catenin mRNA was detected by RT–PCR. Lane M, size markers. Blots are representative of three independent experiments with similar results.

Figure 6
Time- and dose-dependent inhibition of the PI3K/Akt pathway by the infection of L. pneumophila

Jurkat cells were infected with either Corby or flaA mutant and either AA100jm or dotO mutant at an MOI of 100 for various times. Jurkat cells were also infected with Corby strain at different MOIs for 6 h (A, right-hand panels) or 4 h (B, right-hand panels). (A) Cell lysates were prepared and subjected to immunoblotting with the antibodies indicated. Actin was used as a protein-loading control. (B) L. pneumophila-infected Jurkat cells were collected, and β-catenin mRNA was detected by RT–PCR. Lane M, size markers. Blots are representative of three independent experiments with similar results.

A key downstream target of GSK3β is the proto-oncogene β-catenin. Normally, β-catenin is sequestered in the cytoplasm as part of a complex with axin and adenomatous polyposis coli. One pool of GSK3β is also associated with the axin complex and, when active, mediates the serine/threonine phosphorylation of β-catenin. This provides a recognition signal for β-TrCP (β-transducin repeat-containing protein), the receptor component of the Skp1/Cullin/F-box protein–β-TrCP E3 ubiquitin ligase complex, and thus targets the β-catenin complex for proteasomal degradation [22]. Increased phosphorylation and concomitant inhibition of GSK3β would therefore be predicted to prevent β-catenin phosphorylation, resulting in decreased ubiquitin-mediated proteolysis and increased levels of signalling-competent β-catenin. Corby and AA100jm strains, but not the flaA and dotO mutants, inhibited GSK3β phosphorylation, thereby maintaining GSK3β in its active form. Levels of β-catenin in Jurkat cells infected with Corby and AA100jm strains were therefore low (Figure 6A), and these effects of Corby were dose-dependent. RT–PCR of Jurkat cells infected with Corby and flaA mutant revealed that Legionella infection did not inhibit the expression of β-catenin mRNA (Figure 6B).

Next, we investigated the effect of Legionella on the major upstream event of Akt, PI3K (Figure 6A). The results revealed no significant alteration in the expression of PI3K, the regulatory subunit p85 or the catalytic subunit p110β in Jurkat cells. Akt is phosphorylated at Thr308 in the kinase activation loop mediated by PDK1, although phosphorylation of Akt within the C-terminal tail at Ser473 is required for maximal activation [23]. The mTOR complex 2, which consists of mTOR, mLST8 (mammalian lethal with SEC13 protein 8), mSin1 (mammalian stress-activated-protein-kinase-interacting protein 1) and rictor, is responsible for phosphorylating Ser473 [21]. Corby and AA100jm strains, but not flaA and dotO mutants, inhibited the expression of rictor as well as phosphorylated and non-phosphorylated PDK1 after 2–4 h of infection.

To characterize the inhibition of the Akt pathway and subsequent processing of caspases by L. pneumophila in human lymphocytes, Akt inactivation and subsequent processing of caspases 3 and 9 in PBMCs stimulated with PHA was examined. The Corby strain inhibited the phosphorylation of Akt and the expression of XIAP and β-catenin, and activated Bax in a time-dependent manner, which is similar to the observations for Jurkat cells (Figure 7). Furthermore, Corby strain also induced an increase in cleavage/activation of caspase 3 and 9, as well as PARP degradation (Figure 7). These observations suggest that Legionella induces inhibition of the Akt pathway and subsequent induction of apoptosis in human lymphocytes as well as in a T-cell line.

Time-dependent inhibition of the Akt pathway and subsequent processing of caspases 3 and 9 by the infection of L. pneumophila in PBMCs

Figure 7
Time-dependent inhibition of the Akt pathway and subsequent processing of caspases 3 and 9 by the infection of L. pneumophila in PBMCs

PBMCs stimulated with PHA (10 μg/ml) for 72 h were infected with Corby at an MOI of 50 for various times. Cell lysates were prepared and subjected to immunoblotting with the antibodies indicated. Arrows indicate the cleaved form of caspase 9. Actin was used as a protein-loading control. Blots are representative of three independent experiments with similar results.

Figure 7
Time-dependent inhibition of the Akt pathway and subsequent processing of caspases 3 and 9 by the infection of L. pneumophila in PBMCs

PBMCs stimulated with PHA (10 μg/ml) for 72 h were infected with Corby at an MOI of 50 for various times. Cell lysates were prepared and subjected to immunoblotting with the antibodies indicated. Arrows indicate the cleaved form of caspase 9. Actin was used as a protein-loading control. Blots are representative of three independent experiments with similar results.

Treatment of Jurkat cells with an Akt inhibitor mimics Legionella-mediated inhibition of the Akt pathway

Inhibitor experiments were then conducted to evaluate whether Akt down-regulation is responsible for L. pneumophila-induced cell death. Akt inhibitor IV inhibits Akt phosphorylation/activation by targeting the ATP-binding site of the kinase upstream of Akt, but downstream of PI3K. Cell death was measured by WST-8 assay. As shown in Figure 8(A), Akt inhibitor IV decreased cell viability in a dose-dependent manner, suggesting that Akt down-regulation plays a role in L. pneumophila-induced cell death. At the molecular level, treatment with Akt inhibitor IV also induced a marked increase in cleavage/activation of caspases 3 and 9, as well as PARP degradation.

Treatment with Akt inhibitor IV induces apoptosis of Jurkat cells

Figure 8
Treatment with Akt inhibitor IV induces apoptosis of Jurkat cells

(A) Effects of Akt inhibitor IV on cell viability in Jurkat cells treated with various concentrations of Akt inhibitor IV for 24 h. Viability was measured by WST-8 assay. Results are means±S.D. for three independent experiments. (B) Protein lysates were prepared and subjected to immunoblot analysis using the antibodies indicated. Arrow indicates the cleaved form of caspase 9. Actin was used as a protein-loading control. Blot is representative of three independent experiments with similar results.

Figure 8
Treatment with Akt inhibitor IV induces apoptosis of Jurkat cells

(A) Effects of Akt inhibitor IV on cell viability in Jurkat cells treated with various concentrations of Akt inhibitor IV for 24 h. Viability was measured by WST-8 assay. Results are means±S.D. for three independent experiments. (B) Protein lysates were prepared and subjected to immunoblot analysis using the antibodies indicated. Arrow indicates the cleaved form of caspase 9. Actin was used as a protein-loading control. Blot is representative of three independent experiments with similar results.

We also examined the direct role of Akt signalling in the regulation of XIAP and β-catenin expression and Bax activation in Jurkat cells using Akt inhibitor IV. Treatment of Jurkat cells with this inhibitor inhibited the Akt pathway in a dose-dependent manner (Figure 8B). There was inhibition of phosphorylated Akt and GSK3β in the absence of any inhibition of non-phosphorylated proteins. In addition, treatment of Jurkat cells with Akt inhibitor IV inhibited XIAP and β-catenin expression, and activated Bax in a dose-dependent manner. These findings therefore suggest that Legionella-induced inhibition of the Akt pathway is involved in inhibiting XIAP and β-catenin expression, and Bax activation. Taken together, these data suggest that the major pathway by which Legionella induces apoptosis is down-regulation of Akt phosphorylation.

Treatment of Jurkat cells with a GSK3β inhibitor induced caspase-9-mediated apoptosis

We used a selective GSK3β inhibitor VIII to determine whether this downstream target of Akt was responsible for L. pneumophila-induced cell death. Jurkat cells treated with GSK3β inhibitor VIII showed inhibition of phosphorylated GSK3β in the absence of inhibition of non-phosphorylated protein. To determine the ability of GSK3β inhibitor VIII to inhibit GSK3β activity in Jurkat cells, we assessed β-catenin expression. Treatment with GSK3β inhibitor VIII induced an increase in expression of β-catenin (Figure 9A), thus demonstrating its ability to inhibit GSK3β activity. RT–PCR of Jurkat cells treated with GSK3β inhibitor VIII revealed that this inhibitor did not increase the expression of β-catenin mRNA (Figure 9B).

Treatment with GSK3β inhibitor VIII induced apoptosis of Jurkat cells

Figure 9
Treatment with GSK3β inhibitor VIII induced apoptosis of Jurkat cells

Jurkat cells were treated with various concentrations of GSK3β inhibitor VIII for 24 h. (A) Protein lysates were prepared and subjected to immunoblot analysis using the antibodies indicated. Arrows indicate the cleaved form of caspase 9. Actin was used as a protein-loading control. The vertical line indicates the grouping of images from different parts of the same gel. (B) GSK3β inhibitor VIII-treated Jurkat cells were collected, and β-catenin mRNA was detected by RT–PCR. Blots are representative of three independent experiments with similar results. Lane M, size markers.

Figure 9
Treatment with GSK3β inhibitor VIII induced apoptosis of Jurkat cells

Jurkat cells were treated with various concentrations of GSK3β inhibitor VIII for 24 h. (A) Protein lysates were prepared and subjected to immunoblot analysis using the antibodies indicated. Arrows indicate the cleaved form of caspase 9. Actin was used as a protein-loading control. The vertical line indicates the grouping of images from different parts of the same gel. (B) GSK3β inhibitor VIII-treated Jurkat cells were collected, and β-catenin mRNA was detected by RT–PCR. Blots are representative of three independent experiments with similar results. Lane M, size markers.

Phosphorylated GSK3β attenuates cell death [24,25]. We therefore examined whether treatment with GSK3β inhibitor VIII was able to induce apoptosis. As shown in Figure 9(A), the GSK3β inhibitor VIII induced an increase in cleavage/activation of caspases 3 and 9, as well as PARP degradation. In contrast, it did not appreciably modify XIAP expression and Bax activation.

Lack of involvement of the JNK pathway in Legionella-induced apoptosis

Finally, we examined the effects of infection with Legionella on the expression of survival- and stress-related signalling pathways. Corby strain, but not the flaA mutant, induced a rapid phosphorylation of JNK1/JNK2 at Thr183 and Tyr185, detectable at 1 h, but had no significant effects on total JNK1/JNK2 levels (Figure 10A). The upstream kinases TAK1 and MKK4, and the downstream transcription factor c-Jun, were also phosphorylated at the same time points, indicating that JNK signalling was activated by Legionella infection. Because of its reported involvement in apoptosis [26], the role of JNK in Legionella-induced apoptosis was also studied using Jurkat cells. The JNK inhibitor SP600125 blocked Legionella-induced c-Jun phosphorylation, but did not inhibit Legionella-induced cleavage/activation of caspases 3 and 9 or PARP degradation (Figure 10B). SP600125 did not affect the multiplication of L. pneumophila in Jurkat cells (results not shown). These findings thus indicate that Legionella-induced JNK activation is not a major pathway that mediates Legionella-associated cytotoxicity.

Activation of the JNK pathway by L. pneumophila has no effects on apoptosis

Figure 10
Activation of the JNK pathway by L. pneumophila has no effects on apoptosis

(A) Jurkat cells were infected with the indicated strain of L. pneumophila at an MOI of 100 for various times. Whole-cell lysates were prepared, and the same amount of protein was analysed by immunoblotting using the antibodies indicated. (B) The JNK inhibitor failed to affect cleavage of PARP, caspase 3 or caspase 9. Jurkat cells were incubated with JNK inhibitor (SP600125) at 20 μM for 1 h, and then infected with Corby strain at an MOI of 100 for various times. Cell lysates were prepared and analysed by immunoblotting with the antibodies indicated. Arrows indicate the cleaved form of caspase 9. Actin was used as a protein-loading control. Blots are representative of three independent experiments with similar results.

Figure 10
Activation of the JNK pathway by L. pneumophila has no effects on apoptosis

(A) Jurkat cells were infected with the indicated strain of L. pneumophila at an MOI of 100 for various times. Whole-cell lysates were prepared, and the same amount of protein was analysed by immunoblotting using the antibodies indicated. (B) The JNK inhibitor failed to affect cleavage of PARP, caspase 3 or caspase 9. Jurkat cells were incubated with JNK inhibitor (SP600125) at 20 μM for 1 h, and then infected with Corby strain at an MOI of 100 for various times. Cell lysates were prepared and analysed by immunoblotting with the antibodies indicated. Arrows indicate the cleaved form of caspase 9. Actin was used as a protein-loading control. Blots are representative of three independent experiments with similar results.

DISCUSSION

Modulation of apoptosis is a widely applied strategy among extra- and intra-cellular bacteria. However, the exact mechanisms by which bacteria affect the endogenous cellular suicide programme remain undefined. Two cell death pathways have been found to restrict L. pneumophila replication in macrophages and DCs (dendritic cells). The first pathway involves activation of Naip5 (NLR family, apoptosis inhibitory protein 5) by a process that requires L. pneumophila flagellin in macrophages and DCs [2731], resulting in the activation of caspase 1 [2731], which is a critical mediator of pyroptosis. A second cell death pathway involving Bax and Bak regulation of caspase 3 activation efficiently restricted L. pneumophila replication in DCs [27]. Generally, infected epithelial cells and lymphocytes undergo apoptosis, whereas macrophages and DCs die primarily by pyroptosis, but also, in some cases, by apoptosis [32]. Caspase 1 was not activated in Jurkat cells upon infection by L. pneumophila in the present study (results not shown). In contrast, L. pneumophila activated caspase 9 and caspase 3 in Jurkat cells, and a functional Dot/Icm system and flagellin were essential to transmit the signal that activates caspase 9.

Several pathogens have been reported to trigger apoptosis. This is accomplished through different mechanisms, including the production of bacterial toxins or expression of virulence factors that interact directly with key components of the death machinery [33]. Alternatively, pathogens modulate apoptosis by interfering with NF-κB (nuclear factor κB) and/or MAPK cell survival pathways, which are activated downstream of pattern recognition receptors and are responsible for producing inflammatory mediators as well as survival proteins such as Bcl-XL and c-IAP2 (cellular inhibitor of apoptosis 2). For example, Salmonella AvrA and Yersinia YopJ proteins inhibit NF-κB activation [3436], whereas Bacillus anthracis's protease lethal factor, a component of its lethal toxin, targets MKK6 to dampen MAPK signalling [37]. It is presumed that, through inhibition of these pathways, apoptosis is engaged by default.

The present study investigated the regulation and function of Akt in L. pneumophila-induced T-cell death. First, we demonstrated that phosphorylation of Akt was decreased in L. pneumophila-infected Jurkat cells and PHA-stimulated PBMCs. Secondly, Akt inhibitor IV inhibited Akt phosphorylation and induced apoptosis. These results suggest that L. pneumophila infection decreases Akt activity, and that such down-regulated Akt signalling promotes T-cell death in infected cells. Although Akt has been associated with cell death signalling, this is the first report of Akt as a pivotal kinase in L. pneumophila-induced T-cell death.

We and others [10,11] have found caspase 9 processing in infected Jurkat cells. The only known mechanism for caspase 9 activation is the cytochrome c-induced recruitment to oligomerized Apaf-1 (apoptotic protease-activating factor 1) [38]. Cytochrome c release during apoptosis is achieved by Bax activation, which involves its oligomerization and integration into the mitochondrial membrane, and formation of non-selective channels/lipidic pores [39]. In addition, XIAP is a potent inhibitor of apoptosis via direct binding to caspases 3, 7 and 9, rendering them inactive [4042]. Akt can also be an upstream regulator of XIAP and Bax, which both possess an Akt phosphorylation site [4345]. The conformational change and translocation of Bax may be regulated by the PI3K/Akt pathway, with active PI3K capable of maintaining a cytosolic Bax distribution [43,44]. Furthermore, β-catenin can inhibit Bax by increasing Akt expression and activation [46], whereas phosphorylation at Ser87 stabilizes XIAP, protecting it from degradation [45]. However, whether Akt is involved in regulating XIAP and Bax in Jurkat cells remains to be clarified. In the present study, inhibition of XIAP expression and Bax activation were induced by inhibition of Akt, indicating that Akt signalling occurs upstream of XIAP and Bax in human T-cells.

As a downstream effector of Akt, GSK3β is phosphorylated at Ser9 by Akt, and the phosphorylated GSK3β inhibits the opening of mPTPs (mitochondrial permeability transition pores), thought to be a critical factor mediating cytochrome c release from the mitochondria into the cytosol [24]. The phosphorylated GSK3β interacts with adenine nucleotide translocase, one of the mPTP components, reducing its interaction with cyclophilin D and thus inhibiting the opening of mPTPs [25]. In the present study, inhibition of GSK3β induced cleavage/activation of caspase 9. L. pneumophila may also induce T-cell death through a caspase-9-dependent mechanism that involves the down-regulation of phosphorylated GSK3β via Akt.

We have also shown that inhibiting PDK1 and rictor expression suppresses the Akt pathway in Jurkat cells infected with L. pneumophila. Infection of Jurkat cells with Legionella for 1 h showed a marked reduction of Akt activation, but the expression levels of PDK1 and rictor were reduced at 2–4 h post-infection. Thus these data suggest the existence of a mechanism by which L. pneumophila inhibits Akt phosphorylation that is independent of the regulation of PDK1 and rictor expression. Akt phosphorylation is tightly regulated, representing a balance between kinase-activating and phosphatase-inactivating events. Several protein phosphatases, including the dual-action phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10), which transforms PtdIns(3,4,5)P3 into PtdIns(4,5)P2, canonical PP (protein phosphatase) 1 and PP2A, as well as newly identified Akt phosphatases, were recently reported to bind and dephosphorylate Akt in an agonist-dependent manner [47]. Because PTEN is deficient in Jurkat cells [48], activation of PPs such as PP2A may contribute to Legionella-induced dephosphorylation of Akt. It was found that the endogenous ceramide generated during infection of Leishmania donovani, an intracellular protozoan parasite, in macrophages induces PP2A activation and leads to the dephosphorylation of Akt [49]. Dephosphorylation of Akt may be mediated by ceramide in L. pneumophila-infected T-cells.

Figure 11 illustrates the probable apoptotic pathways induced by infection with L. pneumophila, with the most important candidates including the inhibition of Akt phosphorylation, Bax activation, cytochrome c release from mitochondria, caspase 9 activation and caspase 3 activation. Inhibiting the expression of downstream effectors of Akt such as XIAP and β-catenin may also activate caspase 9, whereas inhibiting phosphorylated GSK3β expression might also stimulate cytochrome c release. Although JNK was activated immediately after L. pneumophila infection, it had no effect on apoptosis.

Schematic diagram of the L. pneumophila-induced apoptosis pathway

L. pneumophila-induced apoptosis in Jurkat cells does not require intracellular bacterial replication. However, the bacterial factor(s) of L. pneumophila that are responsible for induction of apoptosis have yet to be determined. In the present study, flagellin-deficient or Dot/Icm-deficient Legionella neither inactivated Akt nor induced apoptosis, suggesting that contact-mediated export of a bacterial factor such as flagellin after attachment of the bacteria to host cells early in infection and establishment of Legionella-containing vacuole later during the infection may play a role in inducing apoptosis. Further studies are therefore needed to elucidate these apoptosis-inducing bacterial factors.

Abbreviations

     
  • β-TrCP

    β-transducin repeat-containing protein

  •  
  • CFU

    colony-forming unit

  •  
  • DAPI

    4′,6-diamidino-2-phenylindole

  •  
  • DC

    dendritic cell

  •  
  • GSK3β

    glycogen synthase kinase 3β

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MKK

    MAPK kinase

  •  
  • MOI

    multiplicity of infection

  •  
  • mPTP

    mitochondrial permeability transition pore

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • NF-κB

    nuclear factor κB

  •  
  • PARP

    poly(ADP-ribose) polymerase

  •  
  • PBMC

    peripheral blood mononuclear cell

  •  
  • PDK1

    phosphoinositide-dependent kinase 1

  •  
  • PHA

    phytohaemagglutinin

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PP

    protein phosphatase

  •  
  • PTEN

    phosphatase and tensin homologue deleted on chromosome 10

  •  
  • rictor

    rapamycin-insensitive companion of mTOR

  •  
  • RT

    reverse transcription

  •  
  • TAK1

    transforming growth factor β-activated kinase 1

  •  
  • WST-8

    water-soluble tetrazolium 8

  •  
  • XIAP

    X-linked inhibitor of apoptosis

AUTHOR CONTRIBUTION

Reika Takamatsu designed and performed the research, analysed data and wrote the manuscript. Eriko Takeshima, Chie Ishikawa, Kei Yamamoto and Hiromitsu Teruya contributed to the experimental concept and provided technical support. Klaus Heuner, Futoshi Higa and Jiro Fujita provided bacterial strains. Naoki Mori established the research plan, supervised the project and helped to draft the manuscript.

FUNDING

This work was supported by Grants-in-Aid for Scientific Research (C) [grant number 21591211] from the Japan Society for the Promotion of Science; Scientific Research on Priority Areas [grant number 20012044] from the Ministry of Education, Culture, Sports, Science and Technology; and the Takeda Science Foundation.

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