Loss of the TLR adaptor Myd88 reveals TLR-independent MTOR signaling elicited by Dot/Icm+ Legionella

Previously, we reported that TLR-dependent activation of MTOR is suppressed when macrophages become infected with Legionella encoding a functional Dot/Icm system [51]. Thus, the role of the TLR adaptor Myd88, which is required for LPS-induced MTOR signaling [46,48], was investigated in the context of Legionella infection. To eliminate rapid pyroptosis induced by Legionella flagellin, flagellin-deletion mutants (ΔflaA) were used in this study [52]. Also, the cells were serum-starved for 10hrs prior to and for the duration of the infections to avoid growth factor-induced MTOR activation. There was no detectible decrease in cell viability caused by serum withdrawal under these conditions.

To assess mTOR signaling, Myd88^-/- BMMs were infected with a pathogenic Dot/Icm+ strain (ΔflaA) or an avirulent Dot/Icm- strain (ΔdotA) and phosphorylation of the ribosomal S6 protein (rS6p) was measured by immunoblot analysis to assess MTOR activity (Fig 1a). Phosphorylation of rS6p at serine residues 235 and 236 by the S6 kinase 1 (S6K1) was used as readout for mTOR signaling because S6K1 is directly activated by TORC1 [53]. As expected, a ΔdotA strain did not induce rS6p phosphorylation in Myd88^-/- BMMs because TLR signaling is defective; surprisingly, the Dot/Icm+ strain triggered a robust response (Fig 1a). These data are in sharp contrast with the attenuated rS6p phosphorylation elicited by the ΔflaA strain as compared to the ΔdotA strain in BMMs derived from C57BL/6 mice (Fig 1b). To analyze MTOR activity specifically in infected cells we quantitated phospho-rS6p fluorescence intensity using single-cell immunofluorescence analysis. These studies confirmed that robust phospho-rS6p was elicited by a ΔflaA strain in Myd88^-/- BMMs and by a ΔdotA strain in C57BL/6 BMMs (Fig 1c and 1d and S1 Fig). Taken together, these data demonstrate that MTOR suppression in response to virulent Legionella requires MyD88 signaling, and that in the absence of MyD88 signaling virulent Legionella augment MTOR activation.

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Fig 1. Analysis of rS6p phosphorylation in bone marrow-derived macrophages.

Immunoblot analysis of cell lysates from Myd88^-/- (a) or C57BL/6 (b) BMMs that were serum-starved and infected with Legionella (MOI = 20) for 4 hrs as indicated under serum-free conditions. Quantified band intensities are normalized to uninfected conditions (UN) and listed below each blot. Single-cell analysis of rS6p phosphorylation in serum-starved BMMs infected with either ΔflaA (c) of ΔdotA (d). Graph shows means and 95% confidence intervals of phospho-rS6p fluorescent intensity signal for at least 100 infected cells for each condition. * p<0.05, ** p<0.005 (one-way ANOVA). (a-d) A representative of three biological replicates is shown for each experiment.

https://doi.org/10.1371/journal.ppat.1006088.g001

To exclude the possibility of an MTOR-independent phospho-rS6p response to the ΔflaA strain in Myd88^-/- BMMs, MTOR function was inhibited in infected cells through various approaches (Fig 2a). Addition of the MTOR inhibitors PP242 or rapamycin to synchronized Legionella infections of Myd88^-/- BMMs reduced phosphorylation of S6K1 and its substrate rS6p (Fig 2b). Single cells microscopy confirmed that phospho-rS6p signal was diminished in infected macrophages when MTOR inhibitors were added (Fig 2c and 2d). Amino acid starvation (HBSS treatment) or PI3K inhibition also ablated Legionella-induced phospho-rS6p in Myd88^-/- BMMs (Fig 2b–2d). Under these infection conditions, MTOR inhibition was initiated after the infections were synchronized and therefore did not interfere with bacterial uptake. LCV maturation and bacterial viability were also unaffected by MTOR inhibition as evidenced by the ubiquitin accumulation on vacuoles containing bacteria with normal morphology (Fig 2d). Thus, loss of phospho-rS6p under MTOR inhibiting conditions was not a result of bacterial killing by the host cell or perturbations in LCV biogenesis. Together, these data indicate that phosphorylation of rS6p induced by Legionella in Myd88^-/- BMMs requires PI3K/MTOR signaling and is an indicator of MTOR activity in the cell.

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Fig 2. Legionella-induced phosphorylation of rS6p requires MTOR and PI3K activity.

(a) Simplified schematics of the PI3K/MTOR signaling axis indicating the inhibitors used in this study. PI3K is inhibited by LY294002. Rapamycin, PP242 and Torin2 act on MTOR and Hanks' Balanced Salt Solution (HBSS) blocks MTOR by starving cells for amino acids. (b-d) Analyses of serum-starved Myd88^-/- BMMs unstimulated or infected with ΔflaA (MOI = 20) for 5hrs under serum-free conditions. Inhibitors—Rapamycin (200nM), PP242 (5μM), LY294002 (10μM)—or HBSS were added at the time of infection synchronization at 60 min post infection. (b) Immunoblot analysis of S6K1 and rS6p phosphorylation from cell lysates showing quantified band intensities normalized to uninfected conditions (UN). (c) Single cell immunofluorescence analysis of phospho-rS6 positive (MFI>300) Myd88^-/-macrophages exposed to ΔflaA (MOI = 20). Graphed are the means and standard deviations (s.d) of technical triplicates for the two distinct groups within the cell population—infected (LCV present) and uninfected (LCV absent) for each condition. At least 100 cells were analyzed for each condition. ** p<0.005 (one-way ANOVA) (d) Immunofluorescense micrographs of representative infected cells from each condition stained with anti-L. pneumophila (L.p), anti-p-rS6p (S235/236), anti-ubiquitinated proteins (FK2) antibodies and Hoechst 33342. Arrowheads indicate Legionella-containing vacuoles, Bar = 5μm. (b-d) A representative of three biological replicates is shown for each experiment.

https://doi.org/10.1371/journal.ppat.1006088.g002

MTOR activation by Legionella is a Dot/Icm effector-driven process that is independent of intracellular niche biogenesis

To gain insight into the mechanism by which Legionella infections activate MTOR in Myd88^-/- BMMs, the kinetics of MTOR signaling was analyzed using fluorescence microscopy by quantification of phospho-rS6p positive cells at different times post-infection (Fig 3a and 3b). In synchronized infections, at 2hrs post-infection over 40% of infected cells stained positive for phospho-rS6p as compared to only 15% of neighboring uninfected cells (Fig 3b). In a population of unstimulated serum-starved BMMs only 17% of the cells were phospho-rS6p positive (Fig 3b). Over time, the number of phospho-rS6p positive infected cells further increased to over 80% at 5hrs and 8hrs post-infection. In contrast, the number of phospho-rS6p positive uninfected cells remained at basal level. Thus, a significant number of infected cells activated MTOR prior to initiation of bacterial replication and sustained high MTOR activity throughout the Legionella intracellular lifecycle.

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Fig 3. Legionella-induced MTOR activation is a Dot/Icm effector-driven process independent of intracellular niche biogenesis.

(a-h) Show results from synchronized infections (MOI = 20) under serum-free conditions of serum-starved Myd88^-/- BMMs treated as indicated. (a) Immonofluorescent micrograph of ΔflaA-infected (**) or neighboring uninfected (*) cells at 6 hrs post-infection stained with anti-L. pneumophila (L.p), anti-p-rS6p (S235/236) antibodies and Hoechst 33342. Arrowheads indicate Legionella-containing vacuoles, Bar = 10μm. (b-c and g) Single cell immunofluorescence analysis of phospho-rS6 positive (MFI>300) BMMs exposed to various Legionella strains for the indicated times (b) or for 8hrs (c and g). Means ± s.d of technical triplicates for the two distinct groups within the cell population—infected (LCV present) and uninfected (LCV absent) for each condition are shown. (d) Micrographs of cells infected with the indicated strains for 8hrs and stained with anti-L. pneumophila (L.p), anti-p-rS6p (S235/236), anti-ubiquitinated proteins (FK2) antibodies and Hoechst 33342. Arrowheads indicate intracellular bacteria, Bar = 5μm. (e) Quantitation of p-rS6p positive cells (MFI>300) in synchronized infections with a thyA ΔflaA strain in the presence (+thy) or absence (-thy) of thymidine for the indicated time periods. Means ± s.d of technical triplicates are shown. (f-g) Synchronized macrophages infections with different Legionella species are shown. (f) Representative micrographs of cells infected with L. pneumophila or L. dumoffii for 8hrs (MOI = 20) and stained as in (d), which are quantitated in (g). (h-i) Cells were treated with cytochalasin D (5μM) or vehicle (DMSO) for 15 min prior to and for the duration of infections (3hrs) with the indicated bacterial strains. (i) Quantitation of p-rS6p positive cells (MFI>300) in contact with bacteria (ΔflaA and ΔdotA) or uninfected cells (UNi). Means ± s.d of technical triplicates are shown. (h) Representative micrograph of macrophages treated with cytochalasin D (5μM) and infected with ΔflaA and stained as in (d). Arrowhead indicates a bacterium in contact with the macrophage, Bar = 1μm. At least 50 cells (i) or 100 cells (b, c, e and g) were analyzed for each condition. A representative of two (f-h) or three (a-g) biological replicates is shown for each experiment. (b, c, e, g and i) n.s—not significant, ** p<0.005 (unpaired T-test).

https://doi.org/10.1371/journal.ppat.1006088.g003

Because a functional Dot/Icm apparatus is essential for MTOR activation by Legionella (Fig 1a), a strain lacking the chaperone IcmS, which loads effectors into the Dot/Icm apparatus [54,55], was used to determine whether the apparatus or secretion of effectors through the apparatus is important for MTOR activation. The ΔicmS bacteria assemble a functional Dot/Icm apparatus but exhibits a severe defect in translocation of most effectors and therefore fails to establish an intracellular niche that supports bacterial replication in macrophages [56]. Both ΔdotA and ΔicmS strains failed to induce MTOR activity (Fig 3c and 3d) indicating that signaling is unlikely a host response to the Dot/Icm apparatus but rather efficient translocation of effectors is necessary for MTOR activation. Such a requirement indicates that Dot/Icm effectors mediate MTOR activation directly or indirectly through LCV biogenesis.

A potential indirect trigger for MTOR activation could be the need of the infected cell to offset a metabolic demand placed by a rapidly expanding membrane-bound organelle that contains replicating bacteria. Such a mechanism would require bacterial replication; however, a thymidine auxotroph strain activated MTOR irrespective of thymidine presence, indicating that organelle expansion is not the trigger (Fig 3e). Next, MTOR activation by other Legionella species was examined. L. dumoffii, L. longbeachae and L. jordanis have conserved LCV biogenesis programs but considerable diversity in their Dot/Icm effector repertoires. All the tested Legionella species established LCVs that supported bacterial replication, but only L. pneumophila infections activated MTOR (Fig 3f and 3g). In co-infections of Myd88^-/- BMMs, L. dumoffii failed to repress MTOR signaling induced by L. pneumophila indicating that L. dumoffii neither induces nor suppresses MTOR (S2 Fig). Thus, MTOR is activated by a L. pneumophila-specific process and is independent of LCV expansion.

Next, the actin polymerization inhibitor cytochalasin D was used to investigate the possibility that LCV establishment could lead to MTOR induction. Treatment with cytochalasin D blocks LCV formation because phagocytosis is inhibited and the bacteria are retained on the extracellular interface of the plasma membrane. However, cell-surface associated bacteria still inject Dot/Icm effectors in the host cytosol [57]. Macrophages in contact with ΔflaA bacteria, but not ΔdotA bacteria, triggered MTOR irrespective of cytochalasin D treatment (Fig 3h and 3i). Therefore, MTOR induction by L. pneumophila is independent of LCV biogenesis altogether and strongly support the existence of Dot/Icm effector(s) that promote PI3K-dependent MTOR signaling. The pentuple deletion mutant lacking 71 Dot/Icm effector genes [58] still activated MTOR (S2c Fig), therefore a forward genetic screen would be required to identify the Legionella MTOR regulator(s).

Blocking Legionella-induced MTOR activation triggers host cell death

Next, the impact of sustained MTOR activation on Legionella intracellular life cycle was determined in macrophage infections under serum-free conditions that were allowed to progress for less than a single infection cycle (~16hrs) in the presence or absence of MTOR inhibitors (Fig 4a). Inhibitors were added at 4 hrs post infection and timed to coincide with the start of bacterial replication to eliminate potential interference with bacterial uptake and trafficking. A morphologically distinct population of infected cells characterized by condensed nuclei—decreased nuclear volume (from 284 to 141μm³) and increased fluorescence when stained with the DNA dye Hoechst 33342—was detected clearly under all infection conditions (Fig 4b and S3 Fig). Aberrant condensed nuclear morphology is a prototypical hallmark used to identify dying cell [59].

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Fig 4. Loss of MTOR function triggers a host cell death response that requires bacterial replication.

(a) Experimental scheme for the results shown in (b-e and g-j). (b-j) BMMs were serum-starved and infected under serum-free conditions either at MOI = 20 (b-e and g-j) or MOI = 10 (f). (b) Micrographs showing representative of live and dead macrophages infected with Legionella for 12 hrs and stained with anti-L. pneumophila (L.p), anti-ubiquitinated proteins (FK2) antibodies and Hoechst 33342. Arrowheads indicate LCVs, Bar = 5μm. (*) marks the condensed nucleus of the dead cells. The mean nuclear volumes ± s.d of at least 100 live and 100 dead cells are graphed. (c-d) Quantitation of infected and neighboring uninfected Myd88^-/- (c-d), C57BL/6 (d) and Mtor^-/- (d) macrophages with condensed nuclei after infections with ΔflaA (c-d) or ΔdotA (d). Means ± s.d of technical replicates of dead cell as percentage of total cells in each condition are shown. (e) Quantitation of infected Myd88^-/- BMMs with condensed nuclei after infection with L. dumoffii and treatment with inhibitors or vehicle as indicated. (f-g) Kinetics of the cell death response in C57BL/6 BMMs under serum starvation conditions infected as indicated. Quantitation of infected cells with condensed nuclei (f) and LDH released in the culture supernatants (g) are shown. (h-i) Analyses of ubiquitin recruitment (h) and LCV size (i) in live and dead Myd88^-/- BMMs infected with ΔflaA and treated with PP242. (j) Cell death in infected Myd88^-/- BMMs with thyA ΔflaA strain treated with vehicle (DMSO) or PP242 in the presence or absence of thymidine. (c, e, h-j) Rapamycin (250nM), PP242 (2.5μM), LY294002 (10μM), Torin2 (300nM). (c-j) Means ± s.d of technical triplicates for each condition are shown. At least 50 cells (e, h-j) or 200 cells (c-d) were analyzed for each condition. A representative of two (e-j) or three (c-d) biological replicates is shown for each experiment. (b-j) n.s—not significant, ** p<0.005 (unpaired T-test).

https://doi.org/10.1371/journal.ppat.1006088.g004

The number of dead Myd88^-/- BMMs infected with Legionella, identified by their aberrant nuclear morphology, significantly increased from 8% to over 40% when PI3K (LY294002) or MTOR (PP242, Torin2) were inhibited (Fig 4c). This cell-death phenotype was not a result of inhibitor-induced cell toxicity because neighboring uninfected cells were not affected. Interestingly, rapamycin, which inhibits the phosphorylation of a subset of TORC1 substrates, such as rS6p [45], did not induce cell death in infected or uninfected cells (Fig 4c). Taken together, these data demonstrate that suppression of the Legionella-induced PI3K/MTOR signaling triggers a cell death response in infected Myd88^-/- BMMs through a rapamycin-insensitive mechanism.

If Legionella-induced MTOR activity interferes with a host cell death response, it would be predicted that in the presence of TLR-signaling the host Akt/MTOR-ubiquitination pathway will phenocopy the effect of the pharmacological inhibitors in Myd88^-/- BMMs infections. Indeed, a significant increase in the number of dead Legionella-infected cells was observed at 12 hrs post-infection in macrophages derived from C57BL/6 mice (Fig 4d). Similar results were obtained when macrophages derived from mice with MTOR ablation in the myeloid compartment were infected (Fig 4d). Importantly, under serum-free conditions, neighboring uninfected bystander macrophages from infections with either the ΔflaA or the ΔdotA strain did not undergo cell death. Also, Myd88^-/- macrophages, which sustain MTOR activity, remained alive when infected with the ΔflaA strain. Thus, the host Akt/MTOR-ubiquitination pathway as well as pharmacological or genetic ablation of MTOR function triggered a cell-death response specifically in macrophages infected with virulent Legionella encoding functionally intact Dot/Icm system. However, the Dot/Icm apparatus itself did not trigger this cell death response because infections with L. dumoffii, which encodes a homologous Dot/Icm system, did not induce host cell death in Myd88^-/- BMMs even when MTOR inhibitors were present (Fig 4e).

The host cell death in Legionella-infected macrophages triggered by MTOR suppression requires bacterial replication

Multiple bacteria within ubiquitin-positive LCVs were observed frequently within macrophages undergoing MTOR suppression-dependent cell death (similar to Fig 4b); therefore, the kinetics of the cell death response was investigated. Under MTOR suppressing conditions, BMMs with aberrant nuclear morphology infected with ΔflaA bacteria increased gradually starting at 6hrs after bacterial replication is initiated and peaked at 10-14hrs post-infection (Fig 4f). The release in the culture supernatants of the cytosolic enzyme lactate dehydrogenase (LDH) by dying macrophages also increased with similar kinetics (Fig 4g). In contrast, infections with the ΔsdhA mutant strain, which triggers cell death prior to bacterial replication [18], produced distinct kinetics that peaked at 6hrs post-infection (Fig 4f and 4g). Moreover, majority of LCVs within dead macrophages in MTOR-suppressing conditions were mature ubiquitin-positive LCVs that have supported bacterial replication (i.e. contained more than 4 bacteria) (Fig 4h and 4i). Thus, bacterial replication is likely initiated prior to host cell death. To determine whether bacterial replication is required for initiation of the MTOR-regulated host cell death, Myd88^-/- BMMs were infected with a thymidine auxotroph ΔflaA strain. In those infections, host cell death was detected only in the presence of PP242 and thymidine, demonstrating that bacterial replication together with MTOR suppression are required for induction of host cell death (Fig 4j).

Serum-derived lipids rescue the host cell death phenotype brought by MTOR inhibition

The detection of a Legionella-induced host cell death response in C57BL/6 BMMs under serum starvation is intriguing because it is well established that ΔflaA L. pneumophila replicates in these macrophages without initiating a cell death response in the presence of fetal bovine serum (FBS) [52]. Therefore, the possibility that FBS could account for the distinct phenotypes was investigated by infecting macrophages for 12hrs (less than a single replication cycle) with Legionella in the presence/absence of FBS and MTOR inhibitors (Fig 5a). Under these conditions, serum supplementation effectively blocked host cell death in Legionella-infected Myd88^-/- BMMs treated with the MTOR inhibitor PP242 (Fig 5b). Because, FBS did not interfere with PP242 activity (Fig 5c), these data indicate that FBS compensated for the loss of MTOR function. Supplementation with FBS also blocked the Legionella-induced cell death in BMMs derived from C57BL/6 and from Mtor^-/- mice (Fig 5d) providing further evidence that FBS compensates for the reduction in MTOR function brought by multiple MTOR-suppression mechanisms.

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Fig 5. Legionella-dependent cell death triggered by MTOR suppression is blocked by lipids supplementation.

(a) Experimental scheme for the results shown in (b-d, f). (b-c) Myd88^-/- BMMs with condensed nuclei (b) or positive for phospho-rS6p (MFI>300) (c) produced by infections with ΔflaA (MOI = 20) and the indicated treatments. Infected and neighboring uninfected cells were quantified for each category. (d) Infected and neighboring uninfected BMMs with condensed nuclei after infections with ΔflaA or ΔdotA (MOI = 20) in the presence/absence of FBS. (e) Kinetics of LDH release by C57BL/6 BMMs infected as indicated (MOI = 10) in the presence of FBS or dFBS. (f) Infected Myd88^-/- BMMs with condensed nuclei produced by ΔflaA infection (MOI = 20) and the indicated treatments. (b-f) PP242 (2.5μM), LY294002 (10μM), FBS (10%), dFBS (10%), human LDL (10mg/ml). (b-f) Means ± s.d of technical triplicates for each condition are shown. At least 50 cells (c,f) or 100 cells (b,d) were analyzed for each condition. A representative of two (e-f) or three (b-d) biological replicates is shown for each experiment. (b-f) n.s—not significant, ** p<0.005 (unpaired T-test).

https://doi.org/10.1371/journal.ppat.1006088.g005

Although MTOR is a global metabolic regulator, the potential loss of MTOR-dependent lipogenesis was investigated first because: (1) FBS can compensate for loss of MTOR-driven lipogenesis and when FBS is available cells default to lipid uptake over de novo synthesis to reduce energy expenditure [29]; (2) serum is a major source for cellular lipids and lipid-precursors in vertebrates [29]; (3) rapamycin failed to trigger cell death in Legionella infections and MTOR-driven lipogenesis is insensitive to rapamycin in some cell types [36,60,61]. The master lipogenic transcription factors SREBP1 and SREBP2 promote lipogenesis downstream of MTOR by increasing gene expression of lipid uptake regulators and biosynthesis enzymes [33,34,62]. Consistent with induction of a lipogenic transcriptional response, a significant increase in expression of Srebf1 as well as several of its target genes was detected in Myd88^-/- BMMs infected with ΔflaA but not ΔdotA Legionella (S4 Fig). Therefore, a functional Dot/Icm apparatus is critical for initiation of MTOR signaling and the induction of a pro-lipogenic transcriptional program in Legionella-infected macrophages (S4 Fig).

If serum-derived lipids prevented host cell death in infected cells by compensating for the loss of MTOR-driven lipogenesis, the phenotype should be sensitive to depletion of serum lipids. Thus, the cell death response brought by MTOR-inhibition was investigated in the presence of delipidated FBS (dFBS) (cholesterol 2mg/dL, triglycerides 59mg/dL) or complete FBS (cholesterol 34mg/dL, triglycerides 61mg/dL) (Fig 5). Delipidated FBS failed to block the Legionella-induced host cell death brought by PP242-treatment in Myd88^-/- macrophages (Fig 5b) or by the host MTOR-suppression pathway (Fig 5d and 5e), demonstrating that FBS-derived lipids compensate for the lost MTOR function in infected macrophages. Furthermore, addition of purified Low-density lipoprotein (LDL) particles—the main cholesterol transport carriers in serum—was sufficient to block cell death in Legionella-infected Myd88^-/- BMMs triggered by LY294002 (PI3K inhibitor) or PP242 (MTOR inhibitor) (Fig 5f).

Next, the Legionella-induced MTOR-regulated cell death response and its dependency on lipids starvation was investigated in human macrophages. Legionella-infected phorbol-ester differentiated U937 macrophage cells were treated with PP242 in the presence of FBS or dFBS. Similar to the results with mouse BMMs, the number of dead infected U937 increased only when PP242 was added under serum-free conditions or in the presence of delipidated FBS; addition of PP242 and lipids-containing FBS blocked the PP242-induced cell death (S5 Fig). Collectively, these data link MTOR-driven lipogenesis with the Legionella-induced cells death response and indicate that MTOR activation or presence of exogenous lipids prevents a premature death of infected macrophages.

MTOR maintains LCV integrity through activation of de novo lipogenesis

Due to the unique features of the Legionella-induced cell death: (1) requirement for bacterial replication and (2) loss of host lipogenesis, it was investigated whether insufficient supply of lipids could increase membrane tension and lead to LCV rupture during expansion. Elegant infection studies with a ΔsdhA strain, which resides in a rupture-prone LCV, have demonstrated that loss of LCV integrity releases bacterial molecules in the host cytosol leading to flagellin-independent cell death of infected macrophages [18].

To discriminate between intact and leaky LCVs, the vacuole ability to prevent cytosolic anti-Legionella antibody from accessing the LCV lumen was investigated. First, the plasma membrane was selectively permeabilized with the amphipathic glycoside saponin and cells were stained with anti-Legionella chicken antibody as well as anti-ubiquitin antibody. Next, all membrane compartments were permeabilized with methanol and cells were stained with anti-Legionella rabbit antibody (Fig 6a). Thus, bacteria within intact LCVs are detected only after methanol incubation and remain single positive, whereas bacteria in leaky LCVs are accessible after saponin treatment and become double positive (Fig 6b and 6c). Detection of the ubiquitinated proteins that accumulate on the cytosolic interface of the LCV membrane during infection was used to control for saponin activity. In Myd88^-/- BMMs infected with the ΔflaA strain for 7hrs, 16% of the LCVs were leaky as indicated by detection of the vacuole-residing bacteria after saponin treatment (Fig 6c and 6d). The number of leaky LCVs increased to 51% following PP242 treatment indicating that MTOR suppression compromises LCV integrity. Although leaky LCVs were detected in BMMs with normal nuclear morphology (Fig 6c), all LCVs harbored by cells with aberrant nuclear morphology were leaky (Fig 6e and 6f). These data indicate that MTOR suppression destabilizes LCVs.

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Fig 6. MTOR inhibition destabilizes LCVs.

(a) Schematic for detection of leaky LCVs by selective plasma membrane permeabilization of infected cells. Single-positive bacteria reside in stable LCVs; double-positive bacteria reside in disrupted LCVs (b-f) Serum-starved Myd88^-/- macrophages infected with ΔflaA L. pneumophila (MOI = 20) for 7hrs. PP242 was added at the time of synchronization– 60min p.i. (b-c) Micrographs of representative stable (b) or leaky (c) LCVs are shown. Cells were stained as indicated in (a). Arrowheads indicate LCVs. Bar = 5μm (d) Quantitation of destabilized LCVs in cells treated as indicated. (e) Micrographs of representative destabilized LCVs (double-positive) in BMM with aberrant (A) nuclear morphology and stable LCVs (single-positive) in a neighboring cell with normal (N) nuclear morphology Bar = 5μm (f) Quantitation of destabilized LCVs in BMMs with normal or aberrant nuclear morphology. BMMs treated as indicated. (b-f) PP242 (2.5μM). (d-f) Means ± s.d of technical triplicates for each condition are shown. At least 100 LCVs were analyzed for each condition. (b-f) A representative of three biological replicates is shown. (d and f) ** p<0.005 (unpaired T-test).

https://doi.org/10.1371/journal.ppat.1006088.g006

To measure LCV rupture the recruitment of the cytosolic lectin Galectin-3 was assessed. Galectin-3 binds to lumenally occluded glycoprotein epitopes and accumulates onto membrane-bound organelles only when their epitopes become exposed upon membrane disruption [18,63–65]. Unstable LCVs, such as the ones harboring the ΔsdhA mutant, become Galectin-3 positive upon rupture (Fig 7a and 7b) [18]. Galactin-3 preferentially accumulates at the sites of membrane rupture [18,66] thus produces a wide-variety of non-uniform straining pattern onto the LCVs (S6a and S6b Fig). Accumulation of Galectin-3 onto LCVs within infected Myd88^-/- BMMs following MTOR inhibition was investigated. At 12 hrs post infection ~13% of the LCVs in vehicle-control treated cell were decorated with Galactin-3 (Fig 7c and 7d) and treatment with PP242 increased the number of Galactin-3+ LCVs to over 36% (Fig 7d and 7e). However, this number is likely an underestimate because Galactin3 staining was undetectable in dead macrophages and PP242-treatment increased the number of infected cells with condensed nuclei under serum-free conditions. MTOR inhibition did not cause global destabilization of pathogen-containing organelles because the number of Galactin-3+ LCVs harboring L. dumoffii did not increase after PP242 treatment (S6c and S6d Fig). In addition, MTOR inhibitors did not affect L. pneumophila replication and viability in vitro (S7 Fig), thus LCV rupture is unlikely a consequence of an interrupted LCV biogenesis program brought by an inhibitor-induced toxic effect on the residing bacteria.

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Fig 7. Serum lipids, SREPB1/2 and MTOR regulate LCV stability.

Serum-starved Myd88^-/- (a-f) or C57BL/6 (g) BMMs or were infected by the indicated strains (MOI = 20) in synchronized infections for 12 hrs (a-f) or for the indicated time periods (g) in the absence (a-g) or presence (f-g) of FBS. (a-b) Galectin 3 accumulation onto LCVs harboring ΔsdhA ΔflaA. (a) projection micrograph of a representative infected cell with the inset showing the LCV (b) Kinetic analysis of Galectin 3+ LCVs harboring ΔsdhA ΔflaA. (c,e) Representative projection micrographs of Galectin 3 positive (c,e) or negative (c) LCVs harboring ΔflaA after treatments with inhibitors (e) or vehicle alone (c). The insets show all individual planes of the projection image. Quantitation of Galectin 3+ LCVs after the indicated treatments in the absence (d and f) or presence (f) of FBS. (g) Kinetics of emergence of Galectin 3 positive LCV under serum starvation or replete conditions. (c-g) PP242 (2.5μM), fatostatin (4μM), FBS (10%). (b,d,f and g) Means ± s.d of technical triplicates for each condition are shown. At least 100 LCVs were analyzed for each condition. A representative of two (b, f-g) or three (a, c-e) biological replicates is shown for each experiment. (b, d, f and g) n.s—not significant, ** p<0.005 (unpaired T-test) (a, c and e) Cells were stained with anti-galectin3, anti-Legionella antibodies and Hoechst 33342. Arrowheads indicate the LCVs. Bar = 5μm.

https://doi.org/10.1371/journal.ppat.1006088.g007

Inhibition of lipogenesis at a step downstream of MTOR with the SREPB1/2 peptidomimetic inhibitor fatostatin, which specifically prevents the release of SREBP1/2 from the ER [67], destabilized LCV similar to PP242 treatment (Fig 7d and 7e). Importantly, addition of FBS restored LCV integrity even in the presence of PP242 and fatostatin as evidenced by the decreased number of Galactin-3+ LCVs (Fig 7f). Under MTOR suppressing condition, Galactin-3+ LCVs increased after bacterial replication was initiated and the kinetics of Galactin-3 acquisition by LCVs harboring ΔflaA bacteria mirrored the kinetics of host cell death response (Figs 7g vs. 4f and 4g). These data demonstrate that LCV membrane integrity during expansion is maintained through a continuous supply of lipids, either through uptake or de novo synthesis. Thus, interference with MTOR-driven lipogenesis results in LCV rupture during expansion and initiation of host cell death likely through the release of bacterial products in the host cytosol. Unlike MTOR suppression-induced instability, rupture of LCVs harboring ΔsdhA bacteria preceded bacterial replication and was not rescued by FBS supplementation indicating that MTOR and SdhA stabilize the LCVs via distinct mechanisms (Fig 7b).

Lipids supply determines the housing capacity of the Legionella intracellular niche

Because the lipids supply/demand ratio is important for LCV homeostasis, its effects on the housing capacity of the pathogen-containing vacuole were investigated. In Legionella-infected cells, the LCV membrane contours the residing bacteria [15] and therefore the volume of the bacterial mass in the vacuole is proportional to the volume of the LCV membrane. In previous studies, housing capacity of a LCV was determined by manually counting the residing bacteria; however, the accuracy and reliability of this approach deteriorates for vacuoles containing more than 12 bacteria, which forces investigators to group large LCVs in a single category. To overcome this obstacle, an unbiased computer-based 3D image analysis was utilized to enumerate accurately bacteria within vacuoles independent of LCV size and topology. This analysis measures the volume of the bacterial mass within a LCV from reconstructed Z-stacks (0.3μm planes distance) of infected cells. To image the whole cell, cell height determined the Z-stack depth (4.5μm to 13μm). Next, the number of bacteria that can fit within the volume of the bacterial mass was extrapolated mathematically based on an algorithm derived from volume measurements of LCVs in which the bacteria can be counted manually (S8a–S8c Fig). When the algorithm was back tested it exhibited excellent correlation between counted and calculated number of bacteria per cell (R² = 0.9287, p = 2.11X10^-38) (S8d Fig). This automated volume-to-bacteria conversion approach facilitates reliable enumeration of bacteria even in large LCVs containing over a hundred bacteria (S8e and S8f Fig and S1 Video). Most 3D-imaging systems capable of volume measurements of binary masks should be able to enumerate intracellular bacteria using this methodology.

The relationship between lipid supply and LCV size was investigated in C57BL/6 BMMs, which suppress MTOR when infected by Legionella. In this system, exogenous FBS restores the lipids supply lost due to MTOR suppression. Thus, LCVs sizes were measured at the end of a single Legionella intracellular replication cycle (~16 hrs) in the presence/absence of FBS (similar to Fig 5a). In synchronized infections, the LCV sizes formed a Gaussian distribution with a peak at the 20–59 bacteria per LCV size bin irrespective of FBS supplementation (Fig 8a). However, FBS addition increased the population of large LCVs containing more than 60 bacteria (Fig 8a). Comparison between the fifty Iargest LCVs detected in each condition, revealed that FBS increased the average number of bacteria per LCV (from 58 to 102) and the number of LCVs containing more than 60 bacteria (from 17 to 46) (Fig 8b and 8c). FBS did not increase bacterial growth in axenic cultures (Fig 8d). Moreover, during the early stages of intracellular replication the number of bacteria per LCV remained similar in the presence or absence of FBS (Fig 8e) indicating that the LCV size increase observed in the later stages of infection was not a result of accelerated or prematurely initiated bacterial replication but likely due to LCV membrane biogenesis.

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Fig 8. Host lipids dictate the LCV housing capacity.

(a-c, f) Size analysis of LCVs harbored by C57BL/6 BMMs infected with ΔflaA bacteria (MOI = 20) for 15 hrs after 60 min synchronization. Cells were serum-starved prior to infection for 10hrs. LCV sizes were measured through 3D microscopy analysis of infected cells as detailed in S8 Fig. (a) Relative distributions of sizes of LCV harbored by live and dead cells produced by infections in the presence/absence of FBS. Live/dead distinction was determined morphologically by nuclear condensation. (b-c) Size analysis of the 50 largest LCVs produced by ΔflaA infections shown in (a). (d) Legionella growth in axenic cultures supplemented with FBS or delipidated FBS (dFBS) (10% v/v). (e) Kinetic analysis of LCVs that support bacterial replication in C57BL/6 BMMs infected with ΔflaA bacteria. FBS was added or omitted after the infection synchronization at 30min post infection. At least 200 LCVs were scored for each condition. (f) Size analysis of LCVs harbored by cells with condensed nucleus from (a). (g) Percentage of Myd88^-/- BMMs harboring large LCVs (bacteria>20) produced by 12 hrs synchronized infections with ΔflaA bacteria. Cell treatments were initiated at 4 hrs post infection as indicated. (h) L. pneumophila intracellular growth in Acanthamoeba castellanii over 48hrs in the presence of DMSO or Torin2 (300 nM), MOI = 5. (i) Percentage of Myd88^-/- BMMs with condensed nuclei uninfected or infected with ΔflaA bacteria for 9hrs. Infections were synchronized at 60min and various treatments were added at 6hrs as indicated. (j) Model for MTOR-dependent regulation of LCV homeostasis through the lipogenesis and serum-derived lipids. Abbreviations: ubiquitin ligase (UBL), pathogen-associated molecular patterns (PAMPs) (b, f-g, h-i) Means ± s.d of technical triplicates for each condition are shown. (a and g) At least 100 LCVs were analyzed for each condition. A representative of two (d, h-i) or three (a-c, e-g) biological replicates is shown for each experiment. (a, g and h) PP242 (2.5 μM), Brefeldin A (17.8 μM), Nocodazole (20 μM), FBS (10% v/v), dFBS (10% v/v) (b, f-i) * p<0.05, ** p<0.005 (unpaired T-test).

https://doi.org/10.1371/journal.ppat.1006088.g008

Next, the size of LCVs harbored by cells with aberrant nuclear morphology was analyzed because host cell death caused by vacuole rupture is rapid and likely captures the critical inflection point of cellular lipid-exhaustion when supply becomes insufficient. In the presence of FBS, infected dead cells harbored on average 132 bacteria and most of the LCVs exceeded 100 bacteria whereas under serum-free conditions the average size was reduced to 32 bacteria and very large LCVs (bacteria>100) were not detected in the population of dead cells (Fig 8a and 8f). Under serum-free conditions, when membrane biogenesis depends on lipogenesis, the size distribution curve of LCVs harbored by dead cells was broad, indicating that the threshold for LCV rupture varied (Fig 8a and 8f). Conversely, in the presence of FBS, most of the LCVs harbored by dead macrophages were very large, consistent with delayed LCV rupture and prolonged bacterial replication when lipids supply to infected cells is sustained (Fig 8a and 8f).

To determine if Legionella-induced MTOR-dependent lipogenesis similarly prolongs intracellular replication, the number of large LCVs harbored by Myd88^-/- BMMs infected for 12 hrs (as in Fig 5a) in the presence/absence of mTOR inhibitors were analyzed. Inhibition of MTOR with PP242, but not rapamycin, decreased the number of large LCVs consistent with regulation of LCV expansion and stability by Legionella-induced MTOR signaling (Fig 8g). Importantly, addition of FBS, but not delipidated FBS, sustained the number of large LCVs in the presence of PP242. Therefore, lipogenesis and lipid uptake are important functionally redundant determinants of the LCV housing capacity in Legionella-infected cells.

However, some large LCVs remained intact even under lipid-starvation conditions (Fig 8a and 8g), suggesting additional MTOR-independent mechanism can facilitate LCV expansion. Functional redundancy is a well-documented survival strategy by Legionella that facilitates a broad host range [28,58]. Indeed, MTOR inhibition with TORIN2 moderately reduced L. pneumophila intracellular replication in the protozoan host Acanthamoeba castellanii supporting MTOR-dependent and -independent LCV expansion mechanisms (Fig 8h). One potential MTOR-independent LCV expansion mechanism could be a direct membrane acquisition from other intracellular compartments. Indeed, early secretory vesicles tether and directly fuse with the LCV [6,68] thus it was tested whether the secretory compartment contributes to LCV expansion during MTOR suppression. To this end, Myd88-/- BMMs infected with ΔflaA bacteria were treated for 3hrs with PP242 and brefeldin A to disperse the Golgi compartment or nocodazole to block microtubules-dependent vasicular trafficking during the late LCV expansion phase (Fig 8i). Neither inhibitor treatment potentiated the PP242-induced cell death indicating that it is unlikely that the secretory compartment directly contributes membranes for the late LCV expansion.

Taken together, our data support a model in which an MTOR-dependent and an MTOR-independent pathway regulate LCV expansion by Legionella species. In the MTOR-dependent pathway, Legionella pneumophila manipulates MTOR function through secreted Dot/Icm effector(s) to drive lipogenesis and facilitate maximal niche expansion (Fig 8j). Insufficient host lipogenesis during Legionella pneumophila intracellular replication results in premature exhaustion of cellular lipids leading to loss of LCV membrane integrity, release of bacterial products in the cytosol of infected cells and cell death. However, serum-derived lipids can compensate for host-mediated or pharmacological MTOR suppression to facilitate maximal LCV expansion. Nevertheless, we provide evidence that L. pneumophila LCV expansion can be achieved through MTOR-independent mechanism(s) which function does not depend on the secretory pathway and are likely extensively utilized also by species other than L. pneumophila.

Ethics statement

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the Louisiana State University Health Sciences Center-Shreveport. Approval notice (P-15-026).

Bacterial strains

The following strains were used in this study: (1) L. pneumophila serogroup 1 strain JR32 deleted for flaA (ΔflaA) or dotA (ΔdotA) [52]; Lp02 pentuple strain [58]; (2) Lp02 thymidine auxotroph strains lacking flaA (thyA ΔflaA), dotA (thyA ΔdotA), icmS (thyA ΔicmS) [23] and sdhA (thyA ΔsdhAΔflaA) (this study); (3) Lp02 pentuple ΔflaA strain (this study); (4) Legionella longbeachae (ATCC 33462), Legionella jordanis BL-540 (ATCC 33623) and Legionella dumoffii (ATCC 33279) species were kind gift from Dr. Nicholas Cianciotto (Northwestern University). Legionella strains were grown on charcoal yeast extract (CYE) plates [1% yeast extract, 1%N-(2-acetamido)-2-aminoethanesulphonic acid (ACES; pH 6.9), 3.3 mM l-cysteine, 0.33 mM Fe(NO3)₃, 1.5% bacto-agar, 0.2% activated charcoal] [86]. Lp02 derived strains were grown on CYE plates containing thymidine at 100μg/ml. For all experiments, Legionella were harvested from CYE plates after growth for 2 days at 37°C.

Plasmids and strain construction

The ΔsdhA and the ΔflaA allele was generated by recombinant PCR using a ~1kb region upstream of the gene using the following primers for ΔsdhA (forward primer 5’-CGGGATCCCCTGATATAATGAAAAATATAGCCC-3’ and reverse primer 5’-CGCGGCCGCATTTCAATATTAAAAAAATTAACTTGTG-3’), a downstream region of sdhA (forward primer 5’-CGCGGCCGTAAAAATATTTGATTTAATCTTTTAATTTACC-3’ and reverse primer 5’-GCGAGCTCTGTCCATTTTTTCATACGAAAATACC-3’) and for ΔflaA (forward primer 5’-CGGGATCCTTCGTTGAAAGCCTTCTGGC-3’ and reverse primer 5’-CCGCTCGAGGGATGTCGCAATCGAAGTGC-3’), a downstream region of ΔflaA (forward primer 5’-CCGCTCGAGTCTCCTCAGACCTGAATCC-3’ and reverse primer 5’-AAACTGCAGCAGTTAATGAATTCACTCCC-3’). The resulting fragments were linked by a EagI site for ΔsdhA and XhoI site for ΔflaA and cloned into the BamHI/SacI sites of the gene replacement vector pSR47s creating pSR47s-CD0376 and pSR47s-CD1340 respectively. Strain Lp02 ΔflaA ΔsdhA was derived from LP02 ΔflaA and generated by allelic exchange of sdhA with ΔsdhA and the Lp02 pentuple ΔflaA was derived from LP02 pentuple and generated by allelic exchange of flaA with ΔflaA. The allelic exchange was confirmed by genotyping clonal isolates with the following primers in a single PCR reaction for sdhA (5’-CATTGAGCGTATCAAACACG-3’, 5’-GTAAAATGATAATTGAAGAGG-3’ and 5’-CAGTCTGGGGTTGAAATGTCC-3’) or for flaA (5’-TTGTGCAGTCATTCGTTGAG-3’, 5’-GTATTGATTATATTACAATCG-3’ and 5’-TCATCAGAAGGCATTTTACG-3’) that produces a fragment of ~330bp for sdhA/flaA or a fragment of ~500bp for ΔsdhA/ΔflaA.

Reagents

The pharmacological agents used in the study were obtained from the following suppliers Cell Signaling (LY294002), Sigma (PP242), Biolegend (Brefeldin A), Cayman Chemical Company (Torin2, Cytochalasin D and Nocodazole), CalBiochem (InSolutionRapamycin) and R&D Systems (Fatostatin). Primary antibodies were purchased from Cell Signaling– α-Akt(pan) (C67E7), α-rS6p(5G10), α-prS6p(S235/236)(D57.2.2E) and α-pS6K1(T389)(108D12); Enzo Life Sciences—α-Ubiquitinated proteins (FK2); Abcam– α-Galectin-3 (A3A12); Santa Cruz—α-Actin (C-2). The following antibodies were custom produced by Cocalico Biologicals against formalin-killed bacterial strains– α-L. pneumophila (chicken IgY), α-L. dumoffii (chicken IgY), α-L. longbeachae (rabbit IgG) and α-L. jordanis (rabbit IgG). Secondary antibodies and Hoechst 33342 (cat #H3570) were purchased from ThermoFisher Scientific—α-mouse-Alexa488 (cat #A11001), α-mouse-Alexa647 (cat #A21235), α-rabbit-Dylight550 (cat #SA5-10039), α-rabbit-Alexa647 (cat #A21244), α-chicken-IgY-FITC (cat #A16055), α-chicken-IgY-TRITC (cat #A16059). Complete FBS was purchased from Seradigm (cat #1300_500). For the studies with delipidated-serum, FBS was purchased from Gemini Bio-products: complete FBS (cat #100–106), delipidated FBS (cat #900–123). The cholesterol and triglycerides content of FBS was verified using biochemical assays. Purified human LDL was purchased from Intracel (cat #RP-032, Intracel Frederick, MD).

Mice

For these studies bone-marrow was harvested from C57BL/6J (Jackson Labs, cat # 000664), Myd88^-/- (Jackson Labs, cat #009088) and mTOR^fl/flCre+ mice (Mtor^-/-). Mice with Mtor deletion in the myeloid compartment were generated by breeding mTOR^fl/fl (Jackson Labs, cat # 011009) harboring loxP sites flanking exons 1–5 of the Mtor locus with LyzM^Pro Cre+ transgenic mice (Jackson Labs, cat #004781) [51].

BMM and U937 derivation and cell culture

To derive BMMs, bone marrow cells were isolated from the femurs and tibiae of mice, and cultured in RPMI 1640 with L-glutamine (Sigma, cat #R8758) containing 10% FBS (Seradigm), 20% macrophage colony-stimulating factor (M-CSF)-conditioned medium, and 1% primocin (Invivogen) at 37°C with 5% CO₂. Equal volume of media was added on day 4. On day 7, cells were harvested and used for infection assays. M-CSF condition media was obtained from an L-929 fibroblast cell line (ATCC). U937 cells (ATCC) were cultured in RPMI 1640 containing 10% FBS (Seradigm) and 1% Pen-Strep (ThermoFicher) at 37°C with 5% CO₂ and were differentiated with 10ng/ml Phorbol 12-myristate 13-acetate (Adipogen) for 24hrs followed by culture in PMA-free media for 48hrs prior to infections.

Infections with Legionella strains for immunofluorescence analysis

For infections, BMMs were seed on cover slips in re-plating media (RPMI 1640 with L-glutamine, 10%FBS, 10% M-CSF-conditioned media) for 8 hrs prior to serum starvation. U937 were seeded in 24-well plates and PMA-differentiated directly onto cover slips. For all experiments cell were serum starved for 10 hrs by culturing in RPMI 1640 with L-glutamine (serum-free RPMI). BMMs (2.5x10⁵ per well) or PMA-differentiated U937 (5x10⁵ per well) seeded on cover slips in 24-well plates were infected with Legionella at MOIs as indicated for each experiment. At 60 min post infections extracellular bacteria were removed by washing 5X with warm (37°C) phosphate-buffered saline (PBS) and the infected BMMs were cultured in serum-free RPMI. Inhibitors and FBS or dFBS were added as indicated for each experiment. The duration of infection is noted for each experiment.

For immunostaining, coverslips containing cells were washed with 3x PBS and fixed with 2% paraformaldehyde (PFA) for 20 min, permeabilized with cold methanol for 30 seconds, blocked with 2% BSA in PBS for 60 min. Primary antibodies dilutions were 1:500 for α-p-rS6p, 1:200 for α-ubiquitin (FK2), 1:200 for α-Galectin 3 and 1:1000 for all α-Legionella antibodies. Primary antibodies were incubated in PBS containing 1% BSA overnight at 4°C. Secondary antibodies were used at 1:500 dilution and Hoechst 33342 at 1:2000 for 60 min at room temperature. Coverslips were mounted with ProLong Gold antifade reagent (ThermoFisher) onto slides and examined by fluorescence microscopy. For detection of leaky LCVs by selective permeabilization cell were fixed with 2% PFA for 20min, incubated with α-ubiquitin (FK2) and α-L. pneumophila chicken ab in PBS with 0.1% saponin and 2% BSA overnight at 4C. Next, coverslips were extensively washed with PBS, incubated with cold 100% methanol for 30 sec, extensively washed with PBS and incubated with α-L. pneumophila rabbit ab for 2hrs. Secondary antibodies were applied as described above.

Legionella intracellular replication in Acanthamoeba castellanii

A. castellanii (ATCC) was propagated in ATCC Medium 712: PYG w/ Additives at room temperature. For infections, amoebae were seeded at 5X10⁵ cells per well in a 24 well tissue culture plates in A.c. buffer and allowed to settle down for 60 min. Next, amoeba were infected with JR32ΔflaA Legionella in A.c. buffer at MOI = 5 in the presence of DMSO or Torin2 (300nM) in technical triplicates for each condition. Inhibitors were added at the time of infection. At 60min post infection and at 48hrs post-infection, cells were collected and mechanically disrupted by passaging 10 times through a 27G syringe needle and various dilution were plated on CYE agar plates.

Microscopy analyses of infected cells

3D images were acquired with inverted wide-field microscope (Nikon Eclipse Ti) controlled by NES Elements v4.3 imaging software (Nikon) using a 60X/1.40 oil objective (Nikon Plan Apo λ), LED illumination (Lumencor) and CoolSNAP MYO CCD camera. Image acquisition, deconvolution and analysis was completed with NES Elements v4.3 imaging software. Only linear image corrections in brightness or contrast were completed. For all analyses, three-dimensional images of randomly selected fields were acquired and image acquisition parameters were kept constant for all the cover slips from the same experiment.

1. For phospho-rS6p analysis in cells, the ubiquitin signal was utilized to define a binary mask for each cell from which the mean fluorescence intensity of p-rS6p was obtained. Background fluorescence signal from each acquired field was individually determined from a cell-size mask positioned in an unoccupied area and subsequently subtracted from the MFI of each cell in the field.
2. For imaging Galectin 3 LCV recruitment, each plane of the Z-stack was scrutinized to ensure that multiple Galectin 3 puncta localize proximal or co-localized with the bacteria. Because Galectin 3 binds exposed sugar moieties on ruptured membranes, LCVs were considered positive if the galactin 3 wrapped the bacteria or greater than 4 puncta were proximal to the bacteria (< 0.5μm).
3. For cell death analysis, we set binary mask using Hoechst fluorescence to define and measure individual objects larger than 5μm³. Cell with aberrant nuclear morphology were obvious even by manual scoring and were marked by decrease in nuclear volume and increase in Hoechst 33342 fluorescence.
4. For LCV size analysis, 3D images of infected cells stained with purified anti-Legionella IgY chicken antibody were acquired by setting the Z-distance between the immediate out-of-focus planes to capture the whole cell. The space between imaged planes was set at 0.3μm, thus depending on the cell type the number of planes in a given Z-stack varied. 3D binary masks were set for each field of cells individually based on bacterial fluorescence to define objects and to ensure out of focus signal was omitted from the mask. Due to the high fidelity of the anti-Legionella IgY antibody, images were not deconvoluted prior to binary mask definition because deconvolution did not improve data acquisition. The volume of each object was measured with the 3D image volume analysis function of NES Elements (Nikon). To derive the mathematical algorithm for volume-to-bacteria conversion the object volumes of LCVs containing bacteria that can be definitively manually enumerated were determined and plotted against the data from the bacterial count per vacuole. The best linear fit algorithm for the plot was used to derive the conversion equation (Y = 0.5842*X+0.4835, X = object volume [μm³] Y = number of bacteria). The conversion algorithm was back tested by extrapolating bacterial numbers of LCVs in which the number of bacteria can be manually counted definitively. (see S8 Fig)

Infections with Legionella strains for LDH release assay

BMMs were seeded in 48-well plates (1X10⁵ cells per well), serum-starved for 10 hrs and infected as indicated at MOI = 10 in technical triplicates for each time point. Total supernatant volume was 200μl. For the FBS rescue experiments, the serum was added at the time of infection. Supernatants were collected and immediately frozen at -20C. Thawed supernatants (50μl) were assayed for LDH activity using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) according to the manufacturer’s protocol. Supernatants from media only, unifected cell and Triton X-100 lysed cells were used to calculate the dynamic range of the assay as per the manufacturer’s instructions.

Infections with Legionella strains for immunoblot analysis

BMMs were seeded in 12-well plates (8X10⁵ cells per well), serum-starved for 10 hrs and infected as indicated. Cells were washed with cold PBS and lysed with RIPA buffer (20mM Tris-HCl pH7.5, 200mM NaCl, 1mM EDTA, 1% NP-40, 1% sodium deoxycholate, 2.5mM sodium pyrophosphate, 1mM β-glycerophosphate, 1mM Na₃VO₄, 1μg/ml leupeptin). Total cell lysates (20μg) were resolved by SDS-PAGE, transferred onto nitrocellulose membranes blocked with 2% BSA and incubated with primary antibody for either 3 hrs at room temperature or overnight at 4°C for phospho-antibodies. Primary antibody dilutions were used as per manufacturers’ instructions. Secondary horseradish peroxidase-conjugated antibodies (ThermoFisher) were used at 1:5000 in 2 hrs incubations at room temperature. Signals were visualized with ECL (GE Healthcare) and analyzed with ImageJ (NIH).

Quantitative PCR analysis of gene expression

Myd88^-/- BMMs (1X10⁶ cells) were serum starved and infected under serum-free conditions as indicated for 6 hrs. Total RNA and first-strand cDNA synthesis was performed with TaqMan Gene Expression Cells-To-Ct Kit (ThermoFisher) according to the manufacturer’s instructions. mRNA levels were determined by quantitative real-time PCR using the Universal ProbeLibrary (Roche, Life Science) and LightCycler 480 Probes Master (Roche, Life Science). Thermal cycling was carried out using a LightCycler 96 instrument (Roche Diagnostics) under the following conditions: 95°C for 5 min and 45 cycles at 95°C for 10 sec and 60°C for 25 sec. Gene expression was normalized to Canx (Calnexin). The fold increase is shown relative to uninfected cells (value of 1).

Statistical analysis

Calculations for statistical differences were completed by Student’s T-test or ANOVA as indicated using Prism v6 (GraphPad Software).