LegC4 confers a fitness disadvantage on L. pneumophila in cytokine-activated macrophages and in a mouse model of Legionnaires’ Disease

We previously reported that LegC4 confers a fitness disadvantage on L. pneumophila in intranasal C57BL/6 mouse models of Legionnaires’ Disease (LD) and within cytokine-activated macrophages [20,21]. C57BL/6 mice are restrictive to L. pneumophila due to flagellin (FlaA)-mediated activation of the NAIP5/NLRC4 inflammasome [23,24]. Thus, to study FlaA-independent host responses within C57BL/6 mice, we leverage FlaA-deficient L. pneumophila (ΔflaA) strains. We found that L. pneumophila ΔflaAΔlegC4 (herein called ΔΔ) mutant strains outcompeted parental ΔflaA strains in the lungs of C57BL/6 wild-type (WT) mice [20] but a growth phenotype for L. pneumophila ΔΔ was only observed within cultured mouse primary bone marrow-derived macrophages (BMDMs) activated with exogenous recombinant (r) TNF or rIFN-γ [20]. However, autocrine and paracrine TNF produced by cultured BMDMs is sufficient to attenuate L. pneumophila strains induced to overexpress legC4 from a complementing multicopy plasmid in naïve WT BMDMs [20]. Importantly, this phenotype was specific to intracellularly growing bacteria since L. pneumophila growth in broth in vitro is unaffected by overexpression of legC4 [20]. Thus, LegC4 confers a growth defect on L. pneumophila in a dose-dependent manner within cytokine-activated macrophages.

We confirmed our previous results and found that overexpression of legC4 was sufficient to impair L. pneumophila replication within WT BMDMs since L. pneumophila ΔΔ (plegC4) replication was significantly attenuated compared to both the L. pneumophila ΔflaA parental strain and L. pneumophila ΔΔ harboring an empty plasmid vector (pEV) (Fig 1A). Impaired replication was dependent on TNF signaling since overexpression of legC4 did not impair growth within BMDMs unable to produce TNF (Myd88^-/-; Fig 1B) or signal through TNFR1 (Tnfr1^-/-; Fig 1C). IFN-γ signaling is sufficient for LegC4 restriction [20]; however, the LegC4 restriction phenotype was retained within Ifngr1^-/- BMDMs (Fig 1D), indicating that the relatively low levels of IFN-γ secreted by macrophages are not sufficient for restriction. Importantly, we found that LegC4 restriction is not specific to mouse macrophages since both L. pneumophila ΔflaA and ΔΔ (plegC4) were impaired for replication compared to the ΔΔ (pEV) strain in PMA-differentiated human THP-1 macrophages (Fig 1E). Thus, LegC4 confers a dose-dependent virulence defect on L. pneumophila within mouse and human macrophages.

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Fig 1. LegC4 attenuates L. pneumophila replication within cytokine-activated macrophages and the mouse lung.

Growth of indicated L. pneumophila strains within (A) WT BMDMs, (B) Myd88^-/- BMDMs, (C) Tnfr1^-/- BMDMs, or (D) Ifngr1^-/- BMDMs, or (E) THP-1 cells over 48 h. Plasmid expression of legC4 was induced with 1 mM IPTG. Data are shown as mean ± s.d. of pooled results of at least two independent experiments with triplicate wells per condition in each experiment. Asterisks denote statistical significance by Students’ t-test (*P<0.05, **P<0.01). ΔΔ: ΔflaAΔlegC4. (F) Competitive index (CI) values from the lungs of mice infected with a 1:1 mixture of L. pneumophila ΔΔ (pJBlegC4) or ΔflaAΔlegC4 (pJB) with L. pneumophila ΔflaA for 48 h. Each symbol represents an individual animal and data are pooled from at least two independent experiments. Mean CI values are shown and asterisks denote statistical significance by two-way ANOVA (***P<0.0001, **P<0.01). ΔΔ: ΔflaAΔlegC4.

https://doi.org/10.1371/journal.ppat.1011473.g001

Cytokine signaling drives LegC4 growth defects in cultured BMDMs, but the role of cytokine signaling in vivo is unclear. We evaluated bacterial fitness by calculating competitive index values for mice infected with a 1:1 mixture of L. pneumophila ΔflaA and either L. pneumophila ΔΔ (pEV) or ΔΔ (plegC4) for 48 h (see Materials & Methods). We confirmed our previous results showing that LegC4 confers a dose-dependent fitness defect on L. pneumophila in the lungs of WT mice since L. pneumophila ΔflaA was outcompeted by L. pneumophila ΔΔ (pEV) but was able to outcompete L. pneumophila ΔΔ (pJBlegC4; Fig 1F) [20]. We also confirmed that loss of TNFR1 is insufficient to abolish the legC4 mutant fitness advantage (Fig 1F) and obtained similar results in the lungs of IFNGR1- and MyD88-deficient mice (Fig 1F). However, the fitness phenotype associated with loss LegC4 was less pronounced in the lungs of Myd88^-/- mice compared to WT, Tnfr1^-/- and Ifngr1^-/- mice (Fig 1F), which is likely due to the relatively low levels of MyD88-independent IFN-γ produced in the L. pneumophila-infected lung [25]. Furthermore, the fitness disadvantage associated with overexpression of legC4 was less pronounced in the lungs of MyD88-, IFNGR1- and TNFR1-deficient mice compared to WT mice (Fig 1F). These data support our previous work showing that LegC4 augments redundant cytokine-mediated pathogen restriction mechanisms [20]. Overexpression of legC4 is clearly not necessary to observe a phenotype; however, based on the narrow phenotypic window in BMDMs activated with exogenous cytokine, this infection model is useful for our goal to define how LegC4 potentiates cytokine-mediated restriction within macrophages

LegC4 induces robust phagolysosomal fusion with LCVs within BMDMs

LegC4 attenuates L. pneumophila replication within cytokine-activated BMDMs; however, the mechanism of bacterial restriction is unknown. Cytokine signaling enhances phagolysosomal fusion with intracellular pathogens [26–29]; thus, we tested the hypothesis that LegC4 restriction involves lysosomal targeting of L. pneumophila. Virulent L. pneumophila establish a replication-permissive compartment called the Legionella-containing vacuole (LCV) by blocking endocytic maturation of their phagosome [30,31]. Avirulent Dot/Icm-deficient L. pneumophila (ΔdotA) are unable to establish a replicative LCV and phagosomes rapidly mature to endolysosomes, as evidenced by presence of lysosomal membrane protein LAMP1 [30]. We initially tested the hypothesis that LegC4 induces phagolysosomal fusion with LCVs by blinded immunofluorescence scoring of LAMP1⁺ LCVs within BMDMs infected with L. pneumophila strains for 9h. Using our legC4 overexpression model, we observed robust LegC4-dependent phagolysosomal fusion with significantly more LAMP1⁺ L. pneumophila ΔΔ (plegC4) LCVs noted relative to both L. pneumophila ΔflaA and ΔΔ (pEV) control strains (Fig 2A). Strikingly, the abundance of LAMP1⁺ LCVs within WT BMDMs infected with L. pneumophila ΔΔ (plegC4) did not differ from cells infected with the avirulent ΔdotA control strain (Fig 2A). Moreover, robust LegC4-mediated phagolysosomal fusion with LCVs was significantly suppressed within TNFR1-deficient BMDMs (Fig 2A) since Tnfr1^-/- BMDMs infected with L. pneumophila ΔΔ (plegC4) harbored significantly fewer LAMP1⁺ LCVs than those infected with the ΔdotA control strain (Fig 2A). Interestingly, loss of TNFR1-mediated signaling did not fully abolish phagolysosomal fusion with L. pneumophila ΔΔ (plegC4) LCVs, but our growth curve data suggest that this is not sufficient for detectable differences in intracellular replication (Fig 1C).

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Fig 2. LegC4 enhances phagolysosomal fusion with LCVs and restriction is suppressed by inhibition of the vacuolar (v)ATPase.

Abundance of LAMP1+ LCVs within (A) naïve WT or Tnfr1^-/- BMDMs infected with L. pneumophila strains for 9h; or (B) rTNF-treated WT BMDMs (25 ng mL^-1) infected with L. pneumophila strains for 6 h. Infections were performed in triplicates and LAMP1 abundance was scored blind (n = 100 cells per coverslip) by immunofluorescence microscopy (n = 300 per condition per experiment). Plasmid expression of legC4 was induced with 1 mM IPTG. Data shown are mean ± s.e.m. of pooled results from two independent experiments (n = 600 total per condition). Asterisks denote statistical significance by two-way ANOVA (*P<0.05; **P<0.01; ns, not significant). (C) Growth of L. pneumophila strains within WT BMDMs treated with 10 nM bafilomycin A1 (BAF) (left panel) or DMSO (vehicle control; right panel). Plasmid expression of legC4 was induced with 1 mM IPTG. Data shown are mean ± s.d. of pooled results from three independent experiments with triplicate samples per experiment. Asterisks denote statistical significance by Students’ t-test (**P<0.01). ΔΔ: ΔflaAΔlegC4.

https://doi.org/10.1371/journal.ppat.1011473.g002

Since endogenous LegC4 suppresses L. pneumophila replication within BMDMs activated with exogenous rTNF [20], we hypothesized that endogenous LegC4 would also be sufficient to induce phagolysosomal fusion with LCVs in rTNF-treated BMDMs. Indeed, more LAMP1⁺ LCVs were present in rTNF-treated BMDMs infected with L. pneumophila ΔflaA compared to L. pneumophila ΔΔ (Fig 2B). Together, these data show that LegC4 augments phagolysosomal fusion with LCVs in cytokine-activated BMDMs.

Pharmacological inhibition of the H⁺-type vacuolar (v)ATPase abolishes LegC4 restriction in BMDMs

We subsequently tested whether lysosomal acidification contributes to LegC4-mediated restriction by quantifying L. pneumophila replication within BMDMs treated with Bafilomycin A1 (BAF). BAF is a pharmacological inhibitor of the H⁺-type vacuolar (v)-ATPase, which drives vacuolar acidification and phagolysosomal fusion [32]. Thus, we tested a role for vATPase activity in LegC4 restriction by quantifying L. pneumophila replication within BAF-treated BMDMs. LCV acidification is important for late stages of the L. pneumophila lifecycle [33]; however, intracellular replication is minimally impacted within BMDMs treated with a low concentration (≤12.5 nM) of BAF [34]. We found that L. pneumophila ΔΔ (plegC4) replication did not differ from L. pneumophila ΔflaA and ΔΔ (pEV) strains within BMDMs treated with 10 nM BAF (Fig 2C, left). However, as expected, L. pneumophila ΔΔ (plegC4) replication was significantly attenuated compared to ΔflaA and ΔΔ (pEV) within vehicle (DMSO)-treated BMDMs (Fig 2C, right). These data further support a role for lysosomes in LegC4-mediated restriction.

LegC4 interacts with host proteasome activator (PA)28α

To gain insight into the mechanism of LegC4 function, we identified LegC4 binding partners by yeast two-hybrid (Y2H) using LegC4 as bait and 30,000 clones of fragmented genomic (g)DNA from mouse splenocytes as prey (Hybrigenics, Inc., see Materials & Methods). Our screen revealed interactions between LegC4 and several regulators of eukaryotic proteasomes, including proteasome activator (PA)28α, PA28γ, and Ecm29 (S1 Table). The highest-confidence interactor was PA28α with 28 individual clones, all of which encoded a minimal overlapping region in the PA28α C-terminal domain (amino acid residues 105–249) (S1 Table and S1 Fig). These data suggest a direct interaction between LegC4 and PA28α, which is conserved in amoebae and upregulated by cytokine signaling in macrophages.

We sought to validate our Y2H data using reciprocal affinity purification, co-immunoprecipitation (IP), and confocal microscopy. We evaluated direct binding between recombinant PA28α and LegC4 by reciprocal affinity chromatography on GST- or His₆-Myc-tagged fusion proteins produced in E. coli. Using glutathione agarose beads, we found that GST-LegC4 retained Myc-PA28α (Fig 3A) and GST-PA28α retained Myc-LegC4 (Fig 3B). PA28α did not interact with either GST alone or the unrelated L. pneumophila effector Lgt1 (Fig 3A and 3B), known to target eukaryotic elongation factor 1A [35,36], which indicates that PA28α does not bind non-specifically to L. pneumophila effectors. To determine if PA28α and LegC4 interact in mammalian cells, we co-transfected HEK293T cells to transiently produce epitope-tagged 3xFLAG-LegC4 and either PA28α-Myc or GFP-PA28α fusion proteins. Ectopic expression of PA28α was necessary in these experiments since it is expressed only in response to cytokine signaling [37,38]. Using α-FLAG and α-GFP pull-downs, we found that PA28α-Myc co-immunoprecipitated with 3xFLAG-LegC4 (Fig 3C) and that 3xFLAG-LegC4 co-immunoprecipitated with GFP-PA28α (Fig 3D), respectively. Together, these data show that LegC4 binds PA28α.

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Fig 3. LegC4 interacts with and alters the subcellular localization of PA28α.

(A-B) Reciprocal affinity chromatography of recombinant GST-fusion proteins from E. coli lysates bound to glutathione agarose beads and incubated with lysates from E. coli producing His₆-Myc-fusion proteins. Proteins retained on the beads were eluted and visualized by SDS-PAGE and Coomassie brilliant blue (CBB) stain and α-Myc Western blot. Arrows denote distinct GST-fusion proteins (GST, 26 kDa; GST-LegC4, 113 kDa; GST-Lgt1, 85 kDa; GST-PA28α, 55 kDa; His₆-Myc-LegC4, 89 kDa; His₆-Myc-Lgt1, 62 kDa; His₆-Myc-PA28α, 31 kDa). Data are representative of three experiments. (C) Western blot of expression (input) and co-immunoprecipitation of PA28α-Myc with 3xFLAG-LegC4 on α-FLAG conjugated magnetic beads from lysates of co-transfected HEK293T cells. Data are representative of two independent experiments. (D) Western blot of expression (input) and co-immunoprecipitation of 3xFLAG-LegC4 with GFP-PA28α on α-GFP-conjugated magnetic beads from lysates of co-transfected HEK 293T cells. Data are representative of two experiments. (E) Representative confocal micrographs of transfected HeLa cells producing ectopic 3xFLAG-LegC4 and either GFP-PA28α or GFP stained with α-FLAG and α-GFP antibodies. Scale bar represents 10 μm. (F) Pearson Correlation Coefficients (PCC) were calculated for individual cells producing 3xFLAG-LegC4 and either GFP-PA28α or GFP alone (n = 10) using Fiji ImageJ software. PCC ≥ 0.7 (dashed line) indicates a high confidence association. Asterisks denote statistical significance by Students’ t-test (**P<0.01). (G) Confocal micrograph of transfected HeLa cells producing GFP-PA28α (arrowhead) or GFP-PA28α and 3xFLAG-LegC4 (arrows). Insets are zoomed images of cells producing (i) PA28α alone (pink box) or both PA28α and LegC4 (blue box). Scale bar represents 10 μm. (H) Confocal micrographs of WT BMDMs infected with L. pneumophila strains (MOI of 25) for 7h and stained with α-PA28α (red) and α-L. pneumophila (green) antibodies. Cellular nuclei were imaged with Hoechst (blue). White and blue arrowheads indicate BMDM nuclei and intracellular bacteria, respectively. Scale bar represents 10 μm. Inset images of intracellular L. pneumophila are magnified from regions indicated with white boxes. Scale bar represents 1 μm. Images are representative of populations of infected BMDMs over two independent experiments. ΔΔ: ΔflaAΔlegC4.

https://doi.org/10.1371/journal.ppat.1011473.g003

We further evaluated the biological relevance of LegC4-PA28α binding by evaluating protein localization in cells by confocal microscopy. Ectopically produced 3xFLAG-LegC4 co-localized with GFP-PA28α within transiently transfected HeLa cells since calculated Pearson Correlation Coefficients (PCC) indicated a significant positive correlation between 3xFLAG-LegC4 and GFP-PA28α (Mean PCC = 0.798 ± 0.054) compared to 3xFLAG-LegC4 and GFP (Mean PCC = 0.546 ± 0.196) (Fig 3E and 3F). Interestingly, LegC4 modulated PA28α subcellular localization since GFP-PA28α localized to the nucleus in the absence of LegC4 [Fig 3E and 3G, arrowhead, (i)] but was cytosolically localized within cells co-expressing LegC4 [Fig 3E and 3G, arrows, (ii)] [39]. We subsequently tested whether PA28α localization is influenced by Dot/Icm-translocated LegC4 within L. pneumophila-infected BMDMs. WT BMDMs were infected with L. pneumophila ΔflaA, ΔΔ, or ΔdotA for 7.5 h, at which time a replication-competent LCV is established. Fixed cells were permeabilized and stained with α-L. pneumophila and α-PA28α antibodies and Hoechst (DNA) and imaged by confocal microscopy. Endogenous PA28α is constitutively produced in immune cells so can be imaged in BMDMs. We found that PA28α was primarily cytosolic within BMDMs infected with L. pneumophila ΔflaA, but localized robustly to the nucleus of BMDMs infected with either L. pneumophila ΔΔ or ΔdotA (Fig 3H, white arrowheads). Interestingly, PA28α was present on LCVs harboring L. pneumophila ΔΔ but not ΔflaA or ΔdotA control strains (Fig 3H, blue arrowheads and inset). Although altered PA28α localization mediated by translocated LegC4 in naïve infected BMDMs was not sufficient for LegC4 restriction or phagolysosomal fusion in naïve WT BMDMs (Figs 1A and 2B), these data further support a biologically relevant interaction between LegC4 and PA28α, which has not previously identified as a target of bacterial effectors.

LegC4 restriction phenotypes are suppressed in PA28α-deficient BMDMs

Based on the interaction between PA28α and LegC4, we tested the hypothesis that PA28α contributes to LegC4-mediated restriction of L. pneumophila. PA28α-deficient (Psme1^-/-) mice are not commercially available, so we evaluated the role of PA28α using BMDMs derived from commercially-available PA28αβ-deficient (Psme1/2^-/-) mice [5]. PA28α associates with PA28β to form the PA28αβ (11S) proteasome regulator [40]. PA28β is homologous to PA28α but is dependent on PA28α for activity and is hypothesized to enhance the interaction of PA28α with 20S proteolytic proteasome core particles (CPs) [41]. Thus, we evaluated the role of PA28α in LegC4 restriction in BMDMs and in vivo using Psme1/2^-/- mice. We first tested whether loss of PA28αβ relieves the LegC4-mediated growth attenuation within TNF-activated BMDMs. In agreement with our previous work [20], L. pneumophila ΔflaA growth was significantly impaired relative to L. pneumophila ΔΔ in rTNF-activated WT BMDMs (P = 0.015; Fig 4A); however, the L. pneumophila ΔΔ growth advantage was lost in Psme1/2^-/- BMDMs (P = 0.10; Fig 4A). Moreover, overexpression of legC4 was insufficient to significantly impair L. pneumophila replication within naïve Psme1/2^-/- BMDMs (P = 0.15) whereas replication was substantially diminished within WT BMDMs (P = 0.006; Fig 4B). These data support a role for PA28αβ in LegC4 growth restriction in cultured macrophages, although the modest growth defects observed in Psme1/2^-/- BMDMs suggest that LegC4 does not target PA28α exclusively.

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Fig 4. PA28αβ is important for LegC4 restriction phenotypes within cultured BMDMs and the mouse lung in vivo.

Growth of L. pneumophila strains within rTNF-treated (A) or naïve (B) WT (top) or Psme1/2^-/- (bottom) BMDMs. Where indicated, BMDMs were treated with 25 ng mL^-1 rTNF and plasmid expression of legC4 was induced with 1 mM IPTG. Data shown are mean ± s.d. of pooled results from three independent experiments with triplicate samples per condition in each experiment. Asterisks denote statistical significance by Students’ t-test (**P<0.01; *P<0.05). ns, not significant. (C) Competitive index (CI) values from the lungs of Psme1/2^-/- mice infected with a 1:1 mixture of L. pneumophila ΔΔ (pJB) and L. pneumophila ΔflaA for 48 h. Each symbol represents an individual animal and data are pooled from three independent experiments. The mean CI value is shown. ΔΔ: ΔflaAΔlegC4.

https://doi.org/10.1371/journal.ppat.1011473.g004

LegC4 does not confer a fitness disadvantage in the lungs of Psme1/2^-/- mice

Since loss of PA28αβ was sufficient to suppress LegC4-mediated growth attenuation in BMDMs, we hypothesized that LegC4 would not confer a fitness disadvantage on L. pneumophila in the lungs of Psme1/2^-/- mice. We infected Psme1/2^-/- mice with a 1:1 mixture of L. pneumophila ΔΔ (pEV) and ΔflaA and calculated competitive index values from lung homogenates at 48 h post-infection. Strikingly, L. pneumophila fitness was unaffected by LegC4 in the lungs of Psme1/2^-/- mice (CI = 1.09 ± 0.793); Fig 4C). In aggregate, our data suggest that PA28αβ is important for LegC4-mediated restriction in cytokine activated BMDMs and the mouse lung in vivo.

The abundance of carbonylated proteins in macrophages is increased in the presence of LegC4 under oxidative stress conditions

PA28αβ is expressed constitutively in cells of the innate immune system and upregulated to maintain the integrity of the proteome under oxidative stress conditions [2,38,40,42]. PA28αβ engages proteolytic proteasome 20S core particles (CPs) to promote rapid ubiquitin-independent proteolysis of oxidant-damaged (carbonylated) proteins [4,43], the abundance of which increases commensurately with the abundance of reactive oxygen species (ROS) [44,45]. 20S CPs can degrade carbonylated proteins, but their efficiency is substantially increased when in complex with PA28αβ [2,43]. Concomitantly, there is a strong negative correlation between PA28αβ activity and carbonylated protein abundance under oxidative stress conditions [43]. Our Y2H data suggest that LegC4 binds the C-terminal domain of PA28α, which is important for PA28αβ docking on proteasome CPs [46–48]. Thus, we rationalized that carbonylated proteins would be more abundant in the presence of LegC4 under oxidative stress conditions. To directly evaluate LegC4, we generated RAW 264.7 cells stably transfected with 3xFLAG-LegC4 (RAW-LegC4) or empty plasmid vector (RAW-Cntrl). We confirmed 3xFLAG-LegC4 production by confocal microscopy (Fig 5A) and Western blot (Fig 5B). Immunoprecipitation was necessary to visualize LegC4 since it is produced by the macrophages at low abundance. However, the abundance of ectopically produced LegC4 was sufficient to attenuate replication of L. pneumophila ΔflaA (Fig 5C). Attenuated growth was observed at 48 h post-infection, similar to the kinetics of LegC4 restriction within BMDMs (see Fig 1A), which corroborates our microscopy data suggesting that ectopically-produced LegC4 is functional (see Fig 3G and 3H) and demonstrates that RAW-LegC4 cells are suitable to evaluate LegC4 activity. RAW-LegC4 and -Cntrl cells were incubated in the presence or absence of 10 μM H₂O₂ for 24 h and protein carbonyls in cell lysates were quantified by ELISA. We used H₂O₂ since it is membrane permeable, stable in solution, and biologically relevant since L. pneumophila-infected macrophages in the lung would also be exposed to neutrophil-generated extracellular ROS [26,49]. Indeed, H₂O₂-treated RAW-LegC4 cells contained significantly more protein carbonyls than RAW-Cntrl cells (Fig 5D). Under native conditions, the concentration of carbonylated proteins was unaffected by LegC4 (Fig 5D), which suggests that LegC4 does not itself induce oxidative stress. The abundance of protein carbonyls was unaffected by H₂O₂ in RAW-Cntrl cells, likely because upregulation of PA28αβ over the duration of this experiment was sufficient for turnover of damaged proteins.

We subsequently tested whether LegC4 increases the abundance of protein carbonyls within L. pneumophila-infected BMDMs. Tnfr1^-/- BMDMs were used to control for any LegC4-mediated differences in TNF secretion [20] and, consequently, intracellular ROS production [50]. Tnfr1^-/- BMDMs were infected with L. pneumophila ΔflaA, ΔΔ (pEV), or ΔΔ (plegC4) for 4 h in the presence or absence of 50 μM H₂O₂ and protein carbonyls were quantified by Western blot and densitometry. We found that carbonylated proteins were more abundant within BMDMs infected with L. pneumophila ΔΔ (plegC4) compared to L. pneumophila ΔflaA and ΔΔ (pEV)-infected and uninfected BMDMs when oxidative stress was induced with H₂O₂ (Fig 5E and 5F). The abundance of protein carbonyls was not affected by LegC4 within naïve untreated BMDMs, which further suggests that LegC4 itself does not induce oxidative stress. Protein carbonyl abundance was not affected by either LegC4 or L. pneumophila infection within untreated (UT) BMDMs (Fig 5E and 5F), likely because the amount of ROS produced in response to L. pneumophila infection under these conditions is insufficient for detectable differences in protein carbonyls by Western blot. Together, these data support our hypothesis that LegC4 perturbs proteostasis in macrophages under oxidative stress conditions and suggest that LegC4 may function by impairing PA28αβ activity.

Oxidative stress is sufficient for LegC4 restriction in PA28αβ-producing BMDMs

Our current data suggest role(s) for PA28αβ and oxidative stress in LegC4 restriction; thus, we hypothesized that oxidative stress would be sufficient for LegC4-mediated restriction within BMDMs producing PA28αβ. To test this, we quantified replication of L. pneumophila ΔflaA and ΔΔ within WT and Psme1/2^-/- BMDMs treated with 20 μM H₂O₂ to induce oxidative stress. Indeed, oxidative stress was sufficient for LegC4 restriction since L. pneumophila ΔΔ replicated to significantly higher levels than L. pneumophila ΔflaA in WT BMDMs (Fig 6A), showing that oxidative stress is sufficient for LegC4 restriction. Importantly, the growth advantage associated with ΔlegC4 mutation was abolished in PA28αβ-deficient BMDMs, but replication of LegC4-producing bacteria was unaffected (Fig 6A). These data show that LegC4 impairs L. pneumophila replication under oxidative stress conditions via a mechanism involving PA28αβ.

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Fig 5. The abundance of carbonylated proteins is increased in the presence of LegC4 under oxidative stress conditions.

(A) Representative confocal micrographs of RAW-LegC4 and -Cntrl cells stained with α-FLAG antibodies and Hoechst to image nuclei. Scale bar represents 10 μm. (B) Western blot of α-FLAG immunoprecipitation from lysates of RAW-LegC4, RAW-Cntrl or untransfected (UT) RAW cells with α-FLAG primary antibody. Arrow indicates 3xFLAG-LegC4 (~84 kDa). (C) Growth of L. pneumophila ΔflaA within RAW-LegC4 and -Cntrl cells. Data shown are mean ± s.d. of pooled results of three independent experiments with triplicate samples per condition in each experiment. Asterisks denote statistical significance by Students’ t-test (**P<0.01). (D) Quantification of protein carbonyls by ELISA in lysates of RAW-LegC4 and -Cntrl cells incubated in low-serum media for 24 h in the presence or absence of 10 μM H₂O₂. Data shown are mean ± s.e.m. and representative of two independent experiments. Asterisks denote statistical significance by two-way ANOVA (*P<0.05; ns, not significant). UT: untreated. (E) Western blot for protein carbonyls and β-actin (loading control) in lysates of Tnfr1^-/- BMDMs infected for 4 h with the indicated strains (MOI of 50) in the presence or absence of 50 μM H₂O₂. Uninfected (UI) samples are from the same experiment but shown separately to remove irrelevant lanes. ΔΔ: ΔflaAΔlegC4; plasmid expression of legC4 was induced with 1 mM IPTG. Data are representative of three independent experiments. (F) Densitometric quantification of carbonylated proteins from Western blots. Signal intensity from protein carbonyl Western blots was normalized to the β-actin loading control for each sample and averaged ratios were normalized to ΔflaA-infected cells. Data shown are mean ± s.d. of values from three independent experiments. Asterisks denote statistical significance by two-way ANOVA (**P<0.01; *P<0.05). ns, not significant.

https://doi.org/10.1371/journal.ppat.1011473.g005

L. pneumophila replication is impaired in Psme1/2^-/- BMDMs irrespective of LegC4

Our data suggest that LegC4 restriction is mediated by subversion of PA28αβ. Thus, it is possible that the dose-dependency of LegC4 in naïve BMDMs results from stoichiometric differences in its abundance relative to PA28αβ. Thus, we rationalized that L. pneumophila replication would be suppressed in naïve Psme1/2^-/- BMDMs irrespective of LegC4 and found that, indeed, both L. pneumophila ΔflaA and ΔΔ strains were attenuated for replication within Psme1/2^-/- BMDMs (Fig 6B). Importantly, impaired replication in Psme1/2^-/- BMDMs was independent of LegC4 since L. pneumophila ΔflaA and ΔΔ were equally attenuated (Fig 6B). These data show that PA28αβ-deficient macrophages are more restrictive restriction to L. pneumophila and suggest a novel role for PA28αβ in host defense against intracellular pathogens in macrophages.

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Fig 6. LegC4 restriction occurs under oxidative stress conditions and is phenocopied by loss of PA28αβ.

Normalized CFU recovered from (A) 20 μM H₂O₂-treated or (B) naïve WT or Psme1/2^-/- BMDMs infected for 48 h with either L. pneumophila ΔflaA or ΔΔ. CFU of each strain recovered at 48 h was normalized to internalized bacteria at 1h post-infection for each condition. Data shown are mean ± s.e.m. of pooled results from three independent experiments with triplicate samples per condition in each experiment. Asterisks denote statistical significance by two-way ANOVA (**P<0.01, *P<0.05). ns, not significant.

https://doi.org/10.1371/journal.ppat.1011473.g006

Neither TNF production nor cytotoxicity are potentiated by loss of PA82αβ in L. pneumophila-infected BMDMs

Our data suggest that LegC4 enhances phagolysosomal fusion with LCVs via subversion of PA28αβ activity and that loss of PA28αβ is sufficient to impair L. pneumophila intracellular replication. However, impaired bacterial growth may also result from increased TNF production or death of infected BMDMs. We quantified TNF in the supernatants of WT and Psme1/2^-/- BMDMs infected with L. pneumophila ΔflaA, ΔΔ (pEV), ΔΔ (plegC4) and ΔdotA (negative control). At 8h post-infection, there was a modest but statistically significant decrease in TNF secretion from Psme1/2^-/- BMDMs infected with L. pneumophila ΔΔ (plegC4) (P = 0.049) but no other PA28αβ-mediated differences were observed (S2A Fig). At 24 h post-infection, significantly less TNF was present in supernatants of Psme1/2^-/- BMDMs infected with L. pneumophila ΔflaA or ΔΔ (pEV) compared to WT BMDMs but no differences in secretion from BMDMs infected with L. pneumophila ΔΔ (plegC4) (S2B Fig). Furthermore, viability of L. pneumophila-infected was not affected by either LegC4 or PA28αβ since there were no differences in lactate dehydrogenase (LDH) activity in culture supernatants (S3 Fig). These data suggest neither LegC4 nor PA28αβ restriction phenotypes result from increased TNF production or death of infected BMDMs.

Inflammasome activation is dispensable for LegC4-mediated restriction

Our data suggest that LegC4 perturbs proteostasis and potentiates oxidative stress. Sustained oxidative and proteotoxic stress and proteostasis perturbations can activate the NLRP3 inflammasome, which culminates in activation of caspase-1, an effector caspase responsible for pyroptotic cell death and inflammatory cytokine secretion. However, pharmacological inhibition of the NLRP3 inflammasome with MCC950 did not affect LegC4 restriction since L. pneumophila ΔΔ (plegC4) was still attenuated for replication compared to L. pneumophila ΔΔ (pEV) and ΔflaA within WT BMDMs (S4A Fig). We also found that LegC4 restriction was preserved within BMDMs derived from caspase-1-deficient (Casp1^-/-) mice (S4B Fig). Within cultured macrophages, pyroptotic cell death is responsible for L. pneumophila restriction downstream of inflammasome activation. Our data suggest that LegC4 does not enhance LDH release from infected BMDMs (S3 Fig); however, pyroptotic cell death is more pronounced within BMDMs primed with toll-like (TLR) receptor agonists [51,52]. Since TLR2 is important for host defense against L. pneumophila [25], we evaluated LDH release from L. pneumophila-infected BMDMs pretreated with the TLR2 agonist PAM₃CSK₄. Similar to our previous results (S3 Fig), LDH release from BMDMs primed with PAM₃CSK₄ was unaffected by LegC4 (S4C Fig). These data suggest that LegC4-mediated restriction occurs independently of NLRP3- and caspase-1-dependent pyroptotic cell death and support an overarching model whereby subversion of PA28αβ by LegC4 augments lysosomal pathogen killing under inflammatory and oxidative stress conditions.

Ethics statement

All experiments involving animals were approved by the Kansas State University and Michigan State University Institutional Animal Care and Use Committees (KSU Protocols: 4022, 4501, and 4386; MSU protocol: PROTO202200365) and performed in compliance with the Animal Welfare Act and National Institutes of Health guidelines. Animals were euthanized in accordance with American Veterinary Medical Association Guidelines.

Bacterial Strains, plasmids, oligonucleotide primers, and reagents

L. pneumophila strains, plasmids, and primers used in this study are listed in Tables A, B, and C in S1 Text, respectively. L. pneumophila strains were cultured on supplemented charcoal N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (CYE) and grown at 37°C, as described [96]. Liquid cultures were grown overnight with shaking at 37°C in supplemented ACES-buffered yeast extract (AYE) medium, as described [20,97]. CYE was supplemented with 10 μg mL^-1 chloramphenicol for plasmid maintenance and legC4 expression was induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG; GoldBio) where indicated. Escherichia coli strains used for cloning (Top10; Invitrogen) and protein expression [BL21 (DE3); New England Biolabs] were grown at 37°C in Luria-Bertani (LB) medium supplemented with antibiotics as appropriate for plasmid selection [50 μg mL^-1 kanamycin (GoldBio), 25 μg mL^-1 chloramphenicol (GoldBio), or 100 μg mL^-1 ampicillin (GoldBio)]. Protein expression in BL21 (DE3) was induced with 1 mM IPTG. Unless otherwise indicated, all chemicals were obtained from Millipore Sigma (St. Louis, MO).

Mice and bone marrow-derived macrophages

Mice on C57BL/6 background were purchased from the Jackson Laboratories (Bar Harbor, Maine). Wild-type, Tnfr1^-/-, Ifngr1^-/-, and Myd88^-/- mice were purchased from live repositories and Psme1/2^-/- mice were recovered from cryopreserved embryos and bred for homozygotes at the Jackson Laboratories (Bar Harbor, ME). In-house colonies were maintained in specific pathogen-free conditions at Kansas State University and Michigan State University.

Bone marrow was harvested from seven- to twelve-week-old mice as described [98]. BMDMs were generated by differentiation in RPMI supplemented with 20% heat-inactivated fetal bovine serum (HI-FBS) (Biowest, Riverside, MO) and 15% L929 cell supernatant for 6 days prior to seeding for infection. Femurs from C57BL/6 Casp1^-/- mice were a gift from Dr. Russell Vance (University of California, Berkeley).

Competitive index

Six- to twelve-week-old sex- and age-matched mice were anesthetized with ketamine/xylazine and infected via the intranasal route as previously described [20]. Mixed bacterial inoculums (1:1) containing a total of 5 x 10⁶ bacteria were diluted and plated on selective medium (10 μg mL^-1 chloramphenicol for plasmid selection). At 48 h post-infection, mice were euthanized and homogenates from extracted lung tissue were plated on selective media, as described [20]. CFU were enumerated and used to calculate CI values [(CFUcm^R_48h/CFUwt_48h)/(CFUcm^R_IN)/CFUwt_IN)].

Molecular cloning

Plasmids were generated for stable and transient ectopic production in mammalian cells and recombinant protein production in E. coli. For production of 3xFLAG-LegC4, legC4 was amplified from L. pneumophila genomic (g)DNA using LegC4BamHI-F/LegC4NotI-R primer pairs (Table C in S1 Text) and cloned as a BamHI/NotI fragment into 3xFLAG 4/TO (Table B in S1 Text) [99]. To produce GFP-PA28α, psme1 was amplified from pCMV-3Tag-4a::psme1 (purchased from Genscript, Piscataway, New Jersey) using Psme1Sal1-F/Psme1BamHI-R primer pairs (Table C in S1 Text) and cloned as a Sal1/BamH1 fragment into pEGFPC1 (Clontech). For production of GST- and His₆-Myc-fusion proteins, open reading frames were cloned into pGEX-6P-1 and pT7HMT vectors, respectively. psme1 was PCR-amplified from pCMV-3Tag-4a::psme1 and legC4 PCR-amplified from L. pneumophila genomic (g)DNA using primers Psme1-BamHI-F/Psme1-NotI-R and LegC4-BamHI-F3/LegC4-NotI-R, respectively (Table C in S1 Text). Each of the amplified fragments were cloned as a BamHI/NotI fragment into either pGEX-GP-1 or pT7HMT. pGEX::lgt1 and pT7HMT::lgt1 were constructed previously [100]. Confirmation of DNA sequences was done by Sanger sequencing (Eton Biosciences Inc.). Plasmid DNA was transformed into chemically competent E. coli Top10 (Invitrogen) or BL21 (DE3) (New England Biolabs) for plasmid purification and recombinant protein production, respectively. Plasmid DNA was purified for transfection into mammalian cell lines using a ZymoPURE II Plasmid Maxiprep Kit (Zymo Research).

Recombinant protein production and affinity chromatography

E. coli BL21 (DE3) overnight cultures were sub-cultured into fresh LB medium containing the appropriate antibiotics at 1:100 and grown with shaking for 3h at 37°C. Recombinant protein production was induced with 1mM IPTG and cultures were grown overnight with shaking at 16°C. Bacteria were harvested by centrifugation at 1,400 r.c.f. for 10 min at 4°C, washed with ice-cold PBS and incubated in bacterial lysis buffer [50mM Tris pH 8.0, 100mM NaCl, 1mM EDTA, 200μg mL^-1 lysozyme, and complete protease inhibitor (Roche)] for 30 min on ice followed by addition of 2 mM DTT. Bacteria were sonicated and lysates were clarified by centrifugation at 21,000 r.c.f. for 15 min at 4°C, as described [100]. Clarified lysates harboring GST-fusion proteins were incubated with magnetic glutathione agarose beads (Pierce) for 1hr at 4°C with rotation and washed twice in Glutathione wash buffer [125mM Tris pH 7.4, 250mM NaCl, 1mM DTT, 1mM EDTA]. Clarified lysates harboring His₆-Myc-fusion proteins were added to the beads and incubated at 4°C with rotation for 1hr. Beads were washed twice with Glutathione wash buffer and proteins were eluted by boiling in Laemmli sample buffer. Proteins were separated by SDS-PAGE followed by Coomassie brilliant blue staining and Western blot (see below).

Cell culture and transfections

HEK 293T, HeLa, and RAW 264.7 cells (gifts from Dr. Craig Roy, Yale University) were maintained at 37°C and 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% HI-FBS. THP-1 cells were purchased from the American Type Culture Collection (ATCC) and maintained in RPMI supplemented with 10% HIFBS and differentiated with 100 nM phorbal 12-myristate 13-acetate (PMA) for 4 days prior to infection with L. pneumophila (see below). All cell lines were used between passage 4–20.

HEK 293T cells were transfected with purified plasmid DNA (Table A in S1 Text) for 24 h using calcium phosphate as described [101]. HeLa cells were transfected for 24 h using jetPRIME transfection reagent according to manufacturer’s guidelines. Two hours before transfection, HeLa cells were washed and incubated in low-serum media (DMEM/4% HIFBS). Media were replaced with DMEM 10% HIFBS four hours post-transfection and assayed 24 h post-transfection.

To generate RAW 264.7 cells with stable plasmid integrations, cells were transfected with pcDNA::3xflag-legC4 or pcDNA::3xflag vector (Table A in S1 Text) using a Nucleofector 2b electroporator (Lonza, Basel, Switzerland) and cultured over 14 days with Zeocin selection (200–1000 μg mL^-1). 3xFLAG-LegC4 expression was confirmed by Western blot and confocal microscopy using a-FLAG antibodies (see below) and stable RAW 264.7 cells were maintained in culture in the presence of 200 μg mL^-1 Zeocin. For L. pneumophila infections, cells were seeded one day prior to infection and infected in the absence of Zeocin.

Western blot

Boiled protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane using a BioRad TransBlot semidry transfer apparatus. Membranes were incubated in blocking buffer [5% non-fat milk powder dissolved in Tris-buffered saline-0.1% Tween-20 (TBST)]. Primary antibodies [rabbit α-FLAG (Sigma-Aldrich, F1804), rabbit α-Myc (Cell Signaling, 2278S), rabbit α-GFP (Abcam, ab6556)] were used at 1:1,000 in blocking buffer and detected with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000; ThermoFisher). Membranes were washed in TBST followed by addition of enhanced chemiluminescent (ECL) reagent (GE Amersham) and visualization using an Azure c300 Darkroom Replacer or a GE Amersham Imager 600.

Immunoprecipitation

Transiently transfected HEK 293T cells or RAW 264.7 cells were washed with phosphate-buffered saline and lysed in ice-cold NP-40 buffer [1% non-iodet P40 (v/v), 20 mM Tris pH 7.5, 150 mM NaCl, 10 mM Na₄P₂O₇, 50 mM NaF, complete protease inhibitor (Roche)]. Lysates were clarified and proteins were immunoprecipitated using α-FLAG M2 magnetic beads or protein G-conjugated Dynabeads (ThermoFisher) pre-incubated with rabbit α-GFP antibody (Abcam, ab6556), as indicated and according to manufacturer’s instructions. Input samples were collected from cell lysates prior to incubation with beads. Beads and input samples were resuspended in 3x Laemmli sample buffer for SDS-PAGE and Western blot analysis.

Protein carbonyl assays

Protein carbonyls were quantified using the OxiSelect Protein Carbonyl ELISA Kit (Cell Biolabs) following manufacturer’s instructions. Briefly, RAW 264.7 cells were seeded at 2x10⁶ in a 10 cm tissue culture dishes in DMEM 10% HIFBS. The next day, media were aspirated, and cells were incubated in DMEM/2.5% HIFBS in the presence or absence of 10 μM H₂O₂. After 24 h, cells were washed twice in PBS and lysed in ice-cold 500 μL of detergent-free lysis buffer [25 mM HEPES pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 2% glycerol (v/v)] supplemented with complete protease inhibitor cocktail (Roche). Cell lysates were scraped into a pre-chilled microcentrifuge tube and incubated with agitation for 30 min at 4°C followed by sonication at 40% intensity three times in 10 second intervals. Lysates were clarified by centrifugation at 4°C for 20 min at 13,000 r.c.f. Supernatants were transferred to a fresh tube, snap frozen in liquid nitrogen for 2 min and stored at -80°C until use. Protein concentrations were quantified using a Coomassie Plus (Bradford) Protein Assay (Pierce) and diluted to 10 μg mL^-1. Samples and standards were adsorbed to a 96-well protein binding plate and ELISA was performed according to manufacturer’s instructions. Absorbance at 450 nm was quantified on a BioTek Epoch2 microplate reader.

Protein carbonyls were visualized using the OxiSelect Protein Carbonyl Immunoblot Kit (Cell Biolabs). Tnrf1^-/- BMDMs were seeded in 24-well plates at 2.5x10⁵ one day prior to infection with indicated L. pneumophila strains at an MOI of 50 in the presence or absence of 10 μM H₂O₂. One hour after infection, cells were washed 3x with PBS^-/-, media were replaced and cells were incubated for an additional 3 h in the presence or absence of H₂O₂. Cells were washed in ice-cold PBS and lysed in 120 μL ice-cold NP-40 lysis buffer (see above). Lysates were diluted in 3x Laemmli sample buffer and boiled for 10 min. Proteins were separated on a 4–20% gradient SDS-PAGE gel and transferred to PVDF membrane using a BioRad TransBlot semi-dry transfer apparatus. Membranes were processed according to manufacturer’s instructions using 10 min wash steps. Blots were stripped and re-probed with rabbit α-β-actin antibody (1:1000; Cell Signaling Technology) followed by goat-α-rabbit-HRP (1:5000; ThermoFisher). Blots were visualized as described above and densitometry was performed using Fiji ImageJ and Adobe Photoshop software.

Immunofluorescence microscopy

To quantify LAMP1+ LCVs in naïve macrophages, 1x10⁵ BMDMs were seeded on poly-L-lysine (PLL)-coated glass coverslips in 24-well plates and infected with L. pneumophila at a multiplicity of infection (MOI) of 25 in triplicates. At 1h post-infection, cells were washed 3x in PBS to remove extracellular bacteria and cells were incubated for an additional 8 h in fresh media was added. Quantification of LAMP1+ LCVs in rTNF-treated BMDMs was performed as described but media were supplemented with 25 ng mL^-1 rTNF throughout the course of infection, which was allowed to proceed for an additional 5 h after washing in PBS. Coverslips were fixed with 4% paraformaldehyde (PFA; Thermo Scientific), permeabilized in ice cold methanol, and blocked with blocking buffer [0.1% saponin (w/v), 1% HIFBS (v/v), 0.5% bovine serum albumin (BSA; w/v) in PBS]. Coverslips were stained with 1:1,000 rabbit α-L. pneumophila (Invitrogen, PA17227), 1:2,000 rat α-LAMP1 (Developmental Studies Hybridoma Bank) primary antibodies and 1:500 Alexa 488-conjugated goat α-rabbit and Alexa594-conjugated goat α-rat secondary antibodies (ThermoFisher) in blocking buffer. Nuclei were stained with Hoechst (ThermoFisher) at 1:2,000. Coverslips were mounted on glass slides with ProLong Gold Antifade Mountant (ThermoFisher). LAMP1+ LCVs were scored on a Leica DMiL LED inverted epifluorescence microscope (n = 300 cells/strain) and the researcher scoring the samples were blind to their identity.

For confocal microscopy of transfected cells, HeLa cells were seeded on PLL-coated coverslips and transfected as described above. Coverslips were fixed in 4% PFA and processed as described above using mouse α-FLAG M2 (Sigma; 1:1,000) and rabbit α-GFP (Abcam; ab6556; 1:5,000) primary antibodies and Alexa546-conjugated goat α-mouse and Alexa488-conjugated goat α-rabbit antibodies (ThermoFisher; 1:500). Images were captured on a Zeiss LSM-5 PASCAL laser scanning confocal microscope (KSU Division of Biology Microscopy Facility). Co-localization was scored using the Pearsons algorithm for individual cells (n = 10) using the colocalize tool Fiji ImageJ software.

For confocal microscopy of infected macrophages, 1.5x10⁵ cells were seeded on PLL-coated coverslips and infected as described above. Coverslips were fixed with 4% PFA and processed as above using rabbit α-L. pneumophila (Millipore Sigma; 1:1000) and mouse α-PA28α (SantaCruz; 1:100) primary antibodies, Alexa488-conjugated goat α-rabbit and Alexa594-conjugated goat α-mouse secondary antibodies, and Hoechst (ThemoFisher; 1,2000). Images were captured using an Olympus FluoView FV1000 confocal Laser scanning microscope (MSU Center for Advanced Microscopy).

L. pneumophila intracellular growth curves

To quantify L. pneumophila intracellular replication, macrophages were seeded in 24-well tissue culture plates and infected in triplicates at an MOI of 1 the next day. Differentiated BMDMs were seeded at 2.5x10⁵ in seeding media (RPMI, 10% HIFBS, 7.5% L929 cell supernatant), THP-1 cells were seeded at 5x10⁵ and differentiated with 100 nM PMA in RPMI 10% HIFBS for three days. Cells were washed with PBS and incubated in the absence of PMA for one day prior to infection. Stable RAW 264.7 cells were seeded at 2.5x10⁵ in DMEM 2.5%HIFBS. At 1 h post-infection, cells were washed 3x with PBS followed by addition of fresh media. Macrophages were lysed in sterile water and colony forming units (CFU) were enumerated at the indicated time points as described [20]. Where indicated, 25 ng mL^-1 rTNF (Gibco), 20–50 μM H₂O₂ (VWR), 10 nM bafilomycin A1 (BAF; ApexBio), or 1 μM MCC950 (ApexBio) were added at the time of infection and maintained throughout. DMSO was used as a vehicle control where indicated.

Enzyme-linked immunosorbent assay (ELISA)

To quantify TNF secretion, 2.5 x 10⁵ BMDMs were seeded in 24-well tissue culture plates and infected with the indicated L. pneumophila strains at an MOI of 10. After 1 h of infection, cells were washed with PBS, and media were replaced. Supernatants were at 8 h post-infection and used fresh or stored at -20°C for up to 1 week and TNF was quantified using mouse TNF-α ELISA MAX kit (BioLegend) following manufacturer’s instructions. Absorbance at 450 nm was quantified on a BioTek Epoch2 microplate reader.

Cytotoxicity assay

To quantify cell death, lactate dehydrogenase (LDH) activity in cell supernatants was quantified. BMDMs were seeded in triplicates at 2.5x10⁵ in a 24-well tissue culture plate for 24 h and infected with the indicated L. pneumophila strains at an MOI of 10 in 500 μL of seeding media (RPMP/10% HIFBS/7.5% L929 cell supernatant) and incubated for the indicated times. Supernatants were transferred to a 96-well plate and centrifuged at 200 r.c.f. for 10 min. LDH was quantified using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) according to manufacturer’s instructions. Absorbance at 490 nm was quantified on a BioTek Epoch2 microplate reader and percent cytotoxicity was calculated by normalizing absorbance values to cells treated with lysis buffer. Where indicated, cells were primed with 1 μM PAM₃CSK₄ (Tocris) for 24 h prior to infection and plasmid expression of legC4 was induced with 1 mM IPTG.

Statistical analysis

Statistical analyses were performed with GraphPad Prism 9 software using either Students’ t-test, or two-way ANOVA, as indicated, with a 95% confidence interval. Data are presented as mean ± standard deviation (s.d.) or standard error of the mean (s.e.m.) and, unless otherwise indicated, statistical analyses were performed on samples in triplicates.