Abstract
Legionella pneumophila is ubiquitous in freshwater environments where it replicates within unicellular protozoa. However, L. pneumophila is also an accidental human pathogen that can cause Legionnaires’ Disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes, L. pneumophila utilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction of L. pneumophila. We found previously that the effector LegC4 is important for L. pneumophila replication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires’ Disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuates L. pneumophila replication. We found that LegC4 enhanced restriction of L. pneumophila replication within macrophages activated with tumor necrosis factor (TNF) or interferon (IFN)-γ. Specifically, TNF-mediated signaling was required for LegC4-mediated attenuation of L. pneumophila replication within macrophages. In addition, expression of legC4 was sufficient to restrict L. longbeachae replication within TNF- or IFN-γ-activated macrophages. Thus, this study demonstrates that LegC4 contributes to L. pneumophila clearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.
Importance Legionella are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controls Legionella infection due in part to rapid and robust production of pro-inflammatory cytokines resulting from detection of Dot/Icm-translocated substrates, including effectors. Here, we demonstrate that the effector LegC4 enhances pro-inflammatory host restriction of Legionella from macrophages. These data suggest that LegC4 may augment pro-inflammatory signaling or antimicrobial activity of macrophages, a function that has not previously been observed for another bacterial effector. Further insight into LegC4 function will likely reveal novel mechanisms to enhance immunity against pathogens.
Introduction
Legionella are natural pathogens of unicellular protozoa and accidental pathogens of humans that can cause in a severe inflammatory pneumonia called Legionnaires’ Disease, which results from uncontrolled bacterial replication within alveolar macrophages. To replicate within eukaryotic phagocytes, Legionella subvert normal endocytic signaling by establishing a specialized compartment called the Legionella containing vacuole (LCV). To form the LCV and replicate intracellularly, Legionella employ a Dot/Icm type IV secretion system (T4SS) to translocate virulence factors - called effector proteins - into host cells (1). Although >15 Legionella species are capable of causing human disease, the overwhelming majority is caused by L. pneumophila (2, 3). In healthy individuals, L. pneumophila infection is efficiently controlled and human-to-human transmission is incredibly rare (4). This is due to efficient detection and subsequent clearance of L. pneumophila by the mammalian innate immune system. Consequently, L. pneumophila is a well-established model pathogen used to characterize mechanisms of host defense against bacterial pathogens.
Innate immune detection of bacterial pathogens is facilitated by host pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPs). Surface toll-like receptors (TLRs) are PRRs critical for host defense against L. pneumophila. The majority of TLRs signal through the adaptor MyD88 to activate pro-inflammatory gene expression. Mice lacking MyD88 are highly susceptible to L. pneumophila infection, which is mostly due to lack of interleukin (IL)-1 and TLR2-mediated signaling (5-7). Intracellular PRRs such as Nod1, Nod2, and inflammasomes also contribute to innate immune restriction of L. pneumophila in macrophages [reviewed in (8, 9)]. Leakage of PAMPs through the Dot/Icm pore amplifies cell-autonomous restriction of L. pneumophila within immune phagocytes. Specifically, recognition of L. pneumophila flagellin monomers (flaA) by the NAIP5/NLRC4 inflammasome is sufficient to restrict L. pneumophila replication within macrophages (10, 11). In addition, translocation of peptidoglycan, nucleic acids, and lipopolysaccharide into the host cell cytosol through the Dot/Icm pore also enhances restriction of L. pneumophila. Engagement of both extracellular and intracellular PRRs results in a robust pro-inflammatory response mediated by secretion of cytokines by infected and bystander immune phagocytes (5, 12-16). In particular, tumor necrosis factor (TNF) and interferon (IFN)-γ are critical for restriction of pulmonary L. pneumophila infection (17-21). TNF and IFN-γ both promote cell autonomous defense against L. pneumophila within macrophages and mediate bacterial killing by increasing phagolysosmal fusion (22, 23).
In addition to canonical PAMPs, translocated effectors can augment pro-inflammatory responses in L. pneumophila-infected macrophages. For example, effector-mediated inhibition of host protein translation results in increased expression of pro-inflammatory genes in macrophages (24-27). In addition, macrophage pro-inflammatory gene expression was decreased during infection with Legionella that possess a functional Dot/Icm T4SS but are unable to translocate a subset of effectors due to a mutation in the icmS effector chaperone gene (28). These studies elaborate the concept of effector-triggered immunity in animal cells (29) and provide further evidence for the contribution of effectors to innate immune restriction of L. pneumophila.
We recently demonstrated that the effector LegC4 attenuates L. pneumophila fitness in a mouse model of Legionnaires’ disease (30). Loss-of-function mutation of the legC4 gene conferred a fitness advantage on L. pneumophila in the mouse lung as evidenced by increased pulmonary bacterial burden and the ability to outcompete the wild-type strain (30). However, the legC4 mutation had no effect on L. pneumophila replication in primary bone-marrow derived macrophages (BMDMs) and impaired replication within a natural amoeba host, Acanthamoeba castellanii (30). Furthermore, expression of legC4 from a plasmid further attenuated L. pneumophila fitness in the mouse lung compared to the wild-type strain. Thus, we hypothesized that LegC4 is deleterious to L. pneumophila in the presence of cell-mediated innate immunity.
The present study was designed to determine how LegC4 augments restriction of L. pneumophila in mammalian hosts. Expression of legC4 from a plasmid was sufficient to attenuate L. pneumophila replication in BMDMs, which relied on TNF secretion and subsequent signaling. Moreover, a ΔlegC4 mutant exhibited increased replication in cytokine-activated BMDMs. Interestingly, expression of legC4 was sufficient to attenuate L. longbeachae replication within TNF- and IFN-γ-activated BMDMs. These results suggest that LegC4 enhances macrophage cell autonomous defense against Legionella by potentiating cytokine-mediated restriction.
Materials and Methods
Bacterial strains, plasmids, primers, growth conditions
Legionella pneumophila Philadelphia-1 SRS43 (30), SRS43 flaA::Tn (30), Lp02 ΔflaA (10), Lp03 (31), and Escherichia coli strains were gifts from Dr. Craig Roy (Yale University). L. longbeachae NSW 150 was a gift from Dr. Hayley Newton (University of Melbourne). Escherichia coli strains used for cloning (Top10, Invitrogen) and L. pneumophila mutagenesis [DH5α-λpir, (32)] were maintained in Luria-Bertani (LB) medium supplemented with 25 μg mL-1 chloramphenicol (pJB1806 and pSN85) or 50 μg mL-1 kanamycin (pSR47s). Legionella strains were cultured on supplemented charcoal N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (CYE) and grown at 37°C as described (33). L. pneumophila Lp02 strains were maintained on CYE supplemented with 100 μg mL-1 thymidine. Liquid cultures were grown at 37°C with aeration in supplemented ACES-buffered yeast extract (AYE) as described (33, 34). When necessary, media were supplemented with 10 μg mL-1 chloramphenicol (plasmid maintenance), 10 μg mL-1 kanamycin (allelic exchange) or 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG).
Where indicated, recombinant mouse interferon-γ (rIFN-γ; Thermo Fisher Scientific), recombinant mouse tumor necrosis factor (rTNF; Gibco), rat α-mouse TNF antibody (α-TNF; R&D Systems), or normal rat IgG control (Rat IgG; R&D Systems) were used at a concentration of 50ng mL-1.
A complete list of oligonucleotide primers used in this study is shown in Table 1.
Molecular cloning, plasmid construction and generation of Legionella strains
In-frame deletions of legC4 were generated by allelic exchange. Plasmids pSR47s:: ΔlegC4 and pSR47s::ΔflaA, gifts from Dr. Craig Roy, were conjugated into SRS43 or Lp02 followed by selection for L. pneumophila deletions as described (35, 36). Sucrose-resistant, kanamycin-sensitive colonies were screened by PCR using legC4KO-up/legC4KO-down and flaAKO-up/flaAKO-down primer pairs for ΔlegC4 and ΔflaA deletions, respectively.
To express legC4 on a plasmid under its endogenous promoter, legC4 plus the 300 base-pair upstream region was amplified with the LegC4BglII-F/LegC4XbaI-R primer pair and cloned as a BglII/XbaI fragment into BamHI/XbaI-digested pJB1806 (pJB) (37).
For IPTG-mediated expression of legC4, the legC4 gene was amplified using the primer pair LegC4BamHI-F/LegC4XbaI-R and cloned as a BamHI/XbaI fragment into BamHI/XbaI- digested pSN85, a gift from Dr. Craig Roy (pEV; N-terminal 3XFLAG epitope tag fusion (38)). Sequence-confirmed pJB1806::plegC4 (pJB::plegC4) and pSN85::legC4 (plegC4) plasmids and empty vectors were transformed into Legionella strains as previous described (39). IPTG-induced expression of 3xFLAG-legC4 was confirmed by Western blot analysis (data not shown).
Mice and BMDMs
C57BL/6 wild-type, Myd88-/-, and Tnfr1-/- breeding pairs were purchased from the Jackson Laboratories (Bar Harbor, Maine) and in-house colonies were maintained in specific pathogen-free conditions at Kansas State University. All experiments involving animals were approved by the Kansas State University Institutional Animal Care and Use Committee (protocol #4022) and performed in compliance with the Animal Welfare Act and NIH guidelines.
Bone marrow was harvested from mice as previous described (40). Bone marrow-derived macrophages were generated by differentiation in RPMI supplemented with 20% heat-inactivated fetal bovine serum (HI-FBS) (Gibco) and 15% L929 cell supernatant for 6 days prior to seeding for infection.
Competitive index (CI) experiments in mice
Six-to ten-week old age and sex-matched C57BL/6 mice were infected for competitive index experiments as previously described (30). Mixed bacterial inoculums (1:1) were diluted and plated on selective medium (5 μg mL-1 for flaA::Tn and 10 μg mL-1 for plasmid selection). At 48 h p.i., mice were euthanized and whole lung tissue was harvested. Lung tissue was homogenized in 300 μL of sterile water using a Bullet Blender (Next Advance) as described (41) and dilutions were plated on selective medium as above. Colony forming units (CFUs) were enumerated and used to calculate CI values [(CFUcmR48h/CFUwt48h)/(CFUcmRIN/CFUwtIN).
Quantification of Legionella replication within macrophages
Differentiated BMDMs were maintained in RPMI supplemented with 10% HI-FBS (Gibco) and 7.5% L929 cell supernatant. BMDMs were seeded 2.5 x 105/well in 24-well plates one day prior to infection. BMDMs were infected with the indicated strains of L. pneumophila or L. longbeachae at a multiplicity of infection (MOI) of 1 in the presence or absence of 1 mM IPTG and/or recombinant cytokine as indicated. At 1 h p.i., cell monolayers were washed three times with PBS-/- and fresh supplemented medium was added. Infections were allowed to proceed for up to 72 h or for 48 h, as indicated. To enumerate CFUs, BMDMs were lysed in sterile water for 8 mins followed by repeat pipetting. Lysates were diluted as appropriate and plated on CYE agar plates, which were then incubated at 37° for 4 days. For growth curve experiments, bacteria were enumerated after 1 h of infection and every 24 h thereafter for up to 72 h. To quantify fold replication, BMDMs were infected for 1 h and 48 h and fold replication was enumerated by normalization of the 48 h CFU counts to the 1 h CFU counts.
Enzyme-linked immunosorbent assay (ELISA)
BMDMs were seeded in a 24-well plate at 2.5 x 105/well 1 day prior to infection. The indicated Lp02 or SRS43 strains were used to infect the BMDMs (n = 3) at an MOI of 30 or 10, respectively. Infections with Lp02 strains were performed in the absence of exogenous thymidine to prevent bacterial replication. One-hour p.i., media were aspirated and cells were washed 3 times with PBS. Media were replaced and supernatants were collected after 5 h. Supernatants were either used fresh or stored at -20°C for up to 1 week followed by quantification of TNF using a Mouse TNF ELISA Kit (BioLegend) following manufacturer’s instructions.
Western blot
To confirm production of 3XFLAG-LegC4 protein from Legionella, suspensions of strains harboring either pSN85 alone (pEV) or pSN85::legC4 (plegC4) induced with IPTG were lysed by boiling in 3X Laemmli buffer. Proteins were separated by SDS-PAGE followed by transfer to polyvinylidene difluoride (PVDF) membrane (ThermoFisher) using a wet transfer cell (BioRad). Membranes were incubated in blocking buffer [5% non-fat milk dissolved in Tris-buffered saline/0.1% Tween 20 (TBST)]. Anti-FLAG (clone M2, Sigma) was diluted at 1:1000 in blocking buffer and incubated with membranes either overnight at 4°C or at ambient temperature for 3 h with rocking. Wash steps were performed 3 times for 10 min each in TBST. HRP-conjugated goat-α-mouse HRP (Sigma) was diluted in blocking buffer at 1:5000 and incubated with membranes for 1-2 hours at room temperature with rocking. Membranes were washed, incubated with ECL substrate (Amersham) and imaged by chemiluminescence using a c300 Azure Biosystems Darkroom Replacer.
Statistical analysis
Statistics were performed with GraphPad Prism software using either Mann-Whitney U test or Students’ t-test, as indicated, with a 95% confidence interval. In all experiments, error bars denote standard deviation (± S.D.) of samples in triplicates.
Results
LegC4 confers a fitness disadvantage on non-flagellated L. pneumophila in wild-type mice
We found previously that the L. pneumophila effector LegC4 is detrimental to bacterial replication in the mouse lung (30). These experiments were performed using mice and macrophages deficient for production of the NLRC4 inflammasome (Nlrc4-/-) to prevent flagellin-mediated restriction of L. pneumophila replication (10, 30, 42). To confirm that LegC4-mediated phenotypes were not due to loss of NLRC4, we examined fitness of a legC4-deficient L. pneumophila (ΔlegC4) strain in the lungs of wild-type C57BL/6 mice using competitive index (CI) experiments. To prevent NLRC4-mediated restriction of bacterial replication we generated flagellin (flaA) loss-of-function mutations in our wild-type (ΔflaA) and legC4 mutant (ΔflaAΔlegC4) strains (see Materials and Methods). We also used a previously generated flaA::Tn mutant to facilitate selective plating since the transposon confers resistance to chloramphenicol (30). Mice were infected intranasally with a 1:1 mixture of L. pneumophila ΔflaAΔlegC4 and flaA::Tn for 48 h. Lung tissue was subsequently homogenized and plated on selective media for CFU enumeration and calculation of CI values (see Materials and Methods). The ΔflaAΔlegC4 strain significantly outcompeted the flaA::Tn mutant in the lungs of wild-type mice, as evidenced by average CI values significantly greater than 1.0 (P < 0.01; Fig 1A). Our previous study also revealed that expression of legC4 from a multi-copy plasmid conferred a fitness disadvantage on L. pneumophila compared to the wild-type strain (30). To confirm these results in wild-type mice, we generated a strain of L. pneumophila ΔflaAΔlegC4 harboring a plasmid encoding legC4 under control of its endogenous promoter (pJB::plegC4). Plasmid expression of legC4 (pJB::plegC4) resulted in significantly impaired fitness defect compared with the ΔflaA parental strain, which was not observed for a ΔflaAΔlegC4 strain harboring vector alone (pJB) (P<0.05, Fig 1B). These data demonstrate that NLRC4 does not affect LegC4-mediated attenuation of L. pneumophila replication in the mouse lung. To fully evaluate LegC4-mediated phenotypes, the remainder of our study was performed using flaA-deficient strains and bone marrow-derived macrophages (BMDMs) derived from wild-type mice.
Plasmid expression of legC4 attenuates L. pneumophila replication in BMDMs
Since plasmid expression of legC4 attenuated L. pneumophila fitness in the mouse lung, we examined whether this also occurred in macrophages ex vivo using BMDMs derived from wild-type mice. We quantified bacterial replication within BMDMs over 72 h. Consistent with our previous study, loss of endogenous legC4 (ΔflaAΔlegC4) does not affect replication of L. pneumophila within primary mouse BMDMs compared to the parental strain (ΔflaA) (30) (Fig 2A). However, L. pneumophila ΔflaAΔlegC4 (pJB::plegC4) was significantly attenuated for replication in BMDMs at 48 and 72 h p.i. compared to the empty vector control strain (Fig 2B). Furthermore, IPTG-induced expression of legC4 from a plasmid (plegC4) also resulted in impaired L. pneumophila replication compared to the control strain (pEV) (P<0.01, Fig 2C). Fitness defects associated with plasmid expression of legC4 were specific to intracellular replication since replication in rich media in vitro was unaffected (data not shown). These data demonstrate that increased levels of LegC4 are detrimental to L. pneumophila intracellular replication in macrophages.
LegC4-mediated restriction of L. pneumophila replication is dependent on cytokine production
We subsequently investigated the mechanism by which plasmid expression of legC4 attenuates L. pneumophila replication. In BMDMs, L. pneumophila infection results in production of pro-inflammatory cytokines through engagement of TLRs by bacterial ligands. Indeed, we previously reported that BMDMs infected with L. pneumophila expressing legC4 secreted increased levels of interleukin (IL)-12 (30). However, increased levels of IL-12 would likely not be sufficient to attenuate L. pneumophila intracellular replication within BMDMs. Like IL-12, tumor necrosis factor (TNF) is a pro-inflammatory cytokine expressed downstream of toll-like receptors (TLRs) in macrophages. TNF is important for host defense against L. pneumophila in mice and humans and TNF-mediated signaling is sufficient to restrict L. pneumophila intracellular replication within macrophages (17-19, 23, 43). Thus, increased TNF signaling could account for LegC4-mediated attenuation of L. pneumophila intracellular replication.
We hypothesized that plasmid expression of legC4 would be sufficient to increase TNF secretion from L. pneumophila-infected BMDMs. Wild-type BMDMs were infected with ΔflaA, ΔflaAΔlegC4, ΔflaAΔlegC4 (plegC4), or ΔflaAΔlegC4 (pEV) for 6 h and secreted TNF was quantified by ELISA (see Materials and Methods). Significantly greater concentrations of TNF were present in the supernatants of cells infected with L. pneumophila expressing legC4 from a plasmid (P<0.01, Fig 3A). Increased TNF secretion was not due to differences in bacterial replication, since all strains replicated to similar levels at 6 h p.i. (Fig 3B). We observed the same phenotype following infection with strains constructed in the Lp02 background, which is a thymidine auxotroph (thyA-) that is metabolically active but does not replicate in the absence of exogenous thymidine (Fig S1). Thus, overexpression of legC4 results in enhanced TNF secretion from L. pneumophila-infected BMDMs.
To determine if TNF secretion contributed LegC4-mediated attenuation of intracellular replication, we evaluated L. pneumophila replication within Myd88-/- BMDMs. Since attenuated L. pneumophila replication within BMDMs associated with LegC4 were observed at 48 h p.i., we quantified fold replication of the indicated strains at this time point (see Materials and Methods). Plasmid expression of legC4 impaired L. pneumophila intracellular replication within wild-type, but not Myd88-/-, BMDMs (Fig 3B). As expected, TNF was not secreted from Myd88-/- BMDMs under any of our experimental conditions [(44) & data not shown]. Together, these data suggest that pro-inflammatory cytokine production contributes to LegC4-mediated attenuation of L. pneumophila intracellular replication in BMDMs.
To further characterize LegC4-mediated restriction of L. pneumophila replication within BMDMs, L. pneumophila replication was evaluated in the absence of TNF signaling. To determine if TNF signaling contributed to legC4-mediated attenuation of L. pneumophila replication, we neutralized TNF in the supernatants of infected wild-type BMDMs using an α-TNF antibody. Wild-type BMDMs were infected with L. pneumophila ΔflaAΔlegC4 harboring plegC4 or pEV in the presence of either α-TNF, Rat IgG isotype control antibody, or neither and fold replication at 48 h p.i. was quantified. Plasmid expression of legC4 resulted in significantly attenuated L. pneumophila replication within untreated and Rat IgG-treated BMDMs (P<0.05); however, α-TNF antibody neutralization of TNF restored replication of the legC4 overexpressing strain to wild-type levels (Fig 4A). We subsequently examined replication of these strains in BMDMs deficient for signaling from TNF receptor-1 (TNFR1). Tnfr1-/- BMDMs were infected with L. pneumophila ΔflaAΔlegC4 harboring either plegC4 or pEV and fold replication was quantified at 48 h p.i. As previously observed, over-expression of legC4 impaired intracellular replication within wild-type BMDMs (P<0.05, Fig 4B). However, there was no difference in fold replication of the legC4-overexpressing strain compared to the empty vector control within Tnfr1-/- BMDMs (Fig 4B). Interestingly, overexpression of legC4 resulted in significantly increased L. pneumophila replication within Tnfr1-/- BMDMs (P<0.01, Fig 4B). These data demonstrate that TNF signaling contributes to LegC4-mediated attenuation of L. pneumophila replication within BMDMs.
Endogenous LegC4 exacerbates TNF-mediated restriction of L. pneumophila from BMDMs
To further characterize LegC4-mediated restriction of L. pneumophila from BMDMs, we examined replication of L. pneumophila in BMDMs activated with recombinant mouse TNF (rTNF). Wild-type BMDMs were infected with L. pneumophila ΔflaA or ΔflaAΔlegC4 in the presence or absence of 50 ng/mL rTNF and fold replication was quantified at 48 h p.i L. pneumophila ΔflaAΔlegC4 replicated to significantly greater levels than the parental ΔflaA strain in rTNF treated BMDMs (P<0.01, Fig 4C). As reported above, loss of endogenous legC4 does not affect L. pneumophila replication within untreated BMDMs (Fig 4C). These data show that endogenous levels of LegC4 can augment TNF-mediated restriction of L. pneumophila replication.
LegC4 impairs L. pneumophila replication in interferon (IFN)-γ activated BMDMs
Interferon (IFN)-γ plays a major role in host defense against L. pneumophila in the lung (20, 45). To determine if LegC4-mediated impairment of L. pneumophila intracellular replication was specific to TNF, we examined bacterial replication within IFN-γ-activated BMDMs. Fold replication of L. pneumophila ΔflaA, ΔflaAΔlegC4, ΔflaAΔlegC4 (pEV) or ΔflaAΔlegC4 (plegC4) within wild-type BMDMs activated with recombinant mouse IFN-γ (rIFN-γ) was quantified. We found that overexpression of legC4 significantly attenuated L. pneumophila replication within IFN-γ-activated BMDMs (P< 0.05; Fig 5A). The L. pneumophila ΔflaAΔlegC4 mutant replicated to higher levels in IFN-γ-activated BMDMs compared to the parental ΔflaA strain (Fig 5B). Although the replication difference was not statistically significant (P=0.0918), the trend was consistently observed. Together, these data suggest that IFN-γ-mediated restriction of L. pneumophila replication is also augmented by LegC4.
Macrophage activation by IFN-γ results in increased TNF production from macrophages (46, 47). To determine if LegC4-mediated restriction of L. pneumophila within IFN-γ activated macrophages was due to TNF signaling, we quantified L. pneumophila replication within Tnfr1-/- BMDMs treated with IFN-γ. Overproduction of LegC4 resulted in significantly decreased L. pneumophila replication in IFN-γ-activated TNFR1-/- BMDMs compared to the control strain (P<0.001; Fig 5C). Thus, LegC4 can also augment non-TNF-mediated restriction of L. pneumophila replication through IFN-γ.
LegC4 impairs L. longbeachae replication within cytokine-activated BMDMs
Legionella longbeachae replicates within eukaryotic phagocytes using a Dot/Icm secretion system (39). Although the L. longbeachae Dot/Icm secretion system is highly similar to that of L. pneumophila, their effector repertoires are quite distinct and L. longbeachae does not encode a homolog of legC4 (48, 49). Importantly, L. longbeachae is more virulent than L. pneumophila and lethal in a mouse model of infection. To determine if LegC4 could attenuate bacterial replication in non-pneumophila Legionella, we generated L. longbeachae strains either expressing legC4 (plegC4) or harboring the empty vector (pEV). Wild-type BMDMs were infected with these L. longbeachae strains and fold-replication at 48 h was quantified. Expression of legC4 did not impair L. longbeachae replication within BMDMs; however, legC4 expression did result in significantly attenuated L. longbeachae replication within rTNF- and rIFN-γ-treated BMDMs compared to the control strain (P<0.001; Fig 6). These data suggest that LegC4 can augment cytokine-mediated restriction of non-pneumophila Legionella within BMDMs.
Discussion
The data presented in this study support the hypothesis that LegC4 potentiates cytokine-mediated host defense against Legionella. Our previous work (30) identifying LegC4 as contributing to L. pneumophila clearance from the lung was performed using flagellated L. pneumophila in a NLRC4-deficient (Nlrc4-/-) mouse model. To fully evaluate the mechanisms of LegC4-mediated clearance, utilized wild-type mice and BMDMs. Consistent with our previous study (30), we found that loss-of-function mutation in the legC4 gene (ΔlegC4) conferred a fitness advantage on L. pneumophila ΔflaA within the wild-type mouse lung. Moreover, complementation of the ΔlegC4 mutation by a plasmid encoding legC4 in trans conferred a fitness disadvantage on L. pneumophila compared to the parental strain. Also consistent with our previous report, L. pneumophila ΔflaAΔlegC4 replication within BMDMs did not differ from replication of the ΔflaA strain. However, in the present study, we found that plasmid expression of legC4 was sufficient to attenuate L. pneumophila replication within BMDMs. Although legC4 was expressed downstream of its endogenous promoter, an exaggerated phenotype likely occurred due to expression from a multi-copy plasmid, suggesting a potential dose-response. Importantly, this strain provided us with a tool to increase the magnitude of LegC4-mediated fitness attenuation within cultured cells. These phenotypes were corroborated by the observation that endogenous LegC4 was deleterious in cytokine-activated BMDMs. We further found that the fitness disadvantage associated with plasmid expression of legC4 was abolished in macrophages deficient for TNF-mediated signaling, suggesting that LegC4 is able to exacerbate cytokine-mediated antimicrobial responses. Finally, we determined that LegC4 could impair replication of L. longbeachae in cytokine-activated macrophages. Together, these data suggest that LegC4 potentiates cytokine-mediated restriction of L. pneumophila within macrophages.
Inflammation is mediated primarily through cytokine secretion, which is critical for restriction of L. pneumophila replication in vivo. This has been evidenced by the inability of Myd88-/- mice to control L. pneumophila replication. Specifically, Myd88-/- BMDMs will not secrete TNF during infection. The inability of plasmid-expressed legC4 to attenuate L. pneumophila replication in Myd88-/- BMDMs is likely due to lack of TNF signaling. In addition, LegC4-mediated increases in TNF secretion may amplify Tnf expression, which would further restrict L. pneumophila replication.
Pro-inflammatory cytokines contribute to host defense against L. pneumophila in vivo and in cultured macrophages (8, 50). Mice deficient for TNF-mediated signaling have increased pulmonary bacterial burdens and can succumb to infection (23, 43). TNF can signal through both TNFR1 and TNFR2; however, TNFR1-mediated signaling is primarily responsible for L. pneumophila restriction within alveolar macrophages in vivo (23) and is potentiated by LegC4. In the lung, multiple cell types contribute to TNF production, a consequence of which would be higher local TNF concentrations (16, 18, 23, 51). In addition, production of IFN-γ during L. pneumophila infection in vivo is mediated primarily by circulating natural killer (NK) cells (45, 52).
Our observation that the L. pneumophila ΔlegC4 mutant had fitness advantage compared to wild-type in the mouse lung but not in cultured macrophages suggested that LegC4 was detrimental to replication under specific environmental conditions. This was supported by the observation that attenuated L. pneumophila replication was correlated with increased TNF secretion from BMDMs. Since the L. pneumophila-infected lung is an inflammatory environment, we examined whether cytokine-mediated restriction was exacerbated by LegC4. Abrogation of signaling from TNFR1 was sufficient to alleviate LegC4-mediated restriction of intracellular replication. Increased replication of the ΔlegC4 mutant within rTNF-treated BMDMs strongly suggests that pro-inflammatory responses are exacerbated by LegC4. This conclusion was corroborated by the observation that the ΔlegC4 mutant consistently replicated to higher levels within IFN-γ-activated macrophages compared to untreated macrophages.
Similar to L. pneumophila, L. longbeachae replicates within an LCV by employing a Dot/Icm secretion system and a repertoire of translocated effector proteins (39). Despite high levels of homology between the Dot/Icm secretion systems of these two organisms, the effector repertoires are quite diverse and L. longbeachae does not encode a homolog of legC4 (48, 49). In contrast to L. pneumophila, L. longbeachae is highly virulent in a mouse model of Legionnaires’ disease (53, 54). Lethality in mice is likely due to L. longbeachae being poorly immunostimulatory and failing to induce substantial levels pro-inflammatory cytokines during infection. However, pro-inflammatory cytokines contribute to host defense against L. longbeachae in BMDMs and in vivo (53). Since inter-species translocation of Dot/Icm effectors by Legionella has been previously observed (39, 55), we introduced legC4 into L. longbeachae. Production of LegC4 by L. longbeachae resulted in significantly attenuated replication within cytokine treated, but not untreated, BMDMs. These data reinforce our previous observations and demonstrate that LegC4-mediated restriction is not specific to L. pneumophila. Since L. longbeachae infection does not induce appreciable TNF secretion from BMDMs (53), it is likely that the concentration of TNF secreted by these cells is too low to permit LegC4-mediated restriction. Together with relatively low levels of effector translocation by L. longbeachae compared to L. pneumophila (39), the amount of translocated LegC4 may be insufficient to restrict bacterial replication within untreated BMDMs. However, LegC4 is sufficient to attenuate L. longbeachae replication within BMDMs activated with either rTNF or rIFN-γ. Whether LegC4 can protect mice from L. longbeachae-mediated lethality will be the subject of a future study.
Multiple effectors contribute to the innate immune response to L. pneumophila infection [reviewed in (56)]. Together with our data, these studies point to a complex interplay between effectors during Legionella infection of mammalian host. The effectors LnaB and LegK1 enhance NF-κB activation, which augments immune signaling (57, 58). Since mammals are a dead-end host for Legionella, the evolutionary basis for effector modulation of NF-κB is intriguing. Interestingly, the effector EnhC enhances L. pneumophila replication in TNF- activated macrophages (59), the opposite of what we have observed for LegC4. Thus, it is tempting to speculate that there may be interplay between EnhC and LegC4 within L. pneumophila infected cells. Future investigations will reveal whether LegC4-mediated phenotypes are dependent on other Dot/Icm-translocated effectors.
In summary, we found that the Dot/Icm effector LegC4 can augment cytokine-mediated restriction of Legionella replication within macrophages. These data add to the growing body of literature on effector triggered immunity in animal cells. As an accidental pathogen that did not co-evolve under the selective pressure of an innate immune system, L. pneumophila continues to provide insight into novel mechanisms of innate immunity towards intracellular bacterial pathogens. Consequently, further understanding of LegC4 function will reveal strategies to augment pro-inflammatory signaling. Thus, this study has provided the foundation for future investigations into the molecular mechanism by which LegC4 enhances host defense against intracellular bacterial pathogens.
Acknowledgements
We would like to thank Drs. B.A. Montelone and A. L. Passarelli for critical reading of the manuscript. The idea for a portion of this work was conceived in Dr. Craig Roy’s laboratory at the Yale School of Medicine. This work was supported by start-up funds and a Mentoring Award from Kansas State University (to S.R.S.), an NIH K-INBRE Developmental Research Project Award (P20GM103418; to S.R.S), and an NIH K-INBRE Semester Scholar Award (to A.J.H.). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.