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. Author manuscript; available in PMC: 2014 May 1.
Published in final edited form as: Eur J Immunol. 2013 Apr 8;43(5):1333–1344. doi: 10.1002/eji.201242835

A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy

Arwa Abu Khweek 1, Kyle Caution 1, Anwari Akhter 1, Basant A Abdulrahman 1,2, Mia Tazi 1, Hoda Hassan 1, Neal Majumdar 1, Andrew Doran 1, Evelyn Guirado 1, Larry S Schlesinger 1, Howard Shuman 3, Amal O Amer 1
PMCID: PMC3782291  NIHMSID: NIHMS501174  PMID: 23420491

Abstract

Legionella pneumophila (L. pneumophila) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires' disease. In contrast to humans, most inbred mouse strains are restrictive to L. pneumophila replication. We demonstrate that autophagy targets L. pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L. pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/SQSTM1 to ubiquitinated vacuoles. The L. pneumophila legA9 encodes for an ankyrin-containing protein with unknown role. We show that the legA9 mutant is the first L. pneumophila mutant to replicate in wild-type (WT) mice and their bone marrow derived macrophages (BMDMs). Less legA9 mutant- containing vacuoles acquired ubiquitin labeling and p62/SQSTM1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L. pneumophila -containing vacuole for autophagy uptake.

Keywords: Autophagy, Ubiquitination, Lysosomes, Trafficking, Macrophage

Introduction

L. pneumophila, the causative agent of Legionnaires' disease, is a intracellular pathogen that replicates within human alveolar macrophages [1]. Upon internalization by human macrophages, L. pneumophila disrupts normal endocytic pathway trafficking to establish a replicative vacuole, this specialized niche allows the bacterium to multiply exponentially [1, 2]. In contrast to human, most inbred mice strains are restrictive to L. pneumophila replication [3-7]. Autophagy is among various mechanisms that restrict L. pneumophila growth [5-9]. Autophagy is a lysosomal degradation pathway that maintains cellular homeostasis by degradation of the cell's internal components in response to starvation and stress [10, 11]. Further, autophagy contributes to the control of variety of bacterial infections [12-16]. Central to this pathway is formation of the autophagosome, a double membrane sac-containing the sequestered cytoplasmic material. Atg8-microtubule associated protein1 light chain 3 (LC3), is the hallmark of autophagosome formation [12, 17-19]. Autophagolysosomes are generated by fusion of autophagosomes and lysosomes, where the sequestered cargo is degraded [8, 11, 12]. Autophagic clearance of protein aggregates requires ubiquitin-binding proteins such as p62/SQSTM1 [20, 21]. Using separate domains, p62 binds ubiquitin and LC3, therefore delivering ubiquitinated targets to the autophagosome for degradation [11, 21].

The genome of L. pneumophila contains several genes predicted to encode homologous eukaryotic-like protein domains and some of them have been implicated in L. pneumophila pathogenesis [22-24]. Ankyrin proteins are distributed within the nucleus and cytoplasm of eukaryotic cells, and play an essential role in cell cycle, motility, oncogenesis and transcriptional regulation [25-27]. LegA9 was identified among other L. pneumophila eukaryotic-like genes in a bioinformatics screen of the L. pneumophila Philadelphia-1 genome and encodes for an ankyrin-containing protein [28]. However, the role of LegA9 in L. pneumophila pathogenesis is not known.

Here we show that in the restrictive BMDMs, the legA9 mutant replicates substantially. This is the first L. pneumophila mutant to be found to replicate in WT BMDMS. In contrast to WT, legA9 mutant vacuoles showed significant reduction in ubiquitin labeling and colocalization with p62, thereby avoiding uptake by autophagosomes, and enabling intracellular growth. Our data support a mechanism whereby LegA9 facilitates recruitment of the autophagic machinery to L. pneumophila vacuoles resulting in clearance of infection. This study provides a mechanistic link between LegA9 and clearance of L. pneumophila in WT macrophages by autophagy.

Results

WT mice and their BMDMs are restrictive to parent L. pneumophila intracellular replication but permissive to legA9 mutant strain

To characterize the role of LegA9 in L. pneumophila pathogenesis, we examined the intracellular replication of the JR32 and legA9 mutant in WT BMDMs. In contrast to the restriction of JR32 in WT macrophages, the legA9 mutant showed a 10 fold increase in the colony forming units (CFUs) over time (48-72 hr) (Fig. 1A). Complementation of legA9 mutation on LegA9 + LegA pBC-KS+ plasmid restricted the replication of this strain in a comparable manner to JR32 in the presence of IPTG (Fig. 1B).

Figure 1.

Figure 1

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WT mice and their BMDMs are restrictive to parent L. pneumophila intracellular replication but permissive to legA9 mutant strain, (A) BMDMs were infected with L. pneumophila JR32 or the legA9 mutant with MOI of 0.5. CFUs were scored at 1, 24, 48 and 72 hr. (B) BMDMs were infected with JR32 or the legA9 mutant harboring empty plasmid (PL), or legA9+ legA9 pBC-KS+ plasmid. Data are presented as means ± S.D of at least three independent experiments. Asterisks indicate significant differences (*P<0.05, **P<0.01, ***P<0.001). Four female C57BL/6 mice/group received 1×106 of JR32 or legA9 mutant bacteria intratracheally. Lungs were homogenized and plated for CFUs counting at (C) 4 or (D) 48 hr post infection. Data are representative of one of two independent experiments and presented as means ± S.D. Asterisks indicate significant differences (**P<0.01).

Since Legionnaires' disease is caused by replication of L. pneumophila in alveolar macrophages [1, 29], we investigated the role of LegA9 for bacterial replication within the lungs of live mice. C57BL/6 mice were infected with 1×106 JR32 or the legA9 mutant intratracheally [5]. CFUs after 4 hr of infection denoted the initial bacterial load in the lungs (Fig. 1C); whereas the lungs of infected mice harbored 3 fold more legA9 mutant bacteria compared to the JR32 strain 48 hr post infection (Fig. 1D).

Flagellin mediates restriction of L. pneumophila in murine macrophages and flaA mutant bacteria replicate significantly more than the parent strain [5]. Western blot analysis of legA9 mutant bacterial lysate shows that it contains flagellin comparable to WT (Supplementary 1A). Furthermore, the legA9 mutant bacteria express a functional Dot/Icm system as shown by the normal lysis of sheep RBCs (Supplementary 1B). Together, these data indicate that LegA9 plays a role in restricting L. pneumophila infection in vitro and in vivo.

The legA9 mutant replicates substantially in human monocyte-derived macrophages (hMDMs)

Human are permissive to L. pneumophila replication [30]. To characterize the role of LegA9 in the intracellular replication of L. pneumophila in human, we tested the intracellular growth of JR32 and legA9 mutant in acute monocytic leukemia cell line (THP-1). THP-1 derived macrophages are permissive to parental strain JR32 and were more permissive to legA9 mutant at 48 and 72 hr post infection (Supplementary 2A). In addition, we evaluated the intracellular growth of legA9 mutant in hMDMs. The legA9 mutant replicated a 100 fold more than JR32 in hMDMs (Supplementary 2B). Therefore, both JR32 and legA9 mutant replicated in hMDMs yet, legA9 mutant replicated significantly more than the JR32 strain.

Vacuoles harboring legA9 mutant bacteria evade fusion with lysosomes and escape recognition by autophagy in WT BMDMs

It is possible that the legA9 mutant replicated in WT macrophages because it evaded lysosomal fusion [8, 9, 31]. Thus, we examined the colocalization of JR32 and legA9 mutant-containing vacuoles with lysotracker red, a dye that stains acidic compartments in live cells and is retained after aldehyde fixation (Fig. 2A). Approximately, 60% of JR32 bacteria were delivered to lysotracker-labeled compartments within 1 hr of infection. In contrast, only ∼30% of legA9 mutant bacteria colocalized with lysotracker-labeled compartments even at 6 hr post infection (Fig. 2B). Therefore, vacuoles containing legA9 mutant bacteria evade lysosomal fusion.

Figure 2.

Figure 2

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Vacuoles harboring legA9 mutant bacteria significantly evade fusion with lysosomes and escape recognition by autophagy in WT BMDMs. (A) Representative images from 6 hr infected WT BMDMs with JR32 or legA9 mutant bacteria. Nuclei were stained blue with DAPI and L. pneumophila stained green with L. pneumophila antibody. Lyso-tracker red was used to stain acidified lysosomes. White arrows show L. pneumophila colocalization with lysotracker. (B) Percent colocalization of L. pneumophila with lyso-tracker. (C) Representative images from 6 hr infected WT BMDMs with JR32 or legA9 mutant bacteria. LC3 is stained red with LC3 antibody. White arrows show L. pneumophila colocalization with LC3. (D) Percent colocalization of L. pneumophila with LC3. Hundred bacteria were scored from each coverslips. Images were captured with the 60X objective and magnified 3×, scale bar = 10μm. Data are presented as means ± S.D of three independent experiments. Asterisks indicate significant differences (**P<0.01, ***P<0.001).

In restrictive murine macrophages, autophagic machinery recognizes the L. pneumophila phagosome as cargo [8, 32, 33]. Autophagosomes harboring L. pneumophila fuse with lysosomes and within the autophagolysosome, L. pneumophila is degraded and the infection is contained [8]. It is possible that legA9-containing vacuoles escape recognition by the autophagy machinery and therefore are not delivered to lysosomes for degradation. Consequently, acquisition of endogenous autophagy marker LC3 by JR32 or legA9-containing vacuoles was examined. In contrast to JR32, legA9-containing vacuoles showed less colocalization with LC3 (Fig. 2C and D). Approximately 55% of JR32-containing vacuoles acquired LC3 staining; whereas ∼30% of legA9-containing vacuoles acquired the autophagy marker. Together, legA9 mutant vacuoles evade the recognition by autophagy and lysosomal fusion resulting in growth in murine macrophages.

Rapamycin treatment increases LC3 acquisition by the legA9-containing vacuole and restricts its intracellular replication

The above data suggested that the legA9-containing vacuoles are not recognized as readily as the parent strain as autophagic cargo for lysosomal delivery. Accordingly, we determined the effect of autophagy induction on trafficking of legA9-containing vacuoles [8, 34]. Rapamycin inhibits the mammalian target of rapamycin (mTOR) pathway and has been used to stimulate autophagy [35]. BMDMs were pre-treated with rapamycin for 1 hr prior to infection with JR32 and legA9 mutant. CFUs enumerated at 1 hr (Fig. 3A) denote equal initial inocula. Induction of autophagy by rapamycin resulted in a significant decrease in the CFUs of the legA9 mutant as compared to non-treated macrophages at 24 hr (Fig. 3B). The decrease in bacterial number is not a result of rapamycin or DMSO-mediated toxicity (Supplementary 3). Furthermore, rapamycin treatment increased co-localization of the legA9-containing vacuoles with lysotracker (Fig. 3C) and LC3 (Fig. 3D) at 2 hr post infection. To confirm the role of autophagy in L. pneumophila pathogenesis, we examined the effect of bafilomycin, an inhibitor of lysosomal acidification, on L. pneumophila replication. Our data demonstrate that bafilomycin treatment resulted in an increase in JR32 CFUs 72 hr post infection (Supplementary 4). Moreover, 3-methyladenine, an inhibitor of PI3 Kinases increased JR32 CFUs 24 hr post infection (Supplementary 5). Therefore, drugs that induce or inhibit autophagy modulate the ability of L. pneumophila to replicate within macrophages.

Figure 3.

Figure 3

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Induction of autophagy in macrophages restricts legA9 mutant replication, and rescues colocalization with LC3 and fusion with lysosomes. BMDMs were untreated or pre-treated with rapamycin (Rap) for 1 hr and kept throughout the infection. BMDMs were infected with L. pneumophila JR32 or the legA9 mutant for (A) 1 or (B) 24 hr with an MOI of 0.5. CFUs were scored at these time points. (C) Percent co-localization of L. pneumophila with (C) lyso-tracker (D) and (D) LC3. Hundred bacteria were scored from each coverslips. Images were captured with the 60X objective and magnified 3×, scale bar = 10μm. Data are presented as means ± S.D of three independent experiments. Asterisks indicate significant differences (**P<0.01).

To further confirm the role of autophagy in controlling L. pneumophila infection in macrophages, we compared the growth rate of JR32 in WT and Atg5-/- derived BMDMs. Atg5 is an essential molecule required for autophagy [10], and functions as an essential mediator of LC3-I to LC3-II conversion [19]. Atg5-/- cells are unable to execute autophagy as a result of a defect in autophagosome formation [19]. Both the JR32 and legA9 mutant CFUs were higher at 72 hr (Fig. 4A). These data suggest that a defect in autophagy in the Atg5-/- mice compromised the clearance of L. pneumophila. We confirmed the lack of Atg5 in the Atg5-/- macrophages by Western blot (Fig. 4B). Furthermore, treatment of infected Atg5-/- BMDMs with JR32 or the legA9 mutant with rapamycin did not alter the bacterial number at 1 hr (Supplementary 6A and C) or 24 hr (Supplementary 6B and D) post infection. These data suggest that rapamycin is not able to induce autophagy in the absence of the essential autophagy protein Atg5.

Figure 4.

Figure 4

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BMDMs derived from Atg5-/- mice are defective in clearing the WT L. pneumophila and the legA9 mutant. BMDMs derived from WT and Atg5-/-mice were infected with L. pneumophila JR32 or legA9 mutant at an MOI of 0.5 (A). Data are representative of two independent experiments performed in triplicate and presented as means ± S.D. Asterisks indicate significant differences (*P<0.05). (B) Western blot showing the absence of Atg5 in the knockout.

It is possible that LegA9 exert its effect locally on L. pneumophila vacuole or globally affecting autophagy throughout the macrophages. To differentiate between these possibilities, we infected WT macrophages with the JR32 and legA9 mutant bacteria and examined the endogenous LC3-I conversion to LC3-II by Western blotting before and after rapamycin treatment. Rapamycin induced autophagy as determined by increase in the level of LC3-II in 2, and 4 hr post infection in both JR32 and legA9 infected macrophages (Supplementary 7A). Despite the induction of autophagy by rapamycin in both JR32 and legA9 infected cells, there were no difference in the level of LC3-II between the JR32 and the legA9 infected macrophages (Supplementary 7B). These data suggest that rapamycin induced autophagy to the same level in both the JR32 and the IegA9 infected macrophages.

Significantly fewer vacuoles-containing the legA9 mutant acquire ubiquitin compared to vacuoles harboring JR32

Ubiquitinated cargos are delivered to the autophagic machinery through a variety of adaptor proteins [11, 21, 36]. The specificity and targeting mechanisms of L. pneumophila-containing vacuoles by autophagy are yet to be elucidated. L. pneumophila-containing vacuoles are decorated with polyubiquitinated proteins in murine BMDMs [37, 38]. We hypothesize that ubiquitination of L. pneumophila vacuoles tags them for autophagosome uptake. Accordingly, we examined the acquisition of ubiquitin by vacuoles-containing JR32 or legA9 mutant. Approximately 70-75% of vacuoles-containing JR32 associated with ubiquitin in murine BMDMs at all time points. In contrast, only 45% of vacuoles containing legA9 mutant bacteria were associated with ubiquitin (Fig. 5A and B). These data indicate that fewer legA9-containing vacuoles acquire ubiquitin. Since it is possible that infection with the legA9 mutant affects the ubiquitination level within the cell, we examined total ubiquitination via Western blotting (Supplementary 8) and found that the defect in colocalization of the legA9 mutant vacuoles with ubiquitin is not a consequence of ubiquitin depletion.

Figure 5.

Figure 5

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Macrophage vacuoles-containing the legA9 mutant, have reduced ubiquitin compared to vacuoles harboring parent L. pneumophila. (A) Representative images from 6 hr infected BMDMs with JR32 or legA9 mutant bacteria. Poly-ubiquitin is stained red. White arrows show L. pneumophila colocalization with poly-ubiquitin. (B) Percent colocalization of L. pneumophila with poly-ubiquitin. Hundred bacteria were scored from each coverslips. Images were captured with the 60X objective and magnified 3×, scale bar = 10μm. Data are presented as means ± S.D of two independent experiments. Asterisks indicate significant differences (*P<0.05, **P<0.01, ***P<0.01).

To examine the role of the proteosomal pathway is controlling L. pneumophila growth, we inhibited proteasome function with (MG-132). Proteasomal inhibition did not affect CFUs recovered after infection with JR32 and legA9 mutant bacteria at 1hr (Supplementary 9A and C) or 24 hr (Supplementary 9B and D). These data suggest that the proteasomal pathway is not required for clearance of L. pneumophila.

p62/SQSTM1 targets L. pneumophila-containing vacuoles to autophagosomes for degradation in WT BMDMs

Vacuoles-containing JR32 readily colocalize with ubiquitin; whereas the legA9-containig vacuoles do so less frequently. The selectivity of this process can be mediated via several adaptor proteins that link ubiquitination to autophagy [20]. Among these adaptors is the p62/SQSTM1, which is able to bind ubiquitin and LC3, therefore delivering ubiquitinated targets to the autophagosome for degradation [11, 21, 36]. Thus, we examined the colocalization of vacuoles-containing JR32 and legA9 mutant with p62. The legA9-containing vacuoles colocalize less frequently with p62 when compared to the JR32 strain (Fig. 6A and B). It is plausible that infection with the legA9 mutant results in downregulation of p62. Therefore, we examined the level of p62 by ELISA in total cell lysates from BMDMs infected with JR32 or the legA9 mutant for 6 hr. The level of p62 increased following infection with JR32 or the legA9 mutant compared to non-treated macrophages (Fig. 6C). These results indicate that legA9-containing vacuoles are defective in colocalization with p62 despite its abundance in macrophages.

Figure 6.

Figure 6

Figure 6

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p62/SQSTM1 targets L. pneumophila-containing vacuoles to the autophagosomes for degradation in WT murine macrophages. (A) Representative images from 6 hr infected BMDMs with JR32 or legA9 mutant bacteria. P62 is stained red with p62 antibody. White arrows show L. pneumophila colocalization with p62. (B) Percent colocalization of L. pneumophila with p62. Hundred bacteria were scored from each coverslips. Images were captured with the 60X objective and magnified 3×, scale bar = 10μm. Data are presented as means ± S.D of three independent experiments. Asterisks indicate significant differences (*P<0.05, **P<0.01). (C) BMDMs were infected with JR32 or the legA9 mutant for 6 hr and the level of p62 protein was quantified in cell lysates by ELISA (performed in triplicate and the S.D.s are too small to detect). BMDMs were nucleofected with scrambled control (sictrl) or siRNA against p62 (sip62). Twenty four hrs after nucleafection, murine macrophages were infected with JR32 or legA9 mutant with an MOI of 0.5 for 1 hr (D and F) or 24hr (E and G). (H) Western blot analysis showing the downregulation of p62 at 24 and 48 hr post nucleofection. Data are representative of three independent experiments and presented as means ± S.D. Asterisks indicate significant differences (*P<0.05, **P<0.01).

To determine if p62 targets L. pneumophila vacuoles for degradation, p62 level was downregulated in murine macrophages with specific siRNA. Twenty four hr post nucleofection macrophages were infected with the JR32 or the legA9 mutant. Equivalent numbers of bacteria were recovered after one hour of infection (Fig. 6D). After 24 hr of infection, WT BMDMs did not allow L. pneumophila replication, yet, downregulation of p62 resulted in a significant increase in CFUs (Fig. 6E). Downregulation of p62 did not result in an increase in CFUs of the legA9 mutant (Fig. 6F and G). The downregulation of p62 was confirmed by Western blot analysis of SiRNA-treated macrophage with the p62 antibody (Fig. 6H). Therefore, p62/SQSTM1 targets L. pneumophila-containing vacuoles to the autophagosomes to restrict its growth in WT murine macrophages. NDP52 is another adaptor protein that acts cooperatively with other adaptor proteins to drive efficient antibacterial autophagy of Salomonella, Shigella and Lesteria[39-41]. To examine the role of NDP52 in delivering L. pneumophila vacuoles to autophagosomes, we nucleofected WT BMDMs with siRNA against NDP52 prior to infection with JR32 or legA9 mutant bacteria (Supplementary 10). Our data show that down-regulation of NDP52 does not alter CFUs recovered 24 hr post infection.

The permissiveness of BMDMs to legA9 mutant bacteria is independent of caspase-1 or caspase-7 activation

Caspase-1 mediated caspase-7 activation restricts L. pneumophila by promoting fusion of L. pneumophila vacuoles with lysosomes [5, 8, 9]. Replication of the legA9 mutant could be a result of impaired activation of caspase-1. Casp-1-/- BMDMs allow for growth of the JR32 strain [6]. Yet, replication of the legA9 mutant surpassed that of JR32 at 24, 48, and 72 hr post infection (Fig. 7A). Caspase-1 activation is associated with cleavage and maturation of IL-1β. Our data show that there is no significant difference in the level of IL-1β produced from WT macrophages infected with JR32 or the legA9 mutant for 24 hr (Fig. 7B). To determine if the replication of legA9 is due to a lack of caspase-1 activation, detection of the mature subunits of casp-1 was performed by Western blot. Infection of WT BMDMs with JR32 or the legA9 mutant was accompanied by caspase-1 activation (Fig. 7C). Taken together, these data suggest that the ability of the legA9 mutant to replicate within WT macrophages is independent of caspase-1 activation.

Figure 7.

Figure 7

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The permissiveness of BMDMs to legA9 mutant bacteria is independent of caspase-1 or caspase-7 activation. (A) Caspase-1 KO (casp-1-/-) macrophages were infected with L. pneumophila JR32 or legA9 with an MOI of 0.5 for 1, 24, 48 and 72 hr. (B) CFUs were measured at the indicated time points. Levels of IL-1β were detected in supernatants of WT or casp-1-/- BMDMs infected with JR32 or the legA9 mutant after 24 hr. WT BMDMs were either not treated (NT) or infected with L. pneumophila JR32 or legA9 mutant bacteria for 2 hr. Activation of casp-1 or casp-7 was detected in cell extracts using (C) casp-1 or (D) casp-7 antibodies, respectively.

At physiological levels of infection, L. pneumophila activates caspase-7 in a caspase-1-dependent manner [6]. Caspase-7 activation during L. pneumophila infection restricts growth by promoting early macrophage death and efficient delivery of the bacterium to the lysosome [6, 42]. It was possible that replication of the legA9 mutant is due to diminished caspase-7 activation. Therefore, activation of caspase-7 was assessed by Western blot. The absence of legA9 did not compromise caspase-7 activation in WT macrophages (Fig. 7D). Therefore, our data demonstrate that replication of the legA9 mutant occurs irrespective of caspase-1 or caspase-7 activation.

The permissiveness of BMDMs to legA9 mutant is independent of host cell death

Early macrophage death terminates the replication cycle of L. pneumophila and prevents the establishment of infection [43]. It is plausible that the legA9 mutant manipulates the macrophage life span to maintain replication. Accordingly, the role of LegA9 in induction of cell death was examined. We measured apoptosis in the overall population of macrophages by determining the cytoplasmic apoptosis histone-associated-DNA-fragments at MOI of 0.5, 1 and 5. Our results demonstrate that infection by JR32 and the legA9 mutant led to comparable cell death at low and high MOIs (Fig. 8A).

Figure 8.

Figure 8

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Replication of the legA9 mutant is not due to extended macrophage survival. (A) BMDMs were not infected (NT) or infected with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5, 1 or 5 for 24 hr. Apoptosis was quantified by Photometric enzyme immunoassay analysis of cytoplasmic (apoptosis) histoneassociated-DNA fragments (ELISA). (B) BMDMs were either not treated (NT) or treated with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5 or 5 for 24 hr. The fold change in LDH release was measured from the overall population of macrophages.

To understand the role of LegA9 in L. pneumophila-mediated host cell necrosis, we measured the level of LDH release in culture supernatants of WT BMDMs infected with JR32 or the legA9 mutant. Our data demonstrated that at an MOI of 0.5 or 5, comparable LDH is released after 24 hr of infection with JR32 and the legA9 mutant (Fig. 8B). Together, our data demonstrate that the ability of legA9 to replicate in WT macrophages is not due to extension of macrophage survival.

Discussion

LegA9 (lpp2058) was identified among L. pneumophila eukaryotic-like genes in a bioinformatics screen of the L. pneumophila Philadelphia-1 genome and encodes for an ankyrin-containing protein with unidentified role [28]. We showed that L. pneumophila lacking legA9 replicate in WT mice and their derived macrophages. Several eukaryotic pathways contribute to the restriction of L. pneumophila growth in WT BMDMs [5-7], among which WT macrophages efficiently activate autophagy and deliver L. pneumophila vacuoles for lysosomal degradation. However, it is still unclear what tags the L. pneumophila vacuole for autophagy uptake.

Our data suggests that LegA9 protein labeled the L. pneumophila vacuole for autophagy uptake but does not alter cellular autophagy activity since LC3II is produced upon both JR32 and legA9 mutant infections. Additionally, rapamycin treatment activated autophagy in both JR32 and legA9 infected macrophages, denying a role for LegA9 in autophagy activation.

We showed that ubiquitination of L. pneumophila vacuoles targets them to autophagy in a process that is dependent upon recognition of the ubiquitinated vacuoles by the adaptor p62, thus delivering them to autophagosomes for degradation. In contrast to JR32, legA9 mutant vacuoles significantly evade recognition by p62 and autophagosomes thus escaping delivery to lysosomes. AJ derived murine macrophages are permissive to L. pneumophila and used to study pathogenesis. Interestingly, vacuoles harboring WT L. pneumophila derived from AJ macrophages exhibits K48 and K63 polyubiquitinated proteins [37, 38]. Generally, lysine 48 conjugated ubiquitin moieties to proteins target them to proteasomal degradation while lysine 63 modulates protein activity and trafficking in the cell [44, 45]. Host proteasomal degradation of Lys48-linked polyubiquitinated proteins, assembled on the L. pneumophila vacuole generate amino acids required for intracellular bacterial survival and growth [46]. However, we showed that ubiquitination of L. pneumophila vacuoles in restrictive macrophages targets them to autophagy machinery for degradation of their contents. Thus ubiquitination can be useful for the bacteria to provide nutrition within the vacuole but could also be detrimental by tagging the vacuole for autophagosome recognition. Thus it is necessary for L. pneumophila to delay the maturation of its containing vacuole [8] by inhibiting autophagy at a specific stage [47]. L. pneumophila express RavZ, (a cysteine protease that specifically deconjugates Atg8 proteins from phospholipid membranes, thus inhibiting autophagy [47]. We think that LegA9 performs functions beyond vacuole tagging since L. pneumophila lacking legA9 replicates more than the JR32 strain in the Atg5-/- macrophages with defective autophagy system.

It seems counterintuitive that L. pneumophila would retain legA9 in the genome, even though it is not required for replication in Acanthamoeba castellanii. It has been suggested that maintaining redundant genes may prevent single genetic lesions from enabling the bacteria to multiply intracellularly [28]. Our study shows that when L. pneumophila lacks LegA9 it replicates more efficiently than the parental strain in human macrophages, thus making the bacterium more pathogenic. Furthermore, cycling of L. pneumophila through multiple protozoan hosts in the environment prevents L. pneumophila-host specialization by maintaining pathways that are required for growth in macrophages and other hosts. However, growth restriction of L. pneumophila in the murine model or under laboratory conditions is sufficient to change the fitness and host range of this bacterium [48]. Restriction of L. pneumophila replication in murine macrophages by acquisition of the LegA9 factor could provide an example of selective pressure to increase adaptability of this bacterium to infect different hosts.

Materials and methods

Bacterial strains

L. pneumophila JR32 [49] and the legA9 mutant [28] were kindly provided by Dr. Howard Shuman, University of Chicago. L. pneumophila strains were grown as previously described [6, 50].

Cell culture and infections

WT mice were purchased from Jackson laboratory. C57BL/6 BMDMs were prepared from the femurs of five to eight-week-old mice as previously described [6, 7]. Isolation and preparation of the human monocyte-derived macrophages (hMDMs) from peripheral blood was carried out as previously described [50-52]. Acute monocytic leukemia cell line (THP-1) was maintained as previously described [53]. Infections were performed as previously described [5-7, 53]. The Atg5-/- mice [54] were obtained from Herbert W. Virgin from Washington University with permission of Noboru Mizushima.

Western blot analysis

30μg/ml of protein was loaded on 12% SDS polyacrylamide gel and immunoblotted with antibodies that recognize caspase-1, caspase-7 (cell signaling), p62 (Sigma), mono and polyubiquitinated conjugates, mAb FK2 (Enzo) and LC3 (Sigma). The α-flaA polyclonal Ab was obtained from Dr. Howard Shuman, University of Chicago. The primary antibodies were followed by appropriate secondary rabbit or mouse antibodies as described in [6, 7, 53].

In vitro drug treatment of macrophages

Rapamycin (Sigma-Aldrich, R0395) was dissolved in DMSO (Sigma-Aldrich, D2650) at 1 mg/ml. Rapamycin was used at a concentration of 0.2 μg/ml [8]. Murine macrophages were either treated or untreated with rapamycin for 1 hr before they were infected. Rapamycin was maintained throughout the infection. Bafilomycin A1 (sigma) was added 1 hr after infection at final concentration of 12.5 nM. The 3-methyladenine (sigma) was dissolved in heated dH2O at 65°C, added 1hr before infection at a concentration of 10 mM and kept throughout the infection [8]. The proteasome inhibitor MG-132 (Biovision, Mountain view, CA) was dissolved in DMSO and maintained throughout the infection [46].

Macrophage cytotoxicity assay

Quantification of apoptosis was performed using the Cell Death Detection ELISA (Roche Applied Science) to the specification of the manufacturer. The fold change of macrophage necrosis was determined by measuring the release of host cell cytoplasmic lactate dehydrogenase (LDH) using the cytotoxicity detection kit (Roche Applied Science) to the specification of the manufacturer.

Confocal laser scanning microscopy (CLSM)

Colocalization experiments were performed as previously described [5-7]. Antibodies against L. pneumophila (Abcam), Atg8/LC3 (Abgent, Ap1805a), polyubiquitin FK2 (Enzo) or p62 (BD bioscience) were used. Images were captured with the Olympus Fluoview Fv10i.

Contact-dependent hemolysis

One × 108 sheep RBCs (sRBCs) were incubated with L. pneumophila at an MOI of 25 for 3 hrs, as previously described [55, 56].

Enzyme-Linked Immunosorbent Assay (ELISA) for p62

Two × 106 of BMDMs were either un-infected or infected with JR32 or the legA9 mutant for 6 hr. The levels of p62 or IL-1β were determined by a sandwich ELISA according to the manufactures specification.

SiRNA treatment

siRNA treatment was performed using siRNA against P62 (Dharmacon-18412):ACAGAUGCCAGAAUCGGAA, CUGCUCAGGAGGAGACGAU,GAACAGAUGGAGUCGGGAA, CCAUGGGUUUCUCGGAUGA. NDP52 (Dharmacon-76815) CAACACAGAGGGUCAAUAA, CAGAAGAGGACAUCCGGAU, CCAAGGAUGUGGAGCGCUA, GAGUUGAGGUGUCCGUGUA, siRNA was nucleofected into primary macrophages using Lonza Nucleofection kit and Amaxa equipment as we described previously [57].

Statistical analysis

All experiments were performed at least 3 independent times and yielded similar results. Comparisons of groups for statistical difference were done using student's two tailed t test. P value ≤ 0.05 was considered significant.

Supplementary Material

Supporting Information

Acknowledgments

Studies in Dr. Amal Amer's laboratory are supported by grants R01HL094586.

Abbreviations

L. pneumophila

Legionella pneumophila

BMDMs

Bone marrow derived macrophages

hMDMs

human monocyte derived macrophages

CFUs

colony forming units

Footnotes

Conflict of interest: The authors declare no financial or commercial conflict of interest.

Ethic statements: This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of health and The Ohio State University. The Institutional Animal Care and Use Committee (IACUC) have approved our protocol number 2007A0070. Human cells were obtained from anonymous samples from Red Cross.

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Supporting Information
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