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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cell Microbiol. Author manuscript; available in PMC 2010 April 19.
Published in final edited form as:
PMCID: PMC2856068
NIHMSID: NIHMS184211

Modulation of ubiquitin dynamics and suppression of DALIS formation by the Legionella pneumophila Dot/Icm system

Summary

Legionella pneumophila is an intracellular pathogen that uses effector proteins translocated by the Dot/Icm type IV secretion system to modulate host cellular processes. Here we investigate the dynamics of subcellular structures containing ubiquitin during L. pneumophila infection of phagocytic host cells. The Dot/Icm system mediated the formation of K48 and K63 poly-ubiquitin conjugates to proteins associated with L. pneumophila-containing vacuoles in macrophages and dendritic cells, suggesting that regulatory events and degradative events involving ubiquitin are regulated by bacterial effectors during infection. Stimulation of TLR2 on the surface of macrophages and dendritic cells by L. pneumophila-derived molecules resulted in the production of ubiquitin-rich dendritic cell aggresome-like structures (DALIS). Cells infected by L. pneumophila with a functional Dot/Icm system, however, failed to produce DALIS. Suppression of DALIS formation did not affect the accumulation of ubiquitinated proteins on vacuoles containing L. pneumophila. Examining other species of Legionella revealed that L. jordanis was unable to suppress DALIS formation after creating a ubiquitin-decorated vacuole. Thus, the L. pneumophila Dot/Icm system has the ability to modulate host processes to promote K48 and K63 ubiquitin conjugates on proteins at the vacuole membrane, and independently suppress cellular events required for the formation of DALIS.

Keywords: Legionella, DALIS, Ubiquitin

Introduction

Legionella pneumophila is an intracellular pathogen capable of causing a severe pneumonia in humans known as Legionnaire’s disease (Fraser et al., 1977, McDade et al., 1977). Alveolar macrophages engulf aerosolized L. pneumophila that descend in the lumen of the lung alveolar ducts, however, fusion of lysosomes with the vacuole containing L. pneumophila is blocked, which allows bacteria to escape macrophage killing (Horwitz, 1983b, Horwitz, 1983a). Legionella-containing vacuoles (LCVs) recruit endoplasmic reticulum (ER) -derived vesicles and establish a specialized ER-derived organelle that supports bacterial replication (Kagan et al., 2002, Swanson et al., 1995). A critical virulence determinant for L. pneumophila is a type IV secretion system called Dot/Icm (Vogel et al., 1998, Segal et al., 1998), which delivers bacterial proteins into the cytosol of infected cells that directly mediate the subversion of host cellular processes (Ninio et al., 2007). Mutants lacking a functional Dot/Icm system fail to evade endosome-lysosome fusion (Berger et al., 1993, Roy et al., 1998), and do not create an ER-derived vacuole during infection (Nagai et al., 2002, Tilney et al., 2001). As a result, dot and icm mutants are defective for intracellular replication and are avirulent in animal models of disease (Wiater et al., 1998, Archer et al., 2006, Marra et al., 1992).

Following infection of host cells with L. pneumophila, ubiquitinated protein accumulate on vacuoles harboring wild-type bacteria but not on vacuoles containing mutants that have a non-functional Dot/Icm system (Dorer et al., 2006). Protein ubiquitination is a process restricted to eukaryotic organisms. Interestingly, the L. pneumophila genome includes proteins containing domains predicted to modulate the process of ubiquitination (Cazalet et al., 2004). A recent study showed that the L. pneumophila effector protein LubX is an E3 ubiquitin ligase that mediates ubiquitination of the host protein Clk1 (Kubori et al., 2008). These data suggest that L. pneumophila has evolved sophisticated mechanisms for modulating host protein ubiquitination, however, specific cellular processes requiring ubiquitinated proteins that are affected by L. pneumophila have not been identified.

DALIS are structures containing aggregates of ubiquitinated proteins that were initially observed in dendritic cells (DCs) exposed to LPS (Lelouard et al., 2002). Proteins that fail to fold properly in the ER are detected by a protein quality control system, and are translocated in the cytosol, ubiquitinated and degraded by the proteasome (Schubert et al., 2000). These Defective Ribosomal Products (DRiPs) are a source for host and pathogen-derived MHC class I-restricted antigens (Princiotta et al., 2003, Yewdell et al., 1996). DALIS can accumulate DRiPs (Lelouard et al., 2002, Lelouard et al., 2004), and therefore are thought to protect a pool of ubiquitinated proteins from proteasome degradation allowing longer access to a source of antigens while DCs migrates from the site of antigen acquisition in the periphery to the lymph nodes where presentation occurs (Pierre, 2005). DCs infected ex vivo with the influenza virus formed DALIS concomitant with delayed MHC class I antigen presentation as compared to nonprofessional antigen presenting cells (Herter et al., 2005). Macrophages also have been shown to induce DALIS in response to various pathogen-derived molecules including LPS, zymosan and L. monocytogenes extracts (Canadien et al., 2005).

Unlike conventional aggregates of ubiquitinated proteins associated with neurodegenerative pathologies (Johnston et al., 1998, Garcia-Mata et al., 1999), DALIS are mobile and do not localize at the MTOC (Lelouard et al., 2002). It is unknown how DALIS circulate in the cell, because disruption of microtubule or actin networks have no effect on DALIS motility (Lelouard et al., 2004). Pharmacological inhibitors of protein translation, but not mRNA synthesis, can block DALIS assembly and induce DALIS fragmentation (Lelouard et al., 2004, Lelouard et al., 2002). DALIS co-localize with E1, E2 and E3 components of the ubiquitin ligase complex (Lelouard et al., 2004) and exclude components of the proteasome machinery (Canadien et al., 2005) suggesting that protein ubiquitination without subsequent degradation occurs within the structures. Indeed fluorescence recovery after photobleaching analysis confirmed that DALIS continuously acquire ubiquitinated proteins (Lelouard et al., 2004).

Here, we demonstrate that L. pneumophila modulates host processes that involve ubiquitinated proteins and discover that the Dot/Icm system has the capacity to robustly interfere with DALIS formation.

Results

K48 and K63 ubiquitin conjugates rapidly accumulate on the LCV

Ubiquitinated proteins have been detected on LCVs (Dorer et al., 2006). To better characterize the process by which the decoration of the vacuole with ubiquitin occurs we examined the kinetics of this process and the linkages that exist in the associated ubiquitin conjugates. The accumulation of ubiquitinated proteins on the LCV in infected bone marrow macrophages (BMMs) derived from mice was investigated at different times after infection (Fig. 1). Immunofluorescence microscopy of BMMs infected with L. pneumophila serogroup 1 and stained with the antibody FK2 that binds mono- as well as poly-ubiquitinated proteins (Fujimuro et al., 1994) revealed ubiquitinated polypeptides surrounding the LCV (Fig. 1A). Ubiquitinated proteins accumulated at the LCV shortly after L. pneumophila uptake and remained on the mature vacuole containing replicating bacteria. Most of the LCVs in BMMs were FK2-positive at 1hour post infection and nearly all of the vacuoles containing L. pneumophila were FK2-positive at 7 hours (Fig. 1B). At 10 hours, large vacuoles containing replicating L. pneumophila showed less intense staining with FK2 and vacuoles containing single bacteria that were FK2-positive were identified, consistent with bacterial egress and re-infection occurring at 10 hours. Similar results were obtained using the FK1 antibody (data not shown) that detects only poly-ubiquitinated proteins (Fujimuro et al., 1994). Consistent with previous observations (Dorer et al., 2006), vacuoles containing the dotA mutant strain lacking a functional Dot/Icm secretion apparatus were not FK2 positive (data not shown), indicating that the recruitment of ubiquitinated proteins to the LCV is mediated by the Dot/Icm system.

Fig. 1
Ubiquitinated proteins accumulate at the LCV

Although multiple factors govern the functional state of a protein that has been poly-ubiquitinated, a key determinant is the type of linkage between the individual ubiquitin moieties within the poly-ubiquitin chain. In general, poly-ubiquitin conjugates built upon the lysine 48 residue (K48) of the ubiquitin polypeptide typically target proteins to the proteasome for degradation and lysine 63 linkages (K63) are involved in protein regulation (Pickart et al., 2004). To determine the ubiquitin conjugates present at the LCV, tagged ubiquitin variants that contain only K48 or K63 were used (Fig. 2A).

Fig. 2
K48 and K63 ubiquitin conjugates are both found at the LCV

Chinese hamster ovary (CHO) cells producing HA-tagged ubiquitin were infected with L. pneumophila and ubiquitin association with the vacuole was assayed by immunofluorescence microscopy (Fig. 2B,C). These data show wild type ubiquitin, K48-only ubiquitin and K63-only ubiquitin accumulated at the LCV following infection (Fig. 2B). Immunofluorescence images obtained with the antibodies FK1 and FK2 showed similar intensities of vacuole staining in infected cells, suggesting that most of the protein conjugates on the vacuole in these cells are poly-ubiquitinated (data not shown). In contrast, LCVs containing the dotA mutant failed to accumulate ubiquitin chimeras, indicating that a functional Dot/Icm system is required for this process (Fig. 2C). Thus, both K48 and K63 linkages are used to conjugate ubiquitin to proteins on the LCV, suggesting that ubiquitination could play both a role in degrading proteins on the LCV and in regulating the activity of proteins associated with the LCV.

Macrophages form DALIS in response to L. pneumophila

Distinct ubiquitin-containing structures were observed in both BMMs and DCs after the addition of L. pneumophila to cells cultured ex vivo (Fig. 3A). Ubiquitin-containing structures were not readily detected in control cells that were not exposed to L. pneumophila, indicating that FK2-positive structures were formed in response to L. pneumophila-derived molecules. These ubiquitin-containing foci resembled dendritic cell aggresome-like structures called DALIS that form in the cytosol of DCs and macrophages upon stimulation with LPS (Lelouard et al., 2002, Canadien et al., 2005). The FK2-positive foci induced by L. pneumophila were similar in size and shape to LPS-induced DALIS in both DCs and BMMs (Fig. 3A). Ubiquitin-containing structures induced following L. pneumophila exposure were first apparent at 2–3 hours and disappeared after 18–24 hours (Fig. 3B, and data not shown), similar to the kinetics of DALIS formation in cells after a one hour pulse with LPS (Fig. 3B). Wild type and dotA mutant L. pneumophila both induced DALIS, suggesting that formation of these structures does not require translocation of bacterial products into the host cytosol.

Fig. 3
DALIS are produced by macrophages and DCs in response to L. pneumophila

Consistent with the biochemical properties of LPS-induced DALIS, ubiquitinated proteins within the L. pneumophila-induced structures were resistant to Triton X-100 extraction (Fig. 3C). Pharmacological inhibition of protein translation is the only known method for inhibiting DALIS formation (Lelouard et al., 2004, Lelouard et al., 2002). Inhibition of eukaryotic protein translation by cycloheximide (CHX) blocked the formation of FK2-positive foci following L. pneumophila exposure and LPS-mediated DALIS formation in BMMs (Fig. 3D). Although CHX treatment induced some cell death, the percentage of dead cells (18%) was lower than the proportion of DALIS-containing cells in the absence of CHX (35%), indicating that cytotoxic effects did not account for the disappearance of FK2-positive foci. These data demonstrate that the FK2-positive foci induced by L. pneumophila in immune cells are indistinguishable from LPS-induced DALIS. Thus, L. pneumophila products induce the formation of DALIS in BMMs and DCs.

DALIS formation is induced by L. pneumophila stimulation of host TLRs

MyD88 is an adapter protein required for most TLR signaling pathways. BMMs deficient in the adapter protein MyD88 were infected with L. pneumophila to determine whether DALIS formation was being induced in response to TLR activation (Fig. 4). DALIS were not detected in MyD88-deficient BMMs in response to either wild type or dotA mutant L. pneumophila (Fig. 4B and 4C). DALIS formation was also investigated using TLR2-deficient BMMs. As expected, wild type and TLR2-deficient BMMs formed DALIS in response to the TLR4 agonist E. coli LPS and TLR2-deficient BMMs did not form DALIS in response to peptidylglycan (Fig. 4D). DALIS formation in response to L. pneumophila was greatly reduced in TLR2-deficient BMMs (Fig. 4E), indicating L. pneumophila stimulation of TLR2 is important for inducing the formation of DALIS. There were slightly more DALIS-positive cells in the TLR2-deficient BMMs stimulated for 7 hours with L. pneumophila compared to unstimulated cells (10% vs. 3.5%). This slight increase in DALIS formation was not observed after MyD88-deficient BMMs were stimulated with L. pneumophila, suggesting that L. pneumophila stimulation of a second TLR on BMMs can induce the formation of DALIS inefficiently. Examination of the LCVs in BMMs from knockout mice revealed that TLR2 and MyD88 were not required for the accumulation of ubiquitin around vacuoles containing L. pneumophila having a functional Dot/Icm system. Thus, TLR and MyD88 signaling is not required for processes that mediate the accumulation of ubiquitinated proteins on the LCV, suggesting that processes important for DALIS formation are regulated differently from processes required for the accumulation of ubiquitinated proteins on the LCV.

Fig. 4
L. pneumophila stimulation of TLR2 promotes DALIS formation

DALIS formation is blocked by intracellular L. pneumophila

There was not a large difference in the total number of DALIS-positive cells detected when wild type and dotA mutant-exposed BMMs were compared (Fig. 3E); however, most cells analyzed in these assays did not have internalized bacteria. Thus, we next examined only the infected cells to determine whether intracellular L. pneumophila might alter DALIS morphology or formation. When cells containing wild type L. pneumophila were examined, DALIS were not detected in most infected cells. As shown in Fig. 5A, DALIS-negative cells with intracellular L. pneumophila were usually surrounded by uninfected cells containing DALIS (Fig. 5A). Measuring DALIS formation in infected cells revealed that most BMMs containing wild type L. pneumophila did not contain DALIS (Fig 5B). Conversely, BMMs that internalized dotA mutant bacteria formed DALIS (Fig 5A and B). Similar results were obtained using DCs. DALIS were not detected in the majority of DCs infected with wild type L. pneumophila, whereas, DCs infected with a dotA mutant contained DALIS (Fig. 5C and D). These data suggest that BMMs and DCs infected with L. pneumophila are devoid of DALIS because the Dot/Icm system interferes with a process essential for DALIS formation.

Fig. 5
L. pneumophila infected cells suppress the formation of DALIS

L. pneumophila can suppress DALIS formation induced by LPS

The ability of L. pneumophila to suppress DALIS formation in response to a heterologous PAMP was examined by treating BMMs with E. coli LPS 4 hours after infection with L. pneumophila (Fig. 6A). BMMs treatment with LPS alone generated 40% (±2%) DALIS-positive BMMs. Measurements of DALIS formation only in infected cells revealed that BMMs containing wild type L. pneumophila failed to produce DALIS after stimulation with E. coli LPS, whereas, DALIS were produced in BMMs infected with dotA mutants (Fig. 6A). Thus, L. pneumophila suppresses DALIS formation regardless of the TLR pathway used to induce this response.

Fig. 6
L. pneumophila can suppress DALIS formation induced by E. coli LPS

To better understand how L. pneumophila interferes with DALIS formation, BMMs were stimulated with E. coli LPS and cells were then infected with L. pneumophila (Fig. 6B–D). Approximately 72% of BMMs developed DALIS after treatment with E. coli LPS for 3-hour followed by a 9-hour chase with medium alone (Fig. 6C). The overall percentage of DALIS-positive cells after LPS stimulation followed by L. pneumophila infection slowly decreased with time, consistent with the transient nature of the response (Fig. 6C). When BMM were treated with LPS for 3 hours and then infected with L. pneumophila for 3 hours, most of the BMM containing wild type L. pneumophila had both FK2-positive LCVs and DALIS (Fig. 6B). Replicating L. pneumophila were observed 6 hours after infection (Fig 6B) and a drop in the number of cells infected with wild type L. pneumophila that contained DALIS was also observed at 6 hours (Fig. 6D). Infection of BMMs with dotA mutant L. pneumophila did not result in a similar decrease in the percent of infected cells containing DALIS 6 hours after infection (Fig. 6B,D). Although the overall percentage of DALIS-positive cells decreased slightly during the course of infection, a pronounced reduction in DALIS was evident in the L. pneumophila-infected cells at 9 hrs post infection (Fig. 6C). Thus, a host process important for DALIS formation that is suppressed by L. pneumophila is also important for maintaining preexisting DALIS.

L. pneumophila replication is not required for DALIS suppression

To determine whether L. pneumophila replication is essential for DALIS suppression BMMs were infected with a thyA mutant strain that requires the addition of thymidine to the tissue culture medium for replication. Vacuoles containing the thyA mutant bacteria were FK2-positive both in medium containing thymidine and in medium without thymidine (Fig. 7A). Vacuoles containing an isogenic dotA, thyA mutant were FK2-negative. These data confirm that L. pneumophila replication is not essential for Dot/Icm-mediated recruitment of ubiquitined conjugates to the vacuole. As expected, replicating thyA mutants were detected in BMMs when thymidine was added to the culture medium, and DALIS were not detected in these infected host cells. DALIS were not observed in infected BMMs when replication of the thyA mutant was inhibited by withholding thymidine from the culture medium (Fig. 7B). Thus, Dot/Icm-mediated suppression of DALIS occurs independent of bacterial replication.

Fig. 7
L. pneumophila replication is not required for DALIS suppression

L. pneumophila proteins regulate DALIS suppression and ubiquitin accumulation at the vacuole

The observation that Dot/Icm function was required to inhibit DALIS formation suggests that bacterial proteins translocated by this system could be important for DALIS suppression. To determine whether production of proteins by intracellular L. pneumophila is important for DALIS suppression, chloramphenicol (CM) was used to block bacterial protein synthesis during infection (Fig. 8). Control experiments show that CM treatment of BMMs did not inhibit DALIS formation in response to LPS (Fig. 8A). The percentage of CM-treated BMMs containing DALIS after exposure to L. pneumophila was similar to the LPS-treated control (Fig. 8B). FK2 staining of BMMs infected with wild type L. pneumophila showed that the ubiquitin staining surrounding the LCV dissipated upon treatment of cells with CM (Fig. 8C,D). Interestingly, FK2-positive rings that were near, but did not appear to be associated with the LCV, were observed after CM treatment of infected BMMs. As FK2 staining of the LCV became less intense, DALIS appeared in the CM-treated BMMs infected with wild type L. pneumophila (Fig. 8C). DALIS were detected in BMMs infected with L. pneumophila after treatment of cells for 5 hours with CM and the percentage of DALIS-positive cells infected with L. pneumophila increased after treatment with CM for 7 hours (Fig. 8E). These data indicate that de novo synthesis of L. pneumophila proteins after infection of BMMs is required to preserve a block that prevents DALIS formation and to maintain an LCV decorated with ubiquitinated proteins.

Figure 8
Bacterial protein synthesis is important for DALIS suppression

Inhibition of host protein synthesis by the Legionella glycosyltransferase toxins is not required for DALIS suppression

L. pneumophila effectors translocated by the Dot/Icm system include a family of glycosyltransferase toxins (Lgts) that can glycosylate host elongation factor 1α (EF1α) in vitro resulting in inhibition of host protein translation (Belyi et al., 2008, Belyi et al., 2006). Because protein translation is required for DALIS formation (Lelouard et al., 2002), we decided to investigate whether the Lgts were required for DALIS suppression. A mutant strain of L. pneumophila deficient in Lgt1 [lpg1368], Lgt2 [lpg2862] and Lgt3 [lpg1488] (Δlgt1, Δlgt3, lgt2::kanr) was created and tested for DALIS suppression. The lgt triple mutant suppressed DALIS formation to equivalent levels as the isogenic lgt sufficient strain (Fig. 9A and B). Similarly, Legionella longbeachae, a species that is defective for Lgt1 production and deficient in glucosyltransferase activity on EF1α (Belyi et al., 2003), was able to suppress DALIS formation (Fig. 9C). These data demonstrate that the Lgt proteins are not required for DALIS suppression by Legionella. Lastly, because of their possible role in modulating ubiquitin dynamics, we investigated whether three genes encoding L. pneumophila effector proteins with F-box domains (lpg2144, lpg2525 and lpg1488), and the gene encoding the U-box protein LubX, were required for DALIS suppression. Isogenic single mutants deficient for lpg2144, lpg2525, lpg1488 or lubX showed no defect in DALIS suppression or in their ability to recruit ubiquitin conjugates to the vacuole when compared to wild type L. pneumophila (data not shown). Similarly, there were no differences in DALIS suppression or ubiquitin recruitment to the vacuole observed for a triple mutant deficient in lpg2144, lpg2525, and lpg1488, and a quadruple mutant deficient in lpg2144, lpg2525, lpg1488 and lubX (data not shown), suggesting that these genes are not required for DALIS suppression or for ubiquitin accumulation at the LCV.

Figure 9
DALIS suppression and ubiquitin accumulation at the LCV are distinct events

Ubiquitin accumulation at the LCV and DALIS suppression are independent events

To determine whether accumulation of ubiquitinated proteins consistently correlates with DALIS suppression we examined several different Legionella species and the Legionella pneumophila strains Paris and Lens. All L. pneumophila strains analyzed, and L. bozemanii, L. gratiana, and L. longbeachae, created vacuoles that stained positive for ubiquitin and suppressed the formation of DALIS in the infected BMMs (data not shown). By contrast, ubiquitin-rich DALIS were found in the majority of BMMs harboring L. jordanis (Fig. 9D and E). Importantly, ubiquitin staining was clearly visible at the vacuole containing replicating L. jordanis (Fig. 9D), indicating that the defect in DALIS suppression by L. jordanis was not related to a defect in ubiquitin recruitment to the LCV. Thus, the Dot/Icm-dependent events required for DALIS suppression are distinct from the processes that regulate ubiquitin recruitment to the LCV, suggesting that Legionella utilizes different effectors to modulate these events.

Discussion

This study identified several cellular processes involving protein ubiquitination that are modulated by L. pneumophila. Ubiquitinated proteins were found to accumulate on vacuoles containing Legionella, consistent with a previous report (Dorer et al., 2006). Investigating the kinetics of ubiquitin recruitment to the LCV showed that this process is a Dot/Icm-dependent event that precedes bacterial replication and is controlled by proteins produced by Legionella. Thus, it is likely that effectors translocated into host cells by the Dot/Icm apparatus will play an important role in vacuolar recruitment of ubiquitinated proteins.

Several L. pneumophila proteins have regions of amino acid homology that correspond to domains in eukaryotic factors that are involved in controlling ubiquitin-mediated processes (Kubori et al., 2008, Chien et al., 2004, de Felipe et al., 2005, Cazalet et al., 2004). These include L. pneumophila proteins with F-box domains, U-box domains, and ankyrin repeat domains. The prevalence of numerous effectors with the potential to subvert the host ubiquitin machinery suggests that the accumulation of ubiquitinated proteins at the vacuole could be mediated by the independent activities of multiple L. pneumophila proteins. Consistent with this hypothesis, we determined that both K48 and K63 ubiquitin conjugates localize to vacuoles containing L. pneumophila. Generation of these distinct ubiquitin conjugates would likely require L. pneumophila subversion of different ubiquitin ligase components. These data also suggest that L. pneumophila is subverting ubiquitin both for degrading proteins on the vacuole and modulating the activity of proteins associated with the vacuole. Interestingly, the L. pneumophila AM053 strain lacking three F-box-containing proteins and the LubX protein showed no obvious defect in ubiquitin accumulation at the vacuole (data not shown), suggesting that modulation of ubiquitin dynamics involves a subset of L. pneumophila effectors that may not have obvious homology to eukaryotic proteins involved in ubiquitin conjugation.

In addition to the presence of ubiquitinated proteins on the LCV, we found that macrophages and DCs produce ubiquitin-rich structures called DALIS in response to L. pneumophila products. Triggering of a MyD88-dependent signaling pathway downstream of TLR2 stimulated DALIS formation in response to L. pneumophila. DALIS that formed upon exposure of macrophages to L. pneumophila appeared with similar kinetics and were similar morphologically to DALIS generated after LPS exposure, which suggests that the appearance of these structures was a response to TLR activation by extracellular bacteria. This is supported by the observation that both wild type and Dot/Icm-deficient L. pneumophila induced DALIS formation, and that the majority of cells containing DALIS did not harbor intracellular L. pneumophila. Thus, DALIS formation represents a normal response by these professional antigen-presenting cells to bacterial factors produced by L. pneumophila.

Our observation that macrophages and DCs infected with L. pneumophila did not contain DALIS was of particular interest. The finding that DALIS formation was completely blocked in cells infected with L. pneumophila following the stimulation of TLR4 by exogenous LPS indicates that intracellular bacteria interfere with DALIS formation by a mechanism that acts downstream of TLR activation by a cognate ligand. The involvement of Dot/Icm-translocated effectors in DALIS suppression is supported by data showing that most cells infected with dotA mutant bacteria produced DALIS. Recently, it was shown that the intracellular pathogen Brucella has a TIR-domain-containing protein that interferes with TLR2 signaling presumably by interfering with the function of host TIR-domain containing proteins such as MyD88. This Brucella protein, called Btp1, interferes with maturation of DCs, which results in the attenuated production of cytokines, co-stimulatory molecules and DALIS (Salcedo et al., 2008). It is unlikely that L. pneumophila interferes with DALIS formation by a similar mechanism as DC maturation is not affected by L. pneumophila and there is no evidence in support of Dot/Icm-mediated suppression of cytokine production or downregulation of co-stimulatory molecules (Neild et al., 2003). To the contrary, it has been shown that NF-κβ activation is enhanced by Dot/Icm-mediated events (Losick et al., 2006), and that a more robust innate immune response is generated in response to wild type L. pneumophila (Sporri et al., 2006). Thus, it is more likely that L. pneumophila suppresses DALIS formation by interfering with a process important for the assembly of DALIS that occurs after or is independent of NF-κβ activation.

DALIS formation can be suppressed pharmacologically by inhibiting host protein translation following TLR activation. L. pneumophila has the capacity to inhibit host protein synthesis, and one family of proteins that appears to play a role in this process is glycosyltransferase toxins that inactivate the host protein elongation factor 1α (EF-1A) (Belyi et al., 2006, Belyi et al., 2008). Our data indicate, however, that the Lgt toxins are not required for suppression of DALIS formation because L. longbeachae, which lacks glycosyltransferase activity on EF1α, inhibited DALIS formation. Additionally, a mutant L. pneumophila strain deficient in the three expressed Lgt proteins was able to suppress DALIS formation to a level similar to wild type L. pneumophila. Data showing that DALIS are formed in macrophages containing replicating L. jordanis indicates that DALIS suppression can be uncoupled from other Dot/Icm-dependent activities. Thus, it is likely that either loss-of-function screens in L. pneumophila or gain-of-function screens in L. jordanis should lead to the identification of effector proteins required for DALIS suppression.

The benefit of DALIS suppression for L. pneumophila intracellular persistence and survival is unclear. In immune cells, DALIS are thought to serve as temporary antigen storage compartments that are induced after detection of pathogens (Pierre, 2005). This storage compartments would slowly release ubiquitinated proteins for proteasome degradation generating antigen pools for MHC class I presentation. Because antigen presentation requires spatial and temporal coordination to trigger immune responses, the ability of L. pneumophila to disrupt DALIS might result in premature or inefficient antigen presentation. Thus, future studies examining L. pneumophila antigen presentation could provide new insight into the role of DALIS as antigen storage compartments and expand our understanding of the mechanisms of antigen presentation. Moreover, L. pneumophila is the only pathogen shown to inhibit DALIS formation completely without interfering with antigen presentation or the phenotypic maturation of DCs. Thus, L. pneumophila would be ideal to use as a model pathogen to investigate the importance of DALIS as antigen storage compartments in an infection model.

Experimental procedure

Bacterial strains

L. pneumophila serogroup 1, strain Lp01(Berger et al., 1993) and the isogenic dotA mutant strain (Zuckman et al., 1999) were used in all experiments unless otherwise indicated. Legionella longbeachae ATCC 33462 and Legionella jordanis BL-540 (ATCC 33623) species were acquired from Dr. Nicholas Cianciotto (Northwestern University). The thymidine auxotrophs used were the Lp01-derived strains Lp02 (thyA) and Lp03 (thyA dotA) (Berger et al., 1993). Strain AM101 was derived from the L. pneumophila serogroup 1, strain Lp01 and generated by sequential clean deletion of lgt1 and lgt3, followed by insertion mutagenesis of lgt2 (Table 1). Legionella strains were grown on charcoal yeast extract (CYE) plates [1% yeast extract, 1%N-(2-acetamido)-2-aminoethanesulphonic acid (ACES; pH 6.9), 3.3 mM l-cysteine, 0.33 mM Fe(NO3)3, 1.5% bacto-agar, 0.2% activated charcoal] (Feeley et al., 1979). Lp02 and Lp03 strains were grown on CYE plates containing thymidine at 100µg/ml. For all experiments, Legionella were harvested from CYE plates after growth for 2 days at 37°C.

Table 1
Strains

Plasmids and strain construction

The Δlgt1 allele was generated by recombinant PCR using a ~800bp region upstream of lgt1 (generated with primers UR_CD1368 and UL_CD1368), a downstream region of lgt1 (generated with primers DR_CD1368 and DL_CD1368) and the primer pair UL_CD1368/DR_CD1368 (Table 2). The resulting fragment was digested with XbaI and NotI and cloned in the gene replacement vector pSR47s creating pSR47s-CD1368 (Table 3). The Δlgt3 allele was generated analogously to the Δlgt1 allele using the primers UR_CD1488, UL_CD1488, DR_CD1488 and DL_CD1488 and cloned in pSR47s to create pSR47s-CD1488. An internal lgt2 fragment (408bp) was amplified with primers F_IM2862 and R_IM2862 and cloned in the suicide vector pSR47 generating pSR47-IM2862. Strain AM099 was derived from CR39 and generated by allelic exchange of lgt1 with Δlgt1, as previously described (Merriam et al., 1997). Strain AM100 was derived from AM99 by allelic exchange of lgt3 with Δlgt3. Strain AM101 was derived from AM100 by inactivation of lgt2 by co-integration with the plasmid pSR47-IM2862. Strains AM050, AM054, AM055 and AM056 were derived by allelic exchange as described above using their respective primers listed in table 2 and gene replacement vectors listed table 3. The strain AM053 was derived by sequentially clean deletion of lpg2830, lpg2144, lpg2525 and lpg1408.

Table 2
Primers
Table 3
Plasmids

Mice

Bone-marrow was harvested from A/J mice (Harlan Sprague Dawley) unless otherwise indicated. Cells deficient in TLR2 and MyD88 were derived from TLR2−/−, MyD88−/− mice homozygous for the A/J lgn1 allele and their respective heterozygous littermates as described previously (Archer et al., 2006).

Transfection and infection of CHO-FcRγII cells

CHO-FcRγII cells were cultured in DMEM supplemented with 10% FBS (Gibco). For transfection cells were seeded at 6×103 per well in a 24-well plates on coverslips and transfected with 0.3µg plasmid utilizing Fugene6 (Roche). Cells were infected 24 hours post transfection. Expression vectors coding for HA-tagged wild type human ubiquitin, ubiquitin K48-only and ubiquitin K63-only were described previously (Wertz et al., 2004). Bacteria were incubated for 1 hour with anti-L. pneumophila rabbit polyclonal antibody prior to infection. Cells were infected with L. pneumophila (Lpo1) or dotA mutant for 4 hours (MOI = 50) followed by immunofluorescence analysis with monoclonal anti-HA.11 (16B12 clone) (Covance)

BMM and DC cultures

To derive BMMs, bone marrow cells were isolated from the femurs and tibiae of mice, and cultured in RPMI 1640 containing 10% fetal bovine serum (Gibco), 20% macrophage colony-stimulating factor (M-CSF)-conditioned medium and 1% penicillin-streptomycin at 37°C with 5% CO2. Equal volume of media was added on day 4. On day 7, cells were harvested and used for infection assays. M-CSF condition media was obtained from an L-929 fibroblast cell line (ATCC). Bone marrow-derived DCs were prepared as described previously (Inaba et al., 1992).

Infections of BMMs and DCs with Legionella strains

BMMs (2×105 per well) were added to 24-well plates and infected with Legionella at MOI as indicated for each experiment for 60 min then extracellular bacteria were removed by washing 5× with warm (37°C) phosphate-buffered saline (PBS) and the infected BMMs were cultured in RPMI 1640 containing 10% FBS. The duration of infection is noted for each experiment. DCs were seed at 4×106 per well in a 12-well non-tissue culture treated plates and infected with Legionella (MOI = 50) for 60 min, then cells were collected and washed 2× with pre-warmed PBS to remove extracellular bacteria and resuspended in RPMI containing 2% FBS. DCs were seed at 3×105 per well of a 24-well plate on Alcian Blue (Sigma) treated coverslips and the infection was allowed to progress as indicated. To inhibit bacterial protein synthesis during infection, BMMs were infected with Legionella for 60 min, then extracellular bacteria were removed by washing 5× with warm PBS and RPMI 1640 medium containing 10% FBS and chloramphenicol [25 µg/ml] was added for the time periods indicated.

DALIS induction with E. coli LPS and peptidylglycan

BMMs were incubated with E. coli LPS (Sigma) [1µg/ml] or PGN (Sigma) [10µg/ml] for 1 hour then the cells were washed 5× warm PBS and cultured in RPMI 1640 containing 10% FBS. Total incubation periods are indicated for individual experiments. To generate LPS-induced DALIS in Legionella infected BMMs, macrophages were infected with Legionella strains for 1 hour then washed 3× warm PBS and cultured for 3 hours followed by continuous treatment with LPS [1µg/ml] for 4hours.

Immunofluorescence assay for detection of ubiquitinated proteins

Coverslips containing cells were washed with 3× PBS and fixed with 2% paraformaldehyde (PFA) for 10min, permeabilized with ice-cold methanol for 30 seconds, blocked with 2% goat serum in PBS containing 50mM NH4Cl for 60min. Anti-HA.11 was used at 1:1000, anti-L. pneumophila antibody was used at 1:500 dilution all other anti-bodies were used at 2µg/ml diluted in PBS containing 0.02% goat serum for 1 to 3hours. Alexa-fluore conjugated secondary antibodies (Invirtogen) were used at 1:500 dilution for 30 to 60 minutes. After 5× PBS wash coverslips were mounted with ProLong Gold antifade reagent containing DAPI (Invitrogen) slides and examined by fluorescence microscopy. For Triton-x100 extraction, coverslips containing BMMs were incubated with PBS containing 1% Triton X-100 for 20 minutes at room temperature prior to fixation with 2% PFA, then washed 3× PBS fixed and permeabilized as detailed above. Images were acquired with inverted microscope (Nicon Eclipse TE2000-S) using a 100×/1.40 oil objective (Nicon Plan Apo) and Hamamatsu ORCA ER camera. Color was added to images using IPLab software. Images were merged and deconvolution was applied using NIH Image J v1.38. Only linear image corrections in brightness or contrast were completed.

Statistical analysis

Calculations for statistical differences were completed by paired Student's t-test.

Acknowledgements

We are grateful to Dr. N. Cianciotto for the L. longbeachae and L. jordanis strains, E. Campodonico, S. Ninio and E. Cambronne for their suggestions and critical discussion of the manuscript, and K. Archer and C. Case for their technical assistance. This work was supported by NIH awards F32-GM084485, T32-AI07019 (SSI), and R01-AI048770, R01-AI041699 (CRR)

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