Specificity of Legionella pneumophila and Coxiella burnetii Vacuoles and Versatility of Legionella pneumophila Revealed by Coinfection
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感染与免疫杂志 2005年第8期
Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, 903 S. 4th St., Hamilton, Montana 59840
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109
ABSTRACT
Legionella pneumophila and Coxiella burnetii are phylogenetically related intracellular bacteria that cause aerosol-transmitted lung infections. In host cells both pathogens proliferate in vacuoles whose biogenesis displays some common features. To test the functional similarity of their respective intracellular niches, African green monkey kidney epithelial (Vero) cells, A/J mouse bone marrow-derived macrophages, human macrophages, and human dendritic cells (DC) containing mature C. burnetii replication vacuoles were superinfected with L. pneumophila, and then the acidity, lysosome-associated membrane protein (LAMP) content, and cohabitation of mature replication vacuoles was assessed. In all cell types, wild-type L. pneumophila occupied distinct vacuoles in close association with acidic, LAMP-positive C. burnetii replication vacuoles. In murine macrophages, but not primate macrophages, DC, or epithelial cells, L. pneumophila replication vacuoles were acidic and LAMP positive. Unlike wild-type L. pneumophila, type IV secretion-deficient dotA mutants trafficked to lysosome-like C. burnetii vacuoles in Vero cells where they survived but failed to replicate. In primate macrophages, DC, or epithelial cells, growth of L. pneumophila was as robust in superinfected cell cultures as in those singly infected. Thus, despite their noted similarities, L. pneumophila and C. burnetii are exquisitely adapted for replication in unique replication vacuoles, and factors that maintain the C. burnetii replication vacuole do not alter biogenesis of an adjacent L. pneumophila replication vacuole. Moreover, L. pneumophila can replicate efficiently in either lysosomal vacuoles of A/J mouse cells or in nonlysosomal vacuoles of primate cells.
INTRODUCTION
Most intracellular bacterial pathogens inhabit replication vacuoles that exhibit a wide range of interactions with the endocytic pathway (28). Elucidation of bacterial and cellular signaling pathways that promote development of a vacuole permissive for growth is critical to gaining a better understanding of pathogenic mechanisms of intracellular bacteria. Moreover, the biochemical composition of the luminal milieu of vacuoles that promotes pathogen replication is largely undefined. As one approach to improve our understanding of replication vacuole biogenesis and composition, host cells have been doubly infected with pathogens that reside in distinct vacuolar compartments (18, 21, 39, 45). Dual infections can be used to probe the dominance of pathogen signals that direct replication vacuole maturation and the permissiveness of vacuole growth environments (39). The dual infection model used in the present study used Coxiella burnetii and Legionella pneumophila, phylogenetically related macrophage pathogens that have both common and unique associations with the host cell.
C. burnetii is an obligate intracellular gram-negative bacterium and the causative agent of human Q fever (30). C. burnetii is resistant to physical and chemical disruption in the small cell variant form (SCV) of its biphasic life cycle. The SCV is presumably the environmentally stable form of C. burnetii that, when inhaled, infects alveolar macrophages and differentiates into the replicative large cell variant (13). L. pneumophila is a facultative intracellular gram-negative bacterium that parasitizes freshwater amoebas and protozoa that serve as an environmental reservoir. L. pneumophila also undergoes a biphasic life cycle where the transmissive form (TF) (or postexponential phase) is resilient and efficient at infection and the replicative form (RF) (or exponential phase) carries out intracellular growth (48). L. pneumophila can colonize human alveolar macrophages via inhalation of contaminated aerosols and cause a severe pneumonia known as Legionnaires' disease (7).
The establishment of a replication vacuole is absolutely required for infection by L. pneumophila and C. burnetii. Replication vacuoles formed by most intracellular pathogens are permissive for growth because they do not fully mature through the endocytic pathway to fuse with the lysosomal compartment (reviewed in reference 28). Maturation of L. pneumophila and C. burnetii vacuoles through the endocytic pathway is stalled relative to phagosomes containing inert particles or killed organisms (23, 47). The delay in L. pneumophila replication vacuole maturation is thought to allow differentiation of the environmentally stable TF to the replicating but environmentally sensitive RF (33). The functional significance of the delay in C. burnetii replication vacuole maturation is unclear since both SCV and LCV are inherently resistant to long-term exposure to the lysosomes (13, 24). Indeed, the replication vacuole of C. burnetii is unusual in that it ultimately becomes acidified and acquires characteristics of a phagolysosome (21). The maturation of vacuoles harboring L. pneumophila appears to be cell type specific with vacuoles acquiring lysosomal characteristics in primary murine macrophages (47) but not in primary human monocytes (53). L. pneumophila replication vacuoles form by interacting with the endoplasmic reticulum (ER) and the autophagic pathway (1, 49, 50). The C. burnetii replication vacuole also appears to interact with autophagic vesicles during maturation, but specific interactions with the biosynthetic pathway have not been demonstrated (5).
The biogenesis of bacterial replication vacuoles is regulated by the complex interplay of both host and bacterial proteins that modulate vesicular trafficking (28). Bacterial effectors of replication vacuole maturation can be translocated by type III or type IV secretory systems (28). The L. pneumophila type IV secretory apparatus is encoded by 26 dot/icm genes (42, 52). Although an intact Dot/Icm transporter is essential for establishment of the replication vacuole (42, 52), the absence of individual secreted substrates identified thus far does not result in strong defects in intracellular growth, suggesting that Dot/Icm translocated proteins are functionally redundant (14, 29, 35). The C. burnetii genome contains a nearly complete set of the L. pneumophila dot/icm genes with the exception of icmR (44), and C. burnetii homologs of L. pneumophila dotB, icmS, icmW, and icmT complement corresponding mutants in L. pneumophila (56, 57). A role for Dot/Icm in C. burnetii replication vacuole formation has not been established, owing in large part to the lack of workable genetic systems for this bacterium. However, protein synthesis is necessary for maturation and fusogenicity of the C. burnetii replication vacuole, and it is logical to suspect that Dot/Icm translocation substrates govern these events (24). Based on their genetic and cell biological similarities, it is possible that L. pneumophila and C. burnetii share common type IV secretion system-dependent effector molecules that mediate formation of their respective replication vacuoles.
To gain insight into mechanisms mediating the formation of replication vacuoles, C. burnetii-infected cells have been super- and coinfected with other pathogens. Mature C. burnetii replication vacuoles readily fuse with vacuoles harboring the intracellular pathogens Mycobacterium avium (15, 18), Mycobacterium tuberculosis (18), Leishmania amazonensis (51), and Trypanosoma cruzi (2). Of these organisms, M. avium and L. amazonensis efficiently replicate in this environment (18, 51). C. burnetii vacuoles fuse at a very low frequency with replication vacuoles of Toxoplasma gondii (45) and not at all with vacuoles harboring Chlamydia trachomatis (21), two pathogens whose vacuoles appear completely disconnected from the endocytic pathway (21, 25, 45).
C. burnetii and L. pneumophila are related bacterial pathogens that have a superficially similar infectious cycle. To compare and contrast the development of their respective replication vacuoles, we superinfected with L. pneumophila a variety of cell types previously infected with C. burnetii. By this dual-infection approach, we sought to determine whether virulence factors/type IV effectors are restricted to pathogen vacuoles or instead act globally on host cell biology. In particular, we wanted to determine whether maintenance of an existing replication vacuole inhibits subsequent development of a different replication vacuole, whether respective pathogen vacuoles are capable of heterotypic fusion, and whether a coinhabited vacuole is permissive for growth of both pathogens. Our results indicate that preexisting C. burnetii vacuoles do not compete with L. pneumophila for either establishment of its distinct vacuole or its subsequent replication.
MATERIALS AND METHODS
Bacterial strains. C. burnetii (Nine Mile strain in phase I or phase II) were propagated in African green monkey kidney epithelial (Vero) cells (CCL-81; American Type Culture Collection) grown in RPMI medium (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS; Invitrogen). Organisms were purified by renografin density gradient centrifugation as previously described (19) and stored at –80°C. L. pneumophila strain Lp02 (thymine auxotroph), and an isogenic dotA strain defective in type IV secretion, were cultured at 37°C on charcoal-yeast extract (CYE) plates or in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (AYE) broth supplemented with 100 μg of thymidine/ml (4, 16). The TF of L. pneumophila was used for all infections. Briefly, AYE broth was inoculated with L. pneumophila colonies from CYE plates less than 2 weeks old and incubated overnight (16 to 20 h) at 37°C. TF organisms were generated from these cultures by subculturing to fresh AYE broth and incubation until they entered post-exponential phase (optical density at 600 nm of 3.5 to 4.0).
Infection of Vero cells, primary BMDM, and primary human macrophages, and DC. Vero cells were propagated as described above. Murine bone marrow-derived macrophages (BMDM) were isolated from the bone marrow exudates of female A/J mouse femurs and cultured as previously described (49). Human primary macrophages and dendritic cells (DC) were derived from human peripheral blood mononuclear cells. Briefly, peripheral blood mononuclear cells were isolated from buffy coats that were generated by centrifugation through Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden). Cells were enriched for monocytes (CD14+ cells) by using a RossetteSep monocyte enrichment kit (Stem Cell Technologies, Vancouver, British Columbia, Canada). DC were generated by culturing purified monocytes (106 cells per ml) in DC medium (RPMI plus Glutamax, 5% FBS, 15 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 100 μg of penicillin-streptomycin/ml) containing interleukin 4 (10 ng/ml) and granulocyte-macrophage colony-stimulating factor (10 ng/ml) (Peprotech, Rocky Hill, N.J.). Cells were cultured for 6 days with fresh cytokines added every 48 h. Nonadherent cells were harvested and determined to be >95% DC by positive staining for CD209+/CD11c+/CD1a+ as measured by flow cytometry. Macrophages were generated by culturing purified monocytes (106 cells per ml) in macrophage medium (RPMI plus 10% FBS) containing macrophage colony-stimulating factor (10 ng/ml). After culture for at least 7 days, adherent macrophages were harvested by scraping and replated at the desired concentration.
For infections, all host cells were cultivated in 35-mm coverslip-bottom petri dishes (MatTek, Ashland, Mass.) or 24-well tissue culture plates (Corning, Inc., Corning, N.Y.) with or without 12-mm glass coverslips. Vero cells, human DC, and human macrophages were infected with the C. burnetii Nine Mile phase II strain at a multiplicity of infection (MOI) of 10. Murine BMDM were infected with the Nine Mile phase II strain or the Nine Mile phase I strain at MOIs of 10 and 20, respectively. Organisms in tissue culture media were added directly to the host cells and incubated for 2 h. Cell cultures were then washed three times with fresh tissue culture media and replenished with fresh medium. The C. burnetii inoculum was not washed from nonadherent DC cultures. L. pneumophila were used to infect BMDM, human macrophages and DC at an MOI of 2, and Vero cells at an MOI of 100. (The lower MOI used with BMDM, human macrophages, and DC was necessary to minimize L. pneumophila contact-dependent cytotoxicity [27], whereas the higher MOI used with Vero cells was necessary to maximize entry into these nonphagocytic cells, which are not susceptible to L. pneumophila cytotoxicity.) Infections were conducted as described above for C. burnetii. Thymidine was added to all tissue culture media at a final concentration of 100 μg/ml to support L. pneumophila growth. All coinfections were carried out as superinfections; C. burnetii infection preceding L. pneumophila because of its slower growth rate.
Light and fluorescence microscopy. Fixation and staining procedures for indirect immunofluorescence localization of internalized bacteria and lysosomal glycoproteins were conducted as previously described (24). Mouse anti-human lysosome-associated membrane protein 1 (LAMP-1; clone H4A3) was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, Iowa), rat anti-mouse LAMP-2 antibody was acquired from BD Pharmingen (San Diego, Calif.), rabbit anti-L. pneumophila antibody was a generous gift from R. Isberg (Tufts University School of Medicine, Boston, Mass.), and convalescent C. burnetii antiserum was derived from infected guinea pigs. Anti-mouse, anti-rabbit, anti-rat, and anti-guinea pig Alexa Fluor 488, 594, or 647 immunoglobulin G antibodies were purchased from Molecular Probes (Eugene, Oreg.). Images were acquired by using a Perkin-Elmer UltraView spinning disk confocal laser illuminator system connected to a Nikon TE-2000S microscope, a x60 objective lens, and Metamorph software (Universal Imaging Corp., Downingtown, Pa.).
The acidity of bacterium-containing vacuoles was qualitatively determined by acridine orange staining (21). Acidic vesicles were stained with acridine orange at a final concentration of 5 μg/ml in culture medium. After 1 h at 37°C, the stain was removed and the monolayer washed three times with phosphate-buffered saline (150 mM NaCl, 10 mM NaPO4 [pH 7.2]). Phase-contrast and fluorescent images of live acridine orange-stained cells were obtained by using a Nikon TE-2000-E inverted microscope, a MicroPublisher 3.3 RTV digital color camera (QImaging, Burnaby, British Columbia, Canada), and Metamorph software. All images were processed by using Image J (written by Wayne Rasband at the U.S. National Institutes of Health and available by anonymous FTP from zippy.nimh.nih.gov) and Adobe Photoshop software (Adobe Systems, Mountain View, Calif.).
Electron microscopy. Infected cells in six-well cell culture plates were fixed for 5 min with 2.5% glutaraldehyde-4.0% paraformaldehyde (vol/vol) in 0.1 M sodium phosphate buffer at pH 7.0 to 7.2. Adherent cells were detached by gently scraping cells with a cell scraper and transferred with fixative to a 1.5-ml microfuge tube. Samples were pelleted and stored in fixative for 24 to 48 h at 4°C. Fixed pellets were washed with sodium phosphate buffer and then water and then postfixed with 0.5% OsO4-0.8% K4Fe(CN)6 · 3H2O in distilled water for 30 min at room temperature. Pellets were then washed three times with water and treated with 0.1% tannic acid for 5 min. After a three washes with water, pellets were stained en bloc with 1% aqueous uranyl acetate for 30 min at room temperature. Pellets were then dehydrated through a graded ethanol series and embedded in Spurr's low-viscosity resin (Ted Pella, Inc., Redding, Calif.). Thin sections were viewed at 80 kV on a Hitachi H7500 transmission electron microscope. Images were captured by using a Hamamatsu C4742-57-12NR side-mounted charge-coupled device camera and Advantage HR/HR-B digital imaging software (AMT, Danvers, Mass.).
Quantification of L. pneumophila replication. To assess the effect of C. burnetii replication on L. pneumophila replication, CFU growth assays were performed on L. pneumophila as previously described (3). L. pneumophila was used to infect Vero cells, human macrophages, and human DC, previously infected with the C. burnetii Nine Mile phase II strain, which produces a truncated lipopolysaccharide and is more efficiently internalized than the Nine Mile phase I strain (34). Using this approach, >90% of L. pneumophila-infected cells also contained replicating C. burnetii. Growth of L. pneumophila in superinfected murine BMDM was not evaluated since only the poorly internalized C. burnetti phase I variant efficiently replicates in this cell type; consequently, a high percentage of superinfected cells could not be achieved.
Statistical analysis. Comparisons of L. pneumophila growth in singly infected and superinfected cells were performed by using the paired Student t test. The statistical significance was set at a P value of <0.05.
RESULTS
L. pneumophila and C. burnetii replicate within distinct replication vacuoles in superinfected Vero cells. To investigate the degree of similarity of replication vacuoles of L. pneumophila and C. burnetii in an epithelial host cell, we superinfected C. burnetii (phase II)-infected Vero cells with L. pneumophila (Fig. 1). Vero cells are a standard experimental host cell of C. burnetii (21) and have occasionally been used to study L. pneumophila (37). Vero cells singly infected with C. burnetii for 72 h showed replicating organisms within a spacious, acridine orange-positive (acidic), LAMP-1-positive vacuole as previously described (21). Infection of Vero cells with wild-type L. pneumophila for 24 h resulted in robust growth of the pathogen in a large, acridine orange-negative, LAMP-1-negative vacuole. The L. pneumophila vacuolar membrane was derived from the ER, as indicated by its colocalization with calnexin (data not shown). This result corroborates and extends a previous study that showed L. pneumophila replication vacuoles associated with rough ER in Vero cells (38). Vero cells infected with C. burnetii for 48 h, the approximate beginning of exponential growth phase in this cell type (13), were then superinfected with L. pneumophila for 24 h, a protocol that accommodates the slow generation time (12 h) of C. burnetii (13). As observed in the single infections, L. pneumophila resided in a distinct nonacidic, ER-derived vacuole that was typically closely juxtaposed with the acidic, LAMP-1-positive C. burnetii replication vacuole. Colocalization of wild-type L. pneumophila and C. burnetii within the same vacuole was never observed. The nuclear fragmentation of the superinfected cell depicted in the electron micrograph likely reflects L. pneumophila's ability to induce apoptotic host cell death by a caspase-3-dependent pathway (32). In contrast to wild-type L. pneumophila, a dotA mutant defective for type IV secretion did traffic to the C. burnetii replication vacuole. Vacuoles harboring both L. pneumophila dotA and C. burnetii were observed in 60.7% ± 5.0% of cells infected with both pathogens. Thus, avoidance of the lysosome-like compartment of C. burnetii by wild-type L. pneumophila is contingent upon expression of an intact type IV secretion system. Only one dotA mutant organism was typically observed in C. burnetii vacuoles, suggesting the environment is not amenable for growth of L. pneumophila.
L. pneumophila replication is enhanced in C. burnetii-infected Vero cells. We next investigated whether the growth rate of L. pneumophila was altered in Vero cells infected with C. burnetii for 48 h (Fig. 2). If each pathogen uses a similar mechanism(s) for acquisition of nutrients and vacuolar membrane, then exponentially replicating C. burnetii might interfere with subsequent growth of L. pneumophila. Replication of wild-type L. pneumophila was first quantified in singly infected cells. A 53-fold increase in CFU was observed between 2 and 48 h postinfection (p.i.) with growth slowing between 24 and 48 h p.i. Large, distended replication vacuoles were apparent by phase microscopy at 48 h with no obvious cytolysis. Surprisingly, an 86-fold increase in L. pneumophila CFU over the same time course was observed in superinfected cells, which was significantly greater (P < 0.014) than the increase observed in singly infected cells. There was no replication of the dotA mutant in singly infected or superinfected cells as indicated by similar CFU counts at 2 and 48 h p.i. As described above, a high percentage of C. burnetii replication vacuoles harbor dotA mutants in superinfected cells. Consistent with microscopic observations (Fig. 1), growth curve data indicate that these organisms survive but do not replicate. Taken together, these results indicate that replicative vacuoles occupied by dividing C. burnetii do not impede biogenesis of vacuoles harboring L. pneumophila and subsequent replication of the organism.
L. pneumophila and virulent C. burnetii replicate within distinct replication vacuoles in A/J mouse BMDM. A/J mice are susceptible to infection by virulent L. pneumophila, and primary macrophages derived from this strain are commonly used to study this pathogen (54). Of a number of mouse strains tested, A/J mice also rank as one of the more sensitive to C. burnetii infection (41). Therefore, to compare replication vacuole formation in a professionally phagocytic cell type, A/J mouse BMDM infected with C. burnetii for 48 h were superinfected with L. pneumophila for 18 h (Fig. 3). Single infections with C. burnetii were initially conducted with phase II strain, which has a truncated LPS relative to the phase I strain and is considered avirulent for animals (34). Internalized phase II organisms were clearly evident at 66 h p.i. in singly infected cells. However, vacuoles harboring phase II bacteria did not undergo homotypic fusion to form typical spacious replication vacuoles. Instead, small LAMP-2 positive vacuoles were observed scattered throughout the cytoplasm that harbored a nonreplicating, single organism. Infections with C. burnetii were then conducted with the phase I strain. Here replication vacuoles underwent homotypic fusion to form spacious acidic LAMP-2-positive replication vacuoles (ca. one to five per cell) that harbored numerous replicating organisms. At 18 h p.i. in singly infected cells, L. pneumophila also trafficked to an acidic LAMP-2-positive vacuole, where it replicated as previously described (47). As in singly infected cells, L. pneumophila and C. burnetii (phase I) both trafficked to distinct acidic, LAMP-2-positive vacuoles in super-infected cells. However, despite the similar vacuole phenotype, cohabitation of both pathogens within a single vacuole was never observed.
Rare trafficking of L. pneumophila to C. burnetii replication vacuoles occurs in human DC. Control of human infection by intracellular pathogens generally requires a vigorous Th-1 type immune response (31). DC and macrophages, adept at antigen presentation and pathogen killing, respectively, are critical components of this response (31). We have found that, unlike the situation in murine A/J mouse BMDM, phase II C. burnetii replicates in human macrophages and DC with an 10-fold increase in genome equivalents after 6 days of growth (data not shown). Therefore, to compare and contrast L. pneumophila and C. burnetii replication vacuole development in macrophages and DC, human cells infected with phase II C. burnetii for 36 h were superinfected with L. pneumophila for 12 h (Fig. 4 and 5). The shorter incubation time after L. pneumophila infection used here relative to other cell types was necessary to prevent the pronounced cell detachment that was observed for an incubation time of 18 h. In singly infected macrophages at 48 h p.i., replicating C. burnetii localized to multiple large and spacious, acidic (data not shown) LAMP-1 positive vacuoles (Fig. 4). Conversely, at 12 h p.i. L. pneumophila localized to a nonacidic (data not shown) LAMP-1-negative vacuole where robust replication was evident (Fig. 4). The vacuole phenotypes of each pathogen in singly infected macrophages were conserved in superinfected macrophages (Fig. 4). The pathogen vacuoles of superinfected cells were distinct and clearly separate with no cohabitation observed. In singly infected DC, multiple large and spacious, acidic (data not shown) LAMP-1-positive vacuoles containing replicating C. burnetii were observed at 48 h p.i. (Fig. 5). Like the situation in human macrophages, at 12 h p.i. L. pneumophila were harbored in nonacidic (data not shown), LAMP-1-negative vacuoles (Fig. 5). The vacuole phenotypes of each pathogen in singly infected DC were conserved in superinfected DC, with the most common occurrence being the formation of distinct and clearly separate replication vacuoles (Fig. 5). However, unlike the other cell types examined, trafficking of wild-type L. pneumophila to the C. burnetii vacuole was observed, albeit at a very low frequency (<1% of coinfected cells). In coinhabited vacuoles, only C. burnetii appeared to be actively replicating, since only one or two L. pneumophila organisms were present. Thus, although an acidic, LAMP-1-positive vacuole is permissive for growth of wild-type L. pneumophila in murine BMDM, in DC the organism apparently does not replicate in C. burnetii replication vacuoles with the same traits.
Overall, L. pneumophila replication is unaltered in human macrophages and DC infected with C. burnetii. We next investigated whether the growth rate of L. pneumophila was altered in human macrophages and DC infected with C. burnetii (phase II) for 36 h (Fig. 6). Between 2 and 48 h p.i., L. pneumophila replicated at a statistically similar rate in singly infected and superinfected human macrophages that approximated the rate previously described for L. pneumophila in murine BMDM (49). Host cell lysis occurred by 24 h p.i., allowing a secondary round of infection that resulted in 188- and 165-fold increases in CFU in singly infected and superinfected cells, respectively. Between 2 and 72 h p.i., L. pneumophila also replicated in singly infected and superinfected human DC at statistically similar rates. Host cell lysis also occurred by 24 h p.i., allowing a secondary round of infection. In DC, the fold increase in CFU was significantly lower than the yield from macrophages over a shorter time course with 27- and 16-fold increases in CFU in singly infected and superinfected cells, respectively. The lower absolute increase in CFU from DC relative to macrophages likely reflects the fact that the starting inoculum was not removed from infected cells.
DISCUSSION
Signals involved in maturation and maintenance of lysosome-like C. burnetii replication vacuoles do not appear to act in trans to circumvent Dot/Icm-dependent signals that promote formation of L. pneumophila replication vacuoles. Likewise, factors that promote development of L. pneumophila replication vacuoles are apparently specific to this compartment and do not disrupt acidification or the structural integrity of existing C. burnetii vacuoles. Consistent with this model are results showing that L. pneumophila and C. burnetii replicate in distinct vacuoles within superinfected BMDM, human macrophages, and DC, professionally phagocytic cell types, and within Vero epithelial cells. With the exception of murine BMDM, L. pneumophila resides in a nonacidic, LAMP-negative compartment. The occurrence of pathogen-specific replication vacuoles in murine BMDM is somewhat surprising since in this cell type L. pneumophila replicates in acidic, LAMP-positive vacuoles, and the C. burnetii vacuole is promiscuously fusogenic with vacuole compartments having lysosomal characteristics (24). Effectors of C. burnetii vacuole formation also do not disrupt development of T. gondii and C. trachomatis vacuoles in doubly infected cells (21, 45) However, unlike the L. pneumophila vacuole, replication vacuoles of these organisms have negligible interactions with the endocytic pathway (21, 25, 45). Conversely, effectors of C. burnetii replication vacuole formation appear to override regulatory mechanisms that normally stall maturation of vacuoles harboring M. avium or M. tuberculosis at an early endosome stage (11, 15, 18, 46).
The occurrence of distinct replication vacuoles in superinfected cells indicates that L. pneumophila and C. burnetii effectors of vacuole maturation specifically act in cis to mediate their formation. This specificity may reflect the fact that, although the genomes of L. pneumophila and C. burnetii encode similar Dot/Icm secretion systems (42, 44, 52), to date, nearly all defined and putative L. pneumophila type IV effectors lack homologs in C. burnetii (9, 10, 43). Type IV secretion may also be differentially regulated by the two pathogens. In L. pneumophila, type IV secretion is required prior to and immediately after infection for establishment of a replication vacuole in BMDM (40). Once the replication vacuole is formed, continued expression of dot/icm is not required for intracellular growth (12, 40). Conversely, continuous synthesis of C. burnetii protein (possibly translocated by a type IV apparatus) is required for maintenance of the spacious character and fusogenicity of its replication vacuole (24).
Further evidence supporting noncompetitiveness of L. pneumophila and C. burnetii replicative vacuoles are data showing that L. pneumophila replicate at approximately the same rate in singly infected and C. burnetii-infected human macrophages and DC. Indeed, in Vero cells, L. pneumophila grows more robustly in C. burnetii-infected cells. This behavior may be due to nonlytic growth of L. pneumophila in this cell type, combined with the possibility that C. burnetii infection upregulates nutrient trafficking pathways that augment L. pneumophila replication. Our results suggest that host cells can metabolically adapt to support vigorous growth of two intracellular pathogens that possibly exploit a common pathway for nutrient acquisition. Alternatively, pathogens may adapt to exploit noncompetitive nutrient sources.
The idea that the C. burnetii vacuole is toxic to, and/or nutritionally deficient for, L. pneumophila is supported by data showing dotA mutants and wild-type organisms do not replicate in this compartment in Vero cells and human DC, respectively. Lack of replication is likely not due to acidity per se because, as demonstrated here and elsewhere (47), L. pneumophila replicate in acidic vacuoles in BMDM. Moreover, dotA mutants can replicate when presented with an appropriate intracellular environment, since synchronous infection of this strain with wild-type L. pneumophila results in coinhabited vacuoles where both strains replicate (12). Interestingly, the C. burnetii vacuole is permissive for growth of the more distantly related organisms M. avium and L. amazonensis (18, 51). Human DC were the only cell type where trafficking of wild-type L. pneumophila to the C. burnetii replication vacuole was observed, albeit at a very low frequency. This specific trafficking behavior may reflect the dominant antigen presentation pathway of DC (31).
As demonstrated here and elsewhere (22, 53), trafficking of L. pneumophila to an acidic, LAMP-positive vacuole does not occur in primate macrophages, DC, and epithelial cells. Thus, the lysosomal trafficking observed in murine BMDM is likely a host cell-directed process that is not required for pathogen replication. After internalization by BMDM from A/J mice, L. pneumophila initially avoid the endosomal network and delay phagosome maturation by a mechanism that requires dot/icm type IV secretion (4, 6, 22). The L. pneumophila vacuole first acquires ER markers (22, 26, 49) and, after 8 to 12 h, during which time the bacterium differentiates from the TF to RF, the replication vacuole acquires markers of terminal lysosomes, including low pH, LAMP-1, fluid-phase markers, and cathepsin D (47). Replication of L. pneumophila in lysosomal and nonlysosomal compartments also illustrates the metabolic flexibility of the organism. Conversely, C. burnetii absolutely requires an acidic environment for replication (20).
Both replicating virulent phase I C. burnetii and nonreplicating avirulent phase II organisms traffic to LAMP-positive vacuoles in A/J mouse BMDM. The lack of replication of phase II bacteria is consistent with a previous study showing little replication of this variant in BMDM derived from a number of mouse strains (55). In contrast to murine BMDM, we observed vigorous replication of phase II C. burnetii in acidic, LAMP-positive vacuoles of human macrophages and DC. This result contrasts with previous reports showing no replication and subsequent killing of phase II organisms in primary human monocytes and human monocyte-like lines (8, 17). These studies reported that phase II, but not phase I C. burnetii, engage complement receptor 3 to result in delivery of the organism to a microbicidal lysosomal compartment (8, 17). Maturation of vacuoles containing phase I is proposed to stall at a late endosome stage (17). Permissiveness for L. pneumophila growth in DC also appears to be host species specific. Here we show proficient replication of L. pneumophila in human DC, whereas a previous study demonstrated severe growth restriction in murine DC (36). These disparate results may be due to innate differences between human and mouse DC.
In conclusion, both L. pneumophila and C. burnetii appear exquisitely adapted for replication in unique vacuolar environments that noncompetitively coexist within the same cell. L. pneumophila is the more adaptable intracellular pathogen since it can prosper in either a lysosomal or nonlysosomal replication vacuole during its infectious cycle.
ACKNOWLEDGMENTS
We thank Harlan Caldwell, Ted Hackstadt, Olivia Steele-Mortimer, and Ari Molofsky for review of the manuscript and Gary Hettrick for graphics.
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Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109
ABSTRACT
Legionella pneumophila and Coxiella burnetii are phylogenetically related intracellular bacteria that cause aerosol-transmitted lung infections. In host cells both pathogens proliferate in vacuoles whose biogenesis displays some common features. To test the functional similarity of their respective intracellular niches, African green monkey kidney epithelial (Vero) cells, A/J mouse bone marrow-derived macrophages, human macrophages, and human dendritic cells (DC) containing mature C. burnetii replication vacuoles were superinfected with L. pneumophila, and then the acidity, lysosome-associated membrane protein (LAMP) content, and cohabitation of mature replication vacuoles was assessed. In all cell types, wild-type L. pneumophila occupied distinct vacuoles in close association with acidic, LAMP-positive C. burnetii replication vacuoles. In murine macrophages, but not primate macrophages, DC, or epithelial cells, L. pneumophila replication vacuoles were acidic and LAMP positive. Unlike wild-type L. pneumophila, type IV secretion-deficient dotA mutants trafficked to lysosome-like C. burnetii vacuoles in Vero cells where they survived but failed to replicate. In primate macrophages, DC, or epithelial cells, growth of L. pneumophila was as robust in superinfected cell cultures as in those singly infected. Thus, despite their noted similarities, L. pneumophila and C. burnetii are exquisitely adapted for replication in unique replication vacuoles, and factors that maintain the C. burnetii replication vacuole do not alter biogenesis of an adjacent L. pneumophila replication vacuole. Moreover, L. pneumophila can replicate efficiently in either lysosomal vacuoles of A/J mouse cells or in nonlysosomal vacuoles of primate cells.
INTRODUCTION
Most intracellular bacterial pathogens inhabit replication vacuoles that exhibit a wide range of interactions with the endocytic pathway (28). Elucidation of bacterial and cellular signaling pathways that promote development of a vacuole permissive for growth is critical to gaining a better understanding of pathogenic mechanisms of intracellular bacteria. Moreover, the biochemical composition of the luminal milieu of vacuoles that promotes pathogen replication is largely undefined. As one approach to improve our understanding of replication vacuole biogenesis and composition, host cells have been doubly infected with pathogens that reside in distinct vacuolar compartments (18, 21, 39, 45). Dual infections can be used to probe the dominance of pathogen signals that direct replication vacuole maturation and the permissiveness of vacuole growth environments (39). The dual infection model used in the present study used Coxiella burnetii and Legionella pneumophila, phylogenetically related macrophage pathogens that have both common and unique associations with the host cell.
C. burnetii is an obligate intracellular gram-negative bacterium and the causative agent of human Q fever (30). C. burnetii is resistant to physical and chemical disruption in the small cell variant form (SCV) of its biphasic life cycle. The SCV is presumably the environmentally stable form of C. burnetii that, when inhaled, infects alveolar macrophages and differentiates into the replicative large cell variant (13). L. pneumophila is a facultative intracellular gram-negative bacterium that parasitizes freshwater amoebas and protozoa that serve as an environmental reservoir. L. pneumophila also undergoes a biphasic life cycle where the transmissive form (TF) (or postexponential phase) is resilient and efficient at infection and the replicative form (RF) (or exponential phase) carries out intracellular growth (48). L. pneumophila can colonize human alveolar macrophages via inhalation of contaminated aerosols and cause a severe pneumonia known as Legionnaires' disease (7).
The establishment of a replication vacuole is absolutely required for infection by L. pneumophila and C. burnetii. Replication vacuoles formed by most intracellular pathogens are permissive for growth because they do not fully mature through the endocytic pathway to fuse with the lysosomal compartment (reviewed in reference 28). Maturation of L. pneumophila and C. burnetii vacuoles through the endocytic pathway is stalled relative to phagosomes containing inert particles or killed organisms (23, 47). The delay in L. pneumophila replication vacuole maturation is thought to allow differentiation of the environmentally stable TF to the replicating but environmentally sensitive RF (33). The functional significance of the delay in C. burnetii replication vacuole maturation is unclear since both SCV and LCV are inherently resistant to long-term exposure to the lysosomes (13, 24). Indeed, the replication vacuole of C. burnetii is unusual in that it ultimately becomes acidified and acquires characteristics of a phagolysosome (21). The maturation of vacuoles harboring L. pneumophila appears to be cell type specific with vacuoles acquiring lysosomal characteristics in primary murine macrophages (47) but not in primary human monocytes (53). L. pneumophila replication vacuoles form by interacting with the endoplasmic reticulum (ER) and the autophagic pathway (1, 49, 50). The C. burnetii replication vacuole also appears to interact with autophagic vesicles during maturation, but specific interactions with the biosynthetic pathway have not been demonstrated (5).
The biogenesis of bacterial replication vacuoles is regulated by the complex interplay of both host and bacterial proteins that modulate vesicular trafficking (28). Bacterial effectors of replication vacuole maturation can be translocated by type III or type IV secretory systems (28). The L. pneumophila type IV secretory apparatus is encoded by 26 dot/icm genes (42, 52). Although an intact Dot/Icm transporter is essential for establishment of the replication vacuole (42, 52), the absence of individual secreted substrates identified thus far does not result in strong defects in intracellular growth, suggesting that Dot/Icm translocated proteins are functionally redundant (14, 29, 35). The C. burnetii genome contains a nearly complete set of the L. pneumophila dot/icm genes with the exception of icmR (44), and C. burnetii homologs of L. pneumophila dotB, icmS, icmW, and icmT complement corresponding mutants in L. pneumophila (56, 57). A role for Dot/Icm in C. burnetii replication vacuole formation has not been established, owing in large part to the lack of workable genetic systems for this bacterium. However, protein synthesis is necessary for maturation and fusogenicity of the C. burnetii replication vacuole, and it is logical to suspect that Dot/Icm translocation substrates govern these events (24). Based on their genetic and cell biological similarities, it is possible that L. pneumophila and C. burnetii share common type IV secretion system-dependent effector molecules that mediate formation of their respective replication vacuoles.
To gain insight into mechanisms mediating the formation of replication vacuoles, C. burnetii-infected cells have been super- and coinfected with other pathogens. Mature C. burnetii replication vacuoles readily fuse with vacuoles harboring the intracellular pathogens Mycobacterium avium (15, 18), Mycobacterium tuberculosis (18), Leishmania amazonensis (51), and Trypanosoma cruzi (2). Of these organisms, M. avium and L. amazonensis efficiently replicate in this environment (18, 51). C. burnetii vacuoles fuse at a very low frequency with replication vacuoles of Toxoplasma gondii (45) and not at all with vacuoles harboring Chlamydia trachomatis (21), two pathogens whose vacuoles appear completely disconnected from the endocytic pathway (21, 25, 45).
C. burnetii and L. pneumophila are related bacterial pathogens that have a superficially similar infectious cycle. To compare and contrast the development of their respective replication vacuoles, we superinfected with L. pneumophila a variety of cell types previously infected with C. burnetii. By this dual-infection approach, we sought to determine whether virulence factors/type IV effectors are restricted to pathogen vacuoles or instead act globally on host cell biology. In particular, we wanted to determine whether maintenance of an existing replication vacuole inhibits subsequent development of a different replication vacuole, whether respective pathogen vacuoles are capable of heterotypic fusion, and whether a coinhabited vacuole is permissive for growth of both pathogens. Our results indicate that preexisting C. burnetii vacuoles do not compete with L. pneumophila for either establishment of its distinct vacuole or its subsequent replication.
MATERIALS AND METHODS
Bacterial strains. C. burnetii (Nine Mile strain in phase I or phase II) were propagated in African green monkey kidney epithelial (Vero) cells (CCL-81; American Type Culture Collection) grown in RPMI medium (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS; Invitrogen). Organisms were purified by renografin density gradient centrifugation as previously described (19) and stored at –80°C. L. pneumophila strain Lp02 (thymine auxotroph), and an isogenic dotA strain defective in type IV secretion, were cultured at 37°C on charcoal-yeast extract (CYE) plates or in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (AYE) broth supplemented with 100 μg of thymidine/ml (4, 16). The TF of L. pneumophila was used for all infections. Briefly, AYE broth was inoculated with L. pneumophila colonies from CYE plates less than 2 weeks old and incubated overnight (16 to 20 h) at 37°C. TF organisms were generated from these cultures by subculturing to fresh AYE broth and incubation until they entered post-exponential phase (optical density at 600 nm of 3.5 to 4.0).
Infection of Vero cells, primary BMDM, and primary human macrophages, and DC. Vero cells were propagated as described above. Murine bone marrow-derived macrophages (BMDM) were isolated from the bone marrow exudates of female A/J mouse femurs and cultured as previously described (49). Human primary macrophages and dendritic cells (DC) were derived from human peripheral blood mononuclear cells. Briefly, peripheral blood mononuclear cells were isolated from buffy coats that were generated by centrifugation through Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden). Cells were enriched for monocytes (CD14+ cells) by using a RossetteSep monocyte enrichment kit (Stem Cell Technologies, Vancouver, British Columbia, Canada). DC were generated by culturing purified monocytes (106 cells per ml) in DC medium (RPMI plus Glutamax, 5% FBS, 15 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 100 μg of penicillin-streptomycin/ml) containing interleukin 4 (10 ng/ml) and granulocyte-macrophage colony-stimulating factor (10 ng/ml) (Peprotech, Rocky Hill, N.J.). Cells were cultured for 6 days with fresh cytokines added every 48 h. Nonadherent cells were harvested and determined to be >95% DC by positive staining for CD209+/CD11c+/CD1a+ as measured by flow cytometry. Macrophages were generated by culturing purified monocytes (106 cells per ml) in macrophage medium (RPMI plus 10% FBS) containing macrophage colony-stimulating factor (10 ng/ml). After culture for at least 7 days, adherent macrophages were harvested by scraping and replated at the desired concentration.
For infections, all host cells were cultivated in 35-mm coverslip-bottom petri dishes (MatTek, Ashland, Mass.) or 24-well tissue culture plates (Corning, Inc., Corning, N.Y.) with or without 12-mm glass coverslips. Vero cells, human DC, and human macrophages were infected with the C. burnetii Nine Mile phase II strain at a multiplicity of infection (MOI) of 10. Murine BMDM were infected with the Nine Mile phase II strain or the Nine Mile phase I strain at MOIs of 10 and 20, respectively. Organisms in tissue culture media were added directly to the host cells and incubated for 2 h. Cell cultures were then washed three times with fresh tissue culture media and replenished with fresh medium. The C. burnetii inoculum was not washed from nonadherent DC cultures. L. pneumophila were used to infect BMDM, human macrophages and DC at an MOI of 2, and Vero cells at an MOI of 100. (The lower MOI used with BMDM, human macrophages, and DC was necessary to minimize L. pneumophila contact-dependent cytotoxicity [27], whereas the higher MOI used with Vero cells was necessary to maximize entry into these nonphagocytic cells, which are not susceptible to L. pneumophila cytotoxicity.) Infections were conducted as described above for C. burnetii. Thymidine was added to all tissue culture media at a final concentration of 100 μg/ml to support L. pneumophila growth. All coinfections were carried out as superinfections; C. burnetii infection preceding L. pneumophila because of its slower growth rate.
Light and fluorescence microscopy. Fixation and staining procedures for indirect immunofluorescence localization of internalized bacteria and lysosomal glycoproteins were conducted as previously described (24). Mouse anti-human lysosome-associated membrane protein 1 (LAMP-1; clone H4A3) was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, Iowa), rat anti-mouse LAMP-2 antibody was acquired from BD Pharmingen (San Diego, Calif.), rabbit anti-L. pneumophila antibody was a generous gift from R. Isberg (Tufts University School of Medicine, Boston, Mass.), and convalescent C. burnetii antiserum was derived from infected guinea pigs. Anti-mouse, anti-rabbit, anti-rat, and anti-guinea pig Alexa Fluor 488, 594, or 647 immunoglobulin G antibodies were purchased from Molecular Probes (Eugene, Oreg.). Images were acquired by using a Perkin-Elmer UltraView spinning disk confocal laser illuminator system connected to a Nikon TE-2000S microscope, a x60 objective lens, and Metamorph software (Universal Imaging Corp., Downingtown, Pa.).
The acidity of bacterium-containing vacuoles was qualitatively determined by acridine orange staining (21). Acidic vesicles were stained with acridine orange at a final concentration of 5 μg/ml in culture medium. After 1 h at 37°C, the stain was removed and the monolayer washed three times with phosphate-buffered saline (150 mM NaCl, 10 mM NaPO4 [pH 7.2]). Phase-contrast and fluorescent images of live acridine orange-stained cells were obtained by using a Nikon TE-2000-E inverted microscope, a MicroPublisher 3.3 RTV digital color camera (QImaging, Burnaby, British Columbia, Canada), and Metamorph software. All images were processed by using Image J (written by Wayne Rasband at the U.S. National Institutes of Health and available by anonymous FTP from zippy.nimh.nih.gov) and Adobe Photoshop software (Adobe Systems, Mountain View, Calif.).
Electron microscopy. Infected cells in six-well cell culture plates were fixed for 5 min with 2.5% glutaraldehyde-4.0% paraformaldehyde (vol/vol) in 0.1 M sodium phosphate buffer at pH 7.0 to 7.2. Adherent cells were detached by gently scraping cells with a cell scraper and transferred with fixative to a 1.5-ml microfuge tube. Samples were pelleted and stored in fixative for 24 to 48 h at 4°C. Fixed pellets were washed with sodium phosphate buffer and then water and then postfixed with 0.5% OsO4-0.8% K4Fe(CN)6 · 3H2O in distilled water for 30 min at room temperature. Pellets were then washed three times with water and treated with 0.1% tannic acid for 5 min. After a three washes with water, pellets were stained en bloc with 1% aqueous uranyl acetate for 30 min at room temperature. Pellets were then dehydrated through a graded ethanol series and embedded in Spurr's low-viscosity resin (Ted Pella, Inc., Redding, Calif.). Thin sections were viewed at 80 kV on a Hitachi H7500 transmission electron microscope. Images were captured by using a Hamamatsu C4742-57-12NR side-mounted charge-coupled device camera and Advantage HR/HR-B digital imaging software (AMT, Danvers, Mass.).
Quantification of L. pneumophila replication. To assess the effect of C. burnetii replication on L. pneumophila replication, CFU growth assays were performed on L. pneumophila as previously described (3). L. pneumophila was used to infect Vero cells, human macrophages, and human DC, previously infected with the C. burnetii Nine Mile phase II strain, which produces a truncated lipopolysaccharide and is more efficiently internalized than the Nine Mile phase I strain (34). Using this approach, >90% of L. pneumophila-infected cells also contained replicating C. burnetii. Growth of L. pneumophila in superinfected murine BMDM was not evaluated since only the poorly internalized C. burnetti phase I variant efficiently replicates in this cell type; consequently, a high percentage of superinfected cells could not be achieved.
Statistical analysis. Comparisons of L. pneumophila growth in singly infected and superinfected cells were performed by using the paired Student t test. The statistical significance was set at a P value of <0.05.
RESULTS
L. pneumophila and C. burnetii replicate within distinct replication vacuoles in superinfected Vero cells. To investigate the degree of similarity of replication vacuoles of L. pneumophila and C. burnetii in an epithelial host cell, we superinfected C. burnetii (phase II)-infected Vero cells with L. pneumophila (Fig. 1). Vero cells are a standard experimental host cell of C. burnetii (21) and have occasionally been used to study L. pneumophila (37). Vero cells singly infected with C. burnetii for 72 h showed replicating organisms within a spacious, acridine orange-positive (acidic), LAMP-1-positive vacuole as previously described (21). Infection of Vero cells with wild-type L. pneumophila for 24 h resulted in robust growth of the pathogen in a large, acridine orange-negative, LAMP-1-negative vacuole. The L. pneumophila vacuolar membrane was derived from the ER, as indicated by its colocalization with calnexin (data not shown). This result corroborates and extends a previous study that showed L. pneumophila replication vacuoles associated with rough ER in Vero cells (38). Vero cells infected with C. burnetii for 48 h, the approximate beginning of exponential growth phase in this cell type (13), were then superinfected with L. pneumophila for 24 h, a protocol that accommodates the slow generation time (12 h) of C. burnetii (13). As observed in the single infections, L. pneumophila resided in a distinct nonacidic, ER-derived vacuole that was typically closely juxtaposed with the acidic, LAMP-1-positive C. burnetii replication vacuole. Colocalization of wild-type L. pneumophila and C. burnetii within the same vacuole was never observed. The nuclear fragmentation of the superinfected cell depicted in the electron micrograph likely reflects L. pneumophila's ability to induce apoptotic host cell death by a caspase-3-dependent pathway (32). In contrast to wild-type L. pneumophila, a dotA mutant defective for type IV secretion did traffic to the C. burnetii replication vacuole. Vacuoles harboring both L. pneumophila dotA and C. burnetii were observed in 60.7% ± 5.0% of cells infected with both pathogens. Thus, avoidance of the lysosome-like compartment of C. burnetii by wild-type L. pneumophila is contingent upon expression of an intact type IV secretion system. Only one dotA mutant organism was typically observed in C. burnetii vacuoles, suggesting the environment is not amenable for growth of L. pneumophila.
L. pneumophila replication is enhanced in C. burnetii-infected Vero cells. We next investigated whether the growth rate of L. pneumophila was altered in Vero cells infected with C. burnetii for 48 h (Fig. 2). If each pathogen uses a similar mechanism(s) for acquisition of nutrients and vacuolar membrane, then exponentially replicating C. burnetii might interfere with subsequent growth of L. pneumophila. Replication of wild-type L. pneumophila was first quantified in singly infected cells. A 53-fold increase in CFU was observed between 2 and 48 h postinfection (p.i.) with growth slowing between 24 and 48 h p.i. Large, distended replication vacuoles were apparent by phase microscopy at 48 h with no obvious cytolysis. Surprisingly, an 86-fold increase in L. pneumophila CFU over the same time course was observed in superinfected cells, which was significantly greater (P < 0.014) than the increase observed in singly infected cells. There was no replication of the dotA mutant in singly infected or superinfected cells as indicated by similar CFU counts at 2 and 48 h p.i. As described above, a high percentage of C. burnetii replication vacuoles harbor dotA mutants in superinfected cells. Consistent with microscopic observations (Fig. 1), growth curve data indicate that these organisms survive but do not replicate. Taken together, these results indicate that replicative vacuoles occupied by dividing C. burnetii do not impede biogenesis of vacuoles harboring L. pneumophila and subsequent replication of the organism.
L. pneumophila and virulent C. burnetii replicate within distinct replication vacuoles in A/J mouse BMDM. A/J mice are susceptible to infection by virulent L. pneumophila, and primary macrophages derived from this strain are commonly used to study this pathogen (54). Of a number of mouse strains tested, A/J mice also rank as one of the more sensitive to C. burnetii infection (41). Therefore, to compare replication vacuole formation in a professionally phagocytic cell type, A/J mouse BMDM infected with C. burnetii for 48 h were superinfected with L. pneumophila for 18 h (Fig. 3). Single infections with C. burnetii were initially conducted with phase II strain, which has a truncated LPS relative to the phase I strain and is considered avirulent for animals (34). Internalized phase II organisms were clearly evident at 66 h p.i. in singly infected cells. However, vacuoles harboring phase II bacteria did not undergo homotypic fusion to form typical spacious replication vacuoles. Instead, small LAMP-2 positive vacuoles were observed scattered throughout the cytoplasm that harbored a nonreplicating, single organism. Infections with C. burnetii were then conducted with the phase I strain. Here replication vacuoles underwent homotypic fusion to form spacious acidic LAMP-2-positive replication vacuoles (ca. one to five per cell) that harbored numerous replicating organisms. At 18 h p.i. in singly infected cells, L. pneumophila also trafficked to an acidic LAMP-2-positive vacuole, where it replicated as previously described (47). As in singly infected cells, L. pneumophila and C. burnetii (phase I) both trafficked to distinct acidic, LAMP-2-positive vacuoles in super-infected cells. However, despite the similar vacuole phenotype, cohabitation of both pathogens within a single vacuole was never observed.
Rare trafficking of L. pneumophila to C. burnetii replication vacuoles occurs in human DC. Control of human infection by intracellular pathogens generally requires a vigorous Th-1 type immune response (31). DC and macrophages, adept at antigen presentation and pathogen killing, respectively, are critical components of this response (31). We have found that, unlike the situation in murine A/J mouse BMDM, phase II C. burnetii replicates in human macrophages and DC with an 10-fold increase in genome equivalents after 6 days of growth (data not shown). Therefore, to compare and contrast L. pneumophila and C. burnetii replication vacuole development in macrophages and DC, human cells infected with phase II C. burnetii for 36 h were superinfected with L. pneumophila for 12 h (Fig. 4 and 5). The shorter incubation time after L. pneumophila infection used here relative to other cell types was necessary to prevent the pronounced cell detachment that was observed for an incubation time of 18 h. In singly infected macrophages at 48 h p.i., replicating C. burnetii localized to multiple large and spacious, acidic (data not shown) LAMP-1 positive vacuoles (Fig. 4). Conversely, at 12 h p.i. L. pneumophila localized to a nonacidic (data not shown) LAMP-1-negative vacuole where robust replication was evident (Fig. 4). The vacuole phenotypes of each pathogen in singly infected macrophages were conserved in superinfected macrophages (Fig. 4). The pathogen vacuoles of superinfected cells were distinct and clearly separate with no cohabitation observed. In singly infected DC, multiple large and spacious, acidic (data not shown) LAMP-1-positive vacuoles containing replicating C. burnetii were observed at 48 h p.i. (Fig. 5). Like the situation in human macrophages, at 12 h p.i. L. pneumophila were harbored in nonacidic (data not shown), LAMP-1-negative vacuoles (Fig. 5). The vacuole phenotypes of each pathogen in singly infected DC were conserved in superinfected DC, with the most common occurrence being the formation of distinct and clearly separate replication vacuoles (Fig. 5). However, unlike the other cell types examined, trafficking of wild-type L. pneumophila to the C. burnetii vacuole was observed, albeit at a very low frequency (<1% of coinfected cells). In coinhabited vacuoles, only C. burnetii appeared to be actively replicating, since only one or two L. pneumophila organisms were present. Thus, although an acidic, LAMP-1-positive vacuole is permissive for growth of wild-type L. pneumophila in murine BMDM, in DC the organism apparently does not replicate in C. burnetii replication vacuoles with the same traits.
Overall, L. pneumophila replication is unaltered in human macrophages and DC infected with C. burnetii. We next investigated whether the growth rate of L. pneumophila was altered in human macrophages and DC infected with C. burnetii (phase II) for 36 h (Fig. 6). Between 2 and 48 h p.i., L. pneumophila replicated at a statistically similar rate in singly infected and superinfected human macrophages that approximated the rate previously described for L. pneumophila in murine BMDM (49). Host cell lysis occurred by 24 h p.i., allowing a secondary round of infection that resulted in 188- and 165-fold increases in CFU in singly infected and superinfected cells, respectively. Between 2 and 72 h p.i., L. pneumophila also replicated in singly infected and superinfected human DC at statistically similar rates. Host cell lysis also occurred by 24 h p.i., allowing a secondary round of infection. In DC, the fold increase in CFU was significantly lower than the yield from macrophages over a shorter time course with 27- and 16-fold increases in CFU in singly infected and superinfected cells, respectively. The lower absolute increase in CFU from DC relative to macrophages likely reflects the fact that the starting inoculum was not removed from infected cells.
DISCUSSION
Signals involved in maturation and maintenance of lysosome-like C. burnetii replication vacuoles do not appear to act in trans to circumvent Dot/Icm-dependent signals that promote formation of L. pneumophila replication vacuoles. Likewise, factors that promote development of L. pneumophila replication vacuoles are apparently specific to this compartment and do not disrupt acidification or the structural integrity of existing C. burnetii vacuoles. Consistent with this model are results showing that L. pneumophila and C. burnetii replicate in distinct vacuoles within superinfected BMDM, human macrophages, and DC, professionally phagocytic cell types, and within Vero epithelial cells. With the exception of murine BMDM, L. pneumophila resides in a nonacidic, LAMP-negative compartment. The occurrence of pathogen-specific replication vacuoles in murine BMDM is somewhat surprising since in this cell type L. pneumophila replicates in acidic, LAMP-positive vacuoles, and the C. burnetii vacuole is promiscuously fusogenic with vacuole compartments having lysosomal characteristics (24). Effectors of C. burnetii vacuole formation also do not disrupt development of T. gondii and C. trachomatis vacuoles in doubly infected cells (21, 45) However, unlike the L. pneumophila vacuole, replication vacuoles of these organisms have negligible interactions with the endocytic pathway (21, 25, 45). Conversely, effectors of C. burnetii replication vacuole formation appear to override regulatory mechanisms that normally stall maturation of vacuoles harboring M. avium or M. tuberculosis at an early endosome stage (11, 15, 18, 46).
The occurrence of distinct replication vacuoles in superinfected cells indicates that L. pneumophila and C. burnetii effectors of vacuole maturation specifically act in cis to mediate their formation. This specificity may reflect the fact that, although the genomes of L. pneumophila and C. burnetii encode similar Dot/Icm secretion systems (42, 44, 52), to date, nearly all defined and putative L. pneumophila type IV effectors lack homologs in C. burnetii (9, 10, 43). Type IV secretion may also be differentially regulated by the two pathogens. In L. pneumophila, type IV secretion is required prior to and immediately after infection for establishment of a replication vacuole in BMDM (40). Once the replication vacuole is formed, continued expression of dot/icm is not required for intracellular growth (12, 40). Conversely, continuous synthesis of C. burnetii protein (possibly translocated by a type IV apparatus) is required for maintenance of the spacious character and fusogenicity of its replication vacuole (24).
Further evidence supporting noncompetitiveness of L. pneumophila and C. burnetii replicative vacuoles are data showing that L. pneumophila replicate at approximately the same rate in singly infected and C. burnetii-infected human macrophages and DC. Indeed, in Vero cells, L. pneumophila grows more robustly in C. burnetii-infected cells. This behavior may be due to nonlytic growth of L. pneumophila in this cell type, combined with the possibility that C. burnetii infection upregulates nutrient trafficking pathways that augment L. pneumophila replication. Our results suggest that host cells can metabolically adapt to support vigorous growth of two intracellular pathogens that possibly exploit a common pathway for nutrient acquisition. Alternatively, pathogens may adapt to exploit noncompetitive nutrient sources.
The idea that the C. burnetii vacuole is toxic to, and/or nutritionally deficient for, L. pneumophila is supported by data showing dotA mutants and wild-type organisms do not replicate in this compartment in Vero cells and human DC, respectively. Lack of replication is likely not due to acidity per se because, as demonstrated here and elsewhere (47), L. pneumophila replicate in acidic vacuoles in BMDM. Moreover, dotA mutants can replicate when presented with an appropriate intracellular environment, since synchronous infection of this strain with wild-type L. pneumophila results in coinhabited vacuoles where both strains replicate (12). Interestingly, the C. burnetii vacuole is permissive for growth of the more distantly related organisms M. avium and L. amazonensis (18, 51). Human DC were the only cell type where trafficking of wild-type L. pneumophila to the C. burnetii replication vacuole was observed, albeit at a very low frequency. This specific trafficking behavior may reflect the dominant antigen presentation pathway of DC (31).
As demonstrated here and elsewhere (22, 53), trafficking of L. pneumophila to an acidic, LAMP-positive vacuole does not occur in primate macrophages, DC, and epithelial cells. Thus, the lysosomal trafficking observed in murine BMDM is likely a host cell-directed process that is not required for pathogen replication. After internalization by BMDM from A/J mice, L. pneumophila initially avoid the endosomal network and delay phagosome maturation by a mechanism that requires dot/icm type IV secretion (4, 6, 22). The L. pneumophila vacuole first acquires ER markers (22, 26, 49) and, after 8 to 12 h, during which time the bacterium differentiates from the TF to RF, the replication vacuole acquires markers of terminal lysosomes, including low pH, LAMP-1, fluid-phase markers, and cathepsin D (47). Replication of L. pneumophila in lysosomal and nonlysosomal compartments also illustrates the metabolic flexibility of the organism. Conversely, C. burnetii absolutely requires an acidic environment for replication (20).
Both replicating virulent phase I C. burnetii and nonreplicating avirulent phase II organisms traffic to LAMP-positive vacuoles in A/J mouse BMDM. The lack of replication of phase II bacteria is consistent with a previous study showing little replication of this variant in BMDM derived from a number of mouse strains (55). In contrast to murine BMDM, we observed vigorous replication of phase II C. burnetii in acidic, LAMP-positive vacuoles of human macrophages and DC. This result contrasts with previous reports showing no replication and subsequent killing of phase II organisms in primary human monocytes and human monocyte-like lines (8, 17). These studies reported that phase II, but not phase I C. burnetii, engage complement receptor 3 to result in delivery of the organism to a microbicidal lysosomal compartment (8, 17). Maturation of vacuoles containing phase I is proposed to stall at a late endosome stage (17). Permissiveness for L. pneumophila growth in DC also appears to be host species specific. Here we show proficient replication of L. pneumophila in human DC, whereas a previous study demonstrated severe growth restriction in murine DC (36). These disparate results may be due to innate differences between human and mouse DC.
In conclusion, both L. pneumophila and C. burnetii appear exquisitely adapted for replication in unique vacuolar environments that noncompetitively coexist within the same cell. L. pneumophila is the more adaptable intracellular pathogen since it can prosper in either a lysosomal or nonlysosomal replication vacuole during its infectious cycle.
ACKNOWLEDGMENTS
We thank Harlan Caldwell, Ted Hackstadt, Olivia Steele-Mortimer, and Ari Molofsky for review of the manuscript and Gary Hettrick for graphics.
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