Disruption of the Phagosomal Membrane and Egress of Legionella pneumophila into the Cytoplasm during the Last Stages of Intracellular Infection of Macrophages and Acanthamoeba polyphaga

Although the early stages of intracellular infection by Legionellapneumophila are well established at the ultrastructural level,a detailed ultrastructural analysis of late stages of intracellularreplication has never been done. Here we show that the membraneof the L. pneumophila-containing phagosome (LCP) is intact forup to 8 h postinfection of macrophages and Acanthamoeba polyphaga.At 12 h, 71 and 74% of the LCPs are disrupted within macrophagesand A. polyphaga, respectively, while the plasma membrane remainsintact. At 18 and 24 h postinfection, cytoplasmic elements suchas mitochondria, lysosomes, vesicles, and amorphous materialare dispersed among the bacteria and these bacteria are consideredcytoplasmic. At 18 h, 77% of infected macrophages and 32% ofinfected A. polyphaga amoebae harbor cytoplasmic bacteria. At24 h, 99 and 78% of infected macrophages and amoebae, respectively,contain cytoplasmic bacteria. On the basis of lysosomal acidphosphatase staining of infected macrophages and A. polyphaga,the lysosomal enzyme is present among the bacteria when hostvesicles are dispersed among bacteria. Our data indicate thatbacterial replication proceeds despite physical disruption ofthe phagosomal membrane. We also show that an lspG mutant thatis defective in the type II secretion system and therefore doesnot secrete the hydrolytic enzymes metalloprotease, p-nitrophenolphosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipaseA is as efficient as the wild-type strain in disruption of theLCP. Therefore, L. pneumophila disrupts the phagosomal membraneand becomes cytoplasmic at the last stages of infection in bothmacrophages and A. polyphaga. Lysosomal elements, mitochondria,cytoplasmic vesicles, and amorphous material are all dispersedamong the bacteria, after phagosomal disruption, within bothhuman macrophages and A. polyphaga. The disruption of the LCPis independent of the hydrolytic enzymes exported by the typeII secretion system.

INTRODUCTION

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Introduction

The hallmark of Legionnaires' disease is intracellular replicationof Legionella pneumophila in alveolar macrophages (10, 11, 41).L. pneumophila also infects protozoa, which are considered theenvironmental hosts for this intracellular pathogen (41, 42).The intracellular fates of L. pneumophila in both of these evolutionarilydistant host cells are similar, where the bacteria evade fusionof the phagosome to lysosomes (2, 13, 30-32). In addition, theL. pneumophila-containing phagosome (LCP) recruits the endoplasmicreticulum (ER), vesicles, and mitochondria within the first15 min of infection (2, 13, 30, 45). Formation of the rough-ER-surroundedLCP is independent of autophagy (39, 45). After a lag phaseof 4 h, bacterial replication is initiated within both hostcells (30, 41). Upon entry into the postexponential growth phase,L. pneumophila exhibits several virulence traits, includinga pore-forming activity that may allow the bacteria to lysethe host cell (6, 15, 21, 36).

We have previously shown that upon termination of the intracellularinfection, the pore-forming activity of L. pneumophila facilitateslysis of the host cell and bacterial egress (6, 34-36). It hasbeen proposed that bacterial egress may occur in two stagesupon termination of intracellular bacterial replication. First,the phagosomal membrane is disrupted allowing the bacteria toegress to the cytoplasm. The second stage is manifested by lysisof the plasma membrane and bacterial release into the extracellularenvironment (34).

Although numerous studies have examined the early stages ofthis intracellular infection at the ultrastructural level byelectron microscopy, there have been no studies to examine thelate stages of this intracellular infection at the ultrastructurallevel. Therefore, we examined the integrity of the phagosomalmembrane throughout intracellular infection of U937 macrophagesand Acanthamoeba polyphaga at the ultrastructural level. Inaddition, we examined the presence of the lysosomal enzyme acidphosphatase in the phagosome. Our data show that the phagosomalmembrane remains intact for up to 8 h postinfection. While theplasma membrane remains intact, the LCP membrane is disruptedby 12 h. By 18 to 24 h, almost all of the infected cells harboredbacteria free in the cytoplasm, which coincided with the presenceof host cell organelles dispersed between the bacteria, as wellas the lysosomal enzyme acid phosphatase. We show that the typeII secretion system, which is essential for secretion of manyhydrolytic enzymes that can potentially disrupt membranes, isnot involved in the disruption of LCPs within macrophages.

MATERIALS AND METHODS

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Materials and Methods

Bacterial and cell cultures. L. pneumophila serogroup I strain AA100 is a clinical isolatethat has been described previously (4). L. pneumophila strainAA100, the isogenic lspG mutant NU259 (40), and the icmT nullmutant AA100kmT (40) were grown on buffered charcoal-yeast extractplates or in buffered yeast extract broth. U937 cells were culturedin RPMI 1640 medium containing 10% fetal bovine serum (Gibco).U937 cells were differentiated with phorbol 12-myristate 13-acetate(Sigma) for 48 h before use, as previously described (25). A.polyphaga cells were maintained in peptone-yeast extract-glucosemedium.

Electron microscopy. For examination of infected amoebae or macrophages by transmissionelectron microscopy, monolayers in six-well plates were infectedwith L. pneumophila strains at a multiplicity of infection of10 for 1 h, followed by three washes. At 8, 12, 18, and 24 hpostinfection, the infected monolayers were washed with 0.1M Sorenson's phosphate buffer and incubated for 45 min in 0.1M Sorenson's phosphate buffer containing 3.5% glutaraldehyde,pH 7.4, at 4°C. After four washes in Sorenson's phosphatebuffer for 5 min each time, infected cells were postfixed with1% OsO₄ in the same buffer for 45 min. Samples were dehydratedand processed as we described previously (23). Sections werestained with uranyl acetate and lead citrate and examined witha Hitachi H-7000/STEM electron microscope (Hitachi, Ltd., Tokyo,Japan) at 80 kV as described previously (23).

Fusion between lysosomes of the LCP were determined by examinationfor the presence of the lysosomal enzyme acid phosphatase asdescribed previously (13, 35). The monolayers were incubatedwith the acid phosphatase-specific substrate ß-glycerophosphate(0.1 M acetate buffer, 2 mM ß-glycerol phosphate [asthe substrate], 1.2% lead nitrate [as the capture metal]) for1 h at 37°C prior to fixation by osmium tetroxide as describedabove.

RESULTS

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Results

Egress of L. pneumophila into the cytoplasm during the late stages of intracellular infection of macrophages. We examined the integrity of the phagosomal membrane withinU937 macrophages throughout the intracellular life cycle bytransmission electronic microscopy. The phagosomal membranewas considered intact when the membrane had no visible disruptionsof its integrity and no host cell elements, such as mitochondria,cytoplasmic vesicles, or amorphous material, were present withinthe phagosome. The phagosomal membrane was considered disruptedwhen apparent physical disruptions in the integrity of the phagosomalmembrane were present and amorphous material was within thephagosome. The bacteria were considered cytoplasmic when thephagosomal membrane was not continuous and host cell cytoplasmicelements such mitochondria and cytoplasmic vesicles, in additionto amorphous material, were dispersed among the bacteria. Duringthe infection process, lysis of infected cell is initiated by12 to 18 h and the released bacteria initiate secondary infections.To exclude secondary infection from our analysis at 12 h andthereafter, we excluded cells with fewer than 5, 10, and 30bacteria at 12, 18, and 24 h, respectively. In order to ruleout the possibility of deterioration of the membranes of thehost cells by the fixation and staining procedures used, weobserved the plasma membrane at 12 and 18 h postinfection ofcells containing disrupted LCPs.

At 8 h postinfection, all of the phagosomal membranes were thickand did not show any sign of disruption of integrity (Fig. 1and data not shown) and the phagosomal membranes were tightlyclosed around the bacteria, similar to what has been shown bynumerous studies at 2 to 6 h. The lumen of the phagosomes didnot contain any host cell cytoplasmic elements. The phagosomalmembranes were therefore considered intact at 8 h postinfection.ER was also observed around most phagosomes at 8 h (Fig. 1b and c).

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FIG. 1. L. pneumophila bacteria are contained within intact phagosomes at 8 h postinfection. Representative electron micrographs of L. pneumophila-infected macrophages at 8 h postinfection are shown. The membrane of the LCP is intact, as indicated by the thin arrows. Abbreviations: Lpn, L. pneumophila; N, nucleus.

A small portion of the cells had already been lysed by 12 hpostinfection. Only $~$ 2% of the phagosomes had intact membraneswith no signs of disruption of integrity in this time period(Fig. 2A; see also Fig. 8A). The lumen of the phagosomes wasdistended compared to that of 8-h-old phagosomes. Seventy percentof the LCP membranes were disrupted at 12 h (Fig. 2A, part b;see also Fig. 8A). These phagosomes were still free of hostcell cytoplasmic elements such as mitochondria and cytoplasmicvesicles. The rough ER was not visible any more around somephagosomes, but few mitochondria were within the vicinity ofthe phagosomes (data not shown). Twenty-seven percent of theinfected cells harbored cytoplasmic bacteria at 12 h (Fig. 8A).Importantly, the plasma membrane was completely intact in 95%of infected cells harboring disrupted LCPs or cytoplasmic bacteria.

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FIG. 2. Disruption of the integrity of the phagosome membrane at 12 h postinfection (A) and presence of lysosomal contents in the LCP in macrophages at 12 h (B). Representative electron micrographs of L. pneumophila-infected macrophages at 12 h postinfection are shown. The membrane of the LCP is mostly intact, as indicated by the thin black arrows in panel A, part c, but partially disrupted in panel A, part b, and panel B, parts b and c, as indicated by the thick black arrows. Lysosomal staining of acid phosphatase is indicated by the large white arrows. Abbreviations: A, amorphous element; M, mitochondria; Lpn, L. pneumophila; N, nucleus.

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FIG. 8. Quantitative analysis of the integrity of LCPs in macrophages (A) and A. polyphaga (B) at 12, 18, and 24 h postinfection. The percentages of LCPs positive for acid phosphatase were evaluated when the bacteria were within an intact phagosome (IP), a disrupted phagosome (DP), and in the cytoplasm (Cyt). Averages of 60, 50, and 10 to 30 LCPs were analyzed for both macrophages and A. polyphaga at 12, 18, and 24 h, respectively. At 24 h, the number of LCPs analyzed was low because most of the cells had been lysed. Error bars represent standard deviations.

By 18 h, $~$ 50% of the cells in the monolayers were lysed. Amongthe remaining infected cells at 18 h, 22% of the phagosomeswere disrupted (P < 0.01, Student t test) and 77% of theinfected cells harbored cytoplasmic bacteria (P < 0.01, Studentt test), which percentages are significantly different fromthose at the 12-h time point (Fig. 8A). Phagosomal membranesbecame very thin and were discontinuous in many parts (Fig.3A, parts b and c). Vesicles and cytoplasmic amorphous elementsjuxtaposed the bacteria (Fig. 3A, part b). The chromatin inthe nucleus was condensed and was located perinuclearly (Fig.3A and B, parts a), which is a characteristic of apoptosis (22,29) that has been shown to be triggered during late stages ofinfection (37). The plasma membrane was intact in 80% of theinfected cells harboring disrupted LCPs or cytoplasmic bacteria.

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FIG. 3. Disruption of the phagosome and migration of host cell cytoplasmic contents to the phagosome (A) and presence of lysosomal contents in the disrupted LCP in macrophages at 18 h (B). Representative electron micrographs of L. pneumophila-infected macrophages at 18 h postinfection are shown. L. pneumophila (Lpn) bacteria are within disrupted phagosomes (thick black arrows). The intact portions of the membrane are indicated by thin black arrows. White arrows indicate the presence of lysosomal acid phosphatase. In panel A the bacteria surrounded by few vesicles are cytoplasmic whereas in panel B the LCP is disrupted. Note the perinuclear condensation of the chromatin. Abbreviations: A, amorphous material; M, mitochondria; V, vesicles; N, nucleus.

By 24 h postinfection, the majority of the cells had been lysed.In 99% of the remaining infected cells, the bacteria were cytoplasmic,which is significantly different from the percentages at 12and 18 h (P < 0.01, Student t test) (see Fig. 8A). Most ofthe phagosomal membranes disappeared (Fig. 4A, parts b and c).Cytoplasmic elements such as mitochondria, vesicles, and amorphousmaterial were dispersed among the bacteria (Fig. 4A, parts cand d). The bacteria occupied most of the space within the hostcell that did not include the nucleus (Fig. 4A, part a). Perinuclearcondensed chromatin was detected in all cells, a clear signof apoptosis (Fig. 4A, parts a and b).

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FIG. 4. Cytoplasmic L. pneumophila in macrophages at 24 h postinfection (A) and presence of lysosomal contents in the LCP in macrophages at 24 h (B). Representative electron micrographs of L. pneumophila-infected macrophages at 24 h postinfection are shown. L. pneumophila (Lpn) bacteria are cytoplasmic and are surrounded by numerous vesicles (V), lysosomal contents (white arrows), mitochondria (M), and amorphous elements (A). No distinguishable phagosomal membrane surrounds the bacteria. N, nucleus.

Egress of L. pneumophila into the cytoplasm during the late stages of intracellular infection of A. polyphaga. Similar to human macrophages, at 8 h postinfection of A. polyphaga,most of the LCPs were tight and intact with a thick phagosomalmembrane around the bacteria (data not shown). A small portionof cells had already been lysed by 12 h postinfection. At thistime, 74% of the LCPs were disrupted (Fig. 5A and B; see alsoFig. 8B). The lumen of the LCPs was still tight (Fig. 5). Signsof nuclear apoptosis were not visible in A. polyphaga (Fig.5). Thirteen percent of the infected A. polyphaga cells containedcytoplasmic bacteria at 12 h postinfection, where cytoplasmicelements such as mitochondria and amorphous material were alreadydispersed among bacteria (Fig. 5B, parts b, c, and d; see alsoFig. 8B).

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FIG. 5. Presence of lysosomal contents in the LCP in A. polyphaga at 12 h. Shown are representative electron micrographs of L. pneumophila-infected A. polyphaga at 12 h postinfection where the bacteria are in a disrupted phagosome (A) or cytoplasmic (B). Thick black arrows show sites without any visible phagosomal membrane. Thin black arrows show sites where the phagosomal membrane is still visible. Lysosomal acid phosphatase is indicated by the large white arrows. In panel A, the phagosomal membrane is disrupted (large black arrows) and amorphous material (A) is present within the phagosome. In panel B, although parts of the phagosomal membrane are still visible (thin black arrows), L. pneumophila (Lpn) bacteria are mostly cytoplasmic, as shown by the presence of amorphous material (A) and the presence of mitochondria (M) dispersed among the bacteria.

Approximately 50% of A. polyphaga amoebae were lysed by 18 h.At this time, 62% of the LCPs were disrupted and 32% of theinfected cells harbored cytoplasmic bacteria (Fig. 6; see alsoFig. 8B). In the cells harboring cytoplasmic bacteria, the phagosomalmembranes had disappeared almost completely and cytoplasmichost cell elements such as mitochondria, vesicles, and amorphouselements were dispersed among the bacteria (Fig. 6A, parts a,b, and c).

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FIG. 6. Presence of lysosomal contents in the LCP in A. polyphaga at 18 h. (A) Representative electron micrographs of AA100-infected A. polyphaga at 18 h postinfection (A). L. pneumophila (Lpn) bacteria are juxtaposed with mitochondria (M), vesicles (V), and amorphous material (A). Lysosomal acid phosphatase staining (white arrows) is present among the bacteria. N, nucleus. (B) Representative electron micrograph of the positive control for lysosomal acid phosphatase colocalization within the icmT null mutant-containing phagosome. Note the diffuse pattern of the acid phosphatase enzyme in the phagosome that fuses to lysosomes.

At 24 h, similar to infection of macrophages, more than 80%of the cells were lysed. Twenty-three percent of the phagosomeswere disrupted in the remaining infected A. polyphaga amoebae(see Fig. 8B). Seventy-eight percent of the infected cells at24 h harbored cytoplasmic bacteria, which is significantly differentfrom the percentages at the 12- and 18-h time points (P <0.05 and P < 0.01, respectively), where the phagosomal membranewas not visible and mitochondria, vesicles, and amorphous elementswere dispersed among the bacteria (Fig. 7b, c, and d).

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FIG. 7. Presence of lysosomal contents in the LCP in A. polyphaga at 24 h. Representative electron micrographs of L. pneumophila-infected A. polyphaga at 24 h postinfection are shown. L. pneumophila (Lpn) bacteria are cytoplasmic, and mitochondria (M), vesicles (V), and amorphous material (A) are dispersed among the bacteria. Thick black arrows indicate the disrupted areas of the phagosomal membrane. The white arrows show lysosomal staining of acid phosphatase.

We concluded that the phagosomal membrane within infected macrophagesand A. polyphaga was intact for up to 8 h postinfection butclear disruption of the integrity of the phagosomal membranewas apparent at 12 h and thereafter. Bacterial egress into thecytoplasm was observed by 12 h, and by 18 to 24 h all infectedintact cells harbored bacteria in the cytoplasm. Cytolysis ofboth host cells was evident by 12 h.

Presence of lysosomal contents between the bacteria at the end of intracellular infection of U937 macrophages. Since disruption of LCP was accompanied by the presence of hostcell organelles such as mitochondria and vesicles that weredispersed between the bacteria after 12 h postinfection, weexamined whether some of these vesicles were lysosomes. Sincethe LCPs were intact and no cytoplasmic materials were foundwithin the phagosomes at 8 h, we tested for the presence ofthe lysosomal enzyme acid phosphatase within the phagosomesof U937 macrophages at 12, 18, and 24 h postinfection. As mentionedabove, to exclude secondary infection from our analysis at 12h and thereafter, we excluded cells with fewer than 5, 10, and30 bacteria at 12, 18, and 24 h, respectively. We included apositive control for the presence of acid phosphatase in theLCP with an icmT null mutant (AA100kmT) that has been shownto fuse to lysosomes in U937 cells and A. polyphaga (35, 36)(Fig. 6B).

At 12 h, $~$ 48% of the disrupted phagosomes in macrophages containedacid phosphatase (Fig. 8A and 2B). All of the infected cellsthat harbored cytoplasmic bacteria contained lysosomal acidphosphatase concentrated in foci (Fig. 8A), which is most likelywithin vesicles. However, the membrane of lysosomal vesicles,in the phagosome as well as in the cytoplasm, was masked bythe electron-dense substrate. This is in contrast to the diffusepresence of acid phosphatase in the LCP harboring an icmT mutant,which fuses to lysosomes (35, 36) (Fig. 6B).

At 18 and 24 h, cytoplasmic bacteria represented the largestgroup of intracellular bacteria. In all of these cells containingcytoplasmic bacteria, the lysosomal acid phosphatase was presentamong bacteria in concentrated foci (Fig. 3B, part c, 4B, partsb and c, and 8A), suggesting its presence within vesicles.

Presence of lysosomal contents between the bacteria at the end of the intracellular infection of A. polyphaga. Similar to U937 macrophages, at 12 h postinfection of A. polyphaga,lysosomal acid phosphatase was present in concentrated fociamong all of the cytoplasmic bacteria, and 63% of the disruptedphagosomes contained lysosomal acid phosphatase in concentratedfoci (Fig. 5A and B and 8B).

At 18 h postinfection, the lysosomal enzyme was present amongbacteria in all of the infected A. polyphaga amoebae harboringcytoplasmic bacteria (Fig. 6A and 8B). Seventy percent of thedisrupted phagosomes also contained the lysosomal enzyme inconcentrated foci.

At 24 h, most of the bacteria within A. polyphaga amoebae werecytoplasmic and 100% of the cells harboring the cytoplasmicbacteria contained the lysosomal enzyme acid phosphatase inconcentrated foci among the bacteria (Fig. 7 and 8B). About61% of the disrupted phagosomes contained the lysosomal enzyme.

We concluded that in both macrophages and A. polyphaga after12 h, lysosomal acid phosphatase was present in concentratedfoci among the cytoplasmic bacteria. This enzyme was presentin concentrated foci among the bacteria in macrophages and A.polyphaga similar to its vesicular distribution in the cytoplasm.This is in contrast to its diffuse presence in the icmT mutantphagosome, which fuses to lysosomes (Fig. 6B).

Intracellular multiplication of L. pneumophila in the cytoplasm of both macrophages and A. polyphaga. Since L. pneumophila exhibits robust replication within thephagosomes, we examined whether disruption of the phagosomeand bacterial egress into the cytoplasm resulted in terminationof bacterial replication. We performed single-cell analysisto quantitate the number of bacteria per cell at several stagesof the intracellular infection of macrophages and A. polyphaga.

Although the number of bacteria within disrupted phagosomesdid not show a dramatic increase within macrophages between12 and 24 h, it was clear that the average number of cytoplasmicbacteria per macrophage increased from $~$ 17 at 12 h to 35 at 18h and $~$ 58 at 24 h. These results indicated that bacterial growthwas not arrested by disruption of the integrity of the phagosome(Fig. 9A).

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FIG. 9. Numbers of bacteria per phagosome (or per cell when cytoplasmic) within macrophages (A) and A. polyphaga (B) at 12, 18, and 24 h postinfection. The minimum average number of bacteria per phagosome was obtained by single-cell analysis by electron microscopy. Averages of 60, 50, and 10 to 30 LCPs were analyzed for both macrophages and A. polyphaga at 12, 18, and 24 h, respectively. At 24 h, the number of LCPs analyzed was low because most of the cells had been lysed. Error bars represent standard deviations.

In A. polyphaga, the number of bacteria present per disruptedphagosomes increased from an average of 14 at 12 h postinfectionto 25 at 18 h and 45 at 24 h (Fig. 9B). The number of cytoplasmicbacteria per host amoeba increased from an average of 39 at12 h postinfection to 63 at 18 h and 91 at 24 h (Fig. 9B). Notethat the quantitations are based on a single section, and thusthe values obtained represent the minimal numbers of organisms.

Taken together, the intracellular growth kinetics and our enumerationof intracellular bacteria in our single-cell analysis of bothmacrophages and A. polyphaga amoebae indicated that bacterialreplication continued despite disruption of the LCP and migrationof mitochondria and other host cell organelles that become dispersedamong the bacteria.

The type II secretion system is not involved in disruption of the LCP membrane. There are many hydrolytic enzymes secreted by the type II secretionsystem of L. pneumophila, including metalloprotease, p-nitrophenolphosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipaseA (28, 40). Since these hydrolytic enzymes are potential candidatesthat disrupt the LCP, we used the lspG mutant NU259, which isdefective in the type II secretion system owing to a defectin an inner membrane structural protein component of the typeII secretion system (40). In order to determine if the secretionof theses exoproteins is responsible for the disruption of thewild-type LCP, U937 macrophage cell lines were infected withthe lspG mutant NU259 and examined at 8, 12, 18, and 24 h postinfectionby electron microscopy. About 85% of the LCPs were intact at8 h postinfection (Fig. 10). At 12 h postinfection 53% of theLCPs were disrupted, and at 18 h postinfection 87% of the bacteriawere cytoplasmic within U937 macrophages (Fig. 10). At 24 h,most of cells were in a disintegrated state and it was not possibleto analyze the data. Thus, the type II secretion system andthe hydrolytic enzymes secreted through it are not involvedin the disruption of the LCPs.

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FIG. 10. Evaluation of the integrity of the LCPs harboring type II secretion system-deficient lspG mutant strain NU259 within U937 macrophages at 8, 12, and 18 h postinfection. The percentages of bacteria within intact phagosomes, disrupted phagosomes, and the cytoplasm were evaluated among a total of 30 to 50 LCPs. Error bars represent standard deviations.

DISCUSSION

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Discussion

L. pneumophila invades both mammalian and protozoan host cellsand resides within a replicative vacuole, evading phagolysosomalfusion. Late stages of the intracellular life cycle and themechanism of bacterial egress after cessation of intracellularbacterial replication are not well understood. The two-stepegress model that we have previously proposed (34) predictsthat L. pneumophila first egresses from the phagosome into thecytoplasm, followed by lysis of the host cell plasma membrane.Here we show for the first time in both human-derived macrophagesand A. polyphaga amoebae that L. pneumophila is present in anintact phagosomal membrane for up to 8 h postinfection. However,by 12 h and thereafter, physical and structural disruptionsof integrity of the phagosomal membrane are apparent and cytoplasmicorganelles including mitochondria, vesicles, and lysosomes aredispersed among the bacteria.

While ultrastructural studies of early stages of the LCP arerather numerous, LCP integrity during later stages of intracellularinfection has never been examined. Tilney et al. have describedan intact phagosomal membrane at 20 h postinfection of U937macrophages, but the phagosome described contains only two bacteria(45). It is well documented that the bacterial lag phase withinU937 macrophages is $~$ 4 h and the generation time is $~$ 90 to 120min (1, 41, 42). Therefore, phagosomes containing two or fewerbacteria at 20 h are most likely secondary infections or bacteriathat fail to replicate and are not representative of a 20-h-oldLCP. In our study, we have considered that a 12-h LCP may harborat least 5 bacteria, an 18-h LCP may harbor at least 10 bacteria,and a 24-h LCP may harbor at least 30 bacteria. In that manner,we believe that we have excluded from our analysis most LCPsthat are formed as a result of secondary infections.

Single-cell analysis has shown an increasing number of bacteriaper disrupted phagosome in both macrophages and A. polyphagaat 12, 18, and 24 h, which indicates that the bacteria are multiplyingwithin their disrupted phagosomal environment and also whenthey are cytoplasmic. Goetz et al. have shown that upon microinjectionof L. pneumophila into the cytoplasm of epithelial-type Caco-2cells, the bacteria are unable to replicate in the cytoplasm(27). This can be due to one of two reasons. First, legionellaeprimarily infect alveolar macrophages in humans and animal models,and in Caco-2 cells, L. pneumophila is likely to behave differentlythan in macrophages (24, 38). It may therefore be prematureto extrapolate data from macrophages to Caco-2 cells. In supportof this, it has been shown that the Salmonella sifA mutant,which is released into the cytosol of macrophages, fibroblasts,and epithelial cells, replicates in the cytoplasm of epithelialcells (HeLa cells) but not in that of macrophages and fibroblasts(12). Second, L. pneumophila undergoes phenotypic modulationupon intracellular infection accompanied by alterations in thegene expression and ultrastructural characteristics of the bacteria(3, 5, 26, 43). Thus, microinjection bypasses adjustment ofthe bacteria to the phagosomal microenvironment, which may beessential for subsequent adjustment to the cytoplasm. In addition,it is clear that the cytoplasm of macrophages is different fromthat of other types of cells in permissiveness to bacterialreplication.

The cytoplasmic microenvironment that the bacteria inhabit duringthe late stage of infection contains mitochondria, vesicles,and amorphous material. The acid phosphatase enzyme is presentat concentrated foci in the cytoplasm before disruption of thephagosome and later in the vicinity of the cytoplasmic bacteriawithin disrupted phagosomes. The lysosomal membrane is not visiblein either the phagosome or the cytoplasm, most likely becauseof the electron-dense deposits of the substrate that mask thelysosomal membrane. Thus, the bacteria may not be in directcontact with the lysosomal enzyme, which is further supportedby the lack of bacterial degradation. In contrast, fusion ofthe lysosomes with L. pneumophila mutants such as the icmT nullmutant results in diffuse distribution of the acid phosphatasethroughout the phagosome and bacterial degradation (35, 36).This is further supported by our lack of detection of lysosomalvesicles making contact or fusing with the LCP in thousandsof cells that we have examined. In addition, the presence ofmitochondria among the bacteria indicates that intact lysosomes,similar to mitochondria and other vesicles, are present amongthe bacteria. Our data suggest that the lysosomes may migrateto and not fuse with the LCP. Thus, the presence of lysosomalcontents among bacteria is unlikely to be due to a direct fusionof lysosomes to the LCP. However, limited direct lysosomal fusion,in addition to passive migration of the lysosomes through thedisrupted phagosomal membrane, cannot be excluded at this time.By confocal laser scanning microscopy, it has been shown thatduring late stages of infection of A/J mouse macrophages, asignificant proportion of the LCPs colocalizes with lysosomalproteases (43). It is not known whether lysosomal fusion atlate stages also occurs in human cells or whether phagosomaldisruption also occurs in A/J mouse macrophages. Sturgill-Koszyckiand Swanson have also shown by confocal microscopy that bacterialreplication also proceeds in the phagosomes that become colocalizedwith lysosomal contents, which is consistent with our observations(43). Support for lysosomal fusion to the LCP A/J mice macrophageshas been shown by observations that inhibition of the protonpump ATPase activity of infected macrophages with bafilomycinA1 or treatment with ammonium chloride or chloroquine to neutralizethe acidic pH reduce intracellular bacterial replication (43).However, although these treatments have no effect on bacterialreplication in vitro (43), it is not known how these treatmentsaffect bacterial replication within macrophages. Importantly,elevation of the pH in the LCP is expected to block bacterialreplication, since bacterial growth in vitro occurs within avery limited pH range around pH 6.9. Taken together, our datasuggest that colocalization of the LCP with lysosomal contentsduring late stages of infection may be due to migration of lysosomevesicles to the disrupted LCPs. Our data do not exclude directfusion and release of lysosomal contents to the LCP lumen.

L. pneumophila possesses a type II secretion system that allowsthe secretion of many hydrolytic enzymes that have the potentialto degrade membranes, including a metalloprotease, p-nitrophenylphosphorylcholinehydrolase, lipase, phospholipase A, and lysophospholipase A.In this study, we have used an lspG mutant that is defectivein an inner membrane structural protein component of the typeII secretion system (40) in order to determine whether the typeII secretion system and the enzymes secreted through it areinvolved in disruption of the LCP membrane. Our data show thatthe type II secretion system and its secreted enzymes are notrequired for disruption of the LCPs. Thus, these hydrolyticenzymes do not play any detectable role in disruption of theLCPs.

Necrosis is usually the cause of a severe cellular insult, suchas loss of membrane integrity caused by a toxin (33). L. pneumophilahas been shown to have a cytopathogenic effect on host cellsleading to necrosis, which is thought to be mediated by a pore-formingactivity (6, 21, 35, 36). It is likely that the pore-formingactivity during late stages of infection of macrophages contributesto cytolysis of the host cell. However, many other factors maybe involved. First, apoptosis in mammalian cells is likely tobe a contributing factor, since it is triggered during latestages of infection when the LCPs harbor more than 20 bacteria(37). Second, whether other phospholipases secreted by L. pneumophila(7-9, 16-20, 44) also contribute to dismantling of the cellis not known. Third, it is possible that disruption of the phagosomalmembrane may be partially mediated by physical pressure fromthe increasing number of bacteria in the phagosome. Fourth,it is also possible that, similar to S. enterica serovar Typhimurium(14), L. pneumophila is actively involved in maintaining theintegrity of the phagosome during early stages of infectionbut loses that ability later. Therefore, the mechanism of egressof L. pneumophila into the cytoplasm is likely to be complexand multifactorial.

In summary, we have shown that L. pneumophila is within an intactphagosome for up to 8 h in both macrophages and A. polyphaga.The phagosomal membrane is disrupted at 12 h, and the bacteriaegress into the cytoplasm between 12 and 24 h, prior to disruptionand lysis of the host cell plasma membrane. Cytoplasmic elementssuch as mitochondria and vesicles are dispersed among the bacteriaat the end of intracellular replication, and this phagosomaldisruption does not seem to affect bacterial replication. Disruptionof the LCP is not mediated by hydrolytic enzymes secreted bythe type II secretion system.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Room 316, University of Louisville College of Medicine, 319 Abraham Flexner Way 55A, Louisville, KY 40202. Phone: (502) 852-4117. Fax: (502) 852-7531. E-mail abukwaik{at}louisville.edu.

Editor: J. T. Barbieri

REFERENCES

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