Correspondence to: Julie A. Theriot, Department of Biochemistry, Beckman Center, Stanford School of Medicine, Stanford, CA 94305-5307. Tel:(650) 725-7968 Fax:(650) 723-6783 E-mail:theriot{at}cmgm.stanford.edu.
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Abstract |
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The bacterial pathogen, Listeria monocytogenes, grows in the cytoplasm of host cells and spreads intercellularly using a form of actin-based motility mediated by the bacterial protein ActA. Tightly adherent monolayers of MDCK cells that constitutively express GFP-actin were infected with L. monocytogenes, and intercellular spread of bacteria was observed by video microscopy. The probability of formation of membrane-bound protrusions containing bacteria decreased with host cell monolayer age and the establishment of extensive cell-cell contacts. After their extension into a recipient cell, intercellular membrane-bound protrusions underwent a period of bacterium-dependent fitful movement, followed by their collapse into a vacuole and rapid vacuolar lysis. Actin filaments in protrusions exhibited decreased turnover rates compared with bacterially associated cytoplasmic actin comet tails. Recovery of motility in the recipient cell required 12 bacterial generations. This delay may be explained by acid-dependent cleavage of ActA by the bacterial metalloprotease, Mpl. Importantly, we have observed that low levels of endocytosis of neighboring MDCK cell surface fragments occurs in the absence of bacteria, implying that intercellular spread of bacteria may exploit an endogenous process of paracytophagy.
Key Words: Listeria monocytogenes, ActA, actin, epithelial cells, bacterial pathogenesis
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Introduction |
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LISTERIA monocytogenes is a Gram-positive bacterial pathogen that causes food poisoning, meningitis, and spontaneous abortions in its human host. It invades the host through the epithelial lining of the gut (
L. monocytogenes accomplishes intercellular spread by abducting the host cell's actin-based motility components to propel itself into the host cell's plasma membrane and infect adjacent cells. The membrane-associated bacterial protein ActA is necessary and sufficient for such motility (
When these actin comet tails propel the bacterium into its host's plasma membrane, the membrane is distended, forming long protrusions into the extracellular space. In tissue culture cells, these structures are nonproductive and simply extend many microns into the media; however, in vivo, infected tissues are less likely to border on a free space, and thus, these bacterium-containing membrane protrusions have a high probability of entering a neighboring cell. When a protrusion from one cell is taken up by another, intercellular spread of bacteria occurs.
Intercellular spread has not previously been documented in real time, despite extensive observation of intracellular movement of bacteria by video microscopy. Thus, very little is known about the mechanism or kinetics of this critical process of L. monocytogenes infection. Transmission electron micrographs have fortuitously captured a few intermediate steps that show membrane-bound protrusions of bacteria inside neighboring cells. The subsequent uptake of these protrusions, leaving the bacterium enclosed in a secondary vacuole, has been inferred from these images (
This secondary vacuole is distinct from the primary vacuole in that it is surrounded by a double membrane, one from the donor cell and one from the recipient (
To increase our chances of observing intercellular spread by video microscopy, we chose to infect a columnar epithelial cell line. This guaranteed that most of the cellular surface area would be in contact with neighboring cells, thereby promoting the chance of intercellular uptake of bacterium-induced membrane protrusions. MDCK cells form a polarized monolayer of epithelial cells in tissue culture (for review, see
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Materials and Methods |
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Chemicals were purchased from Sigma-Aldrich unless otherwise indicated. All photographic images were compiled using Adobe Photoshop, Adobe Illustrator, and/or Canvas (Deneba Systems) software. The printed images are representative of the original data.
Cell Lines and CDNA Constructs
Human nonmuscle ß-actin (these data are available from GenBank/EMBL/DDBJ under accession number
M10277) was cloned by PCR from a human cDNA library and was fused in-frame with the enhanced GFP gene into the BamHI-XhoI sites of the vector pEGFP-C1 (Clontech Inc.) to generate a fusion between the COOH terminus of EGFP and the NH2 terminus of beta-actin in the expressed protein. PCR-derived sequences were confirmed by dideoxy sequencing. MDCK clone II/G cells were obtained from the laboratory of Gerrit van Meer (Academic Medical Center, University of Amsterdam, Department of Cell Biology and Histology, The Netherlands) and have been described previously (
For experiments, all cells were grown in DME containing 10% FBS and antibiotic-antimycotic (GIBCO BRL) at 37°C in a humidified incubator containing 5% CO2. Antibiotic-antimycotic was removed from cultures used for bacterial infections.
Immunoblotting
For the preparation of lysates, 5 x 105 MDCK cells were plated on 35-mm tissue-culture dishes and extracted after 24 h in culture. For the preparation of SDS lysates, MDCK cells were extracted in hot SDS buffer containing 1% SDS, 10 mM Tris-HCl, pH 7.5, and 2 mM EDTA, and then scraped from the petri dish with a rubber policeman. Samples were boiled for 15 min and insoluble material was removed by centrifugation at 12,000 g for 15 min. For preparation of Triton X-100 lysates, MDCK cells were extracted for 15 min at 4°C with CSK buffer (0.5% Triton X-100, 10 mM Pipes, pH 6.8, 50 mM NaCl, 300 mM sucrose, and 3 mM MgCl2) containing a protease inhibitor mix (1 mM Pefabloc, 1 mM benzamidine, and 10 µg/ml each of aprotinin, pestatin, and leupeptin). Pefabloc, pepstatin, and leupeptin were purchased from Boehringer Mannheim Biochemicals. After extraction, cells were scraped from the petri dish with a rubber policeman and insoluble material was pelleted by centrifugation at 12,000 g for 30 min. Pellets were washed twice with CSK buffer, resuspended in SDS buffer, boiled 10 min, and insoluble material was removed by centrifugation at 12,000 g for 15 min. Protein concentrations were determined using the BCA protein assay reagent kit (Pierce Chemical Co.). Portions of the lysates were boiled for 5 min after adding one-third volume of 4x SDS reducing sample buffer and separated by SDS-PAGE in 7.5% polyacrylamide gels (
Phalloidin Staining
1 x 105 MDCK cells were plated on collagen-coated coverslips in 35-mm tissue-culture dishes and fixed after 24 h in culture. Cells were washed once in Dulbecco's PBS and fixed for 20 min at RT with 2% formaldehyde in Dulbecco's PBS, or cells were washed once, extracted 5 min at 4°C with CSK buffer, washed again, and fixed with 2% formaldehyde. Cells were washed with blocking buffer, incubated 10 min with rhodamine-phalloidin (Molecular Probes; 1 unit per coverslip) in blocking buffer, washed, and processed for microscopy as described previously (
Bacterial Culture and Infection Procedures
L. monocytogenes strain 10403S (
Fluorescence Video Microscopy and Analysis
Infected cells grown on glass coverslips were observed 520 h after infection after mounting on a stage whose temperature was maintained at 37°C. Cells were overlaid with phenol red-free DMEM with 10% FBS and buffered with 20 mM Hepes (pH 7.3), and covered with a thin layer of silicone DC-200 fluid (Serva) to prevent evaporation. Observations were performed on a Nikon Diaphot-300 inverted microscope equipped with phase contrast and epifluorescence optics. Timelapse video microscopy was achieved with an intensified charge-coupled device (CCD) camera (Dage-MTI; GenIISys/CCD-c72) or a cooled CCD camera (NDE/CCD; Princeton Instruments) and Metamorph (Universal Imaging) software. Phase contrast/fluorescence image pairs were recorded every 1030 s, with eight video frames averaged per image (intensified CCD) or 50-ms exposures (cooled CCD).
Immunofluorescence Deconvolution Microscopy
Cells grown on glass coverslips were fixed in 3% formaldehyde for 30 min at room temperature in PBS after 710 h of infection. Cells were then washed twice in PBS at room temperature and extracted with CSK buffer at room temperature for 10 min. Cells were washed twice in PBS at room temperature for 10 min. Cells were blocked in PBS containing 1% BSA, 1% calf serum, and 50 mM NH4Cl for 1 h at room temperature, then in primary antibody solution containing mouse anti-E-cadherin antibody (Transduction Laboratories) for 2 h at room temperature or overnight at 4°C. Cells were then washed three times in PBS with 0.2% BSA and incubated in Texas redconjugated mouse secondary antibody solution for 1 h at room temperature. Cells were washed three times in PBS and 0.2% BSA and mounted in 90% glycerol in 20 mM TRIS.
Three-dimensional images were recorded with a cooled CCD camera (Photometrics Ltd.). Optical sections (1,023 x 1,023 pixels) of thickness 0.100.25 µm were recorded with a 100x oil immersion objective (Olympus Corp.). All aspects of image collection were controlled by a Silicon Graphics workstation (Silicon Graphics Corp.), on a deconvolution microscopy system (Applied Precision).
Metabolic Inhibition
Infected cells were observed on the microscopy workstation described for video microscopy. DMEM + 10% FBS with 0.5% (wt/vol) methyl cellulose was added to the heated chamber and protrusions imaged. Cellular ATP synthesis was inhibited by addition of 50 µM 2,4-dinitrophenol and 10 mM 2-deoxy-D-glucose. Inhibitors were removed by rinsing in fresh DMEM + 10% FBS with 0.5% methyl cellulose.
Fluorescence Labeling of the Plasma Membrane
TRITC.
Cells were incubated 13 h in 110 µM TRITC in DMEM containing 10% FBS and antibiotic-antimycotic (GIBCO BRL). Cells were rinsed 12 times over the next 15 h with fresh media, then trypsinized and replated with unlabeled cells, and grown at least 12 h before infection.
Lipophilic Dye.
Cells were suspended in 2 ml LISS buffer (239 mM sucrose and 5 mM Hepes, pH 7.3), to which was added 410 µl of a 0.25% (wt/vol) stock solution of 3, 3'-dioctadecyloxacarbocyanine perchlorate (DiOC18; Molecular Probes) in dimethylformamide (DMF). Stock solution was prepared by mixing DiO and stearylamine octadecylamine in a 5:1 (wt/wt) ratio and dissolved in chloroform, heated to 50°C, precipitated on ice with two volumes of methanol, and pelleted by centrifugation. The pellet was dried then dissolved in 1,000 µl DMF, heated to 50°C, and centrifuged. Cells were incubated for 20 min with gentle agitation, rinsed twice with LISS, and plated in DMEM + 10% FBS.
Inhibition of Vacuolar Acidification
TRITC-labeled and unlabeled cells were plated together and incubated 1240 h. After infection, DMEM + 10% FBS containing 1 µM bafilomycin A in DMSO was added. This concentration inhibits acidification of endocytic vacuoles in mammalian cells (
Metabolic Labeling
Infection of J774 and metabolic labeling were performed as previously described with some modifications (mpl) (
actA) (
Fluorescence Recovery after Photobleaching
Photobleaching of cells was performed and analyzed on the Nikon microscopy workstation described above. Infected cells were prepared as described in that section. Bleaching was performed by narrowing the field diaphragm on a 100-W mercury arc lamp, exposing the region to be bleached for 35 min through a Chroma FITC filter. For protrusions, bleached regions were chosen so as to exclude the entire cell body except for the protrusion. Clouds were chosen for bleaching based on proximity to cell edges, to minimize photodamage to the cell body and the GFP it contained. Immediately after bleaching, lamp intensity was attenuated to 10% by a neutral density filter and 8-frame averaged images were collected every 7 s to follow recovery.
For analysis, the integrated pixel intensity of each cloud or protrusion was measured using the Measure Brightness function of Metamorph. Additionally, the intensity of a nearby unbleached region of cytoplasm was measured for each frame and used to normalize intensities of regions of analysis, to compensate for fluctuations in lamp intensity and photobleaching due to image capture. The standard intensity never fell below 90% of its original value.
To characterize the exponential recovery of fluorescence, normalized intensities were fit to the following perturbation-relaxation equation:
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(1) |
after is the asymptotic value to which fluorescence intensity recovers after bleaching. Data was fitted to Equation 1 and the half-time (t1/2) to recovery from F0 to F
was calculated using the equation:
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(2) |
Analysis of Infectious Foci
Infected cells grown on glass coverslips were fixed in 3% formaldehyde for 30 min at room temperature and then incubated in FITC-phalloidin in PBS for 30 min at room temperature. They were then rinsed with PBS for 5 min and mounted on slides in 90% glycerol in 20 mM Tris-HCl. Cells were viewed with the Nikon microscopy workstation described above, using a 100x oil immersion objective. Primarily infected cells were defined as (in descending order of importance): (a) cells with the most bacteria, (b) cells in the center of an infectious focus, and (c) cells with the greatest fraction of bacteria possessing tails. Secondarily infected cells were cells with fewer bacteria surrounding the perimeter of the primarily infected cell, and tertiarily infected cells were at least one cell away from the primarily infected cell and contained fewer bacteria than other cells in the focus. After 8 h, primary, secondary, and tertiary infections could no longer be unambiguously defined.
Online Supplemental Material
Video 1. Supplement to Figure 1 c. MDCK canine kidney cells, which constitutively express green fluorescent protein fused with actin, are infected with Listeria monocytogenes. Available at http://www.jcb.org/cgi/content/full/146/6/1333/F1/DC1
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Video 2. Supplement to Figure 3. Ricochets and protrusions can be seen in this monolayer, which was confluent for ~72 h preinfection. Ricochets are marked by yellow R's and protrusions by red P's. Available at http://www.jcb.org/cgi/content/full/146/6/1333/F3/DC1
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Video 3. Supplement to Figure 4. The majority of fitful movement occurs along the axis of the protrusion. Speeded up 120x. Available at http://www.jcb.org/cgi/content/full/146/6/1333/F4/DC1
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Video 4. Supplement to text. In the presence of methyl cellulose, addition of ATP synthesis inhibitors quickly stops all protrusion movement. One protrusion still drifts; however, the chamber was not free of flow, and on average protrusions moved less than 1 µm in 10 min. Washing with inhibitor-free media restores fitful movement. Available at http://www.jcb.org/cgi/content/full/146/6/1333/DC2
Video 5. Supplement to Figure 6. Collapse of the protrusion from an ovoid to spherical geometry. A long protrusion moving into a non-GFP-expressing cell collapses. Available at http://www.jcb.org/cgi/content/full/146/6/1333/F6/DC1
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Video 6. Supplement to Figure 8 (ac). Vacuole lysis. Three protrusions/vacuoles can be seen spreading from one cell to another. Shortly after collapse of the protrusion (especially apparent in the lowest protrusion), the membrane signal disappears but the actin signal remains. Phase contrast: lower right corner. Available at http://www.jcb.org/cgi/content/full/146/6/1333/F8/DC1
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Video 7. Supplement to Figure 8 d. Actin flash. Here, a protrusion is formed, undergoes fitful movement, stillness and collapse, followed shortly by a bright flash of actin intensity temporally coincident with average times for vacuole lysis. The membrane was not labeled in this case. Available at http://www.jcb.org/cgi/content/full/146/6/1333/F8/DC1
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Results |
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MDCK Cells Expressing GFP-Actin Provide an Experimentally Tractable System for Study of L. monocytogenes Intercellular Spread
Since any pathogenic relationship requires contributions from both the pathogen and the host, selection of a model host system is important. We chose to study intercellular spread of L. monocytogenes in MDCK cells, whose columnar epithelial morphology makes them ideal subjects both practically (given their extensive cell-cell contacts) and physiologically (since L. monocytogenes initially invades its human host across the columnar epithelium of the intestine). However, bacteria inside these cells are difficult to observe by phase-contrast microscopy, since the cells are tall and contain many intracellular phase-dense structures.
To solve this problem, an MDCK cell line expressing GFP-actin was selected based on its normal cytoskeletal organization and gross morphology (Figure 1). Examination by Western blot revealed that the amount of GFP-actin expressed was <2% of total monomeric actin (Figure 1 a), although expression levels varied from cell to cell. Upon cell extraction in buffer containing Triton X-100 (Figure 1 a, lower blot), GFP-actin was more abundant in the Triton X-100 soluble fraction than the pellet (Figure 1 a, upper blot) compared with endogenous actin. Nevertheless, we detected GFP-actin incorporation into actin filaments that stained with phalloidin. The level of GFP-actin incorporated into filaments was low enough that it did not appear to disrupt the structure of the filament, but was sufficient to observe actin filament structure and dynamics in living cells (Figure 1 b).
The GFP-actin enabled simultaneous phase-contrast and fluorescence video microscopic observation of L. monocytogenesinduced actin comet tail formation and movement (Figure 1 c), and actin-dependent bacterial motility in these cells was similar to that previously described in other cells not expressing GFP-actin (
Formation of an Intercellular Membrane-bound Protrusion Depends on Permissive Host Cell Structure
The first hypothesized step in intercellular spread is formation of a membrane-bound protrusion into a neighboring cell. We frequently observed this step in infected MDCK cells. Here, L. monocytogenescontaining membrane protrusions are defined as extracellular if formed from a host surface free of cell-cell contacts, and intercellular if formed from one cell and protruding into an adjacent cell. Intercellular protrusions must therefore distend two membranes from the point of their inception, one from the recipient and the other from the donor cell. Only these protrusions permitted the ultimate release of a bacterium into another cell. Intercellular protrusions ranged in length from 3 to 18 µm (average length: 8.5 µm, SD = 4.3, n = 16). Since the host cells were many focal planes deep, intercellular protrusions into neighboring cells were easy to distinguish from those protruding apically into the extracellular medium.
In making a protrusion, the bacterium did not simply distend the lipid bilayer: it pulled the associated cellular structure along with it (Figure 2, a and b). Adherens junctions, mediated by the transmembrane protein, E-cadherin, line the lateral plasma membrane in the form of puncta in MDCK cells partially polarized by growth on coverslips (
Surprisingly, a protrusion did not form on every occasion that a bacterium was propelled by its actin tail into the plasma membrane (Figure 3 a). Instead, the bacterium occasionally ricocheted off the membrane and continued to race around the cell. A single bacterium might ricochet off one face of the plasma membrane and immediately form a membrane-bound protrusion from another, demonstrating that the ricochet events were dependent on properties of the host cell membrane, rather than a bacterial deficiency.
We examined cells to learn more about the basis for ricochet versus membrane protrusion events. There did not appear to be particular regions of the cell membrane that exhibited a predilection for a ricochet or protrusion event, and neither the instantaneous velocity nor angle with which the bacterium struck the membrane was predictive of which fate would result. Bacteria moving at rates >0.02 µm/s and striking the membrane at an angle 30° were equally likely to have been members of the protrusion or ricochet subsets.
However, the probability of a ricochet was highly dependent upon the age of the MDCK monolayer (Figure 3 b). The percentage of membrane contacts resulting in ricochets increased with monolayer age. A possible cause of this change could be structural alterations commensurate with development of cell polarity. MDCK cells are known to undergo structural changes during maturation of the monolayer on solid or permeable substrates (
Intercellular Protrusion Dynamics Are Biphasic
We observed that membrane-bound intercellular protrusions frequently underwent a period of erratic motility (which we termed fitful movement) followed by a period of stillness before their uptake (see next section). 62% (n = 29) of intercellular protrusions exhibited fitful movement in the recipient cell's cytoplasm (Figure 4, a and b). The remaining 38% exhibited little or no movement throughout their duration. Of the protrusions that underwent fitful movement, 78% moved erratically for 715 min (average = 10.3 min, SD = 2.3, n = 15) followed by an additional 1025 min of stillness. 60% of that population was stalled for 23 min before beginning fitful movement (Figure 4). The remaining protrusions which moved (n = 4) exhibited fitful movement throughout the duration of their time in the protrusion.
This fitful movement is consistent with continued polymerization of actin at the bacterial surface, as most of the motion occurs in a vector along the protrusion's axis of formation (Figure 4). We plotted the bacterial path as a function of time with respect to axes defined by the three time points which constituted the initial direction of the intrusive pathway as it crossed the cell border (x axis) (Figure 4 a). We found that 73% (SD = 9, n = 17) of the motility was along the protrusion axis (Figure 4 c), strongly suggesting that the bacterium was largely responsible for fitful movement of the protrusion.
Extracellular membrane-bound protrusions into free space exhibit rapid erratic movement (Figure 5 a). When we inhibited the Brownian component of that movement by increasing extracellular fluid viscosity, we observed fitful movement of the extracellular protrusions similar to that of intercellular protrusions (Figure 5 b). Under these experimental conditions, bacteria moved more slowly and in a directed fashion, occasionally taking steps backward, as intercellular protrusions do, but did not stop moving completely. We monitored these protrusions for motility and found that they displaced an average of 11.1 µm over 10 min (SD = 8.1, n = 10) with an average instantaneous velocity of 0.021 µm/s (SD = 0.018). When host cell ATP production (and therefore actin polymerization) was inhibited by addition of 50 µM 2,4-dinitrophenol and 10 mM 1-deoxyglucose, cytoplasmic bacteria stopped moving in <90 s. Likewise, extracellular protrusions stopped virtually all movement, displacing an average of 1.1 µm over 10 min (SD = 1.5) with an average instantaneous velocity of 0.0018 µm/s (SD = 0.0022; video available at http://www.jcb.org/cgi/content/full/146/6/1333/DC2). 5 min after washing out these inhibitors, the average displacement of the protrusions returned to 10.0 µm over 10 min (SD = 9.6), with average instantaneous velocity 0.016 µm/s (SD = 0.015), which is similar to movements of protrusions in untreated cells (see above).
Taken together, these experiments demonstrate that the fitful movement of membrane-bound protrusions is most likely due to bacterially directed actin polymerization, though it is possible that the recipient cell may make a minor contribution by tugging on the protrusion. Since polymerization-based movement of protrusions requires ATP, it is probable that cessation of that movement following entry of the protrusion into a recipient cell corresponds to the pinching off and sealing of the donor cell's protrusion membrane. This would prevent further generation of actin-based force as it denies bacterial access to the cellular pool of ATP.
Recipient Cell Dictates L. monocytogenes Protrusion Uptake
Extracellular protrusions were not taken up by nearby cells, even when constrained by them. We captured occasional (n = 4) video sequences of membrane-bound protrusions that lay confined in the intercellular space between two neighboring cells. After >1.5 h observation, they were never taken up by these cells, though they occasionally twitched back and forth along the protrusion axis. Furthermore, we never observed a protrusion that was initially extracellular taken up by a neighboring cell, indicating that classical, actin-based phagocytosis of membrane-bound protrusions does not occur frequently, if at all, in these cells. Since we never observed bacterial escape from extracellular protrusions (n > 300), we concluded that the receiving cell must actively pinch off a protrusion that has intruded into its cytoplasmic space. In other words, the bacterium initially plays the active role, pushing the protrusion directly into the cytoplasm of the neighboring cell, which then takes over the active role to engulf the protrusion.
Approximately 3040 min (average = 34 min, SD = 7, n = 14) after the start of membrane-bound protrusion into the recipient cell (corresponding to 1525 min after the cessation of fitful movement), a distinct morphological change in protrusion geometry was apparent in all but one case (Figure 6). Initially, membrane protrusions in recipient cells were an elongated ovoid in shape. Subsequently the protrusion appeared to shrink, and then snap to a roughly spherical morphology over a period of 30150 s. Visually, the protrusion appeared to have been released from tension, as if it had been pinched off.
Of the four protrusions that exhibited constant fitful movement, two underwent this geometric collapse after >1 h of observation. The other two failed to collapse even after 90 min.
We did not observe any correlation between entry speed and protrusion length, or entry speed and time before the change in geometry, suggesting that these variables did not depend on the activity of the bacterium (Figure 7). These results, combined with the properties of fitful movement (see above) lead us to conclude that protrusion uptake occurs in two steps, the timing of which are primarily determined by the recipient host cell. In the first, the supply of ATP to the bacterium is cut off, as the bacterium becomes sealed in a single membrane structure. In the second, which occurs ~20 min later, tension is released as the second membrane closes off and the protrusion snaps to a spherical shape. Shortly after this second step, the vacuole lyses.
Efficient Vacuolar Lysis by the Bacterium Depends on Acidification by the Recipient Cell
Interactions between the membranes supplied by the donor and recipient cells and bacterial lysis of the double membrane vacuole are important steps in intercellular spread, but difficult to observe using the heretofore described system of GFP-actin expressing MDCK cells. Therefore, we added a plasma membrane label to the donor cell. Cells were incubated with TRITC, which covalently and nonspecifically labels membrane proteins on lysine residues, and were then coplated with unlabeled cells. Protrusions in the act of spreading from labeled into unlabeled cells were selected for observation. Simultaneous timelapse recording of phase contrast and the two fluorescent channels, GFP-actin and TRITC, was performed.
In every case examined (n = 9), the membrane TRITC signal vanished 15 min (average = 2.4, SD = 1.4) after the change in geometry, always fading over a time interval <50 s (Figure 8, ac). In the presence of bafilomycin A1, which inhibits vacuolar acidification (
Throughout the period after cessation of fitful movement, but preceding the abrupt loss of the membrane TRITC signal, we noticed that the strength of the GFP-actin signal declined (Figure 8 d). This could represent photobleaching, but could also in part be due to vacuolar acidification, since GFP intensity declines with decreasing pH (
Association of the L. monocytogenes Comet Tail with the Plasma Membrane Stabilizes Actin Filaments
Since we detected a GFP signal for up to 30 min after intercellular spread around the bacterium in recipient cells that did not express detectable levels of GFP-actin, we conclude that a fraction of the GFP-actin from the donor cell depolymerized very slowly after escape from the secondary vacuole. This was unexpected, as actin turnover in L. monocytogenes tails is rapid, with an average half-life of 33 s in PtK2 cells (
We hypothesized that the stabilization of actin around the bacterium is the legacy of the lysed protrusion. Therefore, we examined actin turnover in extracellular and intercellular membrane protrusions. The rate of actin turnover in intercellular and extracellular protrusions was examined by FRAP. For these measurements, cells were grown at medium density in the presence of 0.5% methyl cellulose, which permitted formation of extracellular membrane-bound protrusions and actin polymerization-dependent movement, but inhibited Brownian motion (see above). Neither extracellular (n = 5) nor intercellular protrusions (n = 10) exhibited any measurable recovery of fluorescent actin along the length of the protrusion >10 min of observation after complete photobleaching (27% of photobleached protrusions exhibited some very gradual recovery within 1 µm around the bacterial surface, particularly the tail-associated end, where new polymerization is expected to occur). These results indicate that actin filaments in protrusions, like those in clouds surrounding bacteria that have just escaped from a secondary vacuole, are highly stabilized, with a half-life significantly >10 min. This is consistent with our unpublished observations that bacteria-containing extracellular membrane-bound protrusions persist for >30 min and continue to react with phalloidin along their length, demonstrating the presence of persistent actin filaments, even when the protrusion is extending very slowly.
Bacteria associated with uniform actin clouds in the cytoplasm were also photobleached and their fluorescence recovery measured. Unbleached clouds were observed to be at steady state (their fluorescence intensity and area neither increased nor decreased), demonstrating that the rate of actin polymerization must equal that of depolymerization. Hence FRAP, which measures polymerization rates, allowed us to calculate filament half-life. Unlike clouds surrounding bacteria which have just escaped from the secondary vacuole, filaments in ordinary cytoplasmic clouds turn over at rates close to that in actin tails (average = 45 s, SD = 15, n = 8).
Recovery of Bacterial Motility Is Delayed after Escape from the Secondary Vacuole
Bacteria that had escaped from a secondary vacuole did not recover motility over a period of >30 min of observation (n = 13). After initial invasion, bacteria require 13 h to acquire tails and begin movement (
To observe effects of pH on ActA stability, we turned to the well-characterized model of J774 cells (
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To further examine the kinetics of the recovery of motility in epithelial cells, we examined individual infectious foci in MDCK cells fixed at different time points after infection (Figure 10; see Materials and Methods). Most intercellular spread into secondary cells occurred between 4 and 5 h after infection, with tertiary infections following 2.5 h later at a similar rate (Figure 10 a). The initial baseline percentage of foci with secondary infections (23 h) is probably due to primary infections occurring simultaneously in neighboring cells, as insufficient numbers of bacteria have acquired tails at this time for intercellular spread to have occurred (Figure 10 b). Interestingly, as only 6.9% of cells are infected at t = 2 h, it is highly improbable (P < 0.01 by chi-squared test) that primary infections of neighboring cells (apparent in 12% of foci) are independent events. This may be due to a sensitization effect. It has been observed that transient calcium spikes occur during L. monocytogenes invasion (Wadsworth et al., 1999) and also during invasion by at least one other invasive bacterial species (
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The increase in secondary infections was not followed by immediate recovery of bacterial motility (Figure 10 b). The fraction of moving bacteria in secondary cells followed a curve almost identical to that obtained from primarily infected cells, but with a shorter lag time and slightly steeper slope. To determine how many bacterial divisions were required before initiation of motility, we counted the total number of bacteria in cells that contained 12 bacteria with tails. To exclude the possibility that other, motile bacteria had already left the cell, primary cells with infected neighbors were not counted. For secondarily infected cells, spreading to a tertiary cell or back to the primary cell are equally probable; thus, we excluded secondary cells after 7 h infection, when tertiary infections were first observed. Primary cells contained an average of 6.3 bacteria per cell when 12 tails were observed (SD = 2.6, n = 18), suggesting approximately three divisions were required for acquisition of motility. Long-term video observation in PtK2 cells also indicates that an average of three divisions are required before bacterial motility is initiated (Theriot, J.A., unpublished results). Secondarily infected cells, however, required only two bacterial divisions, with an average bacterial population of 4.0 per cell hosting 12 bacterial tails (SD = 1.5, n = 28). This difference may reflect the time required to upregulate transcription of ActA after initial infection (
Out of 711 cells observed, only one was inhabited by a single, moving bacterium, and it was unclear whether this bacterium's sister could have recently spread to a neighboring cell. All other cells with tail-associated bacteria possessed at least one other bacterium, strongly suggesting that motility cannot initiate except after at least one bacterial division. The near-absolute dependence of motility on bacterial division is consistent with the model that division is responsible for the establishment of ActA polarity and therefore actin polarity (Tilney et al., 1992;
Membrane Engulfment by Neighboring Cells Occurs in the Absence of Bacteria
We were surprised to observe that when TRITC-labeled cells were incubated with unlabeled cells in the absence of L. monocytogenes, 518% of the unlabeled cells adjacent to labeled cells contained 12 TRITC-labeled vacuoles (Figure 11). Percentages varied greatly according to plating density and seed ratio, and did not appear to increase over time (2072 h). Vacuoles ranged in diameter up to ~0.5 µm, and exhibited some saltatory movement inside cells.
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To confirm that these vacuoles did not merely contain labeled extracellular matrix proteins or products of trypsinization, we colabeled TRITC-dyed cells with the dialkylcarbocyanine DiO, a lipophilic tracer. Multiple DiO-containing vesicles were observed inside unlabeled cells, and a subset of these, usually those largest in size, were also TRITC-labeled.
To our knowledge, this is the first demonstration that MDCK cells engulf portions of their neighbors' membranes. L. monocytogenes intercellular spread may capitalize on this natural paracytophagic behavior.
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Discussion |
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Temporal and Physical Kinetics of Intercellular Spread
Since the columnar epithelium of the intestine is the initial target of L. monocytogenes infection, monolayers of MDCK columnar epithelial cells (albeit derived from kidney) provide a useful model system in which to study pathogenicity at the cellular level. The extensive characterization of this cell line as a polarized epithelium increases its utility. It is clear from our work that the host cell is not merely a passive victim of L. monocytogenes infection, but extensively cooperates in perpetuating the infection as an accomplice in intercellular spread.
Intercellular spread by L. monocytogenes can be described by a stereotyped sequence of contingent events that are highly reproducible. Figure 12 a shows the timeline for spread of a typical bacterium, using average times for each event. First, the bacterium is propelled by actin-based motility into a membrane-bound protrusion, which undergoes bacterially directed fitful movement for ~15 min. Cessation of this movement represents cutoff of the bacterium from the donor cell's pool of ATP or possibly other factors. The protrusion's morphology is maintained for some time, then is suddenly released from tension. Within minutes, the vacuole is lysed in an acidification-dependent manner, leaving the bacterium free in the cytoplasm of the recipient cell. Recovery of motility occurs much later, after at least one bacterial division. The process requires approximately one bacterial generation time (~4050 min;
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A Two-Step Model for Uptake of Bacterium-containing Membrane-bound Protrusions
Uptake of membrane-bound protrusions has been described as being phagocytic in nature by analogy to phagocytic uptake of bacteria by macrophages (
Since bacteria are never observed to escape from or lyse extracellular protrusions despite many hours of observation, the recipient cell must play an active role in pinching off that protrusion and permitting vacuolar lysis. In this model, the first host-dependent step is the closure of the donor membrane, which corresponds to the cessation of fitful movement (Figure 12 b, panel 2). This is analogous to the inhibition of fitful movement in extracellular protrusions by host cell ATP depletion.
After the donor membrane is sealed, the protrusion's morphology is maintained under tension by existing cell-cell adherens junctions (Figure 12 b, panel 2). The host proteasomes, which are known to degrade LLO (
Collapse of the protrusion to a roughly spherical geometry marks the closure of the recipient cell membrane (Figure 12 b, panel 3). Now the cell recognizes the endocytosed vacuole as a target for acidification. How this recognition occurs remains an open question. The sudden drop in pH activates LLO and Mpl; the latter cleaves proPC-PLC to its active form and also cleaves ActA. Vacuolar lysis occurs rapidly (Figure 12 b, panel 4).
The temporal coupling between protrusion collapse and vacuolar lysis is striking in that they are consistently separated by <5 min. This timing is consistent with biochemical observations that proteolytic activation of PC-PLC occurs very rapidly, with maximal amounts accumulated within 5 min of acidification (Marquis, H., unpublished observations). The observation that inhibition of acidification decreases the efficiency of vacuolar lysis is consistent with the phenotype of bacteria deficient for the acid-activated PC-PLC which exhibit decreased intercellular spread (
It has been shown that cadherin-cadherin interactions are required for intercellular spread of Shigella flexneri (
The two-step model presented above suggests a plausible resolution to the biochemical problem of regulating two separate membrane fusion events. The model is also consistent with the observation that protrusions which exhibit fitful movement throughout their duration are taken up only after extremely long periods of time or not at all, as it posits their donor membrane was not sealed. Further studies will elucidate the mechanism by which this occurs.
Actin Tail Structure and Dynamics Are Modified by Close Association with Host Cell Plasma Membrane
After escape into the cytoplasm of the recipient cell, actin from the donor cell remains associated with the bacterium for up to 30 min or more, indicating that is not turned over. This is in contrast to cytoplasmic bacteria surrounded by uniform actin clouds in which the actin is rapidly turned over at a rate similar to that in comet tails (half-lives of ~3045 s). However, slow actin turnover around secondarily spread bacteria is consistent with the actin turnover rate in protrusions, which is much slower than that in comet tails. We suggest that intimate association of the bacterial comet tail with the host's membrane in these protrusions forces interactions between actin and other membrane-associated host proteins that stabilize the actin filaments associated with the bacterium.
Characterization of such interactions is beyond the scope of this report, but this hypothesis is supported by previous data. Ultrastructural examination of cytoplasmic comet tails reveals short, randomly oriented actin filaments that are 0.20.3 µm long (1 µm) filaments that are axially oriented along the long axis of the protrusion (
This may also explain the tendency of L. monocytogenes to occasionally slide back out of short extracellular protrusions in PtK2 cells (
Membrane-dependent stabilization of actin filaments along the length of the protrusion does not prevent further polymerization at the tip, which is apparently responsible for the fitful movement of extracellular (Figure 5) and intercellular (Figure 4 b) protrusions. Similar actin-dependent fitful movement has been described for dendritic spines in neurons (
Bacterial Division Is Required before Initiation of Motility
Recovery of motility after intercellular spread is not immediate for two reasons. First, the actin turnover rate is very low in clouds surrounding bacteria that have just spread into a neighboring cell (see above). More importantly, new actin polymerization is inhibited by the acid-dependent cleavage of ActA. Not only does the new bacterium need to replace its ActA, but it must polarize it.
Other studies have reported that ActA is asymmetrically distributed in a gradient along the bacterial surface of bacteria (
Our data also suggest an explanation for the difference in tail acquisition time between primarily and secondarily infecting bacteria. Transcription of the actA gene followed by translation requires more time than just translation to recover sufficient surface ActA density for motility. However, this alone cannot account for the lag in tail recovery, since bacteria begin to nucleate actin at least one generation before they acquire tails (
Who Calls the Shots in Protrusion Uptake: Bacterium or Host?
Our work shows that epithelial cells do not actively internalize extracellular protruding membranes containing bacteria. Instead, cell to cell spread requires that a bacterium actively drive a membrane protrusion to intrude into the cytoplasm of the adjacent cell in order to trigger uptake. After that, the recipient cell takes over, with uptake occurring in two distinct phases, each considerably longer than classical mechanisms of phagocytosis, which normally requires only a few minutes.
It is surprising that an epithelial cell should ingest a membrane-bound protrusion, considering that the only thing that the recipient cells should recognize is the surface of its neighbor. We cannot exclude the possibility of a bacterially produced presentation molecule that lines the external surface of the protrusion and directs the recipient cell to engulf it; however, there is no evidence for such a signal. If such a molecule existed, it would either require posttranslational modification and plasma membrane targeting by the host cell or a multicomponent bacterial secretory system which could insert the signal directly into the host cell membrane during bacterium-membrane contact. The former is unlikely, since it requires secretion in the protrusion but modification and targeting in the cell body, though it is possible the signal is produced in the cytoplasm and then targeted ubiquitously to the host membrane. The latter is unlikely since genetic screens have failed to find a signal, upstream regulatory components, or components of a secretory system with the required properties in L. monocytogenes. It is also possible that the bacterium modifies the interactions of host membrane proteins at the protrusion tip, however, this is unlikely since extracellular membrane-bound protrusions which are confined between cells are not taken up by their neighbors. Finally, unrelated bacterial pathogens, such as S. flexneri, also undergo actin-based motility to spread from cell to cell (
The engulfment and uptake of membrane from one cell into another, as described here for the lateral spread of bacteria, also occurs in several natural processes. One little-understood but well-known example is the formation of dendrites by melanophores to deliver melanin-containing vesicles to numerous nearby keratinocytes (
Another example of plasma membrane exchange includes a set of several cell signaling events in which a transmembrane ligand on the surface of one cell binds to a cognate receptor on an adjacent cell and is internalized. The Drosophila proteins boss and sevenless (
We also note that epithelial cells frequently take on the role of semi-professional phagocytes to digest their apoptosing neighbors, thereby eliminating the need for macrophages to perform this function (
The type of membrane engulfment observed in these genetically characterized pigment delivery, signaling, and apoptotic systems is a normal feature of epithelial cell biology, and we suggest the descriptive term paracytophagy (eating of nearby cells) for this widespread behavior. L. monocytogenes, an excellent bacterial cell biologist, appears to have exploited the tendency of epithelial cells to engulf bits of their neighbors to enable its efficient intercellular spread. Video microscopy has enabled us to observe the dynamic physical and temporal constraints under which this host-pathogen relationship has evolved.
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Footnotes |
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1 The online version of this article contains supplemental material.
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Acknowledgements |
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We gratefully acknowledge Daniel Portnoy for communication of unpublished results and for providing us with bacterial strains. We also thank Sandra McCallum and Raka Mustaphi for helpful comments on the manuscript and thank members of the Theriot and Nelson labs for stimulating discussions throughout the course of this work.
J.R. Robbins was supported by a National Science Foundation Graduate Research Fellowship. This work was supported by National Institutes of Health grants AI3629 (to J.A. Theriot), GM3527 (to W.J. Nelson), and AI42800 (to H. Marquis), and by a fellowship from the David and Lucile Packard Foundation (to J.A. Theriot).
Submitted: 29 June 1999
Revised: 9 August 1999
Accepted: 10 August 1999
1.used in this paper: CCD, charge-coupled device; DiO, dioctadecyloxacarbocyanine perchlorate; DMF, dimethylformamide; LLO, listeriolysin O; Mpl, metalloprotease; p.i., post-infection; PLC, phospholipase C
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