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Infection and Immunity, June 2002, p. 3156-3163, Vol. 70, No. 6
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.6.3156-3163.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Molecular Biology and Biochemistry,1 Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada, V5A 1S62
Received 29 September 2001/ Returned for modification 6 November 2001/ Accepted 31 January 2002
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ABSTRACT |
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INTRODUCTION |
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Many pathogenic microorganisms can enter eukaryotic cells and use this often hostile environment as a niche within which to replicate and/or evade the host immune response. These organisms can enter both professional phagocytes and cells that are not normally phagocytic, such as epithelial or endothelial cells (19). Among the fungi, there are several species that invade mammalian host cells in vitro and in vivo . Candida albicans can survive in macrophages (31) and endothelial cells (18). Following uptake by macrophages, C. albicans forms germ tubes within phagolysosomes, escapes from this acidic compartment, and destroys the macrophage (31). Similarly, phagocytosis of Cryptococcus neoformans by macrophages results in disordered lysosomal trafficking, host cell disruption, and release of the organisms (17).
A. fumigatus conidia have been shown to bind to lung cells (10) and proteins present in the lung (42, 54, 56). In addition, two groups have independently demonstrated internalization of A. fumigatus conidia by A549 cells (10) and primary lung cells (41) using electron microscopy. Preliminary results in our laboratory have confirmed previous reports (10, 41) that A. fumigatus conidia bind to and become internalized by A549 lung epithelial cells. However, neither of these published studies compared conidial uptake to a negative control, nor was the mechanism of uptake elucidated. To facilitate the study of conidia uptake, we developed two strains of A. fumigatus that expressed green fluorescent protein (GFP) for use in cell culture infection models. Initial immunolabeling experiments demonstrated that differentiation of intracellular and extracellular conidia could be achieved in conidium-host cell invasion assays. Therefore, the objectives of this study were threefold: (i) to determine the percent internalization of conidia by three different cultured cell lines relative to a negative control, (ii) to determine whether conidia germinate inside of A549 cells, and (iii) to establish whether the internalization of conidia is accompanied by cytoskeletal arrangements in A549 cells.
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MATERIALS AND METHODS |
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Tissue culture. The type II pneumocyte cell line A549 and murine macrophage line J774 were obtained from the American Type Culture Collection and maintained in RPMI 1640 medium containing 10% fetal bovine serum (Canadian Life Technologies, Burlington, Ontario, Canada), streptomycin (100 mg/liter) and penicillin (16 mg/liter) (both from Sigma-Aldrich Canada, Oakville, Ontario, Canada). Human umbilical vein endothelial (HUVE) cells were isolated from human umbilical veins by the method of Jaffe et al. with modifications (30). Fresh umbilical cords were stored at 4°C and processed within 12 h. The umbilical vein was cannulated with a 14-gauge catheter and secured with a cable tie. The vein was flushed with phosphate-buttered saline (PBS) (until clear), and the other end of the vein was then tied. Each cord was infused with collagenase II (162.5 U/ml; Sigma) in M-199 medium (Canadian Life Technologies) and incubated at 37°C for 8 min. The enzyme solution was removed, and the cells were centrifuged at 800 x g for 5 min. The pellet was gently resuspended in 4 ml of M-199 medium containing 10% (vol/vol) fetal bovine serum (FBS), 10% (vol/vol) fetal calf serum, 2 mM glutamine, penicillin, and streptomycin and then added to gelatin-coated petri dishes. All cells were grown at 37°C in humidified 5% CO2 incubators.
Creation of GFP-expressing A. fumigatus strains. To generate GFP-expressing strains of A. fumigatus, conidia were transformed by electroporation with 0 or 1.5 µg of linearized gGFP plasmid based on the method adapted from Sanchez and Aguirre (48) by Wiedner et al. (57). Reaction mixtures of 200 to 1,000 µl were plated onto YM agar containing hygromycin (250 µg/ml) and incubated for 3 days at 37°C. Conidia from hygromycin-resistant clones were restreaked twice onto YM agar containing hygromycin to check for stable integration.
Detection of GFP expression in hygromycin-resistant transformants. (i) Immunoblotting. To prepare conidial proteins, wild-type and GFP strains were harvested and resuspended in 10 ml of lysis buffer (100 mM Tris-Cl [pH 7.4], 2.5 mM EDTA, 5 mM dithiothreitol) and a protease inhibitor cocktail containing 40 µM 4-(2-aminoethyl) benzenesulfonyl fluoride, 20 µM sodium EDTA, 2.6 mM bestatin, 0.28 mM trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64), 20 µM leupeptin, and 6 µM aprotinin (Sigma). An equal volume of 0.5-mm-diameter acid-washed glass beads was added, and the suspension was vortexed at 30-s intervals (followed by 1 min of cooling) until cell breakage was observed by microscopy. The glass beads were removed, and the suspension was centrifuged at 1,500 x g for 10 min. The supernatant fraction consisted of the whole conidial homogenate. To test for expression of the GFP protein, conidial proteins from the homogenate were separated by sodium dodecyl sulfate-12% polyacrylamide gel (SDS-12% PAGE) (6 µg/lane) and transferred to polyvinylidene difluoride membrane using a submerged tank transfer apparatus according to the manufacturer's directions (Bio-Rad Laboratories, Mississauga, Ontario, Canada). The membrane was blocked overnight in BLOTTO (PBS-5% [wt/vol] skim milk powder). The next day the blot was incubated for 2 h with a polyclonal rabbit anti-GFP antibody (diluted to 1:100; Clontech Laboratories Inc., Palo Alto, Calif.) in BLOTTO and then rinsed four times for 5 min each with PBS-0.05% Tween 20 (PBST). The blot was incubated for 1 h in a solution containing goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech Inc., Baie d'Urfé, Québec, Canada) (diluted 1:1,000 in BLOTTO), rinsed five times with PBST as described above, and developed with DAB substrate (6 mg diaminobenzidine tetrahydrochloride (Sigma) in 10 mM Tris-Cl [pH 7.6] containing 0.3% NiCl2 and H2O2 [1 µl/ml]).
(ii) Fluorescence microscopy. To detect whether transformants expressing GFP protein emitted green fluorescence when excited with blue (488 nm) light, conidia were harvested and 2 x 107 spores were added to 12-mm-diameter poly-L-lysine coated coverslips, fixed with paraformaldehyde, mounted onto slides, and sealed with nail polish. Samples were viewed on a Zeiss Axioplan 2 microscope equipped for epifluorescence with fluorescein filters.
Preparation of an anti-Aspergillus antibody. A. fumigatus mycelial cell wall proteins were used as antigens for the production of a polyclonal antibody that recognizes A. fumigatus conidia. Fernbach flasks containing 800 ml of YM medium were inoculated with 2.5 x 106 A. fumigatus ATCC 13073 conidia/ml and incubated at 150 rpm for 16 h. Mycelial proteins were prepared using the method of Puente et al. (44) and quantitated by a Bradford assay (5). Female New Zealand White rabbits were injected with 1 mg of A. fumigatus mycelial cell wall proteins in 1.5 ml PBS plus 250 µl of TiterMax adjuvant (Sigma). To test serum for the presence of antibodies recognizing A. fumigatus, conidia from strain 13073 were added to an eight-well chamber slide (Becton Dickinson) and reacted with various dilutions of serum prepared in PBS-10% (vol/vol) goat serum. Bound primary antibody was detected by a goat anti-rabbit Texas Red secondary antibody (Molecular Probes, Eugene, Oreg.) diluted 1:100 in PBS-goat serum. Conidia were fixed with 4% (wt/vol) paraformaldehyde in PBS and viewed with an Olympus AHBS3 Vanox microscope equipped for epifluorescence microscopy.
Immunofluorescence-confocal microscopy. (i) Phagocytosis assays. A549 and HUVE cells were seeded at 2.5 x 105 cells/well on 12-mm-diameter number 1 coverslips in 24-well plates (Falcon, Becton-Dickinson Canada Inc., Mississauga, Ontario, Canada) and grown for 16 h. J774 cells were seeded at 2.5 x 105 cells/well for 2 h at 37°C. Following cell growth, wells were blocked for 1 h in minimum essential medium (MEM) (Canadian Life Technologies) containing 0.1% (wt/vol) bovine serum albumin (ICN Pharmaceuticals, Montreal, Canada) at 37°C. Cells were infected with 1 ml of 106 spores/ml in MEM-10% (vol/vol) FBS for the indicated times at 37°C. To prepare heat-killed spores, conidia from strain 13073 gGFP4 were first autoclaved for 20 min at 121°C. As a control for nonspecific uptake, A549 and HUVE cells were also incubated with 1-µm-diameter fluorescein-labeled biotinylated polystyrene beads (5 x 106/ml; Molecular Probes) diluted in MEM-10% FBS for 3 h at 37°C. A fivefold higher multiplicity of infection (compared to infection with conidia) was required in order to get a reasonable number of beads bound to a field of cells. After incubation, unbound spores or beads were removed by washing wells three times with PBST. Extracellular spores were labeled with the rabbit antibody raised against conidial proteins. Primary antibody was diluted 1:50 in PBS-10% (vol/vol) goat serum (Canadian Life Technologies) and added to samples on ice for 1 h. Wells were washed three times with PBS and then incubated for 45 min with a goat anti-rabbit Alexa Fluor 594-conjugated secondary antibody (Molecular Probes) diluted 1:400 in PBS-10% (vol/vol) goat serum. Wells were washed with PBS again and fixed for 1 h with PBS-4% (wt/vol) paraformaldehyde (pH 7.4), and the coverslips were mounted onto slides with ProLong antifade from Molecular Probes. To label extracellular beads, samples were incubated for 1 h with a 1:100 dilution of Streptavidin-Alexa Fluor 594 secondary reagent and then fixed and mounted as described above. Samples were viewed with a Zeiss Axioplan 2 microscope equipped with epifluorescence filters using a 63x lens. To analyze uptake of conidia, 10 fields per coverslip were captured (5 for J774 samples) with a Sony DXC-950P 3CCD camera using Eclipse image capturing software (Empix Imaging Inc., Mississauga, Ontario, Canada). The extracellular and intracellular conidia were enumerated after merging the red and green channels in Eclipse.
(ii) Actin-rearrangement assays. Cells were incubated with strain 13073 gGFP for 2 h and 40 min (rather than 3 h) in order to capture the rearrangement process as conidia were being internalized. Following infection, wells were washed with PBST and fixed with paraformaldehyde, and the cells were permeabilized by incubating for 1 h in PBS-10% (vol/vol) goat serum-0.5% (wt/vol) saponin. Coverslips were incubated for 20 min at room temperature with rhodamine phalloidin (Molecular Probes) diluted 1:50 in PBS to stain cellular actin filaments. Samples were washed with PBS, mounted in ProLong, and viewed with a Zeiss LSM-410 confocal microscope equipped with a krypton-argon laser (Omnichrome), using a 63 x 1.4 numerical aperture lens. Green fluorescence was captured with a 515- to 540-nm band pass filter, and red fluorescence was capture with a 590- to 610-nm band pass filter. Images were processed in Adobe PhotoShop 6.0 (Adobe Systems Incorporated, San Jose, Calif.).
Nystatin protection assay. Cells were seeded into 24-well plates and infected with conidia as described above for the immunofluorescence. Following infection with GFP strains, wells were washed three times with PBST and then incubated with nystatin at 0, 50 (for J774 cells), or 100 µg/ml (for A549 cells) (250 and 500 U/ml, respectively; Sigma) in MEM for 3 h at 37°C. This amount of nystatin was the minimum fungicidal concentration required to kill 106 conidia (data not shown). A lower amount of nystatin was used with J774 cells (250 U/ml), as higher concentrations were cytotoxic to this cell line. Nystatin at this concentration was sufficient to kill 5 x 105 conidia (the number of conidia bound to J774 cells was always less than 5 x 105 conidia/well) but did not affect the viability of either J774 or A549 cells as determined by crystal violet staining (data not shown). After 3 h, monolayers were lysed by incubating in 0.5% Triton X-100 for 10 min with shaking. Released conidia were diluted, plated onto YM agar (two to three replicate plates/well) and incubated at 37°C. After 24 h colonies were counted to determine total bound and intracellular conidia. The binding of the GFP-transformant and the parental 13073 or CHUV strains to A549 cells was similar when examined by differential interference contrast (DIC) microscopy (data not shown). In addition, the GFP transformants behaved similarly to the parental strains in the nystatin protection assay. For example, the invasion index of the 13073 parental strain was 2.7% ± 1.2% of the initial inoculum, compared to the GFP-transformed strain at 4.0% ± 0.8% (P > 0.05).
Invasion assay in the presence of cytoskeletal inhibitors. Actin polymerization inhibitors such as cytochalasin D and the microtubule inhibitors colchicine and nocodazole can be used to determine whether actin filaments or microtubules are required for pathogen entry into host cells. Inhibitors were made as stock solutions in dimethyl sulfoxide (cytochalasin D) or deionized distilled H2O (colchicine) and added to the invasion medium at the indicated concentrations. The concentrations of inhibitors (and dimethyl sulfoxide) used did not compromise A549 cell viability as determined by trypan blue exclusion or crystal violet staining (data not shown). Morphology of the cells was also normal as determined by bright-field microscopy (data not shown). Assays were performed with strain 13073 gGFP4. Addition of colchicine or cytochalasin D to conidia alone at the concentrations used in the assay did not cause any defects in conidia germination or growth. The results are representative of two independent experiments and are expressed as the mean ± standard deviation of three replicates.
Statistics. The Student t test was used for statistical analysis of data.
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RESULTS |
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Phagocytosis of polystyrene beads by A549 and HUVE cells. To measure the extent of nonspecific phagocytosis, we incubated A549 cells with 1 µm fluorescein-labeled biotinylated polystyrene beads (A. fumigatus conidia are 2 to 3 µm in diameter) and measured the invasion index by immunostaining. Previous studies using polystyrene beads have shown that they are internalized by A549 (21) and HUVE (58) cells under certain conditions. In these studies, internalization was determined by subtracting the number of particles bound to the cell surface at 4°C from the number of microspheres associated with the cells at 37°C (21). In contrast, our assay used immunostaining to differentiate between external and internalized beads. Polystyrene beads adhered to A549 and HUVE cells at a frequency of 10 to 20 beads/50 cells; however, the cells did not internalize the bound beads (Table 1). Therefore, under the conditions used in our assay, uptake of A. fumigatus conidia by A549 and HUVE cells was specific, as these cells did not appear to be randomly internalizing particles.
Inhalation of Aspergillus conidia leads to the deposition of spores on the alveolar surface, and the cells most likely to initially interact with conidia are type I and type II pneumocytes. Therefore, for the rest of our experiments we chose A549 cells as model alveolar epithelial cells and further investigated the interaction of A. fumigatus conidia with this cell line.
Phagocytosis of dead conidia by A549 cells. To determine whether internalization of conidia by A549 cells required viable conidia, we compared the rates of uptake of live and heat-killed conidia into A549 cells by immunostaining. The invasion index of viable and heat-killed conidia was 28% ± 7% and 37% ± 9 (P > 0.05), respectively. These data suggest that A549 cells internalize viable and heat-killed conidia to an equal extent, which implies that internalization may occur via recognition of a non-heat-labile surface antigen, such as a polysaccharide.
Phagocytosis of conidia by A549 cells: Effect of serum. To determine whether serum components were required for uptake of conidia by A549 cells, invasion assays were performed in the presence of MEM or MEM plus serum (FBS), and uptake was determined by the nystatin protection assay. There were no significant differences in invasion frequencies between these samples (data not shown) suggesting that the recognition of conidia by A549 surface receptors does not require serum components.
Delay of germination in phagocytosed conidia. To determine whether conidia germinate inside A549 cells, conidia were incubated with cells for 6 h. The A549 cell monolayer was not damaged by this extended incubation time. Visualization by immunostaining revealed that all of the extracellular conidia had swollen and most had germ tubes ranging from 5 to 10 µm in length, whereas a vast majority of the intracellular conidia had not germinated (Fig. 4). We could not determine whether any of the extracellular germinated conidia had penetrated the cell layer. These data suggest, along with the data from the nystatin protection assay, that internalized conidia remain viable inside A549 cells and that germination is severely restricted for at least 6 h. Whether conidia can be retained in a similar state within alveolar epithelial cells in vivo is unknown.
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Internalization of A. fumigatus conidia induces localized actin rearrangement. Many microbial pathogens such as Salmonella enterica serovar Typhimurium (20) and C. albicans (18) that invade nonprofessional phagocytic cells with rigid cytoskeletons induce visible alterations in the underlying host cytoskeletal structure. Since cytochalasin D inhibited uptake of A. fumigatus conidia by A549 cells, we investigated whether conidia internalization would lead to actin rearrangement which could be visualized in A549 cells. Uninfected A549 cells showed intense staining of cortical and cellular actin filaments (data not shown). In infected cells, approximately 10% of the conidia were found inside vacuoles coated with polymerized actin (Fig. 6). However, as phagocytosis assays had demonstrated that the invasion index of conidia by A549 cells was approximately 30%, the remaining 20% must have already shed their phagosome-actin coat, or the microfilaments were only polymerizing around those organisms that were actively becoming phagocytosed the moment that the fixative was applied. Alternatively, some conidia may have been phagocytosed by a non-actin-dependent mechanism.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this report, we transformed two strains of A. fumigatus with gGFP and used them to investigate internalization of conidia by three cultured cell lines. We used differential immunolabeling and a nystatin protection assay to quantify the percent internalization of A. fumigatus conidia by host cells. Previous studies have reported that A. fumigatus conidia are internalized by A549 lung epithelial cells (10) and primary airway type II cells (41). In our study, we confirmed that A549 cells internalize A. fumigatus conidia, and in addition, we determined that the invasion index was approximately 30% and the invasion frequency (relative to the initial inoculum) was approximately 2%. No uptake of 1-µm-diameter polystyrene beads was observed over the assay period. The invasion frequency of conidia into A549 parallels uptake of other pathogenic organisms by these cells: A549 cells internalized 5.7% of the initial inoculum of Mycobacterium tuberculosis (3), 0.2% of the protozoan parasite Encephalitozoon cuniculi (9), and 0.15% of Burkholderia cepacia (36). In contrast, Martin and Mohr (36) observed a 0.008% invasion frequency of the nonpathogenic E. coli HB101 into A549 cells. As a positive control for internalization, we measured uptake of conidia by J774 cells and observed that these professional phagocytes internalized approximately 90% of the bound conidia. Similarly, Káposzta et al. observed that 98% of macrophage-associated C. albicans were phagocytosed by primary macrophages (31). The invasion index for J774 cells calculated by the nystatin protection assay was much lower than that obtained by immunolabeling (27 versus 87% average for the two strains). This was most likely due to killing of the conidia within J774 cells during the nystatin assay incubation period. In contrast, all of the conidia internalized by A549 cells were viable after 6 h. Finally, we measured internalization of conidia by HUVE cells and found that 50% of the bound spores were phagocytosed. This was similar to the uptake of C. neoformans by these cells; Ibrahim et al. (29) showed that 77% of the bound yeasts were internalized by HUVE cells.
The development of the nystatin protection assay allowed us to confirm the internalization results obtained by differential immunolabeling. Since both techniques produced similar internalization rates with A549 cells, we were confident that the nystatin protection assay gave an accurate and objective measurement of invasion. This technique is simple and sensitive and should be applicable to other pathogenic fungi as nystatin is fungicidal toward both C. albicans and C. neoformans (6). Furthermore, comparing the results of immunostaining and nystatin protection may provide quantitative information on the extent of killing by host cells.
To determine whether A. fumigatus conidia germinated following entry into A549 cells, we extended the invasion incubation period from 3 to 6 h. Germination was severely delayed for phagocytosed conidia and this differs from uptake of Candida albicans by HUVE cells. In this case, the yeast cells germinated during the first hour, and by 2 h most of the germlings were internalized by the endothelial cells (18). In their studies of A. fumigatus, DeHart et al. (10) observed that direct hyphal penetration of A549 cells occurred after a twelve h incubation period. Our data suggest that the hyphal penetration of A549 cells observed by Dettart et al. probably occurred via extracellular germlings.
Microbial pathogens use several tactics to gain access to host cells including phagocytosis (9, 37), macropinocytosis (20, 22), receptor-mediated endocytosis (38) and microtubule-dependent internalization (24, 39). Uptake of A. fumigatus conidia by A549 cells occurred via actin-dependent phagocytosis but also required host cell microtubules. Internalization of A. fumigatus conidia did not induce the massive cytoskeletal rearrangements seen in Salmonella uptake into epithelial cells, which enters via membrane ruffling and macropinocytosis (22). Instead, the process of A. fumigatus internalization most likely occurs via engagement of ligands with host cell receptors and resembles the zipper uptake model (53). The identity of these ligands is currently unknown. The microfilament dependence of A. fumigatus uptake resembles epithelial cell invasion by other bacterial pathogens such as M. tuberculosis (3) and S. enterica serovar Typhimurium (20). Although most invasive microbes use host actin to enter nonprofessional phagocytes, there are some organisms that also use microtubules. For example, some strains of Campylobacter jejuni require microtubules but not microfilaments for entry into intestinal cells (4, 39). Following adherence to intestinal epithelial cells, C. jejuni trigger the formation of a microtubule-based membrane extension that meets the invading bacterium (32). In contrast, phagocytosis of A. fumigatus by A549 cells involves both cytoskeletal components which is similar to M. tuberculosis entry into A549 cells (3) and C. albicans uptake by endothelial cells (18).
In summary, we have used several techniques to demonstrate that A. fumigatus conidia are internalized by lung epithelial and endothelial and murine macrophage cells in vitro. Examination of conidium uptake by A549 cells showed that the process was analogous to phagocytosis and utilized host microfilaments and microtubules. Internalization of conidia by nonprofessional phagocytes may be important in the development of aspergillosis in vivo, as sequestration inside these cells may allow the conidia to escape the immune response of the host. Future experiments will investigate the location of the conidium in the endosomal pathway and will determine whether uptake is a defense mechanism of the host or a virulence trait of the fungus.
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ACKNOWLEDGMENTS |
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We thank C. Breuil (Department of Wood Science, University of British Columbia) for the gift of the gGFP plasmid, Loekie van der Wal (Animal Care Facility, Simon Fraser University) for performing animal immunizations, and Wendy Olson for coordinating umbilical cord collections at Royal Columbian Hospital, New Westminster, British Columbia, Canada. We are grateful to Anna H. T. Gifford for critical reading of the manuscript.
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FOOTNOTES |
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