INFECTION AND IMMUNITY, June 2002, p. 3156–3163 Vol. 70, No. 60019-9567/02/$04.00�0 DOI: 10.1128/IAI.70.6.3156–3163.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Uptake of Aspergillus fumigatus Conidia by Phagocytic andNonphagocytic Cells In Vitro: Quantitation Using Strains Expressing

Green Fluorescent ProteinJulie A. Wasylnka1 and Margo M. Moore2*

Department of Molecular Biology and Biochemistry1 and Department of Biological Sciences,2 Simon Fraser University,Burnaby, British Columbia, Canada, V5A 1S6

Received 29 September 2001/Returned for modification 6 November 2001/Accepted 31 January 2002

Several pathogenic fungal organisms enter eukaryotic cells and manipulate the host cell environment tofavor their own growth and survival. Aspergillus fumigatus is a saprophytic fungus that causes invasive lungdisease in the immunocompromised host. To determine whether A. fumigatus could enter eukaryotic cells, westudied the uptake of two different GFP-expressing A. fumigatus strains into A549 lung epithelial cells, humanumbilical vein endothelial (HUVE) cells, and J774 murine macrophages in vitro. A549 cells internalized 30%of the bound conidia whereas HUVE and J774 cells internalized 50 and 90%, respectively. Conidia within A549cells remained viable for 6 h; however, 60 to 80% of conidia within J774 cells were killed after only 4 h. Liveand heat-killed conidia were internalized to the same extent by A549 cells. After 6 h, almost none of the conidiainside A549 cells had germinated, whereas extracellular conidia had developed germ tubes. Internalization ofconidia by A549 cells was a temperature-dependent process and required rearrangement of the underlying hostcell cytoskeleton; uptake was inhibited by 75% with 0.5 �M cytochalasin D and by 65% with 5 �M colchicine.Fluorescent labeling of infected A549 cells with rhodamine phalloidin provided visible evidence of cytoskeletalalteration as many of the intracellular conidia were contained in actin-coated phagosomes. These data provideevidence that significant numbers of A. fumigatus conidia can be internalized by nonprofessional phagocytes invitro and these cells may serve as reservoirs for immune cell evasion and dissemination throughout the host.

Fungal infections cause significant morbidity and mortalityin both animals and humans. Over the past 2 decades there hasbeen a substantial increase in human fungal infections amongimmunocompromised individuals (15, 26). The growth of thishigh-risk group has been attributed to more aggressive cyto-toxic chemotherapy, an increase in the number of bone mar-row and organ transplant recipients and the emergence ofAIDS (15). Aspergillus fumigatus is a saprophytic fungus whichcauses the most common mold infection worldwide (43). In-halation of infectious conidia and their deposition in the alve-oli may lead to germination and growth of the fungus in thelung, and in severely immunocompromised individuals, thismay develop into a potentially fatal infection known as invasiveaspergillosis (13). Amphotericin B is still the drug of choice forthe treatment of invasive aspergillosis, despite its severe sideeffects (33). The newer generation triazole drugs (voricon-azole, posaconazole) and antifungal peptides such as the echi-nocandins are showing promising results in clinical trials; how-ever, their long-term efficacy remains to be determined (14, 23,55). Early diagnosis of invasive aspergillosis is crucial (12), andeven with treatment, the mortality rate exceeds 65% (11).Basic research into the pathogenesis of A. fumigatus remains apriority and may help identify new drug targets which aredesperately needed.

Many pathogenic microorganisms can enter eukaryotic cellsand use this often hostile environment as a niche within which

to replicate and/or evade the host immune response. Theseorganisms can enter both professional phagocytes and cellsthat are not normally phagocytic, such as epithelial or endo-thelial cells (19). Among the fungi, there are several speciesthat invade mammalian host cells in vitro and in vivo . Candidaalbicans can survive in macrophages (31) and endothelial cells(18). Following uptake by macrophages, C. albicans formsgerm tubes within phagolysosomes, escapes from this acidiccompartment, and destroys the macrophage (31). Similarly,phagocytosis of Cryptococcus neoformans by macrophages re-sults 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 internalizationof A. fumigatus conidia by A549 cells (10) and primary lungcells (41) using electron microscopy. Preliminary results in ourlaboratory have confirmed previous reports (10, 41) that A.fumigatus conidia bind to and become internalized by A549lung epithelial cells. However, neither of these published stud-ies compared conidial uptake to a negative control, nor was themechanism of uptake elucidated. To facilitate the study ofconidia uptake, we developed two strains of A. fumigatus thatexpressed green fluorescent protein (GFP) for use in cell cul-ture infection models. Initial immunolabeling experimentsdemonstrated that differentiation of intracellular and extracel-lular conidia could be achieved in conidium-host cell invasionassays. Therefore, the objectives of this study were threefold:(i) to determine the percent internalization of conidia by threedifferent cultured cell lines relative to a negative control, (ii) todetermine whether conidia germinate inside of A549 cells, and

* Corresponding author. Mailing address: Department of BiologicalSciences, 8888 University Dr., Simon Fraser University, Burnaby, Brit-ish Columbia V5A 1S6, Canada. Phone: (604) 291-3441. Fax: (604)291-3496. E-mail: mmoore@sfu.ca.

3156

on March 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

(iii) to establish whether the internalization of conidia is ac-companied by cytoskeletal arrangements in A549 cells.

MATERIALS AND METHODS

A. fumigatus strains and growth conditions. Two strains of A. fumigatus wereused in this study: A. fumigatus ATCC 13703 (American Type Culture Collection,Manassas, Va.) and CHUV (gift from M. Monod, Laboratoire de Mycologie,Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland). Fungi weregrown on YM agar (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, and0.5% dextrose) for 3 days at 37°C until conidia were fully mature. Conidia wereharvested as described previously (56).

Tissue culture. The type II pneumocyte cell line A549 and murine macrophageline J774 were obtained from the American Type Culture Collection and main-tained in RPMI 1640 medium containing 10% fetal bovine serum (Canadian LifeTechnologies, Burlington, Ontario, Canada), streptomycin (100 mg/liter) andpenicillin (16 mg/liter) (both from Sigma-Aldrich Canada, Oakville, Ontario,Canada). Human umbilical vein endothelial (HUVE) cells were isolated fromhuman umbilical veins by the method of Jaffe et al. with modifications (30). Freshumbilical cords were stored at 4°C and processed within 12 h. The umbilical veinwas cannulated with a 14-gauge catheter and secured with a cable tie. The veinwas flushed with phosphate-buttered saline (PBS) (until clear), and the other endof 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°Cfor 8 min. The enzyme solution was removed, and the cells were centrifuged at800 � g for 5 min. The pellet was gently resuspended in 4 ml of M-199 mediumcontaining 10% (vol/vol) fetal bovine serum (FBS), 10% (vol/vol) fetal calfserum, 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 incuba-tors.

Creation of GFP-expressing A. fumigatus strains. To generate GFP-expressingstrains of A. fumigatus, conidia were transformed by electroporation with 0 or 1.5�g of linearized gGFP plasmid based on the method adapted from Sanchez andAguirre (48) by Wiedner et al. (57). Reaction mixtures of 200 to 1,000 �l wereplated onto YM agar containing hygromycin (250 �g/ml) and incubated for 3days at 37°C. Conidia from hygromycin-resistant clones were restreaked twiceonto YM agar containing hygromycin to check for stable integration.

Detection of GFP expression in hygromycin-resistant transformants. (i) Im-munoblotting. To prepare conidial proteins, wild-type and GFP strains wereharvested and resuspended in 10 ml of lysis buffer (100 mM Tris-Cl [pH 7.4], 2.5mM EDTA, 5 mM dithiothreitol) and a protease inhibitor cocktail containing 40�M 4-(2-aminoethyl) benzenesulfonyl fluoride, 20 �M sodium EDTA, 2.6 mMbestatin, 0.28 mM trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64), 20 �M leupeptin, and 6 �M aprotinin (Sigma). An equal volume of 0.5-mm-diameter acid-washed glass beads was added, and the suspension was vor-texed at 30-s intervals (followed by 1 min of cooling) until cell breakage wasobserved by microscopy. The glass beads were removed, and the suspension wascentrifuged at 1,500 � g for 10 min. The supernatant fraction consisted of thewhole conidial homogenate. To test for expression of the GFP protein, conidialproteins from the homogenate were separated by sodium dodecyl sulfate–12%polyacrylamide gel (SDS-12% PAGE) (6 �g/lane) and transferred to polyvinyli-dene difluoride membrane using a submerged tank transfer apparatus accordingto 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 polyclonalrabbit anti-GFP antibody (diluted to 1:100; Clontech Laboratories Inc., PaloAlto, Calif.) in BLOTTO and then rinsed four times for 5 min each withPBS–0.05% Tween 20 (PBST). The blot was incubated for 1 h in a solutioncontaining goat anti-rabbit horseradish peroxidase-conjugated secondary anti-body (Amersham Pharmacia Biotech Inc., Baie d’Urfé, Québec, Canada) (dilut-ed 1:1,000 in BLOTTO), rinsed five times with PBST as described above, anddeveloped with DAB substrate (6 mg diaminobenzidine tetrahydrochloride (Sig-ma) in 10 mM Tris-Cl [pH 7.6] containing 0.3% NiCl2 and H2O2 [1 �l/ml]).

(ii) Fluorescence microscopy. To detect whether transformants expressingGFP protein emitted green fluorescence when excited with blue (488 nm) light,conidia were harvested and 2 � 107 spores were added to 12-mm-diameterpoly-L-lysine coated coverslips, fixed with paraformaldehyde, mounted ontoslides, and sealed with nail polish. Samples were viewed on a Zeiss Axioplan 2microscope equipped for epifluorescence with fluorescein filters.

Preparation of an anti-Aspergillus antibody. A. fumigatus mycelial cell wallproteins were used as antigens for the production of a polyclonal antibody thatrecognizes A. fumigatus conidia. Fernbach flasks containing 800 ml of YM me-dium were inoculated with 2.5 � 106 A. fumigatus ATCC 13073 conidia/ml and

incubated at 150 rpm for 16 h. Mycelial proteins were prepared using the methodof Puente et al. (44) and quantitated by a Bradford assay (5). Female NewZealand White rabbits were injected with 1 mg of A. fumigatus mycelial cell wallproteins in 1.5 ml PBS plus 250 �l of TiterMax adjuvant (Sigma). To test serumfor the presence of antibodies recognizing A. fumigatus, conidia from strain13073 were added to an eight-well chamber slide (Becton Dickinson) and reactedwith various dilutions of serum prepared in PBS–10% (vol/vol) goat serum.Bound primary antibody was detected by a goat anti-rabbit Texas Red secondaryantibody (Molecular Probes, Eugene, Oreg.) diluted 1:100 in PBS-goat serum.Conidia were fixed with 4% (wt/vol) paraformaldehyde in PBS and viewed withan Olympus AHBS3 Vanox microscope equipped for epifluorescence micros-copy.

Immunofluorescence-confocal microscopy. (i) Phagocytosis assays. A549 andHUVE cells were seeded at 2.5 � 105 cells/well on 12-mm-diameter number 1coverslips in 24-well plates (Falcon, Becton-Dickinson Canada Inc., Mississauga,Ontario, Canada) and grown for 16 h. J774 cells were seeded at 2.5 � 105

cells/well for 2 h at 37°C. Following cell growth, wells were blocked for 1 h inminimum essential medium (MEM) (Canadian Life Technologies) containing0.1% (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 fromstrain 13073 gGFP4 were first autoclaved for 20 min at 121°C. As a control fornonspecific uptake, A549 and HUVE cells were also incubated with 1-�m-diameter fluorescein-labeled biotinylated polystyrene beads (5 � 106/ml; Molec-ular Probes) diluted in MEM–10% FBS for 3 h at 37°C. A fivefold highermultiplicity of infection (compared to infection with conidia) was required inorder to get a reasonable number of beads bound to a field of cells. Afterincubation, unbound spores or beads were removed by washing wells three timeswith PBST. Extracellular spores were labeled with the rabbit antibody raisedagainst conidial proteins. Primary antibody was diluted 1:50 in PBS–10% (vol/vol) goat serum (Canadian Life Technologies) and added to samples on ice for1 h. Wells were washed three times with PBS and then incubated for 45 min witha goat anti-rabbit Alexa Fluor 594-conjugated secondary antibody (MolecularProbes) diluted 1:400 in PBS–10% (vol/vol) goat serum. Wells were washed withPBS 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 Mo-lecular Probes. To label extracellular beads, samples were incubated for 1 h witha 1:100 dilution of Streptavidin-Alexa Fluor 594 secondary reagent and thenfixed and mounted as described above. Samples were viewed with a Zeiss Axio-plan 2 microscope equipped with epifluorescence filters using a 63� lens. Toanalyze uptake of conidia, 10 fields per coverslip were captured (5 for J774samples) with a Sony DXC-950P 3CCD camera using Eclipse image capturingsoftware (Empix Imaging Inc., Mississauga, Ontario, Canada). The extracellularand intracellular conidia were enumerated after merging the red and greenchannels in Eclipse.

(ii) Actin-rearrangement assays. Cells were incubated with strain 13073 gGFPfor 2 h and 40 min (rather than 3 h) in order to capture the rearrangementprocess as conidia were being internalized. Following infection, wells werewashed with PBST and fixed with paraformaldehyde, and the cells were perme-abilized 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 rho-damine phalloidin (Molecular Probes) diluted 1:50 in PBS to stain cellular actinfilaments. Samples were washed with PBS, mounted in ProLong, and viewed witha Zeiss LSM-410 confocal microscope equipped with a krypton-argon laser(Omnichrome), using a 63 � 1.4 numerical aperture lens. Green fluorescencewas captured with a 515- to 540-nm band pass filter, and red fluorescence wascapture with a 590- to 610-nm band pass filter. Images were processed in AdobePhotoShop 6.0 (Adobe Systems Incorporated, San Jose, Calif.).

Nystatin protection assay. Cells were seeded into 24-well plates and infectedwith conidia as described above for the immunofluorescence. Following infectionwith GFP strains, wells were washed three times with PBST and then incubatedwith nystatin at 0, 50 (for J774 cells), or 100 �g/ml (for A549 cells) (250 and 500U/ml, respectively; Sigma) in MEM for 3 h at 37°C. This amount of nystatin wasthe minimum fungicidal concentration required to kill 106 conidia (data notshown). A lower amount of nystatin was used with J774 cells (250 U/ml), ashigher concentrations were cytotoxic to this cell line. Nystatin at this concentra-tion was sufficient to kill 5 � 105 conidia (the number of conidia bound to J774cells was always less than 5 � 105 conidia/well) but did not affect the viability ofeither J774 or A549 cells as determined by crystal violet staining (data notshown). After 3 h, monolayers were lysed by incubating in 0.5% Triton X-100 for10 min with shaking. Released conidia were diluted, plated onto YM agar (twoto three replicate plates/well) and incubated at 37°C. After 24 h colonies werecounted to determine total bound and intracellular conidia. The binding of the

VOL. 70, 2002 PHAGOCYTOSIS OF A. FUMIGATUS CONIDIA 3157

on March 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

GFP-transformant and the parental 13073 or CHUV strains to A549 cells wassimilar when examined by differential interference contrast (DIC) microscopy(data not shown). In addition, the GFP transformants behaved similarly to theparental strains in the nystatin protection assay. For example, the invasion indexof the 13073 parental strain was 2.7% � 1.2% of the initial inoculum, comparedto the GFP-transformed strain at 4.0% � 0.8% (P � 0.05).

Invasion assay in the presence of cytoskeletal inhibitors. Actin polymerizationinhibitors such as cytochalasin D and the microtubule inhibitors colchicine andnocodazole can be used to determine whether actin filaments or microtubulesare required for pathogen entry into host cells. Inhibitors were made as stocksolutions in dimethyl sulfoxide (cytochalasin D) or deionized distilled H2O (col-chicine) and added to the invasion medium at the indicated concentrations. Theconcentrations of inhibitors (and dimethyl sulfoxide) used did not compromiseA549 cell viability as determined by trypan blue exclusion or crystal violet stain-ing (data not shown). Morphology of the cells was also normal as determined bybright-field microscopy (data not shown). Assays were performed with strain13073 gGFP4. Addition of colchicine or cytochalasin D to conidia alone at theconcentrations used in the assay did not cause any defects in conidia germinationor growth. The results are representative of two independent experiments andare expressed as the mean � standard deviation of three replicates.

Statistics. The Student t test was used for statistical analysis of data.

RESULTS

Expression of gGFP in A. fumigatus conidia. To better un-derstand the interactions between A. fumigatus conidia andhost cells we constructed two GFP-expressing strains of A.fumigatus using the gGFP plasmid. In some fungi, the unmod-ified wild-type GFP protein is not fluorescent (8). gGFP con-tains the sGFP (S65T) plant codon-optimized gene (7, 27)transcriptionally fused to the Aspergillus nidulans promoter Pgpd, known to provide a high level of constitutive expression inascomycete fungi (35, 49). Conidia from two different A. fu-migatus strains, ATCC 13073 and CHUV, were transformedwith the gGFP plasmid by electroporation, and five hygromy-cin-resistant transformants were obtained with both strains. Todemonstrate that GFP was expressed in these hygromycin-resistant clones, conidia from wild-type or GFP strains werelysed and the homogenates were run on an SDS-PAGE gel.Immunoblotting of the membrane with an anti-GFP antibodydetected a band of the anticipated molecular weight in thetransformed strains which was absent in the wild-type strain(Fig. 1). To determine whether this GFP protein was func-tional, the conidia were observed for green fluorescence underblue light. Under these conditions, the conidia emitted greenfluorescence (Fig. 2). In addition, germinating conidia andhyphae were also fluorescent (data not shown). Of the total

hygromycin transformants, two from each of the strains wereGFP positive; however, only a single clone from each strainwas used in the cell uptake studies.

Phagocytosis of conidia by A549, HUVE, and J774 cells. (i)Measurement by immunostaining. Inhalation of A. fumigatusconidia leads to deposition of spores on the bronchial andalveolar surface (33), and invasion of the pneumocytes liningthe alveoli may allow the conidia to breach the epithelial bar-rier. Previous studies have demonstrated that A. fumigatus canbecome internalized by several cell types: the type II alveolarcell line A549 (10), primary alveolar type II cells, and byHUVE and tracheal epithelial cells (41) in vitro. However,these studies used electron microscopy to observe internaliza-tion and did not measure percent uptake, which is commonlyreported when investigating phagocytic uptake of pathogens byhost cells (3, 9, 36). Since A549 cells and HUVE cells arecapable of internalizing nonspecific particles such as latexbeads (21, 25, 58), we investigated whether uptake of A. fu-migatus conidia by these cells occurred at a significantly greaterrate than nonspecific phagocytosis. We measured the percentinternalization of A. fumigatus conidia by three different cul-tured cells in vitro: the transformed type II pneumocyte cellline A549 (as a model for alveolar epithelial cells); humanumbilical vein endothelial cells (as a model for endothelial celluptake) and the murine macrophage cell line J774 (as a posi-tive control for phagocytosis) (46). Phagocytic uptake was firstdetermined using immunostaining (Fig. 3). As shown in Table1, the highest invasion index was seen with J774 cells, followedby HUVE and A549 cells. Both GFP strains behaved similarlyin the invasion assays.

(ii) Measurement by nystatin protection assay. To confirmthe results we obtained by immunostaining, we developed anystatin protection assay, which is modeled on the gentamicinresistance assay used in bacterial pathogenesis studies (16). Weused the antifungal agent nystatin, which is fungicidal to ger-minating conidia (34, 40) but has a low toxicity to mammalian

FIG. 1. Detection of GFP protein in conidia of transformants byimmunoblotting. Wild-type (a) or gGFP-transformed (b) conidia fromstrain 13073 were lysed, and proteins were separated on an SDS–12%PAGE gel and transferred to polyvinylidene difluoride membrane. Themembrane was probed with an anti-GFP antibody diluted 1:100 fol-lowed by a goat anti-rabbit peroxidase-labeled secondary antibodydiluted 1:1,000. Development of the membrane with the DAB sub-strate revealed a band of 32 kDa in the transformed strain. Molecularmass indicators (in kilodaltons) are labeled on the left.

FIG. 2. Detection of GFP fluorescence in conidia by epifluores-cence. Wild-type (a) or gGFP-transformed (c) conidia from strain13073 were fixed onto poly-L-lysine-coated coverslips and viewed byepifluorescence using fluorescein filters. (b and d) The correspondingDIC images are shown. Bars � 10 �m.

3158 WASYLNKA AND MOORE INFECT. IMMUN.

on March 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

cells and does not penetrate cell membranes (2). In A549 cells,the nystatin protection assay yielded a similar invasion indexfor the two A. fumigatus strains (Table 1). Moreover, the nys-tatin protection assay generated statistically similar invasionindices to the values obtained with immunolabeling for both

Aspergillus strains. In contrast, the invasion index of conidia byJ774 cells was 19% for strain 13073 and 35% for strain CHUVaccording to the nystatin protection assay (Table 1)—muchlower than that obtained by immunostaining. This discrepancywas likely the result of killing of the internalized conidia by the

FIG. 3. Immunofluorescence microscopy demonstrates phagocytosis of A. fumigatus conidia by A549, HUVE, and J774 cells. A549 (A), HUVE(B), or J774 (C) cells were seeded overnight (A549 and HUVE cells) or for 2 h (J774 cells only) onto 12-mm-diameter coverslips in 24-well plates.Cells were incubated with 106 A. fumigatus 13073 conidia/ml in MEM–10% FBS for 3 h (A549 and HUVE cells) or 1 h (J774 cells) at 37°C.Following infection, samples were washed with PBST and processed for immunofluorescence. From left to right, panels show DIC image,fluorescence image showing the green channel (total bound conidia), fluorescence image of the red channel (extracellular conidia), and a mergedoverlay of all images. The results are representative of two independent experiments. Bars � 10 �m.

TABLE 1. Comparison of the extent of invasion of A549, J774, and HUVE cells by two strains of A. fumigatus (ATCC 13073 and CHUV)using fluorescence microscopy and a nystatin protection assaya

A. fumigatusstrain

Extent of invasion of cells by indicated assay

A549 J774 HUVE

Microscopy(invasion indexb)

Nystatin protectionMicroscopy

(invasion index)b

Nystatin protectionMicroscopy

(invasion index)dInvasionindexc

% of initialinoculumd

Invasionindexc

% of initialinoculumc

13073 39 � 11 35 � 7 2.9 � 0.5 91 � 8 19 � 4 6.0 � 1.4 48 � 5CHUV 30 � 3 29 � 7 1.3 � 0.3 85 � 10 35 � 4 6.6 � 1.5 50 � 7

Beads 0 NDe 0

a A549, J774, or HUVE cells were infected with 106 conidia/ml and then analyzed by immunofluorescence microscopy and the nystatin protection assay (A549 andJ774 cells only). Polystyrene beads were added to A549 and HUVE cells as a control for nonspecific phagocytosis, and uptake was determined by microscopy. The resultsare representative of two independent experiments and are expressed as means � standard deviations of three replicates.

b The invasion index determined by microscopy is the number of internalized conidia (green channel � red channel) divided by the number of bound conidia (greenchannel) per field � 100.

c The invasion index determined by the nystatin protection assay is the number of internalized conidia (number of conidia grown in the presence of nystatin) dividedby the number of bound conidia (number of conidia grown in the absence of nystatin) � 100.

d The percentage of initial inoculum (nystatin protection assay only) is the number of conidia enumerated in the presence of nystatin divided by the initial inoculum� 100.

e ND, not determined.

VOL. 70, 2002 PHAGOCYTOSIS OF A. FUMIGATUS CONIDIA 3159

on March 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

J774 cells during the 3-h nystatin incubation step. Therefore,the uptake of conidia by J774 cells was threefold greater thanA549 cells (as measured by immunostaining), but after 3 h, 60to 80% of the conidia within J774 cells had been killed,whereas all of the A549-internalized conidia remained viable.In addition to the invasion index, we also calculated the inva-sion frequency (percent conidia internalized relative to theinitial inoculum). A. fumigatus strain 13073 entered A549 andJ774 cells with invasion frequencies of 2.9 and 6.0%, respec-tively, while the CHUV strain entered A549 and J774 cells ata frequency of 1.3 and 6.6% (Table 1).

Phagocytosis of polystyrene beads by A549 and HUVE cells.To measure the extent of nonspecific phagocytosis, we incu-bated A549 cells with 1 �m fluorescein-labeled biotinylatedpolystyrene beads (A. fumigatus conidia are 2 to 3 �m indiameter) and measured the invasion index by immunostain-ing. Previous studies using polystyrene beads have shown thatthey are internalized by A549 (21) and HUVE (58) cells undercertain conditions. In these studies, internalization was deter-mined by subtracting the number of particles bound to the cellsurface at 4°C from the number of microspheres associatedwith the cells at 37°C (21). In contrast, our assay used immu-nostaining to differentiate between external and internalizedbeads. Polystyrene beads adhered to A549 and HUVE cells ata frequency of 10 to 20 beads/50 cells; however, the cells didnot internalize the bound beads (Table 1). Therefore, underthe conditions used in our assay, uptake of A. fumigatus conidiaby A549 and HUVE cells was specific, as these cells did notappear to be randomly internalizing particles.

Inhalation of Aspergillus conidia leads to the deposition ofspores on the alveolar surface, and the cells most likely to initiallyinteract with conidia are type I and type II pneumocytes. There-fore, for the rest of our experiments we chose A549 cells as modelalveolar epithelial cells and further investigated the interaction ofA. fumigatus conidia with this cell line.

Phagocytosis of dead conidia by A549 cells. To determinewhether internalization of conidia by A549 cells required via-ble conidia, we compared the rates of uptake of live and heat-killed conidia into A549 cells by immunostaining. The invasionindex of viable and heat-killed conidia was 28% � 7% and37% � 9 (P � 0.05), respectively. These data suggest that A549cells internalize viable and heat-killed conidia to an equalextent, which implies that internalization may occur via recog-nition of a non-heat-labile surface antigen, such as a polysac-charide.

Phagocytosis of conidia by A549 cells: Effect of serum. Todetermine whether serum components were required for up-take of conidia by A549 cells, invasion assays were performedin the presence of MEM or MEM plus serum (FBS), anduptake was determined by the nystatin protection assay. Therewere no significant differences in invasion frequencies betweenthese samples (data not shown) suggesting that the recognitionof conidia by A549 surface receptors does not require serumcomponents.

Delay of germination in phagocytosed conidia. To deter-mine whether conidia germinate inside A549 cells, conidiawere incubated with cells for 6 h. The A549 cell monolayer wasnot damaged by this extended incubation time. Visualizationby immunostaining revealed that all of the extracellular conidiahad swollen and most had germ tubes ranging from 5 to 10 �min length, whereas a vast majority of the intracellular conidiahad not germinated (Fig. 4). We could not determine whetherany of the extracellular germinated conidia had penetrated thecell layer. These data suggest, along with the data from thenystatin protection assay, that internalized conidia remain vi-able inside A549 cells and that germination is severely re-stricted for at least 6 h. Whether conidia can be retained in asimilar state within alveolar epithelial cells in vivo is unknown.

Internalization of A. fumigatus conidia by A549 cells is anactive process that requires microfilaments and microtubules.Phagocytosis is the temperature-dependent uptake by cells ofparticles (usually greater than 0.5 �m in diameter) and leads tothe polymerization of actin at the site of entry (45). To deter-mine whether internalization of A. fumigatus conidia into A549cells required polymerization of actin filaments or microtu-bules, invasion assays were performed using the nystatin pro-tection assay in the presence and absence of cytochalasin D orcolchicine. Cytochalasin D prevents addition of actin mono-mers to the fast-growing plus ends of filaments (50), and col-chicine binds to and prevents polymerization of tubulin (47,51). The internalization of conidia into A549 cells was inhib-ited by 75% in the presence of 0.5 �M cytochalasin D and by65% in the presence of 5 �M colchicine (Fig. 5). These con-centrations of cytochalasin D and colchicine had no effect onthe germination and growth of conidia alone (data not shown).Thus, both microfilaments and microtubules are utilized dur-ing internalization of conidia by A549 cells.

Phagocytosis is a temperature-dependent process and parti-cle uptake has been shown to be severely restricted at 4°C (1,52). To determine whether phagocytosis of conidia by A549

FIG. 4. Internalized conidia do not germinate in A549 cells. A549 cells were infected with 106 A. fumigatus 13073 conidia/ml in MEM–10% FBSfor 6 h at 37°C and then fixed with 4% paraformaldehyde. The following day, samples were immunolabeled and then viewed by DIC andfluorescence microscopy. From left to right, panels show DIC image, fluorescence image showing the green channel (total bound conidia),fluorescence image of the red channel (extracellular conidia/germlings), and a merged overlay of all images. Bars � 10 �m.

3160 WASYLNKA AND MOORE INFECT. IMMUN.

on March 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

cells was also temperature dependent, invasion assays werecarried out at 4°C and uptake was measured by the nystatinprotection assay. Internalization of conidia was inhibited by95% when infections were performed at 4°C versus the controlat 37°C (Fig. 5). Taken together, these data suggest that inter-nalization of A. fumigatus conidia by A549 cells occurs by atemperature-dependent phagocytic process, which is depen-dent on the host cell microfilaments and microtubules.

Internalization of A. fumigatus conidia induces localized ac-tin rearrangement. Many microbial pathogens such as Salmo-nella enterica serovar Typhimurium (20) and C. albicans (18)that invade nonprofessional phagocytic cells with rigid cy-toskeletons induce visible alterations in the underlying hostcytoskeletal structure. Since cytochalasin D inhibited uptake ofA. fumigatus conidia by A549 cells, we investigated whetherconidia internalization would lead to actin rearrangementwhich could be visualized in A549 cells. Uninfected A549 cellsshowed intense staining of cortical and cellular actin filaments(data not shown). In infected cells, approximately 10% of theconidia were found inside vacuoles coated with polymerizedactin (Fig. 6). However, as phagocytosis assays had demon-strated that the invasion index of conidia by A549 cells wasapproximately 30%, the remaining 20% must have alreadyshed their phagosome-actin coat, or the microfilaments wereonly polymerizing around those organisms that were activelybecoming phagocytosed the moment that the fixative was ap-plied. Alternatively, some conidia may have been phagocy-tosed by a non-actin-dependent mechanism.

DISCUSSION

Life-threatening diseases due to opportunistic fungi such asA. fumigatus have increased over the past 2 decades (15).Although A. fumigatus makes up less than 1% of all airborneconidia, it remains the most-common invasive mold infectionamong immunocompromised patients worldwide (33). Otherspecies in the genus, namely, Aspergillus terreus and Aspergillus

auricomus, possess similar physical attributes (small spore size,ability to grow at 37°C); however, these species cause infectionmuch more rarely (43). Therefore, it has been postulated thatA. fumigatus must possess unique virulence factors that allow itto colonize the host (28).

In this report, we transformed two strains of A. fumigatus withgGFP and used them to investigate internalization of conidia bythree cultured cell lines. We used differential immunolabelingand a nystatin protection assay to quantify the percent internal-ization of A. fumigatus conidia by host cells. Previous studies havereported that A. fumigatus conidia are internalized by A549 lungepithelial cells (10) and primary airway type II cells (41). In ourstudy, we confirmed that A549 cells internalize A. fumigatusconidia, and in addition, we determined that the invasion indexwas approximately 30% and the invasion frequency (relative tothe initial inoculum) was approximately 2%. No uptake of 1-�m-diameter polystyrene beads was observed over the assay period.The invasion frequency of conidia into A549 parallels uptake ofother pathogenic organisms by these cells: A549 cells internalized5.7% of the initial inoculum of Mycobacterium tuberculosis (3),0.2% of the protozoan parasite Encephalitozoon cuniculi (9), and0.15% of Burkholderia cepacia (36). In contrast, Martin and Mohr(36) observed a 0.008% invasion frequency of the nonpathogenicE. coli HB101 into A549 cells. As a positive control for internal-ization, we measured uptake of conidia by J774 cells and observedthat these professional phagocytes internalized approximately90% of the bound conidia. Similarly, Káposzta et al. observed that98% of macrophage-associated C. albicans were phagocytosed byprimary macrophages (31). The invasion index for J774 cells cal-culated by the nystatin protection assay was much lower than thatobtained by immunolabeling (27 versus 87% average for the twostrains). This was most likely due to killing of the conidia withinJ774 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 cellsand found that 50% of the bound spores were phagocytosed. This

FIG. 5. Internalization of A. fumigatus conidia in the presence of phagocytosis inhibitors. A549 cells were infected with 106 A. fumigatus 13073conidia/ml in MEM–10% FBS containing no inhibitors (37°C) or 0.5 �M cytochalasin D or 5 �M colchicine. Invasion assays were also carried outwith no inhibitors at 4°C to determine whether uptake was temperature dependent. The number of conidia internalized by the A549 cells wasdetermined using the nystatin protection assay (see Materials and Methods). The results are representative of two independent experiments andare expressed as the means � standard deviations (error bars) of three replicates. �, P � 0.05.

VOL. 70, 2002 PHAGOCYTOSIS OF A. FUMIGATUS CONIDIA 3161

on March 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

was similar to the uptake of C. neoformans by these cells; Ibrahimet al. (29) showed that 77% of the bound yeasts were internalizedby HUVE cells.

The development of the nystatin protection assay allowed usto confirm the internalization results obtained by differentialimmunolabeling. Since both techniques produced similar in-ternalization rates with A549 cells, we were confident that thenystatin protection assay gave an accurate and objective mea-surement of invasion. This technique is simple and sensitiveand should be applicable to other pathogenic fungi as nystatinis fungicidal toward both C. albicans and C. neoformans (6).Furthermore, comparing the results of immunostaining andnystatin protection may provide quantitative information onthe extent of killing by host cells.

To determine whether A. fumigatus conidia germinated fol-

lowing entry into A549 cells, we extended the invasion incu-bation period from 3 to 6 h. Germination was severely delayedfor phagocytosed conidia and this differs from uptake of Can-dida albicans by HUVE cells. In this case, the yeast cells ger-minated during the first hour, and by 2 h most of the germlingswere internalized by the endothelial cells (18). In their studiesof A. fumigatus, DeHart et al. (10) observed that direct hyphalpenetration of A549 cells occurred after a twelve h incubationperiod. Our data suggest that the hyphal penetration of A549cells observed by Dettart et al. probably occurred via extracel-lular germlings.

Microbial pathogens use several tactics to gain access to hostcells including phagocytosis (9, 37), macropinocytosis (20, 22),receptor-mediated endocytosis (38) and microtubule-dependentinternalization (24, 39). Uptake of A. fumigatus conidia by A549cells occurred via actin-dependent phagocytosis but also requiredhost cell microtubules. Internalization of A. fumigatus conidia didnot induce the massive cytoskeletal rearrangements seen in Sal-monella uptake into epithelial cells, which enters via membraneruffling and macropinocytosis (22). Instead, the process of A.fumigatus internalization most likely occurs via engagement ofligands with host cell receptors and resembles the zipper uptakemodel (53). The identity of these ligands is currently unknown.The microfilament dependence of A. fumigatus uptake resemblesepithelial cell invasion by other bacterial pathogens such as M.tuberculosis (3) and S. enterica serovar Typhimurium (20). Al-though most invasive microbes use host actin to enter nonprofes-sional phagocytes, there are some organisms that also use micro-tubules. For example, some strains of Campylobacter jejunirequire microtubules but not microfilaments for entry into intes-tinal cells (4, 39). Following adherence to intestinal epithelialcells, C. jejuni trigger the formation of a microtubule-based mem-brane extension that meets the invading bacterium (32). In con-trast, phagocytosis of A. fumigatus by A549 cells involves bothcytoskeletal components which is similar to M. tuberculosis entryinto A549 cells (3) and C. albicans uptake by endothelial cells(18).

In summary, we have used several techniques to demonstratethat A. fumigatus conidia are internalized by lung epithelial andendothelial and murine macrophage cells in vitro. Examination ofconidium uptake by A549 cells showed that the process was anal-ogous to phagocytosis and utilized host microfilaments and mi-crotubules. Internalization of conidia by nonprofessional phago-cytes may be important in the development of aspergillosis invivo, as sequestration inside these cells may allow the conidia toescape the immune response of the host. Future experiments willinvestigate the location of the conidium in the endosomal path-way and will determine whether uptake is a defense mechanism ofthe host or a virulence trait of the fungus.

ACKNOWLEDGMENTS

This work was supported by a grant from the Natural Sciences andEngineering Research Council of Canada (NSERC) to M.M.M. J.A.W.was supported by an NSERC predoctoral scholarship.

We thank C. Breuil (Department of Wood Science, University ofBritish Columbia) for the gift of the gGFP plasmid, Loekie van derWal (Animal Care Facility, Simon Fraser University) for performinganimal immunizations, and Wendy Olson for coordinating umbilicalcord collections at Royal Columbian Hospital, New Westminster, Brit-ish Columbia, Canada. We are grateful to Anna H. T. Gifford forcritical reading of the manuscript.

FIG. 6. Rearrangement of actin during the internalization of A.fumigatus conidia by A549 cells. A549 cells were infected with 106

conidia/ml for 160 min. Following infection, actin microfilaments werestained with rhodamine phalloidin. Fluorescence from the red (a) andgreen (b) channels was collected separately on a Zeiss LSM confocalmicroscope and then overlaid in Adobe PhotoShop (c). Bars � 2 �m.

3162 WASYLNKA AND MOORE INFECT. IMMUN.

on March 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

REFERENCES

1. Band, H., A. Bhattacharya, and G. P. Talwar. 1986. Mechanism of phago-cytosis by Schwann cells. J. Neurol Sci. 75:113–119.

2. Bennett, J. E. 1990. Antifungal agents, p. 1165–1181. In A. G. Gilman, T. W.Rall, A. S. Nies, and P. Taylor (ed.), Goodman and Gilman’s the pharma-cological basis of therapeutics, 8th ed. Pergamon Press Inc., Elmsford, N.Y.

3. Bermudez, L. E., and J. Goodman. 1996. Mycobacterium tuberculosis invadesand replicates within type II alveolar cells. Infect. Immun. 64:1400–1406.

4. Biswas, D., K. Itoh, and C. Sasakawa. 2000. Uptake pathways of clinical andhealthy animal isolates of Campylobacter jejuni into INT-407 cells. FEMSImmunol. Med. Microbiol. 29:203–211.

5. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye bind-ing. Anal. Biochem. 72:248–254.

6. Carrillo-Munoz, A. J., G. Quindos, C. Tur, M. T. Ruesga, Y. Miranda, O. delValle, P. A. Cossum, and T. L. Wallace. 1999. In-vitro antifungal activity ofliposomal nystatin in comparison with nystatin, amphotericin B cholesterylsulphate, liposomal amphotericin B, amphotericin B lipid complex, ampho-tericin B desoxycholate, fluconazole and itraconazole. J. Antimicrob. Che-mother. 44:397–401.

7. Chiu, W., Y. Niwa, W. Zeng, T. Hirano, H. Kobayashi, and J. Sheen. 1996.Engineered GFP as a vital reporter in plants. Curr. Biol. 6:325–330.

8. Cormack, B. 1998. Green fluorescent protein as a reporter of transcriptionand protein localization in fungi. Curr. Opin. Microbiol. 1:406–410.

9. Couzinet, S., E. Cejas, J. Schittny, P. Deplazes, R. Weber, and S. Zimmerli.2000. Phagocytic uptake of Encephalitozoon cuniculi by nonprofessionalphagocytes. Infect. Immun. 68:6939–6945.

10. DeHart, D. J., D. E. Agwu, N. C. Julian, and R. G. Washburn. 1997. Bindingand germination of Aspergillus fumigatus conidia on cultured A549 pneumo-cytes. J. Infect. Dis. 175:146–150.

11. Denning, D. W. 1996. Therapeutic outcome in invasive aspergillosis. Clin.Infect. Dis. 23:608–615.

12. Denning, D. W. 2000. Early diagnosis of invasive aspergillosis. Lancet 355:423–424.

13. Denning, D. W. 1998. Invasive aspergillosis. Clin. Infect. Dis. 26:781–803.14. De Pauw, B. 2000. Is there a need for new antifungal agents? Clin. Microbiol.

Infect. 6(Suppl. 2):23–28.15. Dixon, D. M., M. M. McNeil, M. L. Cohen, B. G. Gellin, and J. R. La

Montagne. 1996. Fungal infections: a growing threat. Public Health Rep.111:226–235.

16. Elsinghorst, E. A. 1994. Measurement of invasion by gentamicin resistance.Methods Enzymol. 236:405–420.

17. Feldmesser, M., S. Tucker, and A. Casadevall. 2001. Intracellular parasitismof macrophages by Cryptococcus neoformans. Trends Microbiol. 9:273–278.

18. Filler, S. G., J. N. Swerdloff, C. Hobbs, and P. M. Luckett. 1995. Penetration anddamage of endothelial cells by Candida albicans. Infect. Immun. 63:976–983.

19. Finlay, B. B., and S. Falkow. 1997. Common themes in microbial pathoge-nicity revisited. Microbiol. Mol. Biol. Rev. 61:136–169.

20. Finlay, B. B., S. Ruschkowski, and S. Dedhar. 1991. Cytoskeletal rearrange-ments accompanying Salmonella entry into epithelial cells. J. Cell Sci. 99:283–296.

21. Foster, K. A., M. Yazdanian, and K. L. Audus. 2001. Microparticulate uptakemechanisms of in-vitro cell culture models of the respiratory epithelium.J. Pharm. Pharmacol. 53:57–66.

22. Francis, C. L., T. A. Ryan, B. D. Jones, S. J. Smith, and S. Falkow. 1993.Ruffles induced by Salmonella and other stimuli direct macropinocytosis ofbacteria. Nature 364:639–642.

23. Gigolashvili, T. 1999. Update on antifungal therapy. Cancer Pract. 7:157–159.24. Grassme, H. U., R. M. Ireland, and J. P. van Putten. 1996. Gonococcal

opacity protein promotes bacterial entry-associated rearrangements of theepithelial cell actin cytoskeleton. Infect. Immun. 64:1621–1630.

25. Griese, M., and D. Reinhardt. 1998. Smaller sized particles are preferentiallytaken up by alveolar type II pneumocytes. J. Drug Target. 5:471–479.

26. Groll, A. H., P. M. Shah, C. Mentzel, M. Schneider, G. Just-Nuebling, andK. Huebner. 1996. Trends in the postmortem epidemiology of invasive fungalinfections at a university hospital. J. Infect. 33:23–32.

27. Haas, J., E. C. Park, and B. Seed. 1996. Codon usage limitation in theexpression of HIV-1 envelope glycoprotein. Curr. Biol. 6:315–324.

28. Hogan, L. H., B. S. Klein, and S. M. Levitz. 1996. Virulence factors ofmedically important fungi. Clin. Microbiol. Rev. 9:469–488.

29. Ibrahim, A. S., S. G. Filler, M. S. Alcouloumre, T. R. Kozel, J. E. Edwards,Jr., and M. A. Ghannoum. 1995. Adherence to and damage of endothelialcells by Cryptococcus neoformans in vitro: role of the capsule. Infect. Immun.63:4368–4374.

30. Jaffe, E. A., R. L. Nachman, C. G. Becker, and C. R. Minick. 1973. Cultureof human endothelial cells derived from umbilical veins. Identification bymorphologic and immunologic criteria. J. Clin. Investig. 52:2745–2756.

31. Káposzta, R., L. Marodi, M. Hollinshead, S. Gordon, and R. P. da Silva.1999. Rapid recruitment of late endosomes and lysosomes in mouse macro-phages ingesting Candida albicans. J. Cell Sci. 112:3237–3248.

32. Kopecko, D. J., L. Hu, and K. J. Zaal. 2001. Campylobacter jejuni: microtu-bule-dependent invasion. Trends Microbiol. 9:389–396.

33. Latge, J. P. 1999. Aspergillus fumigatus and aspergillosis. Clin. Microbiol.Rev. 12:310–350.

34. Manavathu, E. K., G. J. Alangaden, and P. H. Chandrasekar. 1998. In-vitroisolation and antifungal susceptibility of amphotericin B-resistant mutants ofAspergillus fumigatus. J. Antimicrob. Chemother. 41:615–619.

35. Maor, R., M. Puyesky, B. A. Horwitz, and A. Sharon. 1998. Use of greenfluorescent protein (GFP) for studying development and fungal-plant inter-action in Cochliobolus heterostrophus. Mycol. Res. 102:491–496.

36. Martin, D. W., and C. D. Mohr. 2000. Invasion and intracellular survival ofBurkholderia cepacia. Infect. Immun. 68:24–29.

37. Maruta, K., M. Ogawa, H. Miyamoto, K. Izu, and S. I. Yoshida. 1998. Entryand intracellular localization of Legionella dumoffii in Vero cells. Microb.Pathog. 24:65–73.

38. Mengaud, J., H. Ohayon, P. Gounon, R. M. Mege, and P. Cossart. 1996.E-cadherin is the receptor for internalin, a surface protein required for entryof L. monocytogenes into epithelial cells. Cell 84:923–932.

39. Oelschlaeger, T. A., P. Guerry, and D. J. Kopecko. 1993. Unusual microtu-bule-dependent endocytosis mechanisms triggered by Campylobacter jejuniand Citrobacter freundii. Proc. Natl. Acad. Sci. USA 90:6884–6888.

40. Osherov, N., and G. May. 2000. Conidial germination in Aspergillus nidulansrequires RAS signaling and protein synthesis. Genetics 155:647–656.

41. Paris, S., E. Boisvieux-Ulrich, B. Crestani, O. Houcine, D. Taramelli, L.Lombardi, and J. P. Latge. 1997. Internalization of Aspergillus fumigatusconidia by epithelial and endothelial cells. Infect. Immun. 65:1510–1514.

42. Penalver, M. C., J. E. O’Connor, J. P. Martinez, and M. L. Gil. 1996. Bindingof human fibronectin to Aspergillus fumigatus conidia. Infect. Immun. 64:1146–1153.

43. Pitt, J. I. 1994. The current role of Aspergillus and Penicillium in human andanimal health. J. Med. Vet. Mycol. 32:17–32.

44. Puente, P., N. Fernandez, M. C. Ovejero, and F. Leal. 1992. Immunogenicpotential of Aspergillus nidulans subcellular fractions and their polypeptidecomponents. Mycoses 35:235–241.

45. Rabinovitch, M. 1994. Professional and non-professional phagocytes: anintroduction. Trends Cell Biol. 5:85–87.

46. Rodriguez, E., F. Boudard, M. Mallie, J. M. Bastide, and M. Bastide. 1997.Murine macrophage elastolytic activity induced by Aspergillus fumigatusstrains in vitro: evidence of the expression of two macrophage-induced pro-tease genes. Can. J. Microbiol. 43:649–657.

47. Sackett, D. L., and J. K. Varma. 1993. Molecular mechanism of colchicineaction: induced local unfolding of beta-tubulin. Biochemistry 32:13560–13565.

48. Sanchez, O., and J. Aguirre. 1996. Efficient transformation of Aspergillusnidulans by electroporation of germinated conidia. Fungal Genet. Newsl.43:48–51.

49. Santerre Henriksen, A. L., S. Even, C. Muller, P. J. Punt, C. A. van denHondel, and J. Nielsen. 1999. Study of the glucoamylase promoter in As-pergillus niger using green fluorescent protein. Microbiology 145:729–734.

50. Schliwa, M. 1982. Action of cytochalasin D on cytoskeletal networks. J. CellBiol. 92:79–91.

51. Skoufias, D. A., and L. Wilson. 1992. Mechanism of inhibition of microtubulepolymerization by colchicine: inhibitory potencies of unliganded colchicineand tubulin-colchicine complexes. Biochemistry 31:738–746.

52. Steffan, A. M., J. L. Gendrault, R. S. McCuskey, P. A. McCuskey, and A.Kirn. 1986. Phagocytosis, an unrecognized property of murine endothelialliver cells. Hepatology 6:830–836.

53. Swanson, J. A., and S. C. Baer. 1995. Phagocytosis by zippers and triggers.Trends Cell Biol. 5:89–93.

54. Tronchin, G., J. P. Bouchara, G. Larcher, J. C. Lissitzky, and D. Chabasse.1993. Interaction between Aspergillus fumigatus and basement membranelaminin: binding and substrate degradation. Biol. Cell 77:201–208.

55. Walsh, T. J., M. A. Viviani, E. Arathoon, C. Chiou, M. Ghannoum, A. H.Groll, and F. C. Odds. 2000. New targets and delivery systems for antifungaltherapy. Med. Mycol. 38:335–347.

56. Wasylnka, J. A., and M. M. Moore. 2000. Adhesion of Aspergillus species toextracellular matrix proteins: evidence for involvement of negatively chargedcarbohydrates on the conidial surface. Infect. Immun. 68:3377–3384.

57. Weidner, G., C. d’Enfert, A. Koch, P. C. Mol, and A. A. Brakhage. 1998.Development of a homologous transformation system for the human patho-genic fungus Aspergillus fumigatus based on the pyrG gene encoding oroti-dine 5-monophosphate decarboxylase. Curr. Genet. 33:378–385.

58. Zauner, W., N. A. Farrow, and A. M. Haines. 2001. In vitro uptake ofpolystyrene microspheres: effect of particle size, cell line and cell density. J.Control Release 71:39–51.

Editor: T. R. Kozel

VOL. 70, 2002 PHAGOCYTOSIS OF A. FUMIGATUS CONIDIA 3163

on March 27, 2020 by guest

http://iai.asm.org/

Dow

nloaded from