1 Department of Molecular Biology and Biochemistry, Simon Fraser University,
Burnaby, B.C., Canada, V5A 1S6
2 Department of Biological Sciences, Simon Fraser University, Burnaby, B.C.,
Canada, V5A 1S6
* Author for correspondence (e-mail: mmoore{at}sfu.ca)
Accepted 17 December 2002
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Summary |
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Key words: Aspergillus fumigatus, Phagosome, Germination, A549, J774
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Introduction |
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Intracellular pathogens have evolved many mechanisms to survive in the
hostile environment of the host. Invasion of host cells by the pathogen is one
of several possible outcomes once a microbe has successfully colonized a host.
Once internalized, intracellular pathogens are protected from the host immune
system and have access to preformed nutrients. These intracellular microbes
employ several different strategies to grow and reproduce within host cells.
For example, Shigella flexneri and Listeria monocytogenes
degrade the phagosome and gain access to the nutrient rich cytoplasm
(Meresse et al., 1999),
whereas Chlamydia pneumoniae does not enter a phagolysosome but is
intimately associated with early endosomes
(Al-Younes et al., 1999
).
Brucella abortus escapes the endocytic pathway and is found in a
vacuole resembling an autophagosome
(Pizarro-Cerda et al., 1998
).
Finally, Mycobacterium tuberculosis prevents phagosome acidification
by exclusion of the vesicular proton
(Sturgill-Koszycki et al.,
1994
). All of these diverse mechanisms have the common theme of
allowing the pathogen to avoid degradation and create a favorable niche within
which to grow and replicate.
Some pathogenic fungi also evade phagocytic degradation by mechanisms
similar to those used by bacteria. Phagosomes containing Histoplasma
capsulatum fail to acidify while still retaining fusion competence
(Eissenberg et al., 1993). In
macrophages, Candida albicans develops germ tubes within
phagolysosomes and eventually escapes from and destroys the macrophage
(Kàposzta et al.,
1999
). In contrast, in brain endothelial cells, virulent C.
albicans strains invade and transcytose without affecting the integrity
of the endothelial cell monolayer (Jong et
al., 2001
).
Previous work by our laboratory and others has determined that A.
fumigatus conidia are internalized by lung cells
(DeHart et al., 1997;
Paris et al., 1997
;
Wasylnka and Moore, 2002
)
endothelial cells (Paris et al.,
1997
; Wasylnka and Moore,
2002
) and macrophages (Nessa
et al., 1997
; Wasylnka and
Moore, 2002
) in vitro. We have previously demonstrated that 100%
of conidia internalized by A549 lung epithelial cells survive for at least 3
1/2 hours following uptake, whereas 70% of conidia internalized by J774 murine
macrophages are killed within 6 hours. This finding prompted us to determine
whether there were differences in the fate of A. fumigatus conidia
within the endosomal networks of A549 and J774 cells. Therefore, the
objectives of this study were: (1) to determine co-localization of
conidia-containing phagosomes with endosomal/lysosomal proteins in A549 and
J774 cells; (2) to determine the pH of the conidia-containing phagosomes
within these cells, and (3) to determine the viability of conidia within A549
and J774 cells after extended incubation times.
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Materials and Methods |
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Tissue culture
The type II human pneumocyte cell line A549, and murine macrophage line
J774, were obtained from the ATCC and maintained in RPMI 1640 media containing
10% fetal bovine serum (Canadian Life Technologies, Burlington, Ontario,
Canada), 100 mg/l streptomycin and 16 mg/l penicillin (both from Sigma-Aldrich
Canada, Oakville, Ontario, Canada). Cells were maintained at 37°C in
humidified 5% CO2 incubators.
Measurement of conidia survival in A549 and J774 cells
A549 cells and J774 cells were seeded at 5.0x105
cells/well in 24 well plates and grown for 16 hours. Following cell growth,
wells were blocked for 30 minutes in minimum essential media (MEM) (Canadian
Life Technologies) containing 0.1% (w/v) bovine serum albumin (BSA) (ICN
Pharmaceuticals, Montreal, Canada) at 37°C. Cells were infected with a 2:1
multiplicity of infection (M.O.I.) of A. fumigatus gGFP conidia and
incubated in MEM/10% (v/v) FBS for 3 hours at 37°C. After incubation,
unbound spores were removed by washing wells three times with PBS/0.05% Tween
20 (PBS-T). Extracellular conidia were killed with nystatin as described
previously (Wasylnka and Moore,
2002). Cells were lysed with 0.5% Triton X-100 and serial
dilutions of released conidia were plated onto YM agar (three replicate
plates/well). Some samples were incubated in MEM for an additional 6 hours
(both cell lines) or 18 hours (A549 only) at 37°C prior to lysis and
plating. For A549 cell incubations, 5 µg/ml nystatin was added to the
medium. To maintain high viability of the J774 cells, 1% FBS was added to the
incubation media during the extended incubation times. Nystatin was not added
because after 6 hours all spores were intracellular.
Confocal microscopy
Endosomal trafficking assays
A549 and J774 cells were seeded at 5.0x105 cells/well onto
12 mm Number 1 coverslips in 24 well plates (Falcon, Becton-Dickinson Canada,
Mississauga, Ontario, Canada) and grown for 16 hours. Cells were infected with
1 ml of 1-2x106 spores/ml in MEM/10% (v/v) FBS (M.O.I. of 2:1
for A549 and 4:1 for J774 cells) for the indicated times at 37°C and then
washed with PBS-T. A549 cells were treated with nystatin as described above.
Extracellular spores were labelled using an anti-Aspergillus cell
wall polyclonal antibody (Wasylnka and
Moore, 2002). Cells were fixed for 1 hour with PBS/4% (w/v)
paraformaldehyde, pH 7.4 and then permeabilized for 1 hour with PBS/10% goat
serum/0.05% saponin. The A549 antibodies used were anti-human cathepsin D
(Oncogene Research Products, Boston, MA), anti-human CD71 (Sigma), anti-human
LAMP-1 (clone H4A3, Developmental Studies Hybridoma Bank (DHSB), University of
Iowa) and anti-human CD63 (clone H5C6, DHSB). J774 antibodies were anti-murine
LAMP-1 (clone 1D4B, DHSB) and anti-murine CD71 (Sigma). Antibodies were
diluted in PBS/10% goat serum and used at 1:5 (1D4B), 1:100 (cathepsin D,
anti-human CD71 and H4A3), 1:200 (H5C6), or 1:500 (anti-murine CD71). Cells
were incubated with primary antibodies for 60 minutes and washed three times
with PBS. The secondary antibody goat anti-mouse/rat Alexa 647 (Molecular
Probes, Eugene, OR), was diluted 1:100-1:400 in PBS/10% goat serum and
incubated with cells for 45 minutes. Wells were washed with PBS and coverslips
were mounted onto slides with ProLong antifade from Molecular Probes. Cells
were viewed with a Zeiss LSM-410 confocal microscope equipped with a
krypton/argon laser (Omnichrome), using a 63x 1.4 numerical aperture
lens. Green fluorescence was captured with a 515-540 nm band pass filter, red
fluorescence with a 590-610 band pass filter and blue fluorescence with a
670-810 nm bandpass filter. Images were processed in Adobe PhotoShop 6.0
(Adobe Systems Incorporated, San Jose, CA). At least 100 conidia-containing
phagosomes in two separate fields were analyzed for each treatment and
infection time.
LysoTracker co-localization
Cells were seeded at 3x105 cells/well in 8-well chambered
coverslips (Nalge Nunc International Corporation, Naperville, IL) and grown
for 16 hours. Cells were infected for the indicated times with a 2:1 M.O.I. of
A. fumigatus conidia. (A549 cells infected for 24 hours were treated
as in the survival assay). The wells were washed with PBS-T and then incubated
for 30 minutes at 37°C with pre-warmed LysoTracker Red DND-99 (Molecular
Probes) diluted to 50 nM in MEM. The loading solution was replaced with fresh
warm media, and red and green fluorescence was captured on the Zeiss LSM-410
confocal microscope as described above. At least 100 conidia in two separate
fields were analyzed for each treatment and infection time.
Penetration of the A549 epithelial barrier
A549 cells were seeded onto 12 mm diameter No. 1 coverslips at
5.0x105 cells/well in 24-well plates and grown for 16 hours.
Cells were infected with a 2:1 M.O.I. of A. fumigatus gGFP conidia
for 3 hours at 37°C, washed with PBS-T and then incubated with 50 µg/ml
nystatin in MEM for 3 hours. Cells were washed once with PBS and then
incubated for 18 hours with MEM containing 5 µg/ml nystatin. After 24
hours, the cells were washed with PBS and extracellular conidia/hyphae were
either immediately labelled with the anti-Aspergillus cell wall
antibody as described (Wasylnka and Moore,
2002), or incubated a further 12 hours in MEM/2% FBS prior to
labelling. Cells were viewed on the Zeiss LSM-410 confocal microscope as
described above.
Nystatin control assay
Conidia were harvested and 106 spores were diluted in MEM media
and incubated at 37°C for 3 hours to initiate germination. Nystatin
(dissolved in DMSO) was then added to the media at 50 µg/ml and incubated
with the spores for another 3 hours at 37°C. Aliquots were diluted and
plated (along with untreated spores) onto YM agar. The media was then diluted
10-fold to decrease the nystatin concentration to 5 µg/ml and the samples
were incubated for an additional 18 hours. The next day the solution was
diluted and plated to determine the number of surviving spores.
Cytotoxicity of cultured cells during A. fumigatus
infection
To test the viability of the infected and uninfected cells during the
survival assay, 50 µl of supernatant was removed at various time points
post-infection and the level of lactate dehydrogenase (LDH) was determined
using the Cytotox 96 Non-radioactive assay kit from Promega (Madison, WI). The
assay quantitatively measures LDH, a stable cytosolic enzyme that is released
upon cell lysis. Uninfected wells were treated with either 100 µl of 10%
Triton X-100 (1% v/v final concentration) or PBS to serve as positive and
negative controls for LDH release. Samples were incubated with LDH substrate
for 5 minutes and then the absorbance was read on a microplate reader at 490
nm.
The Student t-test was used for statistical analysis of data.
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Results |
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To determine whether the kinetics of conidia transport through the
endosomal network was similar in A549 cells, we infected cells with A.
fumigatus conidia under identical conditions and then labelled them with
CD71 and LAMP-1 antibodies as well as antibodies to CD63 (found on late
endosome/lysosome membranes) and cathepsin D. Cathepsin D is a soluble
lysosomal hydrolase found in late endosomes/lysosomes
(Bohley and Seglen, 1992). Our
previous experiments have shown that internalization of conidia by A549 cells
occurs between 2.5-3 hours post-infection
(Wasylnka and Moore, 2002
).
Since A549 cells are non-professional phagocytes, we hypothesized that
trafficking through the endosomal system would be slower than in J774 cells.
Therefore, we looked at co-localization of conidia phagosomes with endosomal
markers at 3, 6 and 24 hours post-infection. As we had observed with J774
cells, very little co-localization with CD71 occurred
(Fig. 2A,a), but there were
many CD63 (Fig. 2A,b) and
LAMP-1 positive phagosomes at 3 hours (data not shown). Conidia-containing
phagosomes also acquired cathepsin D (Fig.
2A,c). The co-localization of conidia within endosomal/lysosomal
markers was also quantified for A549 cells. Only 5% of conidia were in early
endosomes after 3 hours, whereas 38% were co-localized with LAMP-1 and 51%
with CD63 (Fig. 2B). After 6
hours, only 3% of conidia co-localized with CD71, but 51% and 61% were found
with LAMP-1 and CD63 respectively. These values remained similar up to 24
hours post-infection (2% CD71, 55% LAMP-1, 58% CD63). Cathepsin D was also
found to co-localize early with internalized conidia (76% after 3 hours) and
increased to 87% after 24 hours. The percentage of conidia that associated
with cathepsin D was higher than that seen with LAMP-1 and CD63, as phagosomes
were scored positive for the presence of cathepsin D even when the intensity
of the signal was very weak (Fig.
2A,c). Together, this data suggests that conidia fuse rapidly with
late endosomes/lysosomes following uptake by J774 or A549 cells.
A. fumigatus conidia reside in acidic organelles of A549 and
J774 cells
A large majority of the conidia were co-localized with late
endosome/lysosome markers, and since these organelles typically have a pH of
less than 6, we hypothesized that the phagosomes would also be acidic.
Therefore, we investigated whether the pH of the phagosomes containing conidia
was neutral or acidic. Cells were infected with AfGFP conidia for 1, 2 or 6
hours (J774 cells) or 3, 6 and 24 hours (A549 cells) and then labelled with a
fluorescent probe, LysoTracker, which selectively accumulates in the membranes
of acidic organelles. The LysoTracker probe was observed to co-localize with
GFP conidia as seen by rings of red fluorescence surrounding the green spores
(Fig. 3A,a-c). Germinating
conidia were seen in A549 cells after 6 or 24 hours and after 6 hours in J774
cells, however germination was observed much less frequently in these cells.
We observed that the fluorescence extended around the germlings
(Fig. 3A), suggesting that
these germlings were still contained within membrane-bound acidic organelles.
Occasionally, we also noticed some red phagosomes that had no conidia
associated with them (Fig.
3A,b, blue arrows). These phagosomes may have contained conidia
that had already been degraded.
|
We quantified the proportion of phagosomes that contained conidia that also showed staining with LysoTracker. After 1 hour in J774 cells, 29% of the internalized conidia were in acidic organelles (Fig. 3B) and this number rose to 70% after 6 hours. This data is consistent with the antibody co-localization experiments in which 75% of the conidia co-localized with LAMP-1 after 2 hours.
In contrast, conidia-containing phagosomes within A549 cells were slower to
acidify; only 17% of internalized conidia were in acidic organelles after 3
hours, 24% after 6 hours and this increased to 50% after 24 hours. However,
the data at 3 and 6 hours may underestimate the proportion of conidia in
acidic phagosomes. The number of co-localized conidia was determined by
dividing by the number of ringed conidia by the total conidia/field but at 3
and 6 hours post-infection not all of the conidia had been internalized.
Moreover, this experiment must be done using live cells, so the actual number
of intracellular conidia cannot be determined as the cells cannot be
immunostained. Based on our previous findings, approximately 30% of added
conidia are internalized after 3 hours
(Wasylnka and Moore, 2002).
Thus, the adjusted value of conidia co-localization with LysoTracker after 3
hours is approximately 50%. Therefore, it is probable that up to 50% of
conidia fuse with acidic organelles at 3 hours and this number remains
constant up to 24 hours post-infection.
Thus, A. fumigatus conidia reside in acidic organelles of J774 and A549 cells and these phagosomes are positive for LAMP proteins and lysosomal acid hydrolases.
Survival of A. fumigatus in A549 and J774 cells
The results from the antibody co-localization and LysoTracker experiments
suggested that conidia were trafficking to acidic late endosomes or lysosomes.
Furthermore, conidia were able to germinate within lysosomes in both cell
lines (Fig. 3A). Therefore, it
was of interest to determine the viability of the conidia in A549 and J774
cells over a 6-24 hour time period. Either J774 or A549 cells were incubated
with conidia for 6, 12 or 24 hours and a nystatin protection assay was used to
measure the viability of the internalized spores. After 6 hours, there was a
fourfold difference in the number of conidia recovered from the two cell
lines; 8% of the initial inoculum was still viable in A549 cells, whereas only
2% survived in J774 cells (Table
1). By 12 hours, the difference was tenfold; 6.5% remained in A549
cells versus 0.6% in J774 cells (Table
1).
|
To determine whether conidia could survive for longer periods in A549 cells, the incubation step was increased to 24 hours. (J774 cells were not investigated as essentially all of the conidia were killed after 12 hours.) After 24 hours, 3% of the initial inoculum was still viable (Table 1) and 34±3% of the intracellular conidia had germinated.
To determine whether intracellular conidia had any cytotoxic effect on the cells, we measured the release of the cytosolic enzyme lactate dehydrogenase (LDH) into the supernatant after 6, 12 or 24 hours. In both A549 and J774 cells, there was no difference in LDH release between infected and uninfected cells after 6 or 12 hours (Fig. 4A,B). In contrast, after 24 hours in A549 cells, infected cells displayed a small but significant increase in cytotoxicity over uninfected cells (P<0.05).
|
Intracellular conidia can germinate and penetrate the epithelial
barrier
The finding that conidia could germinate and survive inside acidic
organelles within A549 cells, prompted us to determine whether internalized
germlings could escape the phagosome and penetrate the A549 cell membrane. To
observe this process by confocal microscopy, cells were infected for 24 or 36
hours and stained with an Aspergillus cell wall antibody under
conditions that only label extracellular conidia or hyphae. After 24 hours, we
observed many intracellular conidia and germlings, as well as some
extracellular germlings. The extracellular germlings usually had longer germ
tubes than the intracellular ones (data not shown). We also observed many
germlings that were only partially labelled by the antibody at the ends of the
germ tubes (Fig. 5a, arrows).
Thus, the ends of the germ tube were extracellular and accessible to the
antibody, but the remainder of the germling was still inside the cell. This
data suggests that after 24 hours, germlings begin to penetrate the A549 cell
from within, but the plasma membrane is not so disrupted that antibodies can
access the cytoplasm and stain intracellular germlings.
|
To determine whether these germlings could develop into hyphae, the incubation period was extended to 36 hours and nystatin was removed from the media for the final 12 hours. Infected cells were treated for 3 hours with 50 µg/ml nystatin, for 18 hours with 5 µg/ml nystatin and then the nystatin was removed. In control experiments with no cells, this treatment kills all added conidia by the end of the incubation. Therefore, any extracellular germlings/hyphae present after 24-36 hours should represent conidia that originated from within the A549 cells. As shown in Fig. 5 (b), germlings had grown into extracellular hyphae by 36 hours post-infection. Interestingly, LDH release was not significantly greater than after 24 hours, which suggests that the hyphal extension had not yet caused significant damage to the host cells (data not shown).
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Discussion |
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Uptake of particulate material by cultured cells results in the formation
of a phagosome that can have several fates within the endosomal network,
depending on the nature of the internalized particle. Phagosomes containing
inert materials such as latex beads initially possess early endosome markers
such as rab 5 and gradually accumulate late endosome markers such as rab 7 and
lamp 2 (Desjardins et al.,
1994). Based on elegant video microscopy studies, Desjardins et
al. (Desjardins et al., 1994
)
have suggested that phagolysosome formation is a dynamic process that relies
on the gradual acquisition of proteins from endocytic organelles. Many
microbial pathogens reside in phagosomes that can alter this normal
trafficking behaviour. For example, Salmonella-containing phagosomes
(SPs) possessed significant amounts of rab 5 and N-ethylmaleimide-sensitive
factor and were very fusogenic with early endosomes
(Mukherjee et al., 2000
).
Furthermore, SPs contained no mannose 6-phosphate receptors and only low
amounts of cathepsin D (Garcia-del
Portillo and Finlay, 1995
). Similarly, Mycobacterium
tuberculosis phagosomes (MPs) have properties of early endosomes in that
they contain low amounts of LAMP-1 and CD63 and are not acidic
(Sturgill-Koszycki et al.,
1994
). However, MPs are still fusion competent as demonstrated by
their ability to acquire exogenously added markers such as transferrin
(Clemens and Horwitz,
1996
).
In contrast to the Salmonella or Mycobacteria phagosome, A. fumigatus phagosomes (APs) in J774 and A549 cells are not arrested and acquire the lysosomal membrane proteins CD63 and LAMP-1 as well as cathepsin D. Although staining of APs with the cathepsin D antibody was weak (Fig. 2A), both the co-localization and LysoTracker experiments were consistent with the idea that conidia were trafficking to late endosomes/lysosomes. Furthermore, since APs were acidic, CD63 and LAMP-1 positive, it was not unexpected that cathepsin D would also be present within the lumen.
A. fumigatus phagosomes (APs) fused quickly with late endosome
markers after internalization by A549 and J774 cells. Within 30 minutes after
internalization, APs in A549 cells contained only low levels (3% were
positive) of transferrin receptor (CD71) at the early time points compared to
high levels (51% and 38%) of CD63 and LAMP-1. This rapid movement into
lysosomes resembles a lysosomal recruitment strategy that has been reported to
occur in several other microorganisms. For example, Salmonella
enteritis Serovar Typhimurium fuses with lysosomal membrane proteins 15
minutes after bacterial uptake by HeLa cells
(Garcia-del Portillo and Finlay,
1995) while lysosomes migrate to the entry site of Trypanosoma
cruzi into non-phagocytic cells and actively provide a source of membrane
for the parasite phagosome (Andrews,
1995
). Candida albicans also rapidly attracts late
endosomes following phagocytosis by mouse macrophages
(Kàposzta et al.,
1999
).
The kinetics of transport through the endosomal network was more rapid in
J774 cells than A549 cells. This discrepancy was most likely due to the fact
that J774 cells are professional phagocytes, whereas A549 cells are not. For
example, Oh et al. (Oh et al.,
1996) reported that in mouse bone marrow-derived macrophages,
Salmonella enteritis Serovar Typhimurium fused with lysosomes within
20 minutes of phagocytosis (Oh et al.,
1996
). Moreover, Desjardins et al.
(Desjardins et al., 1994
)
reported that latex beads merged with lysosomal compartments of J774 cells
within 60-90 minutes following uptake. Similarly, movement of A.
fumigatus conidia through the J774 endosomal work was rapid; 75% of APs
were LAMP-1 positive in J774 cells after 120 min, whereas only 55% were LAMP-1
positive in A549 cells after 24 hours.
In addition to these differences in transport rates within the endosomal
network, the viability of conidia within J774 and A549 cells was also markedly
different. After 12 hours post-infection, there was a 10-fold difference in
viability between the conidia in J774 and A549 cells; 99% of the conidia added
to J774 cells had been washed off or killed, whereas significant amounts of
viable conidia remained in the A549 cells after 24 hours (3% of the initial
inoculum). The oxidative metabolism of alveolar macrophages during
phagocytosis of A. fumigatus conidia has been well documented
(Gil-Lamaignere et al., 2001;
Nessa et al., 1997
). In both
cell lines, conidia resided in acidic organelles containing lysosomal
proteins, therefore, the difference in killing rates between the two cell
lines was most likely due to the respiratory burst present in J774 cells. The
finding that the acidic conditions of the AP phagosome did not inhibit
survival or germination of the conidia within A549 cells was not unexpected as
most fungi (including aspergilli) can tolerate low pH (
4)
(Carlile and Watkinson,
1994
).
The composition of the AP phagosome is similar to the intracellular
compartments of other pathogens. The C. albicans phagosome within
murine macrophages is a LAMP-enriched late endosomal vacuole
(Kàposzta et al.,
1999). Similarly, during the growth phase of Legionella
pneumophila in murine macrophages (more than 8 hours post-infection),
Legionella reside in LAMP-1-cathepsin/positive phagosomes with an
average pH of 5.6 (Sturgill-Koszycki and
Swanson, 2000
). Inhibition of phagosome acidification decreased
the intracellular growth of the bacteria within the cell
(Sturgill-Koszycki and Swanson,
2000
).
Others have seen that Amphotericin B, which is structurally related to
nystatin, increased the killing A. fumigatus conidia and C.
albicans yeast cells by macrophages
(Jahn et al., 1998;
Martin et al., 1994
). Jahn et
al. (Jahn et al., 1998
)
reported a killing rate of only 10-15% after a 12 hour incubation of conidia
with monocyte-derived macrophages. Preincubation of the cells for 16 hours
with 1 µg/ml Amphotericin B increased the kill rate to 57%. Martin et al.
(Martin et al., 1994
) observed
a synergistic effect of Amphotericin B with monocyte killing of C.
albicans. However, this effect was not due to amphotericin B-dependent
monocyte activation as the respiratory burst and expression of human leukocyte
antigen-DR was unaltered (Martin et al.,
1994
). In contrast, we determined that 99% of the added conidia
were washed off or killed by 12 hours. Our values were reported as percent
survival relative to the initial inoculum added. Previous experiments in our
lab have demonstrated that 65-80% of the bound conidia are killed by J774
cells in 6 hours (Wasylnka and Moore,
2002
). These values are more in line with those reported by
Schnaffer et al. (Schnaffer et al., 1983); they found that up to 50% of the
conidia internalized by alveolar macrophages were killed after 12 hours. The
differences between our kill rates and those reported by Schaffner et al.
(Schaffner et al., 1983) might be dependent on the anatomical source of the
macrophage. Schaffner et al. found that alveolar macrophages effectively
killed 90% of the ingested conidia by 30 hours, however 85% of conidia
internalized by peritoneal macrophages transformed into mycelia by 24 hours.
Alternatively, the higher kill rates we observed with J774 cells may have been
due to accumulation of nystatin within the cells. However, though the
structure is similar to Amphotericin B, nystatin has different properties in
vitro. Nystatin is more selective than Amphotericin B in binding to ergosterol
over cholesterol (Walker-Caprioglio et
al., 1989
) and a critical nystatin concentration must be reached
before any lipid membrane permeability is observed
(Bolard et al., 1991
).
Furthermore, if some leakage of nystatin did occur during the nystatin-kill
step, then it was likely permeating both A549 and J774 cell lines. Therefore,
the percent survival of conidia in A549 cells estimated from our experiments
may actually underestimate the survival in the cells in vivo.
Germination within phagolysosomes has also been observed during
phagocytosis of C. albicans by murine macrophages
(Kàposzta et al.,
1999). In addition, germ tube formation resulted in the escape of
the fungus from the macrophage, and the frequency of germination was
significantly reduced by neutralization of lysosomal pH
(Kàposzta et al.,
1999
). We observed similar results during internalization of
A. fumigatus conidia by A549 cells. By 6 hours post-infection, we
observed that some intracellular conidia had germinated, and after 24 hours,
more than 30% had formed germ tubes. Immunofluorescence labelling using an
extracellular antibody against fungal cell walls showed that some of the
germlings had penetrated the plasma membrane from within. By 36 hours,
extensive hyphal formation suggested that these germ tubes had elongated.
Interestingly, the data suggested that the hyphal penetration was not
accompanied by the concomitant release of large amounts of cytosolic enzymes
such as LDH. Therefore, we hypothesize that the germlings emerge from the A549
plasma membrane with minimal disruption to the host cell and that hyphal
extension continues once the germling has access to extracellular nutrients.
It is possible that the hyphae are released from the cell membrane at a later
time point.
In summary, our data suggests that A. fumigatus conidia fuse with lysosomes once internalized by A549 and J774 cells; however, their fate within these cells differs markedly. Conidia are rapidly destroyed by murine macrophages but a significant percentage of internalized conidia persist and germinate in A549 epithelial cells. Future experiments will investigate the mechanism of hyphal escape from A549 cells.
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Acknowledgments |
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References |
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