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Antimicrobial Agents and Chemotherapy, January 2001, p. 73-78, Vol. 45, No. 1
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.1.73-78.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Role of P Glycoprotein in the Course and Treatment
of Encephalitozoon Microsporidiosis
Gordon J.
Leitch,1,*
Mary
Scanlon,1
Andrew
Shaw,1 and
Govinda S.
Visvesvara2
Department of Physiology, Morehouse School of
Medicine, Atlanta, Georgia 30310,1 and
Division of Parasitic Diseases, National Center for Infectious
Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
303412
Received 17 July 2000/Returned for modification 11 September
2000/Accepted 5 October 2000
 |
ABSTRACT |
Encephalitozoon microsporidia are obligate
intracellular protozoan parasites that proliferate and differentiate
within a parasitophorous vacuole inside host cells that are usually
epithelial in nature. Isolates of the three species of the
Encephalitozoon microsporidia, E. cuniculi,
E. hellem, and E. intestinalis, were obtained
from AIDS patients and cultured in green monkey (E6) kidney cells. Anti-P-glycoprotein (anti-Pgp) and anti-multidrug resistance-associated protein (anti-MRP) monoclonal antibodies were used to probe for multidrug resistance (MDR) pump epitopes and verapamil- or cyclosporin A- and probenecid-modulated intracellular calcein fluorescence were
used to assess the expression of Pgp and MRP respectively in uninfected
and infected cells. Pgp, but not MRP, was detected immunocytochemically
and by verapamil- and cyclosporin A-potentiated intracellular
fluorescence in both host cells and parasite developing stages. When an
in vitro infection assay was employed, verapamil and cyclosporin A
acted as chemosensitizing agents for the antiparasitic drug
albendazole. These observations suggest that inhibiting host cell and
perhaps parasite MDR pumps may increase the efficacy of antiparasitic
agents in these and other microsporidia species.
| |
INTRODUCTION |
Microsporidia are obligate
intracellular protozoan parasites that constitute the phylum
Microspora. Over 1,200 species have been identified, and to date at
least 13 of these have been shown to infect humans (9,
21). The great majority of the reported clinical cases are in
immunodeficient or immunosuppressed individuals. One agent,
albendazole, has been found effective in the treatment of some forms of
intestinal microsporidiosis and most cases of disseminated
microsporidiosis (7, 21, 27), while the more toxic
fumagillin has been used topically to treat ocular microsporidiosis (10, 21). To date there are no reports of the development of parasite resistance to either agent, but this may not be surprising given the difficulties with diagnosis and identification of parasite species, differing susceptibility of the various microsporidia species
to the agents in question, and the relatively modest number of
microsporidiosis cases reported.
The present study addresses the potential role of host cell and
parasite multidrug resistance (MDR) pumps in microsporidiosis caused by
three species of Encephalitozoon microsporidia, E. hellem, E. intestinalis, and E. cuniculi.
These parasites proliferate and differentiate within a parasitophorous
vacuole. This location within a parasitophorous vacuole means that the
parasite is separated from the host's extracellular fluid by several
membranes. These include the parasite plasma membrane and, in the case
of the mature spore, the environmentally resistant spore coat, the
parasitophorous vacuole membrane, and the host cell plasma membrane
(9). An MDR pump in one or more of these membranes would
significantly affect the concentration of any antiparasitic agent
within the parasite.
In many protozoan parasites genes have been identified that may be
responsible for the expression of membrane pumps that produce MDR by
lowering intracellular drug concentrations (32, 33). Such
pumps are members of the superfamily of ATP binding cassette (ABC)
pumps that are responsible for diverse cellular transport functions
(1, 4, 32). In the present study we have attempted to
determine what roles MDR pumps may have on the course and treatment of
microsporidiosis. The study has (i) determined if infection with the
microsporidia affects host cell clearance of calcein and calcein AM,
transported by MDR-associated protein (MRP) and P-glycoprotein (Pgp)
respectively; (ii) immunocytochemically probed for MRP and Pgp antigens
in host cells and parasite stages; (iii) determined if chemosensitizers
increase the efficacy of albendazole in the treatment of in vitro
microsporidiosis; and (iv) determined if parasite stages express
chemosensitizer-inhibitable ABC pumps.
| |
MATERIALS AND METHODS |
Parasite culture.
E. hellem, E. intestinalis, and E. cuniculi isolates, originally
obtained from AIDS patients, were cultured in green monkey kidney cells
(E6) as described previously (35). All cultures were
maintained in a CO2 incubator at 37°C in Dulbecco's
modified Eagle's medium supplemented with 10% heat-inactivated fetal
calf serum, gentamicin (50 µg/ml), and amphotericin B (5 µg/ml).
Calcein loading and detection.
Infected and uninfected cells
were plated on 35-mm-diameter dishes with no. 1 coverslip bases 2 days
prior to use. Dye loading was performed with cells maintained in growth
medium. In experiments in which verapamil was used, this agent (final
concentration, 10 µM) was added to the medium 45 min prior to the
addition of calcein AM (final concentration, 2 µM) in dimethyl
sulfoxide (DMSO) (final concentration, 0.01%). Fifteen minutes later
the medium was removed and replaced with a 20 mM HEPES-buffered
solution, pH 7.4, containing 135 mM NaCl, 5 mM KCl, 5 mM
NaHCO3, 1.2 mM KH2PO4, 1.2 mM
CaCl2, 1.2 mM MgSO4, and 1 mg each of glucose, bovine serum albumin (BSA), and ascorbic acid per ml. Ascorbic acid was
used as an antiphotooxidation agent. The cells were imaged with an
inverted confocal laser scanning microscope as described previously
(23). Relative fluorescence was measured in cells by
focusing on areas of cytoplasm that were away from the nucleus and free
of vacuoles or areas of sequestered calcein.
The calcein extrusion by E6 cells was variable, presumably reflecting
different degrees of plasma membrane Pgp expression. As a result, the
calcein fluorescence of E6 cell cultures varied between cultures and
between passages, depending on which clonal expansions of cells
dominated the culture. To reduce the variance in relative fluorescence
between treatment groups in an experiment, all cells used in a given
experiment came from the same culture and passage number. Similarly,
while the laser and gain settings were optimized for each experiment,
they were kept constant throughout a given experiment.
In one experiment designed to determine the effect of Pgp and MRP
inhibitors on calcein fluorescence, uninfected cells were
plated as
above and exposed to 10 µM verapamil, 10 µM cyclosporin
A, or 100 µM probenecid. The carrier for cyclosporin A was ethanol
(final
concentration, 0.1%). After 45 min in medium containing
the
transporter inhibitor, calcein AM was added to the medium
for an
additional 15 min as described above. Ethanol and DMSO
carrier controls
were carried out as appropriate. This carrier
did not affect cell
fluorescence at the concentrations
used.
In order to determine if there was a calcein AM or calcein extrusion
pump in the parasite, heavily infected cells were broken
up by passing
a cell suspension through a 26-gauge needle three
times. The most
abundant parasite stage, the mature spore, did
not load with calcein,
presumably due to its complex spore coat.
Meronts and other single
parasite stages were difficult to distinguish
from vesiculated cell
debris. However, chains of sporogonial stages
were readily
distinguished without the need for purification.
Disrupted cells were
therefore exposed to medium or medium containing
one of the transporter
inhibitors for 45 min and to calcein AM
for an additional 15 min as
above. The medium was then removed
by centrifugation in a
microcentrifuge and replaced with the HEPES-buffered
solution as above.
The cell suspension was then placed on the
heated microscope stage, and
the sporogonial stages were allowed
to settle. Due to concerns that
compounds such as polylysine might
affect the membrane integrity of
these small parasite stages (<2
µm in width) the sporogonial chains
were allowed to float freely.
While there was some Brownian movement of
these small parasite
stages, because the chains averaged four cells at
least one parasite
cell was in focus in both the fluorescent and
transmitted-light
images at each
observation.
Infection assay.
A mixture of uninfected and E. hellem-infected cells was plated to confluence in wells of
eight-well coverslip slides. The medium in the wells was then replaced
daily for the next 3 days with medium containing albendazole alone in
DMSO (final concentration, 0.01%), verapamil alone, cyclosporin A
alone, albendazole and verapamil, or albendazole and cyclosporin A,
each at a final concentration of 0.1 µM. Control wells were given
medium alone. All solutions contained the same final concentrations of
the DMSO and ethanol carriers. After 3 days of exposure to these agents
the medium was withdrawn, the monolayers were fixed with 10% neutral
formalin and stained with Giemsa, and the number of infected and
uninfected cells was counted to yield the percentage of infected cells
in each well as described previously (14). The low
concentration of agents was used because while 3 days of exposure to 1 µM albendazole reduced infection by 30 to 60% in these cultures, the
variance of this effect tended to obscure the potentiation effect of
the verapamil and cyclosporin A. The low dose of verapamil was chosen because calcium channel blockers are known to inhibit spore germination (22), and we wished to avoid such a direct effect of
verapamil on the spread of infection.
Immunocytochemistry.
Infected and uninfected cells were
plated onto eight-well chamber slides and 24 h later the medium
was removed and the samples were fixed with acetone at 
20°C for 10 min, followed by 10 min in 2% BSA in Tris-buffered saline (TBS) as a
blocking agent. One of two anti-Pgp monoclonal antibodies or one
anti-MRP monoclonal antibody was then used as a primary antibody. In
one group of experiments mouse anti-Pgp clone F4 (Sigma Chemical Co.,
St. Louis, Mo.) was used diluted 1:40. The second anti-Pgp monoclonal
antibody was clone G/1C obtained from Chemicon International Inc.
(Temecula, Calif.), used at a concentration of 1:40. The anti-MRP
monoclonal antibody was obtained from Alexis, Corp. (San Diego,
Calif.), clone MRPm6, and was used at a dilution of 1:20. All the
primary antibodies were diluted in 1% BSA in TBS and were incubated at 37°C for 1 h. After three TBS washes, the antibody binding sites were visualized by incubating samples in biotinylated goat anti-mouse immunoglobulin G (Jackson Immunoresearch Labs Inc., West Grove, Pa.),
1:300 in 1% BSA in TBS for 1 h at 37°C, followed by
Streptavidin-Oregon Green 488 (Molecular Probes Inc., Eugene, Oreg.),
1:300 in TBS for 45 min at room temperature.
Statistical analyses.
In experiments in which the means of
several values were being compared, the data were first analyzed by a
one-way analysis of variance followed by post hoc Tukey's protected
t tests to determine the significance of differences between
individual mean values. In experiments in which the levels of calcein
fluorescence of sporogonial stages were compared when the parasites
were treated with carriers and with verapamil or cyclosporin A,
Wilcoxon two-group rank tests were used to determine the significance
of differences between means of replicate experiments.
| |
RESULTS |
Green monkey kidney cells were incubated with calcein AM, and
their relative fluorescence was measured by confocal microscopy after
removal of the probe from the medium. This fluorescence provided a
measure of the intracellular concentration of the fluorescent calcein
free acid which resulted from the removal of the acetoxymethyl groups
from the calcein AM by cellular esterases. Calcein AM is extruded from
cells by Pgp (1, 11), while MRP extrudes the free-acid
form of this probe (11, 15). Verapamil and cyclosporin A
were chosen as inhibitors of Pgp (5, 13), and probenecid was chosen as an inhibitor of MRP (13). Figure
1 illustrates the relative fluorescence
of uninfected E6 cells and the effects of 10 µM verapamil and
cyclosporin A and 100 µM probenecid on this fluorescence. The control
values in this figure were measured in cells in carrier-free medium, as
no carrier effect was observed on relative fluorescence. The two Pgp
inhibitors significantly increased cell fluorescence, consistent with
the inhibition of cell membrane Pgp extrusion of entering calcein AM,
while probenecid had no significant effect on the cell fluorescence,
indicating that these cells lacked MRP expression. Higher
concentrations of probenecid (up to 1 mM) were used without significant
effect on cell fluorescence.
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FIG. 1.
Relative fluorescence of intracellular calcein in E6
cells exposed to 10 µM verapamil (Verap), 10 µM cyclosporin A
(Cyclo A), or 100 µM probenecid (Proben) for 1 h. *,
significantly different from control (P < 0.05); error
bar, standard error of the mean.
|
|
Experiments were performed in which the calcein fluorescence was
measured in uninfected and Encephalitozoon-infected E6 cells and in cells plated at the same time and treated in the same manner but
with the additional exposure to 10 µM verapamil for 60 min. Figure
2 illustrates the size of populations of
uninfected and E. intestinalis-infected cells grouped by
their relative fluorescence. These data are representative of multiple
experiments performed using each of the three species of
Encephalitozoon microsporidia. Typically 
20% more of the
infected cells were found to exhibit a lower relative fluorescence than
the uninfected cells (e.g., see Fig. 2a). There are at least three
possible explanations for the observation that there was a larger
subpopulation of infected cells with a lower relative fluorescence. The
first is that the infected cells were losing their esterase activity or
their membrane integrity and were therefore unable to generate or
retain the intracellular fluorescent probe. The second explanation is
that calcein-calcein AM extrusion pump activity is upregulated in
infected cells. The third is that cells with high expression of Pgp
were better able to accommodate the intracellular parasite and were thus more frequently selected for successful infection. The first explanation, that infected cells were deteriorating, was eliminated by
the observation that the Pgp inhibitor, verapamil, increased the
fluorescence of all cell populations and in many cases, such as that
illustrated in Fig. 2b, it eliminated the difference between the number
of uninfected and infected cells exhibiting the lower fluorescence.
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FIG. 2.
Relative fluorescence of intracellular calcein in
uninfected and E. intestinalis-infected E6 cells. (a) Cells
are grouped by relative fluorescence in increments of 10 units, and the
percentage of the uninfected and infected cells falling within each
fluorescence range is shown. (b) A similar experiment was performed
with cells from the same culture that were treated with 10 µM
verapamil prior to exposure to calcein AM.
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|
Two anti-Pgp monoclonal antibodies and one anti-MPR monoclonal antibody
were used to probe for epitopes on host cells and intracellular
parasite stages. Both anti-Pgp antibodies recognized epitopes on the
host cell plasma membrane and intracellular organelles (Fig.
3A and B), presumably the Golgi apparatus
(26). They also recognized epitopes within the parasite.
The numerous refractile spores that are visible in the transmitted
light images (Fig. 3C and D) were not labeled. Antibody labeling of
spores is difficult due to the chitinous and proteinaceous spore coat
which appears to impair antibody penetration. However, both the
merogonial parasite stages adjacent to the parasitophorous vacuole
membrane and chains of sporogonial stages within the vacuole were
labeled with the anti-Pgp antibodies, while the anti-MRP antibody
failed to label epitopes in either the host cell or parasite stages
(data not shown).
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FIG. 3.
Immunofluorescence of E. hellem-infected
cells. (A and B) Confocal images of cells in which host cells and
parasites were probed with a Sigma (A) and a Chemicon (B) anti-Pgp
monoclonal antibody. (C and D) Transmitted-light images of the cells
illustrated in panels A and B, respectively. Arrows indicate chains of
parasite sporogonial stages, arrowheads indicate individual meronts,
and N indicates a host cell nucleus. Bar = 2 µm.
|
|
As the anti-Pgp antibodies recognized epitopes on both host cells and
intracellular parasites while the anti-MRP antibody did not, and as
both Pgp inhibitors, verapamil and cyclosporin A, increased host cell
fluorescence while the MRP inhibitor, probenecid, did not, an infection
assay was used to determine if the Pgp inhibitors could act as
chemosensitizing agents in the treatment of an in vitro infection. The
E. hellem strain used was selected because it yielded the
most consistent and uniform infection. Table
1 summarizes the effects of using the
antiparasitic agent, albendazole, in conjunction with either verapamil
or cyclosporin A, at concentrations that alone had no effect on the
number of cells infected with E. hellem. When either of the
Pgp inhibitors were used in conjunction with albendazole there was a
significant inhibition of infection, indicating that the verapamil and
cyclosporin A were acting as chemosensitizers of albendazole
(12).
Both anti-Pgp monoclonal antibodies recognized epitopes on the
developing stages of E. hellem. To determine if such
developing stages phenotypically expressed Pgp extrusion of calcein AM,
the relative fluorescence of sporogonial stages was measured in cells exposed to calcein AM in the presence and absence of 10 µM verapamil or cyclosporin A. The percentage difference in cell fluorescence when
cells were exposed to the Pgp inhibitors when compared to the
corresponding carrier control was determined. Exposure to verapamil or
cyclosporin A significantly increased fluorescence (45.5% ± 13.1%
and 31.8% ± 11.7%, respectively [mean ± standard error of the
mean]) in these developing parasite stages as judged by the Wilcoxon
signed-rank test (P = 0.028 and P = 0.046, respectively), suggesting that the parasite was able to
extrude calcein AM by a Pgp-like pump. No statistically significant
effect was found when 100 µM probenecid was used in such experiments
(data not given).
| |
DISCUSSION |
Many protozoan parasites have been found to possess MDR-like genes
responsible for the expression of membrane proteins with homology to
mammalian Pgp or MRP (6, 8, 17, 18, 28, 32, 33). The
typical mammalian MDR membrane pump is made up of two homologous
peptide halves, each with an intracellular ATP binding domain and six
membrane-spanning domains (1). While Pgp and MRP pumps
share similar ATP binding sites, their membrane spanning domains are
significantly different from one another. Protozoan parasite MDR-like
membrane proteins are more variable in their structure than their
mammalian counterparts. While some have the predicted Pgp structure
(18), others have only one rather than two peptides
(17), while others have two peptides but fewer than the
total of 12 membrane spanning domains (32). Similarly,
while mammalian Pgp and MRP pumps have been shown to play significant
transport roles and are important in the development of MDR
(1), it has not been possible to definitively ascribe such
roles to similar proteins in many of the protozoan parasites (6,
32, 33). There is some evidence to support the microsporidia resembling fungi more than protozoa (19). ABC proteins
have been extensively studied in yeasts, where they have been found to
be involved in pheromone secretion in sexual reproduction, mitochondrial function, and stress response to cellular detoxification, as well as drug resistance (3). In Saccharomyces
cerevisiae a pleiotropic drug resistance network of genes has been
identified that is comprised of transcriptional regulators that are
responsible for the expression of multiple ABC transporters involved in
the development of drug resistance (20).
The present study points to Encephalitozoon microsporidia
possessing epitopes that are identified by monoclonal antibodies against mammalian Pgp but not by a monoclonal antibody against mammalian MRP. This study also suggests that verapamil and cyclosporin A, two agents that inhibit Pgp pump activity, inhibit
Encephalitozoon Pgp-like extrusion of calcein AM. It is
tempting to suggest that a Pgp-like pump in this microsporidian genus
may extrude chemotherapeutic agents and, if overexpressed, may
contribute to the development of drug-resistant strains. However
similar early observations were made with Plasmodium
falciparum when verapamil was shown to reverse chloroquine
resistance (25) and the pfmdrl gene was observed to be amplified in some chloroquine-resistant strains of the
parasite (36). However, it is more than a decade since these early observations, and the relationship between the
pfmdrl gene product, Pgh1, and drug resistance has not
yet been fully elucidated, although it is known that mutations in
pfmdrl can reduce parasite chloroquine accumulation and
impart resistance (4). The expression of such resistance
may involve mutations in additional genes, however (29).
Similarly in Entamoeba histolytica, multiple Pgps may be
involved in the expression of emetine resistance (8).
We observed that verapamil and cyclosporin A act as chemosensitizing
agents with albendazole to reduce Encephalitozoon infection in E6 cells in culture. These agents have differing mechanisms of
action when inhibiting Pgp pumps (16, 31). This suggests that the albendazole was extruded from either the parasite,
parasitophorous vacuole, or host cell by a Pgp pump and that the
inhibitor or chemosensitizer increased the intracellular concentration
of this agent. Based on the calcein fluorescence studies summarized in Fig. 1 and the immunocytochemical demonstration of Pgp epitopes in the
host cell, it seems likely that the major site of action of these
chemosensitizers is the host cell plasma membrane. The resolution in
the immunocytochemical study did not distinguish between anti-Pgp
epitopes on the parasitophorous vacuole membrane and those on adjacent
host cell organelles and parasite developing stages. As the
Encephalitozoon parasitophorous vacuole membrane appears to
be highly permeable (23), it seems unlikely that this will
be a site of effective drug extrusion, although it is possible that a
parasitophorous vacuole rather than the parasite itself is the site of
a drug efflux pump. There appears to be a MRP-like pump at the
interface between the Cryptosporidium parvum parasitophorous
vacuole and the host cell cytoplasm in the area of the feeder organelle
(28). The parasite plasma membrane would be expected to be
less accessible to chemosensitizer, particularly verapamil, at an
effective concentration. Epithelial cells are a target for
microsporidian infection, and they are known to possess MDR
pumps, often polarized on a given membrane (2, 16). Thus, the use of chemosensitizers with agents such as albendazole may be an
effective mechanism of treating unresponsive microsporidiosis in such
cells. Without knowing the relationships of MDR pumps in the membranes
of multicompartment systems such as Encephalitozoon-infected cells, it is difficult to predict the effect of inhibiting or changing
the expression of these pumps on the efficacy of a particular antiparasitic agent. For example, in the case of P. falciparum, Pgh1 in the parasite food vacuole membrane increases
chloroquine accumulation and drug susceptibility, while its expression
in the parasite plasma membrane reduces susceptibility and increases resistance to aminoalcohols (4).
The observation that at least 20% more cells infected with
microsporidia were found to fall into low calcein fluorescence categories than uninfected cells and that the fluorescence of all
cells, infected and uninfected, was increased by verapamil suggests
either that infection modified the host cell Pgp expression or that
those cells that overexpressed Pgp accommodated the parasite infection
more readily. There is evidence for both of these alternatives with
intracellular parasite infections. Toxoplasma gondii
infection of cells expressing a Pgp pump inhibits extrusion of the
probe, rhodamine-123 (34), apparently due to decreased
expression of host cell membrane Pgp. Also inhibition of the Pgp pump
of this parasite with a cyclosporin A analogue that only inhibits the Pgp but does not bind cyclophilins reduced parasite growth both in vivo
and in vitro (30). The latter observation suggests that Pgp-like pumps may serve parasite metabolic needs and support parasite
growth and differentiation.
The present study indicates that host cells expressing a Pgp pump
protect intracellular stages of Encephalitozoon
microsporidia from agents such as albendazole, probably by limiting
the intracellular concentration of the drug. In addition, the parasite
developing stages also have a Pgp pump which may further contribute to
protecting the parasite from albendazole. A chemosensitizer inhibiting
these pumps would therefore be expected to increase the effectiveness of this antiparasitic agent.
| |
ACKNOWLEDGMENT |
This work was supported in part by U.S. Public Health Service
grant RR03034.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Physiology, Morehouse School of Medicine, 720 Westview Dr., Atlanta, GA
30310. Phone: (404) 752-1681. Fax: (404) 752-1045. E-mail: Leitch{at}msm.edu.
| |
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Antimicrobial Agents and Chemotherapy, January 2001, p. 73-78, Vol. 45, No. 1
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.1.73-78.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
