infection by microsporidia disrupts the host cell cycle

Infection by Microsporidia Disrupts the Host Cell Cycle MARY SCANLON: ANDREW P. SHAW; CHENG J. ZHOU,a.h GOVINDA S. VISVESVARA and GORDON J. LEITCH"

"Department of Physiology and hDepartment of Anatomy, Morehouse School qf Medicine, Atlanta, Georgia 30310, and

LDivision of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, USA

ABSTRACT. Microsporidia of the genus Encephulitozoon infect mammalian cells and have become a source of morbidity and mor- tality in immunocompromised humans. Encephulitozoon microsporidia develop and mature within parasitophorous vacuoles, enlarging the vacuole over time until it eventually occupies most of the cytoplasm of the host cell. The ability of the host cell to accommodate such a large burden for several days suggests that the parasite subverts normal host cell processes to ensure optimal environmental conditions for its growth and development. Since this environment would be threatened if cell division of the host cell occurred, we have formulated the hypothesis that infection with Encephalitozoon microsporidia induces an arrest in the cell cycle of the host cell. In support of this hypothesis, we have found that mitotic index and DNA duplication are reduced in infected cells as compared to uninfected cells. The number of host cell nuclei in S phase is increased. The levels of cyclin D1 and the percentage of cells in G1 are reduced; however, the levels of cyclin B 1 are elevated even though the percentage of cells in G2/M is decreased. These results suggest that host cells infected with Encephalitozoon microsporidia are blocked at multiple points in the cell cycle.

K e y Words. Cyclins, Encephalitozoon, E. cuniculi, E. hellem, E. intestinalis, mitotic index, parasitophorous vacuole.

ICROSPORlDIA are obligate, intracellular protozoan par- M asites that infect many vertebrate and invertebrate hosts (Canning and Lom 1986). Until recently, microsporidia were primarily of interest because of their ability to infect insects and fish, in the former case for their potential use in insect control, and in the latter, because of their impact on the fishing industry (Wittner 1999). Prior to the AIDS epidemic, a few isolated reports of human microsporidiosis were described in the literature (Shadduck 1989; Wittner 1999). However, since the onset of the AIDS epidemic, this situation has changed rad- ically (Bryan, Cali, and Owen 1991; Schwartz et al. 1992; Zen- der et al. 1989). Microsporidiosis is a common problem for patients with AIDS, causing extensive morbidity and some mor- tality. The species that most commonly infects humankind, En- terocytozoon bieneusi, is limited initially to the intestinal mucosal epithelium and has yet to be successfully placed in long- term culture (Desportes et al. 1985; Visvesvara et al. 1995a). On the other hand, three Encephalitozoon species, that infect humans and are used in the studies described here (Encepha- litozoon hellem, Encephalitozoon cuniculi and Encephalitozoon intestinalis), have been placed in long-term culture (Cali, Ko- tler, and Orenstein 1993; DeGroote et al. 1995; Didier et al. 1991; Visvesvara et al. 1991, 1995b).

The genus Encephalitozoon is characterized by the proliferation and development of parasites within a parasitophorous vacuole (PV) within the host cell. The cycle of infection begins when an environmentally resistant spore everts a tube that im- pales a nearby target cell (Canning and Lom 1986). Infective sporoplasm is injected into the target cell via this tube and the cycle of infection is established. The earliest stages of infection are not well-documented. However, within a few hours of infection, a PV forms within the host cell. It is a membrane- bounded organelle that houses developing microsporidial stages (meronts and sporonts) and mature spores in a highly organized manner. Characteristically, developing stages are found near the membrane of the vacuole; the mature spores, which are fully differentiated, lie more centrally. Infected cells house one or more PVs. As the developing stages o f the parasite divide and mature within the PV, the host cell enlarges several-fold to accommodate the proliferating parasites. The PVs frequently occupy most of the host cell cytoplasm, squeezing the host cell nucleus, organelles and cytoskeleton to the periphery (Fig. 1B- D). Infection can last for several days before rupture of the membranes of both the host cell and PV is triggered. The sub-

Corresponding Author: M. Scanlon-Telephone number: 404-752- 1683; FAX number: 404-752-1045; E-mail: [email protected].

sequent dispersal of spores into the extracellular milieu contin- ues the cycle of infection as each mature spore is capable of infecting a neighboring cell. The ability of the host cell to accommodate the parasite without apparent detriment, at least until rupture of the vacuolar membrane, suggests that the parasite must be subverting normal host cell metabolism and cellular processes. One such process is cell division. Because the PVs become so large in these cells, cell division would have a significant, detrimental effect on the integrity of the vacuole and might impact the viability of the developing parasite stages. Since infection with other parasites has been shown to affect cell cycles in the infected host (Matthews and Gull 1994; Mos- ca, Briceno, and Hernandez 1991; Roman, Coriz, and Baca 1986; Vassella et a1 1997), we have hypothesized that infection with Encephalitozoon blocks cell cycling by host cells.

MATERIALS AND METHODS

Cell infection. Each species of microsporidia used in this study (Encephalitozoon hellem, E. cuniculi and E. intestinalis) was isolated from a patient with AIDS; the isolated parasites were then inoculated into African green monkey kidney cells (E6 cells) as previously described (Visvesvara et al. 1999). In- fection was usually well established within two to three days. Infected and uninfected cells were grown in Dulbecco's mod- ified Eagle's medium (Gibco BRL Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 50 kg/ml gentamicin and 5 yg/ml amphotericin B (Gibco BRL Life Technologies, Grand Island, NY) in a 5% CO, incubator at 37 "C. (Unless otherwise stated, reagents were obtained from Sigma Chemical Co., St. Louis, MO.) To guard against cellular transformation, E6 cells were not used past passage 25.

Analysis of the cell cycle of isolated nuclei by flow cytometry. Cells were placed in suspension following a brief trypsinization and washed with ice-cold, phosphate-buffered saline (PBS). Nuclei were isolated by centrifugation after lysis of whole cells with 0.2% NP-40 (LKB, Bromma, Sweden) in 0.5% BSA-containing PBS supplemented with 100 kg/ml RNAse for 30 min at 4 "C in the dark. Isolated nuclei were fixed with ice- cold methanol for 1 h, washed with PBS and stored at 4 "C for up to two days. DNA staining was accomplished by incubation for 30 min with 50 y/ml propidium iodide in PBS containing RNAse. The relative amount of DNA was assayed using standard flow cytometric techniques with a Becton Dickinson FAC- Scan and software packages for data acquisition (Lysis 11) and cell cycle analysis (Cell Fit) (Becton Dickinson, San Jose, CA).

Indirect immunofluorescence and confocal microscopy.

525

5 26 J. EUKARYOT. MICROBIOL., VOL. 47, NO. 6, NOVEMBER-DECEMBER 2000

Fig. 1. Transmitted light images of an uninfected E6 cell (A) and E6 cells infected with Encephulitozoon he//ern (B), Encephulitozoon cuniculi (C), and Enwphalitozoon intestinalis (D). Uninfected E6 cells exhibit the typical morphology of epithelia1 cells in culture. Following infection, parasitophorous vacuoles are formed within the host cell. They contain many meronts, sporonts and spores, and they effectively squeeze the host cell cytoskeleton and organelles to the periphery of the cell. In spite of this accommodation, the host cells look remarkably healthy. Nuclear and plasma membranes are intact; the cells remain adherent to the substrate and nuclear morphology i s indistinguishable from that of uninfected cells. Bar = 2 pm.

Standard indirect immunofluorescent techniques were employed to visualize tubulin and various cell cycle-dependent proteins. Uninfected cells and cells infected with Encephulitozoon were collected from cell culture flasks following a brief trypsinization at 37 "C and then plated overnight in eight-well chamber slides (Nalge Nunc International, Naperville, IL) at a density of approximately 5 X 104 cells/well. Cells were fixed either by ad- dition of ice-cold methanol or 70% acetone on ice for 15 min. Cells were then washed 3X with 50 mM Tris, pH 7.35/0.15 M NaCl (Tris-buffered saline, TBS). Nonspecific antibody-binding

sites were blocked by incubation with TBS containing 2% bovine serum albumin at room temperature for 10 min. Cells were incubated with the appropriate primary antibody in TBS containing 1% bovine serum albumin for 60-120 min at 37 "C. Cells were then washed 3 X with TBS and incubated for 1 h at 37 "C with either Oregon Green 488-conjugated anti-IgG (1: 200 dilution, Molecular Probes, Eugene, OR) or Texas Red- conjugated anti-IgC (1 :200 dilution, Jackson ImmunoResearch, West Grove, PA). In some cases, a three-step procedure was used in which either biotinylated goat anti-mouse or anti-rabbit

SCANLON ET AL.-MICROSPORIDIA AND HOST CELL CYCLING 527

IgG was used as a secondary antibody followed by incubation with streptavidin-conjugated fluorochrome. Three washes with TBS followed. The scaffolding of the chamber slide was re- moved and the slide was mounted in SO% (voVvol) glycerol in TBS containing 0.1 M N-propyl gallate as an anti-bleaching agent.

Fluorescence was visualized within uninfected and Enceph- alitozoon-infected cells using a Multiprobe 200 1 laser scanning confocal microscope (CLSM) (Molecular Dynamics, Sunny- vale, CA). The emission line on the argon laser was set to 488 nm for Oregon Green 488-stained cells and to S 14 nm for Texas Red-stained cells. A Nikon Diaphot inverted epifluorescent microscope was used to select regions of interest prior to collec- tion of confocal images, which were stored, processed and an- alyzed on an Iris Indigo workstation (Silicon Graphics, Moun- tain View, CA) running Imagespace software (Molecular Dy- namics, Sunnyvale, CA). In general, the l O O X objective was used to image cells; with this objective, the vertical resolution of confocal optical sections was approximately 0.25 pm

Assessment of mitotic index. Two methods were used to assess the frequency of mitosis within uninfected and infected populations of nonconfluent cells. In the first, cells were fixed with Carnoy's solution and stained with hematoxylin for visu- alization of metaphase plates. We also used this method on detached cells that were suspended in the media within the tis- sue culture flask in order to assess the percentage of mitosis in infected cells that had lost their adherence to the substrate. De- tached floating cells were layered onto polylysine-coated cov- erslips then fixed and stained with hematoxylin as described above.

In the second method for assessing levels of mitosis, cells were treated for 20 h with 1 FM albendazole, a microtubule inhibitor that blocks chromosome segregation and thus arrests cells in metaphase (Whittaker and Faustman 1991). We expected that in a normally cycling population of cells, the number of cells blocked in mitosis would increase with the duration of albendazole treatment. However, if a population of cells cycled at a reduced rate, the number of spindles observed would be fewer than in the normally cycling population. Following treatment with albendazole, cells were processed for visuali- zation of mitotic spindles using indirect immunofluorescence as above with fixation in ice-cold methanol and staining with an anti-p tubulin monoclonal antibody (clone KMX- 1, 1 :75 dilution, Boehringer Mannheim, Indianapolis, IN).

Immunocytochemical analysis of the cell cycle. The percentages of uninfected and infected cells in GO were determined from confocal analysis of immunocytochemical labeling with an antibody to Ki-67, a nuclear protein that is present in several phases of the cell cycle (Gl, S and G2), but not in GO. Fixation was accomplished with methanol. The three-step procedure was employed with anti-Ki-67 (clone 7B11, Zymed Laboratories, South San Francisco, CA) at a dilution of 1:25, biotinylated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) at 1300 and Oregon Green 488-conjugated streptavidin 1: 200 (Molecular Probes, Inc., Eugene, OR).

The percentages of cells duplicating DNA were determined using a modification of the protocol described for the BrdUr Immunohistochemistry Kit (Oncogene Research Products, Cambridge, MA). Bromodeoxyuridine (BrdU) (10 pM) was added to the incubation medium of uninfected and infected cells for 5 h at 37 "C. The cells were washed to remove unbound analog and fixed with 70% acetone. The remainder of the max- ufacturer's protocol was followed.

Three-step labeling was employed to assess levels of cyclin D1 in methanol-fixed infected and uninfected cells using a poly- clonal antibody to cyclin D1 (Santa Cruz Biotechnology, Santa

Cruz, CA) at a dilution of 1:50, followed by incubations with biotinylated goat anti-rabbit IgG at 1500 and Texas Red-conjugated streptavidin at 1:200 (both from Jackson Immuno- Research, West Grove, PA).

Western blot analysis of intracellular levels of cyclin D1 and cyclin B1. Soluble cellular protein was extracted in cell lysis buffer (50 mM Tris, 1% NP-40, 0.25% sodium deoxycho- late, 150 mM NaCI, 1 mM EGTA, 1 kg/ml leupeptin and pep- statin, 1 mM benzamidine, 0.1 mM AEBSF, 1 mM Na,VO, and 1 mM NaF, pH 7.4) for 20 min on ice, followed by centrifugation for 20 min at 15,000 g and 4 "C. Soluble protein from mature spores, which represent the majority of the parasitic load, was not extracted by this procedure. Identical amounts of protein from uninfected and infected cells were electrophoresed on 10% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were blocked for 25 min with 3% nonfat dry milk in PBS plus 0.05% Tween (PBST) at room temperature, incubated either with anti-cyclin Dl (1: 1000 dilution) or anti-cyclin B1 (mixed cyclin B1 clones OS-373, Upstate Biotechnology, Lake Placid, NY) (1:2000 dilution) in blocking solution overnight at 4 "C, rinsed with PBST, and incubated with horseradish peroxidase-conjugated goat anti- mouse IgG (1:2500) (Amersham, Arlington Heights, IL) in blocking solution for 90 min at room temperature. After a final rinse with PBST, membranes were incubated with ECL detection reagent and exposed to ECL Hyperfilm (Amersham, Ar- lington Heights, IL).

Levels of parasitic cyclin B1 were measured following isolation of mature spores and developing parasitic stages from infected cells by rupture of infected cells through a 22-gauge needle. Three washes with PBS and three washes with 10% glycerol followed. The concentrated spores and developing stages, which were judged to be free of contamination from host cell debris by light microscopy, were then electroporated in 10% glycerol at 4 "C. Approximately SO p1 of sporoplasm was obtained by this method. The sporoplasm was then solu- bilized, electrophoresed and transferred to nitrocellulose as described earlier. The nitrocellulose was probed for parasitic cyclin B1.

Statistical analyses. Data were tested for significance with one-way analysis of variance. If significant differences were found among the means in a group, a post-hoc Tukey's protected t test was used to determine the significance of difference between individual mean values. In the comparison of the cell cycle data generated by flow cytometry, statistical significance was determined with a chi-squared test.

RESULTS Morphology of infection with Encephalitozoon. Infection

of green monkey kidney epithelia1 cells (E6 cells) with each of three species of Encephalitozoon microsporidia (E. hellem, E. cuniculi and E. intestinalis) induced the formation of a PV within the host cell that accommodated the growth and proliferation of the developing stages of the parasite and served as a repos- itory for mature spores. The PVs induced by each of the En- cephalitozoon microsporidia became quite large, containing many meronts, sporonts and mature spores (Fig. 1). The host cell cytoplasm, cytoskeleton and organelles were generally pushed to the periphery of the cell to accommodate the growing PV. Both the plasma and nuclear membranes of infected cells were intact throughout most of the infection. In a previous study (Leitch et al. 1997), it was determined that infected cells re- mained viable as assessed by the cytoplasmic cleavage of ace- toxymethyl groups from the intracellular probe, calcein.

Reduced mitotic frequency in infected cells. Several thou- sand, nonconfluent, hematoxylin-stained cells were examined

528 J. EUKARYOT. MICROBIOL., VOL. 47. NO. 6, NOVEMBER-DECEMBER 2000

B A '*" 1 T

Unlniscled E. hellem- E. intesllnalls- E cunlcull- Unlnfeeted E. hellem- lnlected Infected i n k l e d Infected

Fig. 2. Mitotic frequency in uninfected and Encephulitozoon-infected E6 cells as assessed (A) by counting metaphase figures or (B) by counting mitotic spindles following incubation with albendazole for 20 h. The mitotic frequency, as assessed by both methods, is significantly reduced in Encephalitozoon-infected cells as compared to uninfected cells (A: *, p< 0.01; B: *, p< 0.05).

by light microscopy for evidence of mitosis as indicated by the appearance of condensed chromosomes andlor the dissolution of the nuclear membrane. The percentage of mitotic figures in populations of uninfected cells was 1.32 2 0.57% (mean t s.e.m.). In contrast, the percentage of mitotic cells in populations infected with E. hellem was significantly lower, 0.08 -C 0.05% ( p < 0.01) (Fig. 2a). Statistically significant reductions in mitotic index were also seen in cells infected with E. cuniculi (0.14 2 0.08%) and with E. intestinalis (0.02 +- 0.02%), suggesting that cells infected with Encephalitozoon were less likely to undergo mitosis (Fig. 2a). The reduction in the percentages of infected cells observed in metaphase was not due to the loss of infected cells from the monolayer, as determined by exam- ination of cells that had detached from the substrate (data not shown).

To confirm that there was a difference in mitotic frequency between the two populations, we adopted a second approach in which uninfected and E. hellem-infected, nonconfluent cells were treated with albendazole. Albendazole is an anti-mitotic agent that will arrest both parasites and host cells in mitosis; however, albendazole does not cause resolution of the infection, at least within a short period of treatment. After 20 h of treatment with albendazole, the percentage of uninfected cells blocked in metaphase was 8.9 2 2.3% (Fig. 2b). In contrast, the percentage of E. hellem-infected cells blocked in metaphase was significantly lower (0.5 2 0.2%; p < 0.05), as expected if infected cells cycled at a much slower rate than uninfected cells (Fig. 2b).

Flow cytometric analysis of the cell cycle. The simplest and fastest technique for analyzing the cell cycle is to measure nuclear DNA content in propidium iodide-stained cells using flow cytometry. However, this technique is problematic with infected cells because of the presence of variable amounts of parasitic DNA within the host cell. To overcome this difficulty, flow cytometric analysis was performed on propidium iodide- stained nuclei that had been isolated from either uninfected or E. hellem-infected cells. The distribution of isolated nuclei from E. hellem-infected cells was significantly different from that of uninfected nuclei (X2 = 1580, df = 2, p < 0.0001) (Table 1). The most pronounced difference was between the frequency of the populations in S phase, with the number of infected cells in S phase approximately double that of uninfected cells.

Reduction in BrdIJ incorporation into DNA of infected cells. We used BrdU incorporation into the DNA of E6 cells

Table 1. Distribution of the cell cycle of nuclei isolated from uninfected E6 cells or E6 cells infected with Encephaliro?oon hellern.

G1 S G2lM

Uninfected nuclei" 66.0 2 4.2 14.7 5 4.5 19.3 t- 1.5 E. hellem-infected nucleihL 55.0 2 3.9 28.8 -t 4.7 16.2 t- 0.9

Values represent mean -t s.e.m. of 3 separate experiments. Values represent mean 2 s.e.m. of 4 separate experiments.

' Distribution is significantly different from that of uninfected nuclei ( p < 0.0001).

as a measure of the extent to which infected and uninfected cells were duplicating DNA and progressing through S phase. BrdU is a uridine analogue that incorporates into DNA when cells are duplicating DNA during S phase. Cells that have incorporated BrdU into their genome have duplicated DNA during the period of BrdU availability. Approximately 45% of uninfected cells incorporated detectable levels of BrdU during a five-hour period (Fig. 3 ) . In contrast, the values for infected cells were all significantly reduced (p < 0.01). These values were 8.3%, 16.4% and 19.3%, respectively, for E. hellern-, E. intestinalis- and E. cuniculi-infected cells.

Reduction of BrdU incorporation correlates directly with the size of the PV. In E. hellem-infected cells, an inverse re- lationship was detected between BrdU staining and the degree of infection of the host cell, as extrapolated from the size of the PV(s) within the cell. Cells at early stages of infection with E. hellem, having the smallest PVs, exhibited nearly half the BrdU staining of uninfected cells (23.5% vs. 44.5% in uninfected cells) (Table 2 and Fig. 3) . As the size of the PV within the cell increased, the percentage of infected cells staining with BrdU decreased. No cell with a PV greater than 500 km2 in size stained with detectable levels of BrdU. This inverse correlation between BrdU labeling and the extent of infection could not be detected in E. cuniculi- or E. intestinalis-infected cells.

60 T

*

* *

Uninfected E. hellem- E. intestinalis- E. cuniculi-

BrdU labeling in uninfected E6 cells and in E6 cells infected with Encephalitozoon hellern, Encephalitozoon intestinalis or Enceph- alitozoon cuniculi. Cells were labeled with BrdU for 5 h. Positive nuclear staining for BrdU indicates that a given cell had duplicated DNA at some point during the five-hour time period prior to fixation. There is a significant reduction in BrdU labeling in Encephalitozoon-infected cells ( p < 0.01) as compared to uninfected cells, which suggests that infected cells were less likely to be actively duplicating DNA.

infected infected infected

Fig. 3.

SCANLON ET AL.-MICROSPORIDIA AND HOST CELL CYCLING 529

Table 2. Inverse correlation between extent of Encephalitozoon lirllrrn infection and labeling of E6 cells with BrdU. Cyclin B1 Cyclin D1

Size of parasitophorous vacuole ( p n 2 ) % Nuclei labeled with BrdU

1100 23.5 I0 1-250 13.1 25 1-500 8.2

>500 0

Infected cells are not more likely to be in GO. The reduction in mitotic frequency in infected cells was not due to an increase in the number of cells in GO. There was no statistically significant difference in labeling between uninfected cells (84.5 ? 1.2%) and infected cells (82.3 2 3.2%) using immunocytochemical detection of Ki-67, a nuclear protein present in cycling but not quiescent cells.

Alterations in intracellular levels of cyclins B1 and D1 within infected cells. Levels of cyclin D1 and B1 were measured in uninfected E6 cells and in E6 cells infected with E. hellem using Western blot analysis (Fig.4). Levels of cyclin D1 were consistently lower in infected cells (inf) than uninfected cells (ctl); conversely, levels of B1 were consistently higher in infected cells. To rule out the possibility that the increase in cyclin B1 in infected cells was due to contamination from parasitic cyclin B 1, Western blot analysis was performed on sporoplasm concentrated from a mixture of mature spores and developing stages. Anti-cyclin B1 labeled a spore protein of approximately 30 kDa (data not shown), a lower molecular weight than that of host cell cyclin B1; no band was observed at 55 kDa, the apparent molecular weight of host cell cyclin B1 suggesting that the increase in cyclin B1 in infected cells arises from host cell cyclin B1 and not parasitic cyclin B1.

Reduced levels of cyclin D1 were also measured immuno- cytochemically in E. hellem-infected cells. In six replicate experiments, 29.3 t 2.5% of nonconfluent, uninfected cells exhibited detectable cytoplasmic labeling with cyclin D 1 while labeling in E. hellem-infected cells was significantly lower, 18.9 ? 2.8% (p < 0.05).

DISCUSSION Infection is often accompanied by some derangement in the

cell cycle of the infected cell. Many viruses cause arrest in the host cell cycle, such as HIV (Bartz, Rogel, and Emerman 1996; Jowett et al. 1995), hepatitis B virus (Benn and Schneider 1995), Simian virus 40 (Chang et al. 1997), and cytomegalovirus (Jault et al. 1995). In many cases, a cell cycle arrest in the virally-infected cell correlates with a disruption of genomic stability and chromosomal aberrations that lead to neoplasm or immortalization.

With both intracellular and extracellular parasites, there ap- pear to be diverse effects of infection on the host cell cycle. It is logical to assume that it would be to a parasite’s advantage to cause an arrest in the host cell cycle, thereby producing a stable niche for itself. However, in situ, multiple factors may dictate whether cell cycling is increased, decreased or un- changed. In some cases, such as the attachment of Giardia to intestinal enterocytes, it is not easy to determine if the proliferative response of the mucosal epithelium is a direct result of the protozoan infection or a response to host cytokines (Gillen, A1 Thamery, and Ferguson 1982). The role played by malab- sorption and nutritional derangement associated with this infection has not been clarified either (Leitch et al. 1993). In other cases, such as infection of mouse skeletal muscle cells with Trichinella spiratis, the parasite causes a nearly complete dis-

Fig. 4. A representative Western blot of identical amounts of protein from cytoplasmic extracts of uninfected control (ctl) and Enceph- alitozoon hellern-infected cells (in0 probed for cyclin B 1 and for cyclin D1, Molecular weight markers are to the left. As indicated by the density of the bands, levels of cyclin B1 are higher in infected than uninfected cells while the reverse is true for cyclin Dl .

solution of host myofibrils to produce a large nurse cell lacking the morphology typical of a skeletal muscle cell (Despommier 1998). The nuclei of these multinucleate cells dedifferentiate and incorporate tritiated thymidine; the host cell eventually be- comes arrested at the G2/M border. This arrest, and the niche provided to the larva, may last for several years (Jasmer 1993). When mice are infected with Trypanosoma brucei, their B lymphocytes arrest in GO/Gl (Sacco et al. 1994), which probably accounts for the greatly reduced responsiveness of these im- mune cells during chronic infection of the animal with T. brucei. While many parasitic infections result in an arrest or a slowing of the cell cycle (Mosca, Briceno, and Hernandez 1991; Vassella et al. 1997; Yao, Bohnet, and Jasmer 1998), this is not always the case. When a target cell is infected with Coxiella burnetti, a parasite that resides within a PV, the host cell con- tinues to cycle. During mitosis the vacuole and its contents segregate with one of the daughter cells (Roman, Coriz, and Baca 1986). In the case of Theilaria sp., the parasite coordinates its own cell cycle with that of the host cell (Kramer and John- ston 1997).

The Encephalitozoon microsporidia proliferate and differen- tiate within a PV, enlarging the host cell to several times its original volume (Visvesvara et al. 1999) The distended infected cells remain attached and viable without apparently going into apoptosis or necrosis. Infection with another microsporidian, Nosema algerae (Scanlon et al. 1999), or with Toxoplasma gondii, another parasite that resides within a PV (Nash et al. 1998), appears to impart resistance to apoptotic signals within the host cell. In the Encephalitozoon-infected E6 cells we seldom saw evidence of host cell division. The significant reduction in the number of mitotic spindles seen in infected cells coupled with the reduced incorporation of BrdU into host cell nuclei and the lack of morphological evidence of progression to apoptosis sug- gested that the infected host cells might have entered GO. How- ever, there was no difference in the incidence or extent of immunocytochemical staining of Ki-67 antigen between uninfect-

530 J. EUKARYOT. MICROBIOL.. VOL. 47, NO. 6, NOVEMBER-DECEMBER 2000

ed and E. hellem-infected cells suggesting that infected cells were not more likely to be in GO.

Cell cycle analysis of isolated nuclei showed that the percentage of nuclei in S phase was nearly doubled in E. hellem- infected populations. This increase occurred at the expense of the percentage of nuclei in both G1 and G2. The actual difference in the percentage of nuclei in S phase between infected and uninfected cells was probably even larger than that reported because not all of the cells in a given population are infected. The reduction in BrdU incorporation and the increase in the percentage of infected cells in S phase suggest that Encepha- litozoon infection caused slowing of nucleotide incorporation into DNA and exit from S phase. This slowing could have taken place at several points throughout S phase to account for the observed results. In the case of E. hellem-infected cells, the reduction in BrdU incorporation was directly related to the extent of infection.

Cyclins are cellular proteins that exhibit distinct and char- acteristic changes in concentration during the course of the cell cycle. Cyclin D l is a G l cyclin, so called because its concentration rises over the course of G1. Cyclin B1 is a G 2 N cyclin because its concentration is highest during these periods of the cell cycle. It is known that cyclins work in conjunction with a family of constitutively expressed enzymes to regulate passage through the cell cycle. In some cases of cell cycle arrest, the portion of the cell cycle in which the cell has been arrested has been correlated with elevations in a particular cyclin. For this reason, we compared the relative amounts of cyclin B1 and cyclin D1 in uninfected E6 cells to those in E. hellern-infected cells. The reductions in the levels of cyclin D1 and the percentage of nuclei in G1 are consistent with infection inducing a decrease in mitotic frequency and entry into G1. The increase in the levels of cyclin B 1 within infected cells can be explained by impairment of host cell entry into M. However, this expla- nation is not borne out by the observation that the percentage of nuclei in G2/M was reduced in infected cells (Table 1). Thus, the inhibition of host cell division seen in Encephalitozoon- infected E6 cells cannot be explained by a single block in the cell cycle but may be the result of effects of the infection on multiple points within the cycle. The fact that the morphology of infected cells appears normal except for the presence of a PV and the observation that developing and differentiating stages of the parasite are seen even in very large PVs (Visvesvara et al. 1991) indicate that the factors involved in arresting the host cell cycle at several points do not interfere with the met- abolic support that the host cell provides for the proliferation and differentiation of the parasite.

ACKNOWLEDGMENTS The authors thank Drs. Gene McGrady and Mukaila Akin-

bami for helpful discussions on statistics. This work was sup- ported in part by Public Health Service grant RR03034.

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Received: 0.?/20/00, 06/27/00; accepted 07/04/00

infection by microsporidia disrupts the host cell cycle

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