Simulated Microgravity Reprograms Host Cell Architecture and Mitochondrial Function, Altering Toxoplasma gondii Growth

ABSTRACT

Simulated microgravity (SMG or μG) influences Toxoplasma gondii growth and modifies the cellular structure and function of the host cells. As an obligate intracellular parasite, Toxoplasma gondii depends on the metabolic activity and mitochondrial function of its host. However, the impact of SMG on host–pathogen interactions remains unclear. We investigated the effect of SMG on host cell structure and mitochondrial functions and its impact on Toxoplasma gondii infection. Cultured human foreskin fibroblasts (HFF) and Vero cells under SMG formed stratified cell layers and exhibited morphological mitochondrial abnormalities, such as outer membrane distortion and inner membrane distension. Functional analyses revealed altered mitochondrial membrane potential and intracellular ATP levels without substantial changes in reactive oxygen species. Upon infection, T. gondii showed cell‐type‐dependent growth, increasing in Vero cells but decreasing in HFF at 48 h post‐infection. These findings indicate that SMG‐induced mitochondrial remodeling is associated with altered host cell susceptibility to toxoplasmosis. Our study highlights the role of physical environmental changes in modulating host–pathogen interactions in the context of host cell mitochondrial function.

Simulated microgravity (SMG) induces host‐cell‐specific mitochondrial remodeling that differentially regulates T. gondii intracellular growth. Mitochondrial dysfunction with reduced ATP production is associated with impaired parasite invasion and growth in HFF, whereas mitochondrial adaptation with increased ATP production in Vero cells is associated with enhanced parasite growth.

1. Introduction

Toxoplasma gondii is an obligate intracellular protozoan parasite, recognized as one of the top five economic pathogens [1, 2]. It infects about one‐third of the world's population, causing severe toxoplasmosis, particularly in immunocompromised individuals and congenitally in infants [3, 4]. It has a wide range of intermediate hosts, but the cat is the definitive host [5].

As an obligate intracellular parasite, it depends on its host cells for nutrients and behaves like a scavenger [6]. It uses carbon sources derived from host cells to synthesize some metabolites and is auxotrophic to metabolites such as purines, polyamines, cholesterol, and vitamin precursors like biotin or nicotinamide [7]. Host cell physiology plays a key role in regulating parasite growth, and mitochondria play a central role in coordinating bioenergetic and redox processes during intracellular infections.

Simulated microgravity using a 3D clinostat is becoming a popular approach to mimic the microgravitational environment, as it is more cost effective and less time consuming than doing experiments in actual space [8]. Microgravity is one of the most important characteristics of the space environment, and previous studies have demonstrated that SMG can significantly impact cell morphology, cytoskeletal organization, and cell–cell or cell‐substrate adhesion, leading to three‐dimensional growth and altered tissue‐like architecture [9, 10, 11, 12]. In addition, microgravity influences the molecular responses in plants and mitochondrial function in animal cells [13, 14]. Recent evidence suggests that microgravity can also influence mitochondrial dynamics, including membrane integrity, distribution, and bioenergetic function [15, 16]. Microgravity also influences microbes in the space environment, altering some characteristics of pathogens including their pathogenicity, virulence, growth kinetics, and biofilm formation [17, 18]. We previously found alterations in the proliferation and morphology of Vero cells and HFF in response to SMG (Yamada et al. under publication). In addition, we reported the effect of SMG on the growth of Type II (ME49) Toxoplasma gondii [19]. However, further clarification of the mechanisms of infection under SMG and of host‐pathogen interactions is needed. The changes in host mitochondria may disrupt cellular homeostasis and the metabolic activity required for pathogen replication. Moreover, whether SMG‐induced alterations in host cell architecture and mitochondrial dynamics can modulate T. gondii infection has not been fully explored. Therefore, in the present study, we investigated how SMG reprograms host cell mitochondrial function and metabolic status, and how these changes influence T. gondii growth.

There are three types of T. gondii: type I, II, and III, designated according to their virulence. Type I strains are the most virulent and cause lethal acute infection, type II strains are moderately lethal and have acute tachyzoites and chronic bradyzoites or latent forms of infection, and type III strains are rarely lethal but cause chronic infection [20]. The Vero cell lineage, derived from the kidney tissue of an African green monkey, is widely used to maintain and study Toxoplasma under laboratory conditions. Due to a deficiency in interferon production, these cells are susceptible to a wide range of viruses and pathogens, making them a useful model for studying pathogen replication in the absence of a robust innate immune response [21]. Since Vero cells cannot represent human cells in terms of immune response to pathogenic infections, primary cell human foreskin fibroblasts (HFF) are widely used for experiments with Toxoplasma [22]. We used these cells to observe the growth of a Type I T. gondii (RH‐2F, expressing β‐galactosidase) reporter strain [23].

2. Materials and Methods

2.1. Simulated Microgravity

The microgravitational environment was prepared by using a 3D dual‐axis clinostat (Advanced Engineering Services Co. Ltd., Japan), which was operated at 30 rpm. Medium‐filled culture flasks (iP‐TEC, Sanplatec; cat. 28,642) with semi‐permeable vent caps were placed in centrally and fixed with a band in an incubator at 37°C under 5% CO₂. These flasks were entirely filled with appropriate culture medium for the cell type to avoid disruption in the uniform “free‐fall” condition needed to simulate microgravity. Control flasks were kept in the same incubator in a static position. Before transferring to the clinostat for incubation, the cells were seeded in normal conditions and allowed to form a monolayer. The consistent rotation speed (30 rpm) and configuration of the clinostat were validated for simulating microgravity in cell cultures. The multidirectional continuous rotation randomized the gravity vector over time, leading to a time‐averaged gravitational load approaching microgravity conditions, as described in earlier studies [24, 25].

2.2. Maintenance of the Culture of Host Cells and Parasites

HFF (Human Foreskin Fibroblasts) were cultured in RPMI (Roswell Park Memorial Institute) 1640 medium (cat. 30,264–85, Nacalai Tesque), supplemented with 10% FBS (Fetal Bovine Serum) to supply essential growth factors and nutrients for cell survival and 2% penicillin–streptomycin (cat. 09367–34, Nacalai Tesque) to prevent bacterial contamination. Vero cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium, cat. 08456–65, Nacalai Tesque), supplemented with 10% FBS and 2% penicillin–streptomycin. Vero cells were used to maintain the culture of the type I strain of T. gondii (RH‐2F beta galactosidase), cultured in DMEM with 2% FBS and 2% penicillin–streptomycin. T. gondii was passaged every 3 days. All cultures were maintained at 37°C in a 5% CO₂ incubator.

2.3. Transmission Electron Microscopy (TEM)

Transmission electron microscopy was used to observe the ultrastructure of the cells that were incubated for 7 days in the clinostat. For this experiment, 1 × 10⁶ cells were seeded per flask and incubated for 24 h under normal conditions to allow them to form a monolayer on the bottom surface of the flasks. Then, the culture medium was replaced with new medium, and the flasks were filled entirely to avoid air bubbles. The flasks were then transferred to the clinostat and incubated for 7 days. After certain incubation periods, cells were washed with 1 x PBS and fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (PB, pH adjusted to 7.4) at 4°C. A cell scraper was used to gently remove the fixed cells from the bottom of flasks, which were then centrifuged to obtain a cell pellet. The cell pellets were thoroughly rinsed three times with 0.1 M PB, and then fixed with 1% osmium tetroxide in 0.1 M PB for 2 h at 4°C. After post‐osmication, the samples were immersed and dehydrated through ascending concentrations of ethanol (50%, 70%, 95%, and 100%), transferred into propylene oxide, and then embedded in epoxy resin (Epon 812). An EM UC7i ultra‐microtome (Leica Microsystems) was used to cut ultrathin sections (80‐nm thick) from the samples embedded in Epon 812. The ultrathin sections were not subjected to electron staining with uranyl acetate and lead citrate and were directly examined under an HT7700 transmission electron microscope (Hitachi Co. Ltd.). The brightness and contrast of the captured TIFF data were adjusted by using Photoshop CS6 (Adobe).

2.4. Mitochondrial Membrane Potential

To check the mitochondrial membrane integrity and overall health of the mitochondria, mitochondrial membrane potential was measured using a fluorescent dye, TMRE (Tetramethylrhodamine, ethyl ester), and a membrane‐permeant JC‐1 (5,5,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolcarbocyanine iodide) dye. To measure mitochondrial membrane potential using TMRE, monolayers of Vero cells and HFF were incubated for 7 days under SMG. After incubation, the cells were harvested by trypsinization and centrifuged to obtain the cell pellet. Cell pellets were re‐suspended in medium, and the number of cells was normalized by counting using a cell counter plate (cat. 2–7124‐02, AS ONE AXEL) in a blinded manner. Cells were incubated with 1000 nM TMRE (cat. ab 113,852‐TMRE Mitochondrial Membrane Potential Assay Kit, abcam) for 30 min. After incubation, the cells were washed twice and resuspended in medium, and 5 × 10⁴ cells/100 μL were seeded per well of black 96‐well optical‐bottom plates. Fluorescence intensity was measured using a microplate reader, SH‐9000 Lab (Corona Electric Co. Ltd.), at Ex/Em = 549/575 nm according to the manufacturer's instructions, where it measures the overall mitochondrial membrane potential of the cell population within each well. The representative fluorescence images were captured using Keyence BZ‐X800 to solely illustrate differences in TMRE staining between experimental groups and were not intended for quantitative analysis. Three independent biological replications were conducted with at least three technical replicates. To measure mitochondrial membrane potential using JC‐1 (MitoProbe JC‐1 Assay Kit, cat. M34152, ThermoFisher Scientific) dye (10 μL/mL), HFF were harvested and counted to normalize the number of cells. Cells were incubated for 30 min with medium containing JC‐1 dye. To evaluate the validity of the kits and to confirm the dose of positive control CCCP (carbonyl cyanide m‐chlorophenyl hydrazone), five doses of CCCP (0.25, 0.2, 0.15, 0.1, and 0.05 mM) were used to induce apoptosis 10 min prior to adding JC‐1 dye. Unstained cells were used as a blank to normalize the data. After 30 min of incubation, cells were washed with fresh medium twice and 100 μL of cell suspension was added to each well of 96‐well plates. All the samples were used as triplicates and intensity was measured using SH‐9000 Lab. To check for monomer emission, 490 and 530 nm excitation were used, and to check for polymer emission, 550 nm and 590 nm excitation were used. The ratio of polymer to monomer was used as the membrane potential. For further analysis, 0.05 mM CCCP was used as a positive control. To measure the mitochondrial membrane potential of HFF that were incubated for 7 days by using fluorescence microscopy, cells were harvested, incubated for 30 min, and seeded at 5 × 10⁴ cells/100 μL per well of a black 96‐optical bottom well plate, and more than fifty monomer images from each replication for group were taken with FITC/GFP and TRITC/RFP filters using a Keyence BZ‐X800. The intensity was measured by using Fiji ImageJ software.

2.5. Intracellular ROS

To evaluate cellular damage, intracellular ROS (Reactive Oxygen Species) were measured using the Total Reactive Oxygen Species (ROS) Assay Kit 520 nm (cat. 88–5930‐74, ThermoFisher Scientific). For these experiments, cells were harvested and counted using a cell counter plate; 5 × 10⁴ cells were added per well in a final volume of 100 μL containing reagent (2 μL/mL) and incubated for 60 min in accordance with the manufacturer's instructions. The intensity was checked at 520 nm using the SH‐9000 Lab microplate reader. Three independent experiments were performed, and each sample was used in triplicate.

2.6. Cellular ATP

Metabolic activity and bioenergetic levels of cells were measured using CellTiter‐Glo reagent (cat. G9242, Promega) and Glomax (Promega). For these experiments, cells were collected after 7 days of incubation under SMG, and the number of cells was counted; 5 × 10⁴ cells were seeded per well of an opaque white 96‐well plate and their ATP levels were measured by following the manufacturer's protocol. All samples were used in triplicates, and three independent biological replications were conducted.

2.7. Distribution of Mitochondria

To check the distribution of the mitochondria, 1 × 10⁵ cells were cultured on 13‐mm coverslips for 24 h, transferred to iP‐TEC containers, and incubated for 7 days under SMG. After this incubation, the medium was removed, and cells were washed with 1 x PBS three times and incubated with MitoTracker Red (2 μL/mL; Cat. M22425, Invitrogen, Thermo Fisher Scientific) for 1 h in the dark at 37°C. In the following step, cells were washed with 1 x PBS and co‐stained with Hoechst 33,342 (1 μL/mL, Cat. H3570, Invitrogen, Thermo Fisher Scientific). Samples were fixed with 4% PFA and observed under a Keyence BZ‐X800 microscope, and the distribution of mitochondria was measured per cell using ImageJ software.

2.8. Growth Assay

To investigate the effect of the modulations of host mitochondria on the growth of T. gondii, host cells were collected and seeded on a 96‐well plate for 24 h and then infected with the RH‐2F strain of T. gondii for 48 h. The parasite was quantified using the Beta‐Glo Assay System (cat. E4740, Promega) according to the manufacturer's instructions. Briefly, an equal volume of β‐Glo reagent was added to each well, mixed gently, and incubated at room temperature for 30 min. Luminescence was measured using a microplate reader, Glomax (Promega). Background luminescence from blank control wells was subtracted from all readings. The luminescence intensity was considered proportional to the number of intracellular β‐galactosidase‐expressing parasites and was used as a measure of the parasite. Four independent experiments were conducted with three technical replicates.

2.9. Invasion Assay

The invasion efficiency of T. gondii was evaluated using a β‐galactosidase (β‐Glo) luminescence assay. Host cells were seeded in 96‐well plates for 24 h. Prior to infection, cells were maintained under simulated microgravity for 7 days. Freshly egressed tachyzoites expressing β‐galactosidase were prepared and added to each well at MOI (multiplicity of infection) of 2. Parasites were allowed to invade host cells for 2 h at 37°C in a 5% CO₂ incubator. Following the invasion, non‐invaded extracellular parasites were removed by gently washing the wells with pre‐warmed 1 x PBS. Fresh culture medium was then added to each well, and the plate was incubated for at least 30 min before measurement using the Beta‐Glo Assay System.

For normalization, invasion values were calculated relative to the initial intensity at 0 hpi, which was set to 100%. Each condition was analyzed with three biological and three technical replicates, and data are presented as the mean ± standard error (SE).

2.10. Immunofluorescence Assay (IFA)

An immunofluorescence assay (IFA) was performed to evaluate the intracellular growth of Toxoplasma gondii tachyzoites. The preconditioned HFF (1 × 10⁵) under simulated microgravity for 7 days were seeded onto glass coverslips and cultured for 24 h. The cells were then infected with tachyzoites at a MOI of 1, and incubated for 2 h to allow parasite invasion. After 2 h of invasion, the coverslips were gently washed with 1 x PBS to remove extracellular parasites, and fresh culture medium was added. At 12, 24, and 36 h post‐infection (hpi), the coverslips were collected, washed twice with 1 x PBS, and fixed with 4% paraformaldehyde (PFA). The fixed cells were permeabilized with 0.1% Triton X‐100 in PBS for 10 min at room temperature (RT), followed by two washes with 1 x PBS. Nonspecific antibody binding was blocked by incubating the cells with 3% bovine serum albumin (BSA) in 1 x PBS for 40–45 min at RT.

The cells were then incubated with mouse anti‐SAG1 monoclonal antibody (1:200 dilution) for 2 h at RT, followed by three washes with PBS. Later, the cells were incubated with Alexa Fluor 488‐conjugated secondary antibody (1:1000 dilution) for 40 min at RT in the dark. After three additional PBS washes, the nuclei were co‐stained with Hoechst (2 μg/mL). Finally, the coverslips were mounted onto glass slides, and fluorescence images were acquired using a Keyence BZ‐X800 fluorescence microscope.

2.11. Quantification of Parasite Infection and Intracellular Growth

Immunofluorescence images were analyzed to quantify parasite infection and intracellular replication at 12, 24, and 36 h post‐infection (hpi). For each of the three biological replicates, 50 randomly selected microscopic fields were captured and used for quantitative analysis.

The percentage of infected cells was calculated by dividing the number of infected host cells by the total number of host cell nuclei identified by Hoechst staining and calculated as a percentage. The number of parasitophorous vacuoles (PVs) per infected cell was determined by counting all SAG1‐positive vacuoles within infected host cells. Intracellular parasite replication was assessed by counting the number of tachyzoites in 50 well‐developed PVs per biological replicate for each experimental condition and time point. Images were obtained using a Keyence BZ‐X800 fluorescence microscope, and quantitative data from all biological replicates were used for statistical analysis.

2.12. Statistical Analyses

For all the experiments, three to four biological replications and at least three technical replicates were used. Student's t‐tests assuming equal variance were used for statistical analysis. Results were presented as mean ± standard error (SE). Microsoft Excel and GraphPad Prism 6 were used for statistical analysis and visualization.

3. Results

3.1. Formation of Stratified Cell Layers in HFF and Vero Cells Under Simulated Microgravity

Transmission electron microscopy (TEM) analysis revealed remarkable changes in cellular organization in both HFF and Vero cells following 7 days of exposure to SMG. Under SMG conditions (μG), cells tended to form stratified, multilayered structures consisting of more than five layers. In contrast, 2–3 compact cell layers were observed under normal gravity (1G) conditions (Figure 1).

FIGURE 1.

Transmission Electron Microscopy (TEM) showing that host cells formed stratified cell layers under simulated microgravity (μG) comprising more than five loosely connected cell layers with intercellular cytoplasmic processes, whereas under normal gravity (1G), 2–3 compact, firmly adherent cell layers formed (scale bar = 2 μm).

The stratified arrangement was characterized by loosely connected cell layers with decreased cell–cell contacts and overlapping cytoplasmic extensions, suggesting altered adhesion dynamics and structural organization. Ultrastructural observations revealed that the cells in the upper layers exhibited limited association with the underlying cells, forming disorganized aggregates rather than spreading as a uniform monolayer.

These findings suggest that SMG induces substantial changes in cellular architecture and growth behavior, promoting three‐dimensional organization possibly due to modified cytoskeletal tension, reduced substrate adhesion, or altered mechano‐transduction signaling pathways.

3.2. Alterations in Mitochondrial Membrane Ultrastructure Under Simulated Microgravity

TEM also revealed pronounced ultrastructural alterations in the mitochondria of host cells, especially in HFF following exposure to SMG (μG). Compared to the well‐defined membrane structure observed under normal gravity (1G), the mitochondria in SMG‐treated cells exhibited distinct morphological remodeling of both the outer and inner membranes (Figure 2A). Quantitative analysis of mitochondria revealed that ~6% of abnormal mitochondria were found under simulated microgravity, where ~14% abnormalities were found in cells at normal gravity in Vero cells (Figure 2B). In HFF, ~36% of mitochondria exhibited abnormal features under SMG conditions, compared to ~6% in control cells, suggesting an increase in mitochondrial alterations under SMG (Figure 2C). Data represent a single independent experiment, and fifty mitochondria were evaluated per condition from multiple cells.

FIGURE 2.

Transmission Electron Microscopy (TEM) of Vero cells and HFF under simulated microgravity (μG). (A) Microscopic image revealed mitochondria with deformed, distorted outer membrane and inner membrane distension under simulated microgravity (μG) in HFF, compared to well‐defined mitochondrial membranes under normal gravity (1G) (scale bar = 2 μm). (B, C) Quantification of mitochondrial abnormalities from multiple fields of view across numerous cells ~50 mitochondria were evaluated in each group, which exhibited (B) 6% abnormalities under simulated microgravity and 14% abnormalities in Vero cells under 1G; and (C) 36% abnormalities under SMG and 6% in normal gravity in HFF. Data are derived from a single independent experiment.

Specifically, the outer mitochondrial membrane under SMG (μG) appeared distorted, constricted, or shrunken, whereas the inner mitochondrial membrane was markedly distended or swollen, indicating disruption of the inner membrane architecture in HFF.

Functionally, the outer mitochondrial membrane plays a key role in molecular exchange, allowing the passage of metabolites and ions through channels such as porins [26]. The observed shrinkage may, therefore, indicate restricted permeability and altered metabolite trafficking between the cytosol and mitochondria. The inner mitochondrial membrane is central to redox balance and ATP production as it houses the electron transport chain and maintains the proton gradient required for oxidative phosphorylation [27]. The swelling and structural disorganization of this membrane may reflect impaired electron transport efficiency and disruption of energy metabolism.

Together, our findings indicate that SMG induces mitochondrial dysfunction, where structural alterations in both membranes are closely linked to their respective roles in molecular exchange and bioenergetic regulation.

3.3. Selective Reduction of Mitochondrial Membrane Potential in HFF Confirmed by TMRE and JC‐1 Staining

We used TMRE to measure the overall mitochondrial membrane potential in HFF and observed a significant reduction in mitochondrial membrane potential under SMG. In contrast, no changes were observed in Vero cells (Figure 3A,B). To validate the observation with TMRE, JC‐1 staining was performed in HFF, which showed a decreased ratio of red and green fluorescence intensity, indicating mitochondrial depolarization or decreased mitochondrial integrity (Figure 4C,D).

FIGURE 3.

TMRE staining of Vero cells and HFF under simulated microgravity (μG). (A) Fluorescence microscopy images revealed reduced TMRE signal intensity in HFF, whereas no noticeable change in signal intensity was observed in Vero cells (scale bar = 100 μm). (B) Quantification showing decreased mitochondrial membrane potential in HFF with no significant changes in Vero cells compared to normal gravity (1G) (*p < 0.05).

FIGURE 4.

Staining of HFF with JC‐1 dye under simulated microgravity (μG) compared to normal gravity (1G). (A, B) Validation of the assay using Carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) at different concentrations. (C) Fluorescence microscopy revealed increased proportions of green monomers relative to red aggregated polymers under simulated microgravity, indicating reduced mitochondrial membrane potentials in HFF (scale bar = 100 μm). (D) Quantification of the red/green fluorescence ratio confirmed a significant reduction in mitochondrial membrane potential in HFF under simulated microgravity (μG) (**p < 0.01, and ***p < 0.001).

In line with the TMRE results, JC‐1 analysis confirmed the loss of mitochondrial membrane potential specifically in HFF. JC‐1 staining was not performed in Vero cells, as the TMRE analysis did not indicate any detectable change in mitochondrial polarization in this cell line.

Treatment with carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) effectively induced mitochondrial depolarization, confirming the sensitivity and reliability of the assays used to validate our observations (Figure 4A,B).

3.4. Cell‐Specific Modulation of Oxidative Status and Bioenergetics

Intracellular reactive oxygen species (ROS) levels were assessed to evaluate oxidative status under the experimental conditions. No significant difference in ROS levels was observed in Vero cells. In HFF, a slight increase in ROS was detected; however, this change was not statistically significant (Figure 5A).

FIGURE 5.

Quantification of oxidative damage and bioenergetic status of cells under simulated microgravity (μG). (A) No significant change in intracellular ROS levels was observed in Vero cells or HFF. (B) Cellular ATP levels were increased in Vero cells but decreased in HFF under simulated microgravity (μG) compared to normal gravity (1G) (**p < 0.01, ***p < 0.001, and ****p < 0.0001).

In contrast, cellular bioenergetics, as measured by ATP levels, showed distinct cell type–specific differences. Vero cells exhibited a significant increase in ATP levels, whereas HFF showed reduced ATP production (Figure 5B). These findings indicate that, despite minimal changes in oxidative status, energy metabolism is differentially regulated between the two cell types and bioenergetic status is influenced without severe oxidative damage.

3.5. Differential Remodeling of Mitochondrial Distribution and Content Under Simulated Microgravity

Mitochondrial organization and relative abundance were evaluated using MitoTracker Red staining. In Vero cells, mitochondria exhibited a polarized and organized distribution pattern, which remained unchanged under SMG compared to control conditions (Figure 6A). Quantitative analysis of the fluorescence intensity showed no significant difference in mitochondrial content per cell between the two conditions in Vero cells (Figure 6B).

FIGURE 6.

MitoTracker Red staining of host cells under simulated microgravity (μG). (A) Fluorescence microscopy revealed uniform mitochondrial distribution in Vero cells, but dispersed distribution in HFF (scale bar = 20 μm). (B, C) Quantification of mitochondrial distribution or abundance per cell based on fluorescence intensity measurement in Vero cells (B), and in HFF (C) under simulated microgravity (μG) compared to normal gravity (1G) (****p < 0.0001).

In contrast, HFF displayed a marked alteration in mitochondrial organization under SMG. Mitochondria appeared more dispersed and less polarized throughout the cytoplasm compared to control conditions (Figure 6A). Quantitative analysis further revealed a significant increase in MitoTracker Red fluorescence intensity per cell in HFF under SMG, indicating mitochondrial abundance, accumulation, or fragmentation (Figure 6C).

3.6. Altered Host Cell State Influences T. gondii Replication Under Baseline Culture Conditions

To evaluate the functional consequences of these host cell alterations, HFF and Vero cells were subjected to the experimental conditions and then infected with the RH‐2F strain of T. gondii. Parasite proliferation was quantified at 48 h post‐infection (hpi) using a β‐Glo luminescence assay.

In Vero cells, parasite growth was significantly increased at 48 hpi compared to control conditions. In contrast, in HFF, parasite proliferation was reduced under the same conditions. These findings indicate that prior conditioning of host cells differentially influences T. gondii replication depending on the cell type (Figure 7A).

FIGURE 7.

Simulated microgravity reduces Toxoplasma gondii invasion and intracellular proliferation in HFF cells. (A) Overall parasite growth in preconditioned host cells under simulated microgravity (μG), showing cell‐type dependent growth responses compared to under normal gravity (1G). (B) Quantification of parasite invasion at 2 h post‐infection (hpi) in HFF, presented as the percentage of invaded parasites under 1G and simulated microgravity (μG) conditions. (C) Intracellular replication assessed by counting the number of tachyzoites per parasitophorous vacuole (PV) in HFF at 12, 24, and 36 hpi. (D) Representative immunofluorescence images of T. gondii‐infected HFF cultured under 1G or simulated microgravity at 12, 24, and 36 hpi. Parasites (green, SAG1) and host cell nuclei (blue, Hoechst) are shown. Scale bars = 20 μm. (E) Percentage of infected host cells at 36 hpi. (F) Average number of parasitophorous vacuoles (PVs) per counted cell. Data are presented as the mean ± SE from three independent biological experiments (ns, not significant, ***p < 0.001, and ****p < 0.0001).

To determine whether the reduced parasite growth under simulated microgravity (μG) in HFF was associated with impaired host cell invasion or intracellular replication, invasion assay by luminescence assay and growth assay of T. gondii were conducted by immunofluorescence microscopy. At 2 h post‐infection (hpi), the percentage of invaded parasites was significantly lower under SMG than under normal conditions (Figure 7B), indicating that simulated microgravity reduced the efficiency of parasite invasion into HFF.

Following invasion, intracellular parasite replication was monitored at 12, 24, and 36 hpi by quantifying the number of tachyzoites within each parasitophorous vacuole (PV). No significant difference was observed at 12 hpi; however, parasite replication was significantly reduced under μG at 24 hpi, and this reduction became more pronounced by 36 hpi (Figure 7C). Representative immunofluorescence images confirmed the delayed intracellular proliferation of parasites in μG‐exposed cells compared with the control (Figure 7D).

To further investigate whether the reduction in parasite burden resulted from decreased host cell infection, the percentage of infected cells and the number of PVs per cell were quantified. Simulated microgravity significantly reduced the proportion of infected cells compared with normal gravity (Figure 7E). In contrast, the number of PVs per counted cell did not differ significantly between the two conditions (Figure 7F), indicating that the lower parasite burden under μG was primarily due to reduced invasion and impaired intracellular replication rather than differences in the number of vacuoles established within infected cells.

4. Discussion

Our studies indicate that SMG (or μG) alters the cellular architecture of both HFF and Vero cells, promoting stratified, multilayered growth instead of a typical monolayer. These changes likely result from disrupted cell–cell adhesion and altered cytoskeletal tension, reflecting impaired mechano‐transduction [28, 29]. Actin reorganization and weakened focal adhesions may reduce cell spreading while affecting cell–cell interactions, altering the cytoskeleton and leading to three‐dimensional reorganization [30]. In addition to morphological changes, mitochondrial remodeling was observed, particularly in HFF. The distorted outer mitochondrial membrane suggests restricted metabolite exchange, while distension of the inner membrane indicates impaired oxidative phosphorylation [31, 32]. Together, these results suggest disruption of molecular exchange and energy production. Functionally, HFF exhibited reduced mitochondrial membrane potential and decreased ATP levels without significant changes in ROS levels, indicating mitochondrial dysfunction independent of oxidative stress. In contrast, Vero cells maintained mitochondrial integrity, with stable membrane potential and increased ATP levels, suggesting effective metabolic adaptation to SMG. Differences in mitochondrial organization supported this distinction. Dispersed mitochondrial distribution like that seen in HFF usually reflects enhanced mitochondrial fission leading to fragmentation; however, in certain contexts it also may indicate redistribution or accumulation of mitochondria, particularly during stress or mitophagy [33, 34, 35]. In contrast, no changes in mitochondrial organization were observed in Vero cells. These findings demonstrate structural remodeling with functional impairment in a cell type–specific manner. Importantly, these metabolic differences influenced Toxoplasma gondii replication. Enhanced parasite growth in Vero cells may be driven by increased availability, while the reduced growth observed in HFF likely reflects bioenergetic limitations due to mitochondrial dysfunction.

Our study has several limitations. First, key molecular regulators such as DRP1, MFN1/2, and PINK1/Parkin or any molecular marker of mitophagy were not directly evaluated. Second, our experiments were limited to in vitro cell models, which may not fully reflect physiological host responses. Thirdly, quantitative analysis of mitochondrial morphology including number, size, and fragmentation could not be conducted in the current experimental setup. Lastly, only a Type I strain of T. gondii was used and the mitochondrial function of the parasite was not assessed. Future studies should focus on understanding the molecular mechanisms underlying host–parasite interactions under SMG, especially the relationship between host and parasite mitochondrial function.

Author Contributions

The experiments and the writing of the manuscript were performed by Shirin Musfika Tasnim. Takafumi Yamada conducted the preliminary study with host cells under simulated microgravity and validated the conditions. Daisuke Kondoh and Hiroki Bochimoto carried out the experiments with TEM. Kentaro Kato designed the experiments and provided overall supervision.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request and are restricted from public access due to ethical or privacy concerns.