Introduction
Microsporidian infections affect most invertebrates and vertebrates. They are caused by single-celled spore-forming eukaryotic organisms that are obligate intracellular parasites (Vávra and Lukeš, Reference Vávra and Lukeš2013). In recent years, phylogenetic analyses based on conserved proteins, ribosomal DNA sequences, and whole-genome sequencing have revealed that microsporidia are closely related to fungi and are considered highly derived forms of particular fungi (Han and Weiss, Reference Han and Weiss2017). Some microsporidian infections such as those in economic invertebrates including bees, silkworms, and shrimp cause huge losses, whereas those in Daphnia, nematodes, grasshoppers, and mosquitoes play an important role in regulating their population size (Pan et al., Reference Pan, Bao, Ma, Song, Han, Ran, Li and Zhou2018; Bessette and Williams, Reference Bessette and Williams2022). Nosema bombycis (Nb), the microsporidia that infect silkworms, cause severe losses in silk-producing countries such as China and India (Freeman et al., Reference Freeman, Bell and Sommerville2003; Franzen et al., Reference Franzen, Nassonova, Schölmerich and Issi2006).
As microsporidia are obligate intracellular parasites, all stages of their growth and proliferation occur within the host cell. Studies have revealed that microsporidia have lost many metabolic pathways, including those for the synthesis of most nucleotides and amino acids, and are highly dependent on the host cell (Zhang et al., Reference Zhang, Yao, Zhu, Chen, Chen, Sun, Zhang, Wang and Shen2020). They also lack the protein machinery required for oxidative phosphorylation pathway and typical mitochondria, leaving only mitochondria-related organelles called mitosomes (Weiss, Reference Weiss2015; Hacker et al., Reference Hacker, Sendra, Keisham, Filipescu, Lucocq, Salimi, Ferguson, Bhella, MacNeill, Embley and Lucocq2024). The infection and proliferation of microsporidia are energy-consuming processes that are highly dependent on host-derived adenosine triphosphate (ATP). In the intracellular developmental stage, microsporidia do not use their energy metabolism but steal host ATP through a nucleotide transporter (Dolgikh et al., Reference Dolgikh, Semenov and Grigor'ev2002). Thus, microsporidia create an environment conducive to proliferation by directly connecting with and hijacking the metabolic products of the host mitochondria. Encephalitozoon cuniculi obtains ATP by directly binding to the voltage-dependent anion channel (VDAC) proteins of mitochondria (Hacker et al., Reference Hacker, Howell, Bhella and Lucocq2014; Han et al., Reference Han, Ma, Tu, Tomita, Mayoral, Williams, Horta, Huang and Weiss2019). However, the mitochondria are also a means by which host cells resist parasitic pathogens. For example, direct contact between the mitochondria and Toxoplasma gondii limits the uptake of fatty acids by the pathogen, resulting in the inhibition of replication (Pernas et al., Reference Pernas, Bean, Boothroyd and Scorrano2018). Mitochondria also require a large number of purines and deoxyribonucleoside triphosphates (dNTPs) to replicate the mitochondrial DNA, and some parasitic pathogens, such as T. gondii, Trypanosoma cruzi, and Plasmodium falciparum, also require them. Hence, hosts can enhance the uptake of purines or dNTPs by increasing the expression of mitochondrial transport proteins at the pathogen contact sites, ultimately limiting pathogen proliferation (Lyu et al., Reference Lyu, Chen, Meng, Yang, Ye, Niu, Ei-Debs, Gupta and Shen2023; Wang et al., Reference Wang, Yu, Zhang, Zhou, Sun, Xiao, Zhang, Liu, Li, Li, Luo, Xu, Lian, Lin, Wang, Zhang, Guo, Ren and Deng2023). In addition to dNTPs, glutamine and glucose are other nutrients competitively acquired by the mitochondria and parasitic pathogens (Shah-Simpson et al., Reference Shah-Simpson, Lentini, Dumoulin and Burleigh2017; Xia et al., Reference Xia, Ye, Liang, Chen, Zhou, Fang, Zhao, Gupta, Yang, Yuan and Shen2019). Mitochondria can also inhibit proliferation of parasitic pathogens by translocating anti-parasite molecules at the site of contact; for example, accumulation of mitochondrial reactive oxygen species (ROS) has been found at the mitochondria–Plasmodium contact site, resulting in the inhibition of Plasmodium proliferation (Zuzarte-Luís et al., Reference Zuzarte-Luís, Mello-Vieira, Marreiros, Liehl, Chora, Carret, Carvalho and Mota2017).
Translocator protein (TSPO) is an evolutionarily conserved mitochondrial outer membrane protein found in all organisms from archaea and bacteria to insects, vertebrates, plants, fungi, and humans (Hiser et al., Reference Hiser, Montgomery and Ferguson-Miller2021). TSPO plays an indispensable role in many intracellular processes such as regulation of cholesterol transport, steroid hormone synthesis, and programmed cell death (El Chemali et al., Reference El Chemali, Akwa and Massaad-Massade2022). Studies have also shown that TSPO plays an important role in the regulation of cell metabolism and immune response-related functions in the host defence system (Betlazar et al., Reference Betlazar, Middleton, Banati and Liu2020). Tanimoto et al. found that the immunoregulatory role of TSPO was attributable to its regulation of thymocyte apoptosis, and that enhanced TSPO expression protects newborn mice from fatal viral infections (Tanimoto et al., Reference Tanimoto, Onishi, Sato and Kizaki1999). The pro-apoptotic function of TSPO may help reduce viral infections (Everett et al., Reference Everett, Barry, Sun, Lee, Frantz, Berthiaume, McFadden and Bleackley2002). Moreover, a study on chicken showed that changes in the immune response caused by diazepam treatment were related to TSPO-stimulated immune cells (Morgulis and Palermo-Neto, Reference Morgulis and Palermo-Neto2002). The TSPO ligand, midazolam, alters the ability of immune cells to phagocytose Staphylococcus aureus in horses (Massoco and Palermo-Neto, Reference Massoco and Palermo-Neto2003). In addition, Mühling et al. found that midazolam and Ro5-4864 significantly reduced the formation of the immune function markers O2 and H2O2, which confirmed the connection between TSPO and the host immune response (Mühling et al., Reference Mühling, Gonter, Nickolaus, Matejec, Welters, Wolff, Sablotzki, Engel, Krüll, Menges, Fuchs, Dehne and Hempelmann2005).
In Drosophila, TSPO enhances sensitivity to alcohol, mediates host immune response against bacterial infection, and is associated with wing disc cell apoptosis and lifespan of male flies (Lin et al., Reference Lin, Angelin, Da Settimo, Martini, Taliani, Zhu and Wallace2014, Reference Lin, Rittenhouse, Sweeney, Potluri and Wallace2015; Cho et al., Reference Cho, Park, Chung, Shim, Jeon, Yu and Lee2015). We previously identified genes and proteins that undergo expression changes during Nb infection (Yue et al., Reference Yue, Tang, Xu, Yan, Li, Xiao, Fu, Wang, Li and Shen2015; Tang et al., Reference Tang, Zhang, Zhou, Liu and Shen2020). We found that BmTSPO (XM_004926544.3) underwent significant changes at both the mRNA and protein levels, suggesting that BmTSPO may play a role in Nb proliferation. In the present study, we investigated the role of BmTSPO during Nb infection in silkworms. These results may contribute to improved understanding of the function of TSPO in insects, and provide new strategies and methods for controlling microsporidia proliferation and treating pebrine disease.
Materials and methods
Silkworms, pathogens, and cell lines
The silkworm strain P50 and microsporidia N. bombycis (Zhenjiang strain) were obtained from the Laboratory of Silkworm Physiology and Pathology at the Institute of Sericulture (Chinese Academy of Agricultural Sciences, Zhenjiang, China). The B. mori cell line BmN was cultured in TC-100 insect medium (AppliChem, Darmstadt, Germany) supplemented with 10% foetal bovine serum (Invitrogen, Carlsbad, CA, USA), and 1% penicillin/streptomycin (Invitrogen) at 28°C.
Analysis of BmTSPO sequence
Sequence analysis of BmTSPO was performed using the ExPASy proteomics server. The software and websites used were as follows: InterPro for protein function annotation (http://www.ebi.ac.uk/interpro/), TMHMM Server (ver. 2.0) for transmembrane domain structure prediction (http://www.cbs.dtu.dk/services/TMHMM), and SWISS-MODEL for protein tertiary structure prediction (http://swissmodel.expasy.org/). Amino acid sequences of TSPO from different species were downloaded from the GenBank database (https://cipotato.org/genebankcip/) and multiple sequence alignments were performed using MEGA 11 software to construct a phylogenetic tree (neighbour-joining method). Homology colouring was performed using the GeneDoc software.
Analysis of BmTSPO gene expression profile
Silkworms (strain P50) were reared on fresh mulberry leaves at 27–28°C and 70–80% relative humidity. To analyse the tissue distribution of BmTSPO, the ovaries, testes, Malpighian tubules, skin, silk glands, head, haemolymph, fat body and midgut were collected from 5th instar day-3 silkworm larvae. Ninety silkworms were divided into three groups. For each tissue, ten silkworm samples were mixed into one sample. All experiments were repeated three times. BmTSPO expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and the relative expression level was normalised to that of BmTSPO in the ovary.
The relative expression levels of BmTSPO were analysed in 15 developmental stages: 2nd instar larvae in moulting, 3rd instar day-1 larva, 3rd instar larva close to the moulting stage, 3rd instar larva in moulting, 4th instar day-1 larva, 4th instar larva close to the moulting stage, 4th instar larva in moulting, 4th instar larva close to the moulting stage, 5th instar day-1 larva, 5th instar day-3 larva, day-1 in pupation, day-3 in pupation, day-5 in pupation, day-7 in pupation, day-9 in pupation, and moth at day-1. A total of 135 silkworms were divided into three groups and the whole bodies of the three silkworms were ground into a single sample. All experiments were repeated three times. The relative BmTSPO expression level was normalised to that of BmTSPO in 2nd instar of moulting larvae.
Nb suspension was diluted with sterile water to a concentration of 107 spores ml−1 and sprayed onto fresh mulberry leaves and air-dried at 25°C. The 5th instar day-3 larvae were orally fed the prepared leaves, while the control group was fed normal leaves. Midguts of ten silkworms at 0, 12, 24, 48, 72, 96, 120, 144, 168, and 180 h post-infection were collected from the Nb-infected and control groups. All experiments were repeated thrice. The relative expression level of BmTSPO in Nb-infected group was normalised to that of the control group at the same stage.
Overexpression and RNAi-mediated knockdown of BmTSPO
BmTSPO gene sequence (GenBank: XM_004926544.3) was obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/), and specific primers were designed and synthesised (table S1). BmTSPO was amplified by PCR with primers BmTSPO-F and BmTSPO-R using complementary DNA (cDNA) from the midgut of P50 as the template. The PCR product was digested with EcoR I and Xhol I and then ligated into the plasmid PIZ/V5-mCherry digested with the same enzymes to obtain the plasmid PIZ-mCherry-BmTSPO, which expresses BmTSPO fused with mCherry (Yu et al., Reference Yu, Ling, Li, Guo, Xu, Qian and Li2024).
BmTSPO was overexpressed by transfecting BmN cells with the PIZ-mCherry-BmTSPO plasmid. Briefly, 1 μg of plasmid DNA was transformed into 1 × 106 cells using EntransferTM-H4000 transfection reagent (Engreen Biosystem, Beijing, China). Cells were collected 48 h post-transfection and BmTSPO expression was analysed by western blotting using an anti-mCherry antibody (Beyotime, Beijing, China).
RNAi-mediated knockdown of BmTSPO was performed by transfecting BmN cells with BmTSPO small interfering RNAs (siRNAs; table S1). Three sets of double-stranded siRNAs were designed and synthesised based on the BmTSPO sequence. Each siRNA (40 pmol) was transfected into 1 × 106 BmN cells with Lipo8000™ reagent (Beyotime, Suzhou, China). Forty-eight hours after siRNA transfection, BmTSPO expression was measured by qRT-PCR. The cells were then infected with purified Nb spores (spore: cell ratio, 10:1).
Extraction of total RNA and synthesis of cDNA
Total RNA was extracted from silkworms and BmN cells using the total RNA extraction reagent (Vazyme, Nanjing, China). Then, the total RNA was reverse-transcribed into cDNA using HiScript IIQ RT SuperMix for qPCR (Vazyme). Genomic DNA was extracted from silkworms and BmN cells using a DNA Kit (TaKaRa, Japan) according to the manufacturer's instructions.
Western blot analysis
Protein samples were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride transfer membrane (Immobilon®-PSQ Transfer Membrane, Millipore, Ireland). After blocking the membrane with 5% milk dissolved in a phosphate-buffered saline (PBS) buffer containing 1% Tween-20 (PBST; Diamond, Sangon Biotech, Shanghai, China) at room temperature for 1 h, the membrane was incubated with an anti-mCherry primary antibody (Beyotime, Beijing, China) for 2 h at room temperature. Then, the membranes were incubated with goat anti-mouse IgG (H + L) HRP-conjugated secondary antibody (Multi Sciences, Beijing, China) for 1 h. The protein bands were detected using Tanon™ High-sig ECL Western Blotting Substrate with the Tanon machine (Tanon, Shanghai, China).
Detection of ROS, Ca++, ATP, and apoptosis
BmN cells subjected to different treatments (transfected with siRNA or overexpression vectors for 48 h) were cultured in a 96-well plate until the cells reached 70–80% confluence. (1) ROS were detected using a Reactive Oxygen Species Assay Kit (Beyotime, Suzhou, China). DCFH-DA solution (100 μl) was added to each well (final concentration 2 μM) and incubated at 37°C for 20 min. The cells were then washed thrice with PBS. Serum-free culture medium (100 μl well−1) was added to the wells and absorbance was measured using a microplate reader (Sheng Gong, Shanghai, China) at excitation and emission wavelengths of 488 and 525 nm, respectively. (2) Ca++ were detected using the calcium ion fluorescence probe Fura-2AM (Beyotime, Suzhou, China). Fura-2AM solution (100 μl well−1) was added to each well (final concentration of 1 μM) and incubated at 25°C in the dark for 30 min. The cells were then washed thrice with PBS. Serum-free culture medium (100 μl well−1) was added to the wells and the absorbance was measured with a microplate reader at excitation and emission wavelengths of 340 and 510 nm, respectively. (3) ATP concentration was detected using the CellTiter-Lumi™ Luminescent Cell Viability Assay Kit (Beyotime, Suzhou, China). CellTiter-Lumi™ luminescent assay detection reagent (100 μl) was added to each well and incubated at 25°C for 10 min. The luminescence in each well was measured using a microplate reader. (4) The apoptotic level was detected with CellTiter-Lumi™ Luminescent Cell Viability Assay Kit (Beyotime, Suzhou, China). YPI/PI detection solution (100 μl) was added to each well and incubated at 37°C in the dark for 20 min. The plates were analysed using a microplate reader. YPI-positive cells emitted green fluorescence at excitation and emission wavelengths of 488 and 525 nm, respectively. The PI-positive cells emitted red fluorescence at excitation and emission wavelengths of 535 and 617 nm, respectively. The level of cellular apoptosis was determined based on the ratio of YPI/PI-positive cells. Each experiment was repeated thrice.
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
The primers QRT-BmTSPO-F and QRT-BmTSPO-R were used to detect BmTSPO expression. QRT-BmGADPH-F and QRT-BmGADPH-R were used as internal controls to detect GAPDH expression (GenBank accession no: NM_001043921.1). QRT-Nb β-tubulin-F and QRT-Nb β-tubulin-R were used to detect the genomic copies of Nb. QRT-HOP-F, QRT-HOP-R, QRT-DRK-F, QRT-DRK-R, QRT-STAT-F, QRT-STAT-R, QRT-Domeless-F, and QRT-Domeless-R were used to detect the expression of the immune-related genes (table S1).
SYBR® Green PCR Master Mix (approximately 10 μl; TaKaRa) was added to 20 μl reaction cocktail. All the PCR reactions were performed on an ABI PRISM® 7300 Sequence Detection System (Applied Biosystems, CA, USA) under the following cycling conditions: denaturation at 95°C for 2 min, followed by 45 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 40 s. Each reaction was repeated three times. The relative mRNA expression was calculated using the 2−ΔΔct method (Liu et al., Reference Liu, Gu, Xu, Jiang, Li and Wei2023). The statistical significance of the differences in expression was analysed using SPSS 20.
For relative Nb genomic copy number, a standard curve described with the equation y = −2.33x + 38.20 (R 2 = 0.956) was used to show the copy number of Nb relative to that of β-tubulin. Relative Nb genomic copies of Nb were obtained by normalising the treated group to the control group.
Statistical analyses
Statistical analyses were performed by Student's t-test using GraphPad Prism 8.0 (San Diego, CA, USA). All data are presented as the mean ± standard deviation (SD). Statistical significance was set at P < 0.05. All experiments were repeated thrice.
Results
Sequence characteristics of BmTSPO
Cloning and sequencing of BmTSPO (XM_004926544.3) was performed using PCR with specific primers. Sequencing results showed that the cloned BmTSPO sequence shared 99.9% similarity with XM_004926544.3 sequence in the NCBI database. Sequence analysis showed that the BmTSPO protein contains 161 amino acids with a molecular weight of 18 kDa, consistent with the size of the TSPO protein family. BmTSPO contains five transmembrane domains (fig. 1a), and its 3D structure revealed that the transmembrane domains possess an α-helical structure (fig. 1b). Subcellular localisation prediction using Cell-Ploc showed that BmTSPO mainly localises to the mitochondrial membrane. The phylogenetic tree constructed with TSPOs from different insects consisted of five major branches (subgroups). BmTSPO was located in Branch II, forming the smallest branch along with Bombyx mandarina, indicating that the two organisms were closely related. Branch II included species such as Helicoverpa, Spodoptera, and Melitaea, whereas branch I included species such as Papilio. From a classification perspective, branches II and I belong to Lepidoptera, unlike the species in the other branches. This suggests that TSPO phylogenetic relationships between different species are closely related to their classification status (fig. 1c).
Figure 1. Sequence and characteristics of the translocator protein from Bombyx mori (BmTSPO). (a) Positions of the five transmembrane domains, including TM-1, TM-2, TM-3, TM-4, and TM-5 in the BmTSPO sequence. (b) Three dimensional (3D) structure of BmTSPO constructed using Swiss-Model, showing the α-helical structure of its transmembrane domains. (c) Phylogenetic analysis of the TSPO family members from silkworm and other insects.
Expression profile of BmTSPO
QRT-PCR was performed to characterise the expression profile of BmTSPO in different silkworm tissues. The results revealed that BmTSPO expression was highest in the fat body, followed by the midgut. The Malpighian tubules showed the third highest BmTSPO expression, whereas BmTSPO expression in the ovaries, testis, and blood was relatively low (fig. 2a). In the P50 strain of silkworms, BmTSPO was expressed at very low levels during the embryonic and pre-moulting periods (1st instar to 2nd instar dormant period) and could be detected from the 2nd to the 3rd instar dormant periods; however, its expression level was low. BmTSPO expression gradually increased from the 4th instar to the peak in the 5th instar day-3 larva, but significantly decreased on the first day of pupation and remained stable on the 3rd, 5th, 7th, and 9th days of the pupal stage. Subsequently, BmTSPO expression rapidly increased on the first day after moulting before reaching its highest expression level (fig. 2b).
Figure 2. Expression profile of BmTSPO in different tissues and developmental stages. (a) Relative expression of BmTSPO in tissues from the 5th instar day-3 larvae of Bombyx mori, including fat body, midgut, Malpighian tubule, silk gland, head, epidermis, haematocyte, testis, and ovary. Relative expression level of BmTSPO in the tissue was normalised to that in the ovary. (b) Relative expression of BmTSPO in different developmental stages of B. mori. The table on the right lists the 15 developmental stages. All analyses were repeated three times. Relative expression level of BmTSPO at the specific developmental stage was normalised to that in 2nd instar moulting larva.
BmTSPO regulates intracellular ROS, Ca++, and ATP levels
Studies have shown that the TSPO expression in the mitochondria is related to the intracellular levels of ROS, Ca++, and ATP. Therefore, we measured the levels of ROS, Ca++, and ATP in BmN cells after knockdown or overexpression of BmTSPO. Compared to the control group, the cellular levels of ROS, Ca++, and ATP were significantly lower in BmTSPO knockdown cells (fig. 3a–c), whereas they were higher in BmTSPO overexpressing cells (fig. 3d, f). These results demonstrated that BmTSPO regulates intracellular ROS, Ca++, and ATP levels.
Figure 3. Effects of BmTSPO knockdown and overexpression on ROS, Ca++, and ATP level in BmN cells. (a–c) Effects of RNAi-mediated BmTSPO knockdown on intracellular levels of ROS, Ca++, and ATP at 48 h post-siRNA transfection. (d–f) Effects of BmTSPO overexpression on intracellular levels of ROS, Ca++, and ATP at 48 h post-PIZ-mCherry-BmTSPO transfection. Each bar represents the mean ± standard deviation (SD) of three independent experiments. Differences between the two groups were analysed using Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).
Nb infection induces BmTSPO expression
To analyse the expression of BmTSPO in the midgut of silkworms after Nb infection, total RNA was isolated from midgut tissues at different time points post-infection, and qRT-PCR was performed. The results showed that BmTSPO expression increased significantly (1.65-fold) at 12 h post-infection compared to that in the control group. As the infection progressed, the expression of BmTSPO gradually increased and reached a peak at 144 h post-infection. Although there was a slight decrease at 168 and 180 h, the expression levels remained significantly higher than those in the control group. These results indicate that Nb infection induces BmTSPO expression (fig. 4).
Figure 4. Expression profile of BmTSPO in the midgut of silkworms infected with Nosema bombycis. Midgut samples of infected silkworms were collected 0, 12, 24, 48, 72, 96, 120, 144, 168, and 180 h post-Nb infection and BmTSPO expression was analysed. Relative expression level of BmTSPO was normalised to that in the uninfected group. Each bar represents the mean ± standard deviation (SD) of three independent experiments. Differences between the two groups were analysed using Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Knockdown of BmTSPO promotes Nb proliferation
To determine the effect of BmTSPO knockdown on Nb proliferation, three sets of double-stranded siRNAs were designed and transfected into BmN cells. The siRNA-mediated knockdown efficacy was analysed 48 h after transfection. The results showed that BmTSPO expression was significantly downregulated in all three experimental groups, indicating that all three siRNAs efficiently knocked down BmTSPO (fig. 5a). After transfection with siRNA for 48 h, the cells were infected with Nb and the relative genomic copy number of Nb was detected by qRT-PCR 72 h post-infection. The results showed that the genomic copy number of Nb increased significantly after BmTSPO knockdown (fig. 5b), indicating that inhibition of BmTSPO is beneficial for Nb proliferation.
Figure 5. Knockdown of BmTSPO promotes proliferation of Nosema bombycis. (a) Efficiency of RNAi-mediated BmTSPO knockdown at 48 h post-siRNA transfection. Total RNAs were collected to determine the expression of BmTSPO. The relative expression level of BmTSPO was normalised to that in the negative control siRNA-transfected group. (b) Relative copy number of Nb genome following BmTSPO knockdown at 72 h post-Nb infection, DNA were extracted to detect the Nb genomic copy number. The relative number of copies were normalised to that in the negative control siRNA-transfected group. Each bar represents the mean ± standard deviation (SD) of three independent experiments. Differences between the two groups were analysed using Student's t-test (***P < 0.001, ****P < 0.0001).
Overexpression of BmTSPO inhibits Nb proliferation
To overexpress BmTSPO, BmN cells were transfected with the PIZ/V5-BmTSPO-mCherry plasmid (fig. 6a). Quantitative PCR and western blotting (anti-mCherry antibody) showed that BmTSPO was successfully overexpressed in BmN cells at 48 h post-transfection (fig. 6b, c). Quantitative PCR analysis of the relative copy of Nb to that of β-tubulin in total DNA showed that the relative copy of Nb in the BmTSPO overexpression group was reduced by approximately 80% compared to that in the control group (fig. 6d), indicating that overexpression of BmTSPO inhibits Nb proliferation
Figure 6. Overexpression of BmTSPO inhibits Nosema bombycis proliferation. (a) BmN cells transfected with PIZ/V5-BmTSPO-mCherry plasmid were visualised at 48 h post-transfection. (b) Relative expression of BmTSPO at 48 h post-transfection with PIZ/V5-BmTSPO-mCherry plasmid. Relative expression of BmTSPO was normalised to that in the empty plasmid PIZ/V5-mCherry-transfected cells. (c) After transfection with PIZ/V5-BmTSPO-mCherry plasmid for 48 h, the expression of BmTSPO-mCherry fusion protein was detected using an anti-mCherry antibody. Panel 1: mCherry only. Panel 2: BmTSPO + mCherry. (d) BmN cells were transfected with PIZ/V5-BmTSPO-mCherry plasmid for 48 h, and then infected with Nb. Seventy-two hours post-infection, qRT-PCR was performed to detect the relative copy of the Nb genome. Each bar represents the mean ± standard deviation (SD) of three independent experiments. Differences between the two groups were analysed using Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
BmTSPO increases cell apoptosis
As high levels of intracellular ROS and Ca++ may lead to cell apoptosis, we measured the level of cell apoptosis after overexpression and RNAi-mediated knockdown of BmTSPO. The results showed that cell apoptosis was significantly increased following BmTSPO overexpression but decreased following siRNA-mediated BmTSPO knockdown (fig. 7). We also found that, regardless of overexpression or knockdown, the overall level of cell apoptosis decreased significantly in the Nb-infected groups, indicating that Nb infection inhibits host cell apoptosis, which is consistent with the results observed with protozoan parasites (Heussler et al., Reference Heussler, Kuenzi and Rottenberg2001).
Figure 7. Effect of BmTSPO on apoptosis. (a) Apoptotic levels in BmN cells following knockdown of BmTSPO in Nosema bombycis (Nb) infected (Nb+) and uninfected (Nb–) groups. (b) Apoptotic levels following overexpression of BmTSPO in Nb+ and Nb– groups. Each bar represents the mean ± standard deviation (SD) of three independent experiments. Differences between the two groups were analysed using Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
BmTSPO activates IMD and JAK-STAT signalling pathways
To further understand how BmTSPO inhibits Nb replication, transcriptome sequencing was performed to analyse gene expression profile in BmTSPO-overexpressing BmN cells. The results showed that 131 genes were significantly altered, of which 17 were upregulated and 114 were downregulated (table S2). Among these, death-related ced-3/Nedd3-like protein (Dredd 3) and STAT, key genes in the immune deficiency (IMD) and Janus kinase-signal transducer and the activator of the transcription (JAK-STAT) signalling pathways, respectively, were significantly upregulated. Therefore, we hypothesised that BmTSPO might be involved in the activation of the IMD and JAK-STAT signalling pathways. First, we analysed the expression of genes involved in the IMD and JAK-STAT pathways in Nb-infected silkworms. The results showed that Imd and Dredd3 were upregulated over fivefold, whereas Dredd4 and Relish1 were upregulated over twofold in Nb-infected silkworms. Domeless was upregulated 97-fold, followed by STAT, which was upregulated by more than 30-fold (fig. 8a, b) in Nb-infected silkworms. To confirm the relationship between BmTSPO and the signalling pathways, we measured the expression of these genes in BmTSPO overexpressing and BmTSPO knockdown cells. The results showed a significant increase in Imd, Tak1, Dredd3, Dredd4, Fadd, and Relish2 in the IMD pathway, with Relish2 being upregulated by more than 40-fold (fig. 8c). In the JAK-STAT pathway, HOP, STAT, and DRK showed a significant increase, with STAT being upregulated by more than sevenfold (fig. 8d). In BmTSPO knockdown cells, there was a significant decrease in Imd, Dredd3, Dredd4, and Fadd expression in the IMD pathway. Moreover, STAT and DRK expression in the JAK-STAT pathway decreased nearly 10-fold (fig. 8e, f). These results indicate that Nb infection induces the expression of TSPO, which subsequently activates the IMD and JAK-STAT pathways to inhibit Nb proliferation.
Figure 8. Effects of BmTSPO on immune deficiency (IMD) and JAK-STAT signalling pathway genes. (a, b) Expression of IMD and JAK-STAT immune pathway genes at 72 h post-Nosema bombycis (Nb) infection. The relative expression was compared between infected (Nb+) and uninfected (Nb–) groups. (c, d) Expression of IMD and JAK-STAT immune pathway genes at cells overexpressing BmTSPO. The relative expression was compared between PIZ/V5-BmTSPO-mCherry and PIZ/V5-mCherry transfected groups. (e, f) Expression of IMD and JAK-STAT immune pathway genes in cells with BmTSPO knockdown. The relative expression was compared between negative CK and BmTSPO siRNA3-transfected cells. Each bar represents the mean ± standard deviation (SD) of three independent experiments. Differences between the two groups were analysed using Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant).
Discussion
In the present study, we cloned and analysed TSPO in silkworms. BmTSPO contains five transmembrane helices that form the conserved 3D structure of all TSPOs (Hiser et al., Reference Hiser, Montgomery and Ferguson-Miller2021). TSPO is widely distributed in various tissues and organs (Rupprecht et al., Reference Rupprecht, Rammes, Eser, Baghai, Schüle, Nothdurfter, Troxler, Gentsch, Kalkman, Chaperon, Uzunov, McAllister, Bertaina-Anglade, La Rochelle, Tuerck, Floesser, Kiese, Schumacher, Landgraf, Holsboer and Kucher2009). In mammals, TSPO is highly expressed in steroid-synthesising cells of the adrenal gland, gonads, and placenta (Selvaraj et al., Reference Selvaraj, Stocco and Tu2015). In Lepidoptera insects, TSPO is expressed in the midgut, fat body, and prothoracic gland tissues of Manduca sexta (Snyder and Van Antwerpen, Reference Snyder and Van Antwerpen1998). Our data also showed that BmTSPO is highly expressed in the fat body and midgut, whereas its expression is relatively low in the ovaries and testes in silkworm. TSPO expression has been reported to be consistent with ecdysone production (Smith, Reference Smith1995). The TSPO ligand FGIN-1-27 stimulates ecdysone production (Snyder and Van Antwerpen, Reference Snyder and Van Antwerpen1998). These data indicate that the changes in BmTSPO expression before and after the dormant period in silkworms are most likely due to silkworm moulting activity.
Our results indicate that BmTSPO regulates intracellular ROS, Ca++, and ATP levels. Gatliff et al. found that cytosolic Ca++ levels were higher in cells overexpressing TSPO than in cells with TSPO knockdown because TSPO overexpression affects the Ca++ uptake ability of mitochondria (Gatliff et al., Reference Gatliff, East, Singh, Alvarez, Frison, Matic, Ferraina, Sampson, Turkheimer and Campanella2017). Studies have also shown a close relationship between Ca++ and ROS. TSPO regulates mitochondrial Ca++ signalling, leading to an increase in cytosolic Ca++ levels and activation of NADPH oxidase, thereby increasing ROS levels (Halliwell, Reference Halliwell2011; Winterbourn, Reference Winterbourn2015). In TSPO knockout mice, mitochondrial energy metabolism is altered, and oxygen consumption, membrane potential, and ATP levels are significantly reduced (Orrenius et al., Reference Orrenius, Gogvadze and Zhivotovsky2015; Redza-Dutordoir and Averill-Bates, Reference Redza-Dutordoir and Averill-Bates2016).
Through overexpression and RNAi experiments, we found that BmTSPO inhibits Nb proliferation. In Drosophila, the E3 ubiquitin ligase, parkin, mediates the host immune response to bacterial infection through the TSPO-VDAC complex (Cho et al., Reference Cho, Park, Chung, Shim, Jeon, Yu and Lee2015). TSPO inhibits the synthesis of HIV-1 viral envelope glycoproteins through the endoplasmic reticulum-related protein degradation pathway. TSPO knockout or PK1115 inhibition promotes HIV proliferation (Zhou et al., Reference Zhou, Dang and Zheng2014). Infection with P. falciparum induces high expression of the TSPO-VDAC complex, and inhibitors, such as PK11195 and Ro5-4864, inhibit the proliferation of P. falciparum by stimulating zinc porphyrin absorption and ROS aggregation (Bouyer et al., Reference Bouyer, Cueff, Egée, Kmiecik, Maksimova, Glogowska, Gallagher and Thomas2011; Marginedas-Freixa et al., Reference Marginedas-Freixa, Hattab, Bouyer, Halle, Chene, Lefevre, Cambot, Cueff, Schmitt, Gamain, Lacapere, Egee, Bihel, Le Van Kim and Ostuni2016). Infection with Leishmania amazonensis results in decreased TSPO expression; however, PK11195 inhibits the proliferation of L. parasites (Guedes et al., Reference Guedes, Dias, Petersen, Cruz, Almeida, Andrade, Menezes, Borges and Veras2018). We found that Nb infection induced high expression of BmTSPO, but not BmVDAC, whereas PK11195 promoted Nb proliferation (fig. S1). The results for the three parasitic pathogens are completely different, indicating that TSPO plays different roles in the infection processes of the different pathogens.
Regarding the mechanism underlying the effect of BmTSPO on Nb, we found that BmTSPO overexpression significantly increased the level of apoptosis, whereas its knockdown decreased the overall level of apoptosis. Nb infection inhibits the overall level of apoptosis in the host. Studies have shown that Nb inhibits actinomycin D-induced apoptosis in silkworm BmN cells by upregulating anti-apoptotic genes and downregulating pro-apoptotic genes (He et al., Reference He, Fu, Li, Liu, Cai, Man and Lu2015). Encephalitozoon inhibits host cell apoptosis by inhibiting caspase-3 activation as well as phosphorylation, and nuclear entry of the tumour suppressor gene p53 (del Aguila et al., Reference del Aguila, Izquierdo, Granja, Hurtado, Fenoy, Fresno and Revilla2006). Encephalitozoon cuniculi and Vittaforma corneae inhibit staurosporine-induced apoptosis of human THP-1 macrophages (Didier et al., Reference Didier, Sokolova, Alvarez and Bowers2009), whereas Nosema ceranae reduces apoptosis in honeybees by enhancing the transcription of the inhibitor of apoptosis protein (TAP2) (Kurze et al., Reference Kurze, Le Conte, Dussaubat, Erler, Kryger, Lewkowski, Müller, Widder and Moritz2015). Therefore, we hypothesised that BmTSPO-induced apoptosis may not play a dominant role in inhibition of Nb proliferation. As a housekeeping gene, TSPO is essential for maintaining basic cellular functions, including programmed cell death and the regulation of gene expression, and is expressed in all cells in an organism under normal and pathological conditions (Kusumawidjaja et al., Reference Kusumawidjaja, Kayed, Giese, Bauer, Erkan, Giese, Hoheise, Friess and Kleeff2007; Morrow and Tanguay, Reference Morrow and Tanguay2012). Furthermore, studies have shown that TSPO acts as a mitochondrial signal that regulates the expression of nuclear genes (Caballero et al., Reference Caballero, Veenman and Gavish2013; Yasin et al., Reference Yasin, Veenman, Singh, Azrad, Bode, Vainshtein, Caballero, Marek and Gavish2017). Therefore, we performed transcriptome sequencing of cells overexpressing BmTSPO and observed increased expression of genes related to the IMD and JAK-STAT pathways. The primary defence mechanism of silkworms against pathogens relies on innate immunity, including RNA interference and IMD, Toll, and JAK-STAT pathways. Previous studies have indicated that the IMD pathway effectively combats Gram-negative bacterial, viral, fungal, and parasitic infection (Sonenshine and Macaluso, Reference Sonenshine and Macaluso2017; Zeng et al., Reference Zeng, Jaffar, Xu and Qi2022). JAK/STAT pathway also plays an important role in immune regulation by resisting the invasion of different viruses in Drosophila, Bemisia tabaci, Apis mellifera, and some Lepidoptera insects (Souza-Neto et al., Reference Souza-Neto, Sim and Dimopoulos2009; Chen et al., Reference Chen, Eldein, Zhou, Sun, Gao, Sun, Liu and Wang2018; McMenamin et al., Reference McMenamin, Daughenbaugh, Parekh, Pizzorno and Flenniken2018). Dostert et al. confirmed that the JAK-STAT pathway is necessary for antiviral responses in Drosophila, and is activated by bacterial infections in Gambian mosquitoes (Dostert et al., Reference Dostert, Jouanguy, Irving, Troxler, Galiana-Arnoux, Hetru, Hoffmann and Imler2005). Oral infection with Bacillus thuringiensis in silkworms activates the JAK-STAT pathway, resulting in AMP expression (Huang et al., Reference Huang, Cheng, Xu, Cheng, Fang and Xia2009). Ma et al. demonstrated that infection with Nb in silkworms leads to the activation of the JAK-STAT pathway and causes changes in the expression of immune genes (Ma et al., Reference Ma, Li, Pan, Li, Han, Xu, Lan, Chen, Yang, Chen, Sang, Ji, Li, Long and Zhou2013). Moreover, immune signalling pathways do not function in isolation, and some act synergistically (Liu et al., Reference Liu, Liu, Lu, Gong, Zhu, Chen, Liang, Zhu, Kuang, Hu, Cao, Xue and Gong2015; Zhai et al., Reference Zhai, Huang and Yin2018). For example, components of the IMD pathway can activate the JAK-STAT pathway, leading to transcriptional activation of antimicrobial genes (Boutros et al., Reference Boutros, Agaisse and Perrimon2002). Wu et al. found that genes in the IMD and JAK-STAT pathways are activated by S. aureus and Escherichia coli infections in the gut of silkworms, and have a synergistic regulatory effect on infection (Wu et al., Reference Wu, Zhang, He, Shuai, Chen and Ling2010). Our data showed that key genes in the IMD pathway, such as Imd, Dredd3, Dredd4, and Fadd, and key genes in the JAK-STAT pathway, such as HOP, STAT, and DRK were significantly increased following BmTSPO overexpression, indicating that BmTSPO may inhibit Nb cell proliferation by simultaneously activating both the IMD and JAK-STAT pathways.
In conclusion, Nb infection significantly induced BmTSPO expression, which in turn inhibited the proliferation of Nb by promoting apoptosis and activating the IMD and JAK-STAT pathways (fig. 9).
Figure 9. Mechanism of BmTSPO-mediated inhibition of Nosema bombycis (Nb) proliferation. 
Nb infection induces the expression of BmTSPO. 
Increased BmTSPO levels facilitate the transport of Ca++ into the cytoplasm, which promotes the release of ROS and ATP from the mitochondria. Subsequently, the elevated Ca++ and ROS levels promote the release of Cyto C from mitochondria into the cytoplasm, leading to apoptosis of the infected cells. 
BmTSPO also inhibits Nb proliferation by activating the immune deficiency (IMD) and JAK-STAT signalling pathways.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0007485324000385.
Author contributions
Conceptualisation: X. T. and P. Q. Methodology: F. Z. and Y. Z. Investigation and analysis: M. L., Y. S., B. D., Z. H., and M. L. Writing – original draft: M. L. and Y. S. Writing – review and editing: L. W., Y. S., X. T., and Q. Q. Visualisation: Y. F. Funding acquisition: X. T., F. Z., and P. Q. Resources: Y. Z. and Y. F. Supervision: X. T. All authors contributed to the article and approved the submitted version.
Financial support
This work was supported by the Natural Science Foundation of Jiangsu Province (BK20231254), Technology Innovation Fund Project of Zhenjiang City (NY2023004), Open Project of Key Laboratory of Silkworm and Mulberry Genetic Improvement (KL202204, KL202205), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX23-3840).
Competing interests
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
