A novel ATP-binding cassette protein (NoboABCG1.3) p lays a vital role in the proliferation of Nosema bombycis

A novel ATP-binding cassette protein (NoboABCG1.3) p lays a vital role in the proliferation of Nosema bombycis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A novel ATP-binding cassette protein (NoboABCG1.3) p lays a vital role in the proliferation of Nosema bombycis Shaogang He, Shiyi Zheng, Honglin Zhu, Yuanke Hu, Bin Yu, Junhong Wei, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4793566/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Dec, 2024 Read the published version in Parasitology Research → Version 1 posted 11 You are reading this latest preprint version Abstract ATP-binding cassette (ABC) transporter proteins, one of the largest families of membrane transport proteins, participate in almost all biological processes and widely exist in living organisms. Microsporidia are intracellular parasites, they can reduce crop yields and pose a threat to human health. The ABC proteins are also present in microsporidia and plays a critical role in their proliferation and energy transport. In this study, a novel ABC transporter protein of Nosema bombycis named NoboABCG1.3 was identified. The NoboABCG1.3 protein is comprised of 640 amino acids, which contains six transmembrane domains and one nucleotide-binding domain. After N. bombycis infection of cells or tissues, quantitative reverse transcription polymerase chain reaction analysis revealed a progressive elevation in the transcript levels of NoboABCG1.3 . Downregulation of NoboABCG1.3 expression significantly inhibited N. bombycis proliferation. Subsequently, a transgenic cell line stably expressing an interfering fragment of NoboABCG1.3 was established, which exhibited extreme inhibition on the proliferation of N. bombycis . These findings indicate that NoboABCG1.3 plays a crucial role in the proliferation of N. bombycis and holds promise as a target for developing N. bombycis -resistant silkworms. Nosema bombycis ATP-binding cassette (ABC) transporter RNAi resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Microsporidia are intracellular eukaryotic parasites that can infect both vertebrates and invertebrates, causing a wide range of diseases (Cavalier-Smith 1998 ; Pan et al. 2018 ). Nosema bombycis , the first discovered microsporidia with a protective spore wall and high resistance to the external environment (Bigliardi et al. 1996 ; Bigliardi and Sacchi 2001 ), is the causative agent of pébrine, leading to heavy economic loss in sericulture (Bhat et al. 2009 ). Currently, sericulture relies on disinfectants, such as bleach and formaldehyde, to control pébrine, but these methods are low efficiency and pollute the environment (Wang et al. 2010 ). Therefore, there is an urgent need for new prevention and treatment techniques for microsporidian infections to prevent and treat pébrine effectively. ATP-binding cassette (ABC) transporter proteins encompass a diverse array of membrane protein complexes that utilize ATP hydrolysis to enable the transmembrane transport of a wide range of substances (Theodoulou and Kerr 2015 ). These transporters constitute one of the largest families of transmembrane transporter proteins found in all living organisms. ABC transporters typically consist of two domains: the nucleotide-binding domain (NBD) and the transmembrane domain (TMD) (Dean et al. 2001 ; Beis 2015 ). The NBD contains three conserved motifs, Walker A, Walker B, and Walker C, which is crucial for ATP binding and hydrolysis (Okada and Murakami 2022 ). This NBD domain uses the energy of ATP hydrolysis to drive transport (Beis 2015 ). The TMD comprises multiple α-helices that create a channel across the lipid bilayer, facilitating the passage of substrates through the membrane (Biemans-Oldehinkel et al. 2006 ). Based on the sequence homology of their NBDs, eukaryotic ABC transporter proteins are currently classified into eight subfamilies, designated ABCA to ABCH (Sohma 2013 ). Except for a special class of bacterial-type ABCI subfamily transporter protein, the members of the ABCH subfamily have not been found in plants (Yazaki 2006 ). ABC transporters play crucial roles in diverse physiological processes including metabolic detoxification, internal and external signal transduction, lipid homeostasis, viral defense, and antigenic cascade presentation within organisms (Huang and Ecker 2023 ; Moore et al. 2023 ). Moreover, parasite ABC transporters contribute to the development of antibiotic resistance of pathogens by exporting compounds (Okada and Murakami 2022 ). The life cycle of microsporidia encompasses three distinct phases: infection, proliferation, and sporulation. Microsporidia are capable of completing their life cycle exclusively within infected eukaryotic cells. When N. bombycis spores encounter favorable environmental conditions, they deliver sporoplasm into host cells by ejecting polar tubes, followed by proliferation. (Lv et al. 2024 ). Based on this lifestyle, microsporidia lost the biological pathways for synthesizing ATP and nucleotides through a dramatic streamlining of its genome and organelles (Dean et al. 2016 , 2018 ; Freibert et al. 2017 ). Therefore, microsporidia rely heavily on their host cells to provide the energy, cofactors, and nucleic acid components necessary to complete their life cycle (Nakjang et al. 2013 ; Dean et al. 2016 ). Parasite transporter proteins are critical in providing energy and metabolites required for parasite growth and replication, but data on the function of microsporidian transporter proteins are limited (Heinz et al. 2014 ; Dean et al. 2018 ). Previous studies on the ABC transporter of N. bombycis have shown that the NoboABCG1.1 is present on the plasma membrane of mature spores and plays a significant role in pathogen proliferation (He et al. 2019 ). Researches on the ABCGs family of transporter proteins of N. bombycis will fill a gap in this area and has the potential to enhance silkworm resistance to N. bombycis . The application of RNA interference (RNAi) technology to enhance insect resistance to parasites is a proven method. Anti-malarial studies in Anopheles stephensi have shown that the inhibition of key components of the immunoregulatory signaling pathway or energy transmission process by RNAi technology can significantly reduce the number of Plasmodium oocysts, thereby inhibiting the proliferation of Plasmodium (Hentzschel et al. 2020 ; Barletta et al. 2024 ). In anti- Leishmania protozoa studies, silencing the Lutzomyia longipalpis protein tyrosine phosphatase SHP by RNAi significantly reduced the parasite load of L. protozoa in sand flies (Telleria et al. 2023 ). The RNAi technology has also been widely applied in microsporidia researches. In Heterosporis saurida , the infection level in EK-1 cells was reduced by 40% and 60%, respectively, following siRNA-mediated silencing of the ATP/ADP reverse transporter protein 1 and methionine aminopeptidase II (Saleh et al. 2016 ). In Nosema ceranae , feeding in vitro-synthesized dsRNA targeting the ATP/ADP transporter protein gene inhibited microsporidian proliferation (Paldi et al. 2010 ). In addition, the researchers constructed tail-to-tail interference vectors targeting genes related to energy metabolism, material transport, and spore formation of N. bombycis . Transfection of these vectors into cells inhibited the proliferation of N. bombycis . (Huang et al. 2018b ; Zheng et al. 2021 ; Zhu et al. 2022 ). The above studies indicate that RNAi technology has great potential to enhance the resistance of silkworms against N. bombycis . In this study, NoboABCG1.3 , a novel ABC transporter gene from N. bombycis was cloned. Then, the sequence characterization, phylogenetic and transcriptional analysis of NoboABCG1.3 was performed. Additionally, we transiently silenced its expression using RNAi. Our findings indicate that NoboABCG1.3 could serve as a target to inhibit the proliferation of N. bombycis . Furthermore, we established a RNAi-mediated transgenic cell line targeting NoboABCG1.3 and observed significant inhibition of N. bombycis proliferation. Materials and methods Microsporidia and host The N. bombycis were obtained from the China Veterinary Strain Conservation Centre (CVCC 102059) and purified as described previously (Yu et al. 2023 ). The Bombyx mori D9L were reared at 25°C under a 12-h photoperiod and fed fresh mulberry leaves. The Sf9-III cells were cultured in Sf-900™ III SFM (Thermo, USA) and maintained at 28°C. Cloning of NoboABCG1.3 gene The DNA of N. bombycis was isolated using a DNA extraction kit (Omega, USA). Specific primers were designed based on the nucleotide sequence of NBO_7g0021 (GenBank: EOB15203.1). The 5' flanking sequence of NBO_7g0021 was amplified using a Genome Walking kit (Takara, Japan). Subsequently, the obtained 5' flanking sequences were underwent bioinformatic analysis using TBtools software (Chen et al. 2020 ). RNA from N. bombycis -infected Sf9-III cells was extracted using the Total RNA Kit II (Omega, USA), followed by cDNA synthesis using the EvoScript Universal cDNA Master Kit (Roche, China). Specific primers were designed to determine the open reading frame (ORF) of NBO_7g0021 based on the predicted maximum complete ORF sequence. PCR was conducted using the cDNA as a template for amplification. The resulting PCR products were separated on 1% agarose gels and purified using the Gel Extraction Kit (Omega, USA). These products were subsequently ligated into the TOPO vector using the TOPO-Blunt Cloning Kit (Yeasen, China) and sequenced by Sangon (Shanghai, China). Detailed primer sequences can be found in Table S1 . Sequence characterization and phylogenetic analysis Online prediction of the isoelectric point and Molecular masses of the NoboABCG1.3 protein using ExPASy Proteomics ( https://web.expasy.org/protparam/ ). Then, phosphorylation and glycosylation sites of NoboABCG1.3 were anticipated using DTU Health Tech ( https://services.healthtech.dtu.dk/ ) (Krogh et al. 2001 ). TMHMM-2.0 ( https://www.cbs.dtu.dk/services/TMHMM/ ) and SMART ( http://smart.embl ) were used to forecast the transmembrane regions and functional structural domains, respectively. ABC transporter protein sequences from microsporidia were downloaded from NCBI, and multiple sequence alignments were performed using ClustalX 2.1. Phylogenetic trees of NoboABCG1.3 were constructed using MEGA 11.0.10 according to the Poisson correlation model generated using the maximum likelihood method. Phylogenetic trees were landscaped using Evolview 3.0 ( https://www.evolgenius.info/ ) (Subramanian et al. 2019 ). Transcription level analysis of NoboABCG1.3 Fifth instar silkworms were inoculated with N. bombycis spores (1 × 10 5 spores per larva). Midgut samples were dissected at 8 h, 16 h, 1 d, 2 d, 3 d, 4 d, 5 d, and 6 d post-infection and immediately frozen at -80°C for preservation. Sf9-III cells were cultured with N. bombycis spores pre-treated with 0.1 M KOH (spores: cell, 5:1). The cells were harvested at 8 h, 16 h, 1 d, 2 d, 3 d, 4 d, 5 d, and 6 d post-infection and stored at -80°C. RNA from infected Sf9 cells and silkworm midguts was extracted using the E.Z.N.A.™ Total RNA Kit II (OMEGA, USA). Subsequently, the GoScript™ Reverse Transcription System Kit (Promega, USA) was employed to convert 1 µg of total RNA into cDNA following DNase I treatment for DNA removal. For the assessment of NoboABCG1.3 gene expression, RT-PCR was conducted using F-q-NoboABCG1.3 and R-q-NoboABCG1.3 primers, alongside reference gene primers F-q-NoboSSU and R-q-NoboSSU (Table S1 ). RT-PCR cycling conditions included initial denaturation at 95°C for 5 min, followed by 40 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 40 s. NoboABCG1.3 transcription levels were determined using the 2 −ΔΔt method across three independent experiments. RNAi An interfering fragment of 340 bp was designed based on the BLOCK-iT™ RNAi Designer ( https://rnaidesigner.thermofisher.com/rnaiexpress/ ). Fragments were amplified using the F-RI-NoboABCG1.3-T7 and R-RI-NoboABCG1.3-T7 primers (Table S1 ) by PCR. Subsequently, double-stranded RNA (dsRNA) was synthesized using the T7 RiboMAX™ Express Large-Scale RNA Production System (Promega, USA). Sf9-III cells were seeded into 12-well plates (5×10 5 cells/well) and cultured at 28°C for 12 h. Following this, dsRNA targeting NoboABCG1.3 (2 µg) was transfected into the cells, with dsRNA targeting EGFP serving as a negative control (Huang et al. 2018b ). After 6 hours, the culture medium was replaced. Spores germinated in 0.1 M KOH solution were added to the Sf9-III cells at a ratio of 5:1 (spores:cells). Cell samples infected with the spores were collected in phosphate-buffered saline (PBS) or TRIZOL (Invitrogen, USA) at 3 and 5 days post-infection (dpi) and immediately stored at -80°C. Vector construction The NoboABCG1.3 interference fragment was obtained by PCR amplification with primers F-NoboABCG1.3- K-B and R-NoboABCG1.3- S-N , using cDNA of N. bombycis as template. The PCR product was inserted into a pESI vector using the TOPO-TA Cloning Kit (Yeasen, China) to obtain pESI[ K - B -dsNoboABCG1.3- S - N ]. The sense fragment, obtained by digesting pESI[ K - B -dsNoboABCG1.3- S - N ] with Bam HI and Sma I, was inserted into the pSL[IE1-MCS-SV40] vector to construct pSL[IE1-Sense-A3intron-SV40].The antisense fragment, obtained by digesting pESI[ K - B -dsNoboABCG1.3- S - N ] with Kpn I and Not I, was inserted into the pSL[IE1-Sense-A3intron-SV40] vector to construct pSL[IE1-Sense-A3intron-Antisense-SV40]. The expression cassette was digested with Asc I and inserted into piggyBac[A3-Neo + IE2-DsRed] to obtain piggyBac[A3-Neo + IE1-dsNoboABCG1.3 + IE2-DsRed] (dsABCG1.3), so as to construct a stable cell line expression interference fragment. Transgenic cell line establishment and microsporidian infection Sf9-III cells were plated in six-well plates (10 6 cells per well). After incubation at 28°C for 12 hours, the cells were co-transfected with the dsABCG1.3 plasmid (3 µg) and the helper plasmid pHA3PIG (3 µg) using the Cellfectin II DNA Transfection Reagent (Gibco, USA). The medium was replaced with fresh medium after 6 h. After 3 days, the cells were cultured in Sf-900 III SFM supplemented with 1 mg/ml geneticin (G-418; Merck, Germany), with medium changes every 2 days. Transgenic cell lines was obtained after continuous screening for 4 months. Next, both the transgenic Sf9-III cells and control Sf9-III cells were infected with N. bombycis (spores: cells, 5:1). Genomic DNA was isolated from infected Sf9-III cells using the E.Z.N.A.™ Tissue DNA Kit (OMEGA, USA) to evaluate parasite infection. Quantitative Real-time PCR The genome harboring N. bombycis infection served as the template for evaluating parasite presence through quantitative real-time PCR (qPCR). A qPCR standard curve spanning six orders of magnitude (1.0 × 10 2 -10 7 copies) was established as described previously (Huang et al. 2018a ). The qPCR reactions were conducted in 10µL mixtures containing 1µL of standard template or genomic DNA (30ng/µL), 0.2µL of each q-Nobo-β-tubulin primer(10 mM; Table S2 ), 5µL SYBR Green Master Mix (Yeasen, China), and 3.6µL ddH 2 O. The reaction procedure was consistent with previous experiments. Each experiment was repeated three times, with three samples detected each time. Statistical analysis Statistical analysis and data visualization were conducted using GraphPad Prism version 9.5.0 (GraphPad Software, USA). Significance levels were denoted as follows: *p < 0.05 for statistically significant differences, **p < 0.01 for highly significant differences, and ***p < 0.001 for extremely significant differences, determined through multiple t-tests. Results are expressed as means ± standard deviation based on at least three independent experiments. Results Cloning and sequence analysis of NoboABCG1.3 Based on unpublished transcriptome data from the silkworm midgut infected with N. bombycis , the transcription level of NBO_7g0021 gene (GenBank No. EOB15203.1) was found to be high. BLAST analysis showed a 97% identity between the amino acid sequences of NBO_7g0021 and ABCG1 protein of Nosema granulosis (GenBank No. KAF9762884.1). However, NBO_7g0021 lacks multiple transmembrane regions, and its NBD structural domain is incomplete, distinguishing it from typical ABC proteins. The upstream fragment of NBO_7g0021 could not be efficiently amplified by PCR based on the available genomic data, possibly due to incomplete splicing of the genomic database. Subsequently, the upstream sequence of NBO_7g0021 was successfully cloned using the Genome Walking Kit, leading to the amplification of a 1923 bp coding sequence (CDS) of NBO_7g0021 (Fig. 1 a). The amplified products were then sequenced, and the resulting CDS sequences have been uploaded to the China National Center for Bioinformation (Accession Number: C_AA052047.1). Sequence analysis confirmed that the cloned gene contained an ORF of 1923 bp, encoding a polypeptide composed of 640 amino acids with a predicted molecular mass of 73.6 kDa. The protein has a theoretical pI of 8.31. It has no signal peptide and contains six transmembrane regions. There are 82 O-glycosylation sites, 2 N-glycosylation sites, and 55 phosphorylation sites on this protein. The domains of this protein were predicted by SMART, the results showed that it has a nucleotide-binding domain (NBD) located at 39–227 aa. Transmembrane topological analysis indicated that the NBD domain is an extracellular transporter (Fig. 1 b). The above analysis shows that this protein may be an ABC transporter. Multiple sequence alignment revealed high homology of this protein with other ABCGs of N. bombycis . Specifically, it showed 93.05% homology with NoboABCG1.1 (Fig. 2 a). Given its similarity to the ABCG1 protein of N. granulosis , we conducted multiple sequence alignment and phylogenetic analysis involving this protein and 97 other members of the ABCG subfamily retrieved from the TCDB database. The evolutionary tree analysis placed this protein closer to NoboABCG1.1 and NoboABCG1.2, within the same branch as NoboABCG1.1 (Fig. 2 b). Consequently, we named this protein NoboABCG1.3. Transcriptional profile of NoboABCG1.3 in infected cells and silkworm midguts RT-qPCR was employed to assess the transcriptional activity of the NoboABCG1.3 gene in both N. bombycis -infected Sf9-III cells and the silkworm midgut, utilizing NoboSSU as an internal reference. After N. bombycis infected Sf9-III cells, expression of NoboABCG1.3 was detected early during infection and showed continuous increase from 1 to 6 dpi, with a decrease only at 4 dpi. This expression pattern may correlate with the formation of mature spores (Fig. 3 a). In the midgut of silkworms infected with N. bombycis , NoboABCG1.3 expression was initially low during the pre-infection phase, but began to consistently increase after 3 dpi (Fig. 3 b). Transcriptional analysis reveals that NoboABCG1.3 dynamically participates in the proliferation process of N. bombycis . Silencing of NoboABCG1.3 inhibits the proliferation of N. bombycis After introducing dsRNA targeting NoboABCG1.3 into Sf9-III cells, samples were collected at 3 and 5 dpi following infection with N. bombycis . RT-qPCR analysis showed that the transcript levels of NoboABCG1.3 were downregulated in infected cells by approximately 40% and 50% compared to control cells (dsEGFP groups), respectively (Fig. 4 a). The qPCR results showed that N. bombycis can proliferate in both the dsABCG1.3 and dsEGFP groups. However, the pathogen load in cells transfected with dsABCG1.3 was significantly reduced at both 3 and 5 dpi (Fig. 4 b). These findings indicate that NoboABCG1.3 exerts a pivotal role in the proliferation of N. bombycis and represents a promising target for suppressing its growth. To assess the effect of the stabilizing interference system on the proliferation of N. bombycis , the piggyBac plasmid NoboABCG1.3 -dsRNA (Fig. 5 a) was co-transfected with a helper plasmid into Sf9-III cells. After screening with G-418 for 4 months, a red fluorescent transgenic cell line was obtained (Fig. 5 b). Then, the proliferation of N. bombycis in NoboABCG1.3 interfering cell line and the control Sf9-III cell was assessed by qPCR. The results showed that the proliferation of N. bombycis in the NoboABCG1.3 interfering cell line was significantly inhibited compared to Sf9-III cells (Fig. 5 c). This result indicates that silencing NoboABCG1.3 inhibits the proliferation of N. bombycis. Discussion Microsporidia lack mitochondria and numerous essential genes for independent energy metabolism, rendering them reliant on host nutrients for growth and development (Weidner et al. 1999 ; Pan et al. 2013 ). To compensate for these deficiencies, many microsporidia have acquired transporter genes through horizontal gene transfer, facilitating nutrient acquisition and adaptation to parasitism (Nakjang et al. 2013 ; He et al. 2019 ). Among these transporter genes, ABC transporters are particularly significant. ABC transporters typically consist of TMDs and NBD domains (Beis 2015 ). In this study, we cloned NoboABCG1.3 , which contains six TMDs and an NBD domain characteristic of ABC transporters. Previous research has shown that the NBD of NoboABCG1.1 protrudes into the host cytoplasm, likely facilitating ATP binding and hydrolysis for substrate transport (He et al. 2019 ). Phylogenetic analysis indicates that NoboABCG1.3 shares high amino acid sequence similarity with NoboABCG1.1 and brancher similarly, suggesting that NoboABCG1.3 may play a comparable role in the growth and proliferation of N. bombycis . NoboABCG1.3 exhibited consistent expression throughout the infection period, particularly during the proliferative cleavage phase, suggesting its potential significance in material transport. This study demonstrates that interference with NoboABCG1.3 significantly impairs the proliferation of N. bombycis . Therefore, further investigation into the critical role of N. bombycis ABCG in substrate transport is warranted. In Leishmania species, drug resistance often arises due to changes in plasma membrane structure caused by gene amplification or point mutations in the ABCG protein family (Bigot et al. 2023 ). Additionally, PfABCG in Plasmodium falciparum is located on the plasma membrane and plays a role in lipid transport (Edaye and Georges 2015 ). These findings offer valuable insights into substrate transport, drug resistance mechanisms, and energy metabolism in N. bombycis . To date, stable gene editing techniques for N. bombycis have remained elusive. However, a stable RNAi strategy has been established (Saleh et al. 2016 ). Previous studies have successfully developed silkworms resistant to B. mori nucleopolyhedrovirus using RNAi (Jiang et al. 2013 ). In this study, the Sf9-III cell line was induced to be resistant to N. bombycis by RNAi targeting the NoboABCG1.3 gene. Nonetheless, the utility of transgenic cell lines for pathogen resistance is limited under high pathogen loads. Previous research has demonstrated that single-chain antibodies against N. bombycis spore wall proteins, membrane proteins, and secretory proteins can significantly inhibits spore proliferation at the cellular level (Huang et al. 2018b ; Zheng et al. 2021 ; Yu et al. 2023 ). Furthermore, knockdown of key proteins using antibody-guided Trim21 or NSlmb degradation systems effectively inhibits spore growth in cells (Sun et al. 2022 ). These studies suggest that future research should focus on generating single-chain antibodies against NoboABCG1.3 and expressing both dsRNA of NoboABCG1.3 and single-chain antibodies in individual silkworms using transgenic technology. This approach will enable exploration of NoboABCG1.3 function and promoter silkworm resistance to N. bombycis . Conclusions In this investigation, we characterized an ABC transporter within the microsporidian N. bombycis . Following infection of Sf9-III cells and silkworms with N. bombycis , NoboABCG1.3 exhibited heightened expression during the proliferative phase. Knockdown of NoboABCG1.3 in the Sf9-III cell line significantly impeded the proliferation of N. bombycis . These observations underscore the pivotal role of NoboABCG1.3 in the growth dynamics of N. bombycis and set the stage for developing transgenic silkworms resistant to this pathogen. Declarations Acknowledgments We thank Dr. Yun Wang (Department of Cell Biology, College of Basic Medical Sciences, Army Medical University, China) for critical reading of the manuscript. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. Ethics approval and consent to participate This study received approval from the Laboratory Animals Ethics Review Committee of Southwest University (Chongqing, China), ensuring adherence to the Guidelines for Ethical Review of Experimental Animal Welfare (GBT35892-2018) throughout all animal experiments. Consent to participate All authors confirm their participation in the study. Consent for publication All authors consent to publication of the manuscript. Competing interests All authors declare no conflicts of interest. Authors' contributions Shaogang He, Data curation, Investigation, Methodology, Software, Validation, Visualization, Drafting the original manuscript, Reviewing and editing the manuscript. Shiyi Zheng, Data curation, Investigation, Methodology, Software, Validation, Visualization. Honglin Zhu, Investigation, Methodology, Validation. Yuanke Hu, Conceptualization, Investigation, Methodology. Bin Yu, Conceptualization, Formal analysis, Drafting the original manuscript, Reviewing and editing the manuscript. Junhong Wei, Conceptualization, Formal analysis, Reviewing and editing the manuscript. Guoqing Pan, Conceptualization, Data curation, Funding acquisition, Project administration, Supervision. Zeyang Zhou, Conceptualization, Formal analysis, Funding acquisition, Project administration, Resources, Supervision. Chunfeng Li, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Visualization, Drafting the original manuscript, Reviewing and editing the manuscript. Funding This research was funded by the Chongqing Modern Agricultural Industry Technology System (CQMAIT202311) and Natural Science Foundation of Chogqing, China (cstc2021jcyj-cxttX0005). Availability of data and materials The conclusions drawn in this article are substantiated by the integrated data within the text. 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J Mol Biol 305:567–580. https://doi.org/10.1006/jmbi.2000.4315 Lv Q, Hong L, Qi L, et al (2024) Microsporidia dressing up: the spore polaroplast transport through the polar tube and transformation into the sporoplasm membrane. mBio 15:e0274923. https://doi.org/10.1128/mbio.02749-23 Moore JM, Bell EL, Hughes RO, Garfield AS (2023) ABC transporters: human disease and pharmacotherapeutic potential. Trends Mol Med 29:152–172. https://doi.org/10.1016/j.molmed.2022.11.001 Nakjang S, Williams TA, Heinz E, et al (2013) Reduction and expansion in microsporidian genome evolution: new insights from comparative genomics. Genome Biol Evol 5:2285–2303. https://doi.org/10.1093/gbe/evt184 Okada U, Murakami S (2022) Structural and functional characteristics of the tripartite ABC transporter. Microbiology (Reading) 168:. https://doi.org/10.1099/mic.0.001257 Paldi N, Glick E, Oliva M, et al (2010) Effective Gene Silencing in a Microsporidian Parasite Associated with Honeybee (Apis mellifera) Colony Declines. Applied and Environmental Microbiology 76:5960–5964. https://doi.org/10.1128/AEM.01067-10 Pan G, Bao J, Ma Z, et al (2018) Invertebrate host responses to microsporidia infections. Dev Comp Immunol 83:104–113. https://doi.org/10.1016/j.dci.2018.02.004 Pan G, Xu J, Li T, et al (2013) Comparative genomics of parasitic silkworm microsporidia reveal an association between genome expansion and host adaptation. BMC Genomics 14:186. https://doi.org/10.1186/1471-2164-14-186 Saleh M, Kumar G, Abdel-Baki A-A, et al (2016) In Vitro Gene Silencing of the Fish Microsporidian Heterosporis saurida by RNA Interference. Nucleic Acid Ther 26:250–256. https://doi.org/10.1089/nat.2016.0613 Sohma Y (2013) [ABC transporter superfamily]. Nihon Yakurigaku Zasshi 141:222–223. https://doi.org/10.1254/fpj.141.222 Subramanian B, Gao S, Lercher MJ, et al (2019) Evolview v3: a webserver for visualization, annotation, and management of phylogenetic trees. Nucleic Acids Res 47:W270–W275. https://doi.org/10.1093/nar/gkz357 Sun X, Yu B, Zhang R, et al (2022) Generation of Resistance to Nosema bombycis (Dissociodihaplophasida: Nosematidae) by Degrading NbSWP12 Using the Ubiquitin-Proteasome Pathway in Sf9-III Cells. J Econ Entomol 115:2068–2074. https://doi.org/10.1093/jee/toac145 Telleria EL, Tinoco-Nunes B, Forrest DM, et al (2023) Evidence of a conserved mammalian immunosuppression mechanism in Lutzomyia longipalpis upon infection with Leishmania. Front Immunol 14:. https://doi.org/10.3389/fimmu.2023.1162596 Theodoulou FL, Kerr ID (2015) ABC transporter research: going strong 40 years on. Biochem Soc Trans 43:1033–1040. https://doi.org/10.1042/BST20150139 Wang Z, Liao F, Lin J, et al (2010) Inactivation and mechanisms of chlorine dioxide on Nosema bombycis. J Invertebr Pathol 104:134–139. https://doi.org/10.1016/j.jip.2009.11.007 Weidner E, Canning EU, Rutledge CR, Meek CL (1999) Mosquito (Diptera: Culicidae) host compatibility and vector competency for the human myositic parasite Trachipleistophora hominis (Phylum Microspora). J Med Entomol 36:522–525. https://doi.org/10.1093/jmedent/36.4.522 Yazaki K (2006) ABC transporters involved in the transport of plant secondary metabolites. FEBS Lett 580:1183–1191. https://doi.org/10.1016/j.febslet.2005.12.009 Yu B, Zheng R, Bian M, et al (2023) A monoclonal antibody targeting spore wall protein 1 inhibits the proliferation of Nosema bombycis in Bombyx mori. Microbiol Spectr 11:e0068123. https://doi.org/10.1128/spectrum.00681-23 Zheng S, Huang Y, Huang H, et al (2021) The role of NbTMP1, a surface protein of sporoplasm, in Nosema bombycis infection. Parasit Vectors 14:81. https://doi.org/10.1186/s13071-021-04595-8 Zhu F, Xiao S, Qin X, et al (2022) Identification and subcellular localization of NbIAP in the microsporidian Nosema bombycis. J Invertebr Pathol 195:107846. https://doi.org/10.1016/j.jip.2022.107846 Additional Declarations No competing interests reported. Supplementary Files FigureS1uncroppedGels.tif TableS1.OligonucleotideprimersusedforvectorcloningandqPCRanalysis.docx Cite Share Download PDF Status: Published Journal Publication published 19 Dec, 2024 Read the published version in Parasitology Research → Version 1 posted Editorial decision: Revision requested 24 Sep, 2024 Reviews received at journal 20 Sep, 2024 Reviews received at journal 28 Aug, 2024 Reviews received at journal 13 Aug, 2024 Reviewers agreed at journal 11 Aug, 2024 Reviewers agreed at journal 08 Aug, 2024 Reviewers agreed at journal 07 Aug, 2024 Reviewers invited by journal 05 Aug, 2024 Editor assigned by journal 30 Jul, 2024 Submission checks completed at journal 29 Jul, 2024 First submitted to journal 24 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4793566","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":341912265,"identity":"9f45eff7-37e6-4fd4-8fc9-f7a216f44139","order_by":0,"name":"Shaogang He","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Shaogang","middleName":"","lastName":"He","suffix":""},{"id":341912266,"identity":"9c29cacb-a2ce-4f16-b090-9d92c53d3ef4","order_by":1,"name":"Shiyi Zheng","email":"","orcid":"","institution":"Zhejiang University of Medicine, Jinhua Municipal Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shiyi","middleName":"","lastName":"Zheng","suffix":""},{"id":341912267,"identity":"afd80065-2691-4ae4-a9ae-47fad17db8bf","order_by":2,"name":"Honglin Zhu","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Honglin","middleName":"","lastName":"Zhu","suffix":""},{"id":341912268,"identity":"07507c91-8298-4fa2-a3df-4f9dc77ba9ef","order_by":3,"name":"Yuanke Hu","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Yuanke","middleName":"","lastName":"Hu","suffix":""},{"id":341912269,"identity":"993a0931-aeec-4d9a-a7d2-6f2e5b8ca71c","order_by":4,"name":"Bin Yu","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Yu","suffix":""},{"id":341912270,"identity":"2462b36b-97ac-40a9-af88-d15ce8b79a8e","order_by":5,"name":"Junhong Wei","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Junhong","middleName":"","lastName":"Wei","suffix":""},{"id":341912271,"identity":"827118c1-f4d0-4728-b565-fd6be281770b","order_by":6,"name":"Gu oqing Pan","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Gu","middleName":"oqing","lastName":"Pan","suffix":""},{"id":341912272,"identity":"1fd5f49b-e87e-4534-b79d-572bef8b4ad8","order_by":7,"name":"Zeyang Zhou","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Zeyang","middleName":"","lastName":"Zhou","suffix":""},{"id":341912273,"identity":"ed6b971d-b7c5-4a4d-92ac-19e8b84b4333","order_by":8,"name":"Chunfeng Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqElEQVRIiWNgGAWjYBACPmYGhg8fIGwD4rSwMTMwzpxBmhYGBsbZPKRpYWd+2Gzz53BiA3vzNgmGmjvEOIzNsDm3DaiF51iZBMOxZ8Ro4WF/nNtwO7FBIsdMgrHhMFFaGJst/gC1yL8hRQsDG8gWHqK1sBk29rb9N27jSSu2SDhGhBZ+/sMPG378SZPtZz+88caHGiK0IKwDEQkkaBgFo2AUjIJRgAcAAOxTMogUu2TgAAAAAElFTkSuQmCC","orcid":"","institution":"Southwest University","correspondingAuthor":true,"prefix":"","firstName":"Chunfeng","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-07-24 08:21:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4793566/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4793566/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00436-024-08440-6","type":"published","date":"2024-12-19T15:57:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63242574,"identity":"1846d5f7-9f1a-442c-80ce-517f3e2d951f","added_by":"auto","created_at":"2024-08-26 05:02:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1566710,"visible":true,"origin":"","legend":"\u003cp\u003eIdentifying of NoboABCG1.3. (a) Clone of \u003cem\u003eNoboABCG1.3\u003c/em\u003e. The cDNA and genomic DNA of \u003cem\u003eN. bombycis\u003c/em\u003e were used as amplification templates, and the genome of Sf9-III cells was used as a negative control for cloning. (b) Schematic map of the conserved domains of NoboABCG1.3. NBD indicates a nucleotide-binding domain and TMD indicates a transmembrane domain.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4793566/v1/1ee6d75e46c3dca5a5b0f5a5.png"},{"id":63244282,"identity":"1c14b6c3-26f9-4e47-add7-2f92890883b7","added_by":"auto","created_at":"2024-08-26 05:26:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4353885,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid alignment and\u003cstrong\u003e \u003c/strong\u003ephylogenetic analysis of NoboABCG1.3. (a) Amino acid alignment of ABCG transporter family of \u003cem\u003eN. bombycis. \u003c/em\u003eRed box: NBD domain. (b) A maximum-likelihood phylogenetic tree was constructed using sequences from the ABCG transporter family found in the genomes of 17 microsporidian species. NoboABCG1.3 from \u003cem\u003eN. bombycis\u003c/em\u003e is marked with a red star, while other ABCG transporter family members from \u003cem\u003eN. bombycis\u003c/em\u003e are denoted by brown dots.\u003cem\u003e \u003c/em\u003eThe microsporidian species are indicated with the following abbreviations: Anal, \u003cem\u003eAnncaliia algerae\u003c/em\u003e; Encu, \u003cem\u003eEncephalitozoon cuniculi\u003c/em\u003e; Enhe, \u003cem\u003eEncephalitozoon hellem\u003c/em\u003e; Enin, \u003cem\u003eEncephalitozoon intestinalis\u003c/em\u003e; Enro, \u003cem\u003eEncephalitozoon romaleae\u003c/em\u003e; Edae, \u003cem\u003eEdhazardia aedis\u003c/em\u003e; Nobo, \u003cem\u003eNosema bombycis\u003c/em\u003e; Noap, \u003cem\u003eNosema apis\u003c/em\u003e; Noce, \u003cem\u003eNosema ceranae\u003c/em\u003e; Nepa1, \u003cem\u003eNematocida parisii\u003c/em\u003e ERTm1; Nepa3, \u003cem\u003eNematocida parisii\u003c/em\u003e ERTm3; Nesp6, \u003cem\u003eNematocida\u003c/em\u003e sp. 1 ERTm6; Orco, \u003cem\u003eOrdospora colligate\u003c/em\u003e; Psne,\u003cem\u003e Pseudoloma neurophilia\u003c/em\u003e; Trho, \u003cem\u003eTrachipleistophora hominis\u003c/em\u003e; Vacu, \u003cem\u003eVavraia culicis \u003c/em\u003esubsp. floridensis; Vico, \u003cem\u003eVittaforma corneae\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4793566/v1/c12832952de8a94f268bd3d6.png"},{"id":63242567,"identity":"4c036b27-8b87-477a-b74e-6e29c7f4a45e","added_by":"auto","created_at":"2024-08-26 05:02:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":97692,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptional profiling of NoboABCG1.3 involved RT-PCR analysis, utilizing \u003cem\u003eN. bombycis\u003c/em\u003e small subunit ribosomal RNA as an internal standard. (a) Transcription level of \u003cem\u003eNoboABCG1.3\u003c/em\u003e in infected Sf9-III cells. (b) Transcription levels of \u003cem\u003eNoboABCG1.3\u003c/em\u003e in the midgut of infected silkworms. Vertical lines indicate mean ± standard error (\u003cem\u003en \u003c/em\u003e= 3).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4793566/v1/495913ef2b94446ada8c8999.png"},{"id":63242569,"identity":"f138d4ff-b350-410c-9402-b3df6d8422ba","added_by":"auto","created_at":"2024-08-26 05:02:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":196687,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of \u003cem\u003eN. bombycis\u003c/em\u003e proliferation after depression of NoboABCG1.3 via RNAi. (a) Relative expression levels of \u003cem\u003eNoboABCG1.3\u003c/em\u003e in infected Sf9-III cells at 3 and 5 dpi were detected by qRT-PCR. (b) Comparisons the number of \u003cem\u003eN. bombycis\u003c/em\u003e between the dsEGFP group and dsNoboABCG1.3 group by qPCR. The data were analyzed using Student’s t-test, *p \u0026lt; 0.05, **p \u0026lt; 0.01. Vertical lines indicate mean ± standard error (\u003cem\u003en \u003c/em\u003e= 3). EGFP: Enhanced green fuorescent protein.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4793566/v1/8390d57d747f6b3682fed328.png"},{"id":63242571,"identity":"41cbb847-4ae6-429f-a561-8c0378f60311","added_by":"auto","created_at":"2024-08-26 05:02:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5848871,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of NoboABCG1.3 on \u003cem\u003eN. bombycis\u003c/em\u003e proliferation. (a) Schematic diagram of interference vector piggyBac[A3-Neo+IE1-dsNoboABCG1.3+IE2-DsRed] construction. (b) Establishment of transgenic cell lines. Sf9-III cells were co-transfected with RNAi-NoboABCG1.3 plasmid and helper plasmid. The fluorescence of Sf9-III cells was observed using an inverted fluorescence microscope. (c) Comparisons the number of \u003cem\u003eN. bombycis\u003c/em\u003e between the Sf9-III cells and NoboABCG1.3 interfering cell line by qPCR. The data were analyzed using student's t-test, ***p \u0026lt; 0.001. Vertical lines indicate mean ± standard error (n = 3).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4793566/v1/6a92188e629296a94baf96f0.png"},{"id":72202093,"identity":"897dbf5d-7a78-4dc1-8765-36119995f9d2","added_by":"auto","created_at":"2024-12-23 16:14:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12243689,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4793566/v1/1b9dcaaf-fccc-4a3b-b942-5399d40dde66.pdf"},{"id":63242573,"identity":"f113a989-9e5e-40ec-bcf3-b4e1387e963c","added_by":"auto","created_at":"2024-08-26 05:02:43","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":49714054,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1uncroppedGels.tif","url":"https://assets-eu.researchsquare.com/files/rs-4793566/v1/ee2fbd954d1872889d1edadc.tif"},{"id":63242568,"identity":"e45a6ffa-b7c5-4315-b3f7-d6d6fc642eec","added_by":"auto","created_at":"2024-08-26 05:02:42","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14504,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.OligonucleotideprimersusedforvectorcloningandqPCRanalysis.docx","url":"https://assets-eu.researchsquare.com/files/rs-4793566/v1/28fdab740fdd6962741e14be.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel ATP-binding cassette protein (NoboABCG1.3) p lays a vital role in the proliferation of Nosema bombycis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicrosporidia are intracellular eukaryotic parasites that can infect both vertebrates and invertebrates, causing a wide range of diseases (Cavalier-Smith \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Pan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eNosema bombycis\u003c/em\u003e, the first discovered microsporidia with a protective spore wall and high resistance to the external environment (Bigliardi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Bigliardi and Sacchi \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), is the causative agent of p\u0026eacute;brine, leading to heavy economic loss in sericulture (Bhat et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Currently, sericulture relies on disinfectants, such as bleach and formaldehyde, to control p\u0026eacute;brine, but these methods are low efficiency and pollute the environment (Wang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Therefore, there is an urgent need for new prevention and treatment techniques for microsporidian infections to prevent and treat p\u0026eacute;brine effectively.\u003c/p\u003e \u003cp\u003eATP-binding cassette (ABC) transporter proteins encompass a diverse array of membrane protein complexes that utilize ATP hydrolysis to enable the transmembrane transport of a wide range of substances (Theodoulou and Kerr \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These transporters constitute one of the largest families of transmembrane transporter proteins found in all living organisms. ABC transporters typically consist of two domains: the nucleotide-binding domain (NBD) and the transmembrane domain (TMD) (Dean et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Beis \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The NBD contains three conserved motifs, Walker A, Walker B, and Walker C, which is crucial for ATP binding and hydrolysis (Okada and Murakami \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This NBD domain uses the energy of ATP hydrolysis to drive transport (Beis \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The TMD comprises multiple α-helices that create a channel across the lipid bilayer, facilitating the passage of substrates through the membrane (Biemans-Oldehinkel et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Based on the sequence homology of their NBDs, eukaryotic ABC transporter proteins are currently classified into eight subfamilies, designated ABCA to ABCH (Sohma \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Except for a special class of bacterial-type ABCI subfamily transporter protein, the members of the ABCH subfamily have not been found in plants (Yazaki \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). ABC transporters play crucial roles in diverse physiological processes including metabolic detoxification, internal and external signal transduction, lipid homeostasis, viral defense, and antigenic cascade presentation within organisms (Huang and Ecker \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Moore et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, parasite ABC transporters contribute to the development of antibiotic resistance of pathogens by exporting compounds (Okada and Murakami \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe life cycle of microsporidia encompasses three distinct phases: infection, proliferation, and sporulation. Microsporidia are capable of completing their life cycle exclusively within infected eukaryotic cells. When \u003cem\u003eN. bombycis\u003c/em\u003e spores encounter favorable environmental conditions, they deliver sporoplasm into host cells by ejecting polar tubes, followed by proliferation. (Lv et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Based on this lifestyle, microsporidia lost the biological pathways for synthesizing ATP and nucleotides through a dramatic streamlining of its genome and organelles (Dean et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Freibert et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, microsporidia rely heavily on their host cells to provide the energy, cofactors, and nucleic acid components necessary to complete their life cycle (Nakjang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Dean et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Parasite transporter proteins are critical in providing energy and metabolites required for parasite growth and replication, but data on the function of microsporidian transporter proteins are limited (Heinz et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Dean et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Previous studies on the ABC transporter of \u003cem\u003eN. bombycis\u003c/em\u003e have shown that the NoboABCG1.1 is present on the plasma membrane of mature spores and plays a significant role in pathogen proliferation (He et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Researches on the ABCGs family of transporter proteins of \u003cem\u003eN. bombycis\u003c/em\u003e will fill a gap in this area and has the potential to enhance silkworm resistance to \u003cem\u003eN. bombycis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe application of RNA interference (RNAi) technology to enhance insect resistance to parasites is a proven method. Anti-malarial studies in \u003cem\u003eAnopheles stephensi\u003c/em\u003e have shown that the inhibition of key components of the immunoregulatory signaling pathway or energy transmission process by RNAi technology can significantly reduce the number of \u003cem\u003ePlasmodium\u003c/em\u003e oocysts, thereby inhibiting the proliferation of \u003cem\u003ePlasmodium\u003c/em\u003e (Hentzschel et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Barletta et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In anti-\u003cem\u003eLeishmania protozoa\u003c/em\u003e studies, silencing the \u003cem\u003eLutzomyia longipalpis\u003c/em\u003e protein tyrosine phosphatase SHP by RNAi significantly reduced the parasite load of \u003cem\u003eL. protozoa\u003c/em\u003e in sand flies (Telleria et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The RNAi technology has also been widely applied in microsporidia researches. In \u003cem\u003eHeterosporis saurida\u003c/em\u003e, the infection level in EK-1 cells was reduced by 40% and 60%, respectively, following siRNA-mediated silencing of the ATP/ADP reverse transporter protein 1 and methionine aminopeptidase II (Saleh et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In \u003cem\u003eNosema ceranae\u003c/em\u003e, feeding in vitro-synthesized dsRNA targeting the ATP/ADP transporter protein gene inhibited microsporidian proliferation (Paldi et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In addition, the researchers constructed tail-to-tail interference vectors targeting genes related to energy metabolism, material transport, and spore formation of \u003cem\u003eN. bombycis\u003c/em\u003e. Transfection of these vectors into cells inhibited the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e. (Huang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The above studies indicate that RNAi technology has great potential to enhance the resistance of silkworms against \u003cem\u003eN. bombycis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn this study, \u003cem\u003eNoboABCG1.3\u003c/em\u003e, a novel ABC transporter gene from \u003cem\u003eN. bombycis\u003c/em\u003e was cloned. Then, the sequence characterization, phylogenetic and transcriptional analysis of NoboABCG1.3 was performed. Additionally, we transiently silenced its expression using RNAi. Our findings indicate that NoboABCG1.3 could serve as a target to inhibit the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e. Furthermore, we established a RNAi-mediated transgenic cell line targeting NoboABCG1.3 and observed significant inhibition of \u003cem\u003eN. bombycis\u003c/em\u003e proliferation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMicrosporidia and host\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eN. bombycis\u003c/em\u003e were obtained from the China Veterinary Strain Conservation Centre (CVCC 102059) and purified as described previously (Yu et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eBombyx mori\u003c/em\u003e D9L were reared at 25\u0026deg;C under a 12-h photoperiod and fed fresh mulberry leaves.\u003c/p\u003e \u003cp\u003eThe Sf9-III cells were cultured in Sf-900\u0026trade; III SFM (Thermo, USA) and maintained at 28\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCloning of\u003c/b\u003e \u003cb\u003eNoboABCG1.3\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe DNA of \u003cem\u003eN. bombycis\u003c/em\u003e was isolated using a DNA extraction kit (Omega, USA). Specific primers were designed based on the nucleotide sequence of NBO_7g0021 (GenBank: EOB15203.1). The 5' flanking sequence of NBO_7g0021 was amplified using a Genome Walking kit (Takara, Japan). Subsequently, the obtained 5' flanking sequences were underwent bioinformatic analysis using TBtools software (Chen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). RNA from \u003cem\u003eN. bombycis\u003c/em\u003e-infected Sf9-III cells was extracted using the Total RNA Kit II (Omega, USA), followed by cDNA synthesis using the EvoScript Universal cDNA Master Kit (Roche, China). Specific primers were designed to determine the open reading frame (ORF) of NBO_7g0021 based on the predicted maximum complete ORF sequence. PCR was conducted using the cDNA as a template for amplification. The resulting PCR products were separated on 1% agarose gels and purified using the Gel Extraction Kit (Omega, USA). These products were subsequently ligated into the TOPO vector using the TOPO-Blunt Cloning Kit (Yeasen, China) and sequenced by Sangon (Shanghai, China). Detailed primer sequences can be found in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSequence characterization and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eOnline prediction of the isoelectric point and Molecular masses of the NoboABCG1.3 protein using ExPASy Proteomics (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Then, phosphorylation and glycosylation sites of NoboABCG1.3 were anticipated using DTU Health Tech (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Krogh et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). TMHMM-2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cbs.dtu.dk/services/TMHMM/\u003c/span\u003e\u003cspan address=\"https://www.cbs.dtu.dk/services/TMHMM/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl\u003c/span\u003e\u003cspan address=\"http://smart.embl\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used to forecast the transmembrane regions and functional structural domains, respectively. ABC transporter protein sequences from microsporidia were downloaded from NCBI, and multiple sequence alignments were performed using ClustalX 2.1. Phylogenetic trees of NoboABCG1.3 were constructed using MEGA 11.0.10 according to the Poisson correlation model generated using the maximum likelihood method. Phylogenetic trees were landscaped using Evolview 3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.evolgenius.info/\u003c/span\u003e\u003cspan address=\"https://www.evolgenius.info/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Subramanian et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscription level analysis of\u003c/b\u003e \u003cb\u003eNoboABCG1.3\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFifth instar silkworms were inoculated with \u003cem\u003eN. bombycis\u003c/em\u003e spores (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e spores per larva). Midgut samples were dissected at 8 h, 16 h, 1 d, 2 d, 3 d, 4 d, 5 d, and 6 d post-infection and immediately frozen at -80\u0026deg;C for preservation. Sf9-III cells were cultured with \u003cem\u003eN. bombycis\u003c/em\u003e spores pre-treated with 0.1 M KOH (spores: cell, 5:1). The cells were harvested at 8 h, 16 h, 1 d, 2 d, 3 d, 4 d, 5 d, and 6 d post-infection and stored at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eRNA from infected Sf9 cells and silkworm midguts was extracted using the E.Z.N.A.\u0026trade; Total RNA Kit II (OMEGA, USA). Subsequently, the GoScript\u0026trade; Reverse Transcription System Kit (Promega, USA) was employed to convert 1 \u0026micro;g of total RNA into cDNA following DNase I treatment for DNA removal. For the assessment of \u003cem\u003eNoboABCG1.3\u003c/em\u003e gene expression, RT-PCR was conducted using F-q-NoboABCG1.3 and R-q-NoboABCG1.3 primers, alongside reference gene primers F-q-NoboSSU and R-q-NoboSSU (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). RT-PCR cycling conditions included initial denaturation at 95\u0026deg;C for 5 min, followed by 40 cycles at 95\u0026deg;C for 10 s, 60\u0026deg;C for 20 s, and 72\u0026deg;C for 40 s. \u003cem\u003eNoboABCG1.3\u003c/em\u003e transcription levels were determined using the 2\u003csup\u003e\u0026minus;ΔΔt\u003c/sup\u003e method across three independent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNAi\u003c/h2\u003e \u003cp\u003eAn interfering fragment of 340 bp was designed based on the BLOCK-iT\u0026trade; RNAi Designer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rnaidesigner.thermofisher.com/rnaiexpress/\u003c/span\u003e\u003cspan address=\"https://rnaidesigner.thermofisher.com/rnaiexpress/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Fragments were amplified using the F-RI-NoboABCG1.3-T7 and R-RI-NoboABCG1.3-T7 primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) by PCR. Subsequently, double-stranded RNA (dsRNA) was synthesized using the T7 RiboMAX\u0026trade; Express Large-Scale RNA Production System (Promega, USA). Sf9-III cells were seeded into 12-well plates (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) and cultured at 28\u0026deg;C for 12 h. Following this, dsRNA targeting NoboABCG1.3 (2 \u0026micro;g) was transfected into the cells, with dsRNA targeting EGFP serving as a negative control (Huang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e). After 6 hours, the culture medium was replaced. Spores germinated in 0.1 M KOH solution were added to the Sf9-III cells at a ratio of 5:1 (spores:cells). Cell samples infected with the spores were collected in phosphate-buffered saline (PBS) or TRIZOL (Invitrogen, USA) at 3 and 5 days post-infection (dpi) and immediately stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eVector construction\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eNoboABCG1.3\u003c/em\u003e interference fragment was obtained by PCR amplification with primers F-NoboABCG1.3-\u003cem\u003eK-B\u003c/em\u003e and R-NoboABCG1.3-\u003cem\u003eS-N\u003c/em\u003e, using cDNA of \u003cem\u003eN. bombycis\u003c/em\u003e as template. The PCR product was inserted into a pESI vector using the TOPO-TA Cloning Kit (Yeasen, China) to obtain pESI[\u003cem\u003eK\u003c/em\u003e-\u003cem\u003eB\u003c/em\u003e-dsNoboABCG1.3-\u003cem\u003eS\u003c/em\u003e-\u003cem\u003eN\u003c/em\u003e]. The sense fragment, obtained by digesting pESI[\u003cem\u003eK\u003c/em\u003e-\u003cem\u003eB\u003c/em\u003e-dsNoboABCG1.3-\u003cem\u003eS\u003c/em\u003e-\u003cem\u003eN\u003c/em\u003e] with \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eSma\u003c/em\u003eI, was inserted into the pSL[IE1-MCS-SV40] vector to construct pSL[IE1-Sense-A3intron-SV40].The antisense fragment, obtained by digesting pESI[\u003cem\u003eK\u003c/em\u003e-\u003cem\u003eB\u003c/em\u003e-dsNoboABCG1.3-\u003cem\u003eS\u003c/em\u003e-\u003cem\u003eN\u003c/em\u003e] with \u003cem\u003eKpn\u003c/em\u003eI and \u003cem\u003eNot\u003c/em\u003eI, was inserted into the pSL[IE1-Sense-A3intron-SV40] vector to construct pSL[IE1-Sense-A3intron-Antisense-SV40]. The expression cassette was digested with \u003cem\u003eAsc\u003c/em\u003eI and inserted into piggyBac[A3-Neo\u0026thinsp;+\u0026thinsp;IE2-DsRed] to obtain piggyBac[A3-Neo\u0026thinsp;+\u0026thinsp;IE1-dsNoboABCG1.3\u0026thinsp;+\u0026thinsp;IE2-DsRed] (dsABCG1.3), so as to construct a stable cell line expression interference fragment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTransgenic cell line establishment and microsporidian infection\u003c/h2\u003e \u003cp\u003eSf9-III cells were plated in six-well plates (10\u003csup\u003e6\u003c/sup\u003e cells per well). After incubation at 28\u0026deg;C for 12 hours, the cells were co-transfected with the dsABCG1.3 plasmid (3 \u0026micro;g) and the helper plasmid pHA3PIG (3 \u0026micro;g) using the Cellfectin II DNA Transfection Reagent (Gibco, USA). The medium was replaced with fresh medium after 6 h. After 3 days, the cells were cultured in Sf-900 III SFM supplemented with 1 mg/ml geneticin (G-418; Merck, Germany), with medium changes every 2 days. Transgenic cell lines was obtained after continuous screening for 4 months. Next, both the transgenic Sf9-III cells and control Sf9-III cells were infected with \u003cem\u003eN. bombycis\u003c/em\u003e (spores: cells, 5:1). Genomic DNA was isolated from infected Sf9-III cells using the E.Z.N.A.\u0026trade; Tissue DNA Kit (OMEGA, USA) to evaluate parasite infection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real-time PCR\u003c/h2\u003e \u003cp\u003eThe genome harboring \u003cem\u003eN. bombycis\u003c/em\u003e infection served as the template for evaluating parasite presence through quantitative real-time PCR (qPCR). A qPCR standard curve spanning six orders of magnitude (1.0 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e-10\u003csup\u003e7\u003c/sup\u003e copies) was established as described previously (Huang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). The qPCR reactions were conducted in 10\u0026micro;L mixtures containing 1\u0026micro;L of standard template or genomic DNA (30ng/\u0026micro;L), 0.2\u0026micro;L of each q-Nobo-β-tubulin primer(10 mM; Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), 5\u0026micro;L SYBR Green Master Mix (Yeasen, China), and 3.6\u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO. The reaction procedure was consistent with previous experiments. Each experiment was repeated three times, with three samples detected each time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis and data visualization were conducted using GraphPad Prism version 9.5.0 (GraphPad Software, USA). Significance levels were denoted as follows: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for statistically significant differences, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for highly significant differences, and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for extremely significant differences, determined through multiple t-tests. Results are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation based on at least three independent experiments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCloning and sequence analysis of\u003c/b\u003e \u003cb\u003eNoboABCG1.3\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on unpublished transcriptome data from the silkworm midgut infected with \u003cem\u003eN. bombycis\u003c/em\u003e, the transcription level of NBO_7g0021 gene (GenBank No. EOB15203.1) was found to be high. BLAST analysis showed a 97% identity between the amino acid sequences of NBO_7g0021 and ABCG1 protein of \u003cem\u003eNosema granulosis\u003c/em\u003e (GenBank No. KAF9762884.1). However, NBO_7g0021 lacks multiple transmembrane regions, and its NBD structural domain is incomplete, distinguishing it from typical ABC proteins. The upstream fragment of \u003cem\u003eNBO_7g0021\u003c/em\u003e could not be efficiently amplified by PCR based on the available genomic data, possibly due to incomplete splicing of the genomic database. Subsequently, the upstream sequence of \u003cem\u003eNBO_7g0021\u003c/em\u003e was successfully cloned using the Genome Walking Kit, leading to the amplification of a 1923 bp coding sequence (CDS) of \u003cem\u003eNBO_7g0021\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The amplified products were then sequenced, and the resulting CDS sequences have been uploaded to the China National Center for Bioinformation (Accession Number: C_AA052047.1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSequence analysis confirmed that the cloned gene contained an ORF of 1923 bp, encoding a polypeptide composed of 640 amino acids with a predicted molecular mass of 73.6 kDa. The protein has a theoretical pI of 8.31. It has no signal peptide and contains six transmembrane regions. There are 82 O-glycosylation sites, 2 N-glycosylation sites, and 55 phosphorylation sites on this protein. The domains of this protein were predicted by SMART, the results showed that it has a nucleotide-binding domain (NBD) located at 39\u0026ndash;227 aa. Transmembrane topological analysis indicated that the NBD domain is an extracellular transporter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The above analysis shows that this protein may be an ABC transporter.\u003c/p\u003e \u003cp\u003eMultiple sequence alignment revealed high homology of this protein with other ABCGs of \u003cem\u003eN. bombycis\u003c/em\u003e. Specifically, it showed 93.05% homology with NoboABCG1.1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Given its similarity to the ABCG1 protein of \u003cem\u003eN. granulosis\u003c/em\u003e, we conducted multiple sequence alignment and phylogenetic analysis involving this protein and 97 other members of the ABCG subfamily retrieved from the TCDB database. The evolutionary tree analysis placed this protein closer to NoboABCG1.1 and NoboABCG1.2, within the same branch as NoboABCG1.1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Consequently, we named this protein NoboABCG1.3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptional profile of\u003c/b\u003e \u003cb\u003eNoboABCG1.3\u003c/b\u003e \u003cb\u003ein infected cells and silkworm midguts\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRT-qPCR was employed to assess the transcriptional activity of the \u003cem\u003eNoboABCG1.3\u003c/em\u003e gene in both \u003cem\u003eN. bombycis\u003c/em\u003e-infected Sf9-III cells and the silkworm midgut, utilizing \u003cem\u003eNoboSSU\u003c/em\u003e as an internal reference. After \u003cem\u003eN. bombycis\u003c/em\u003e infected Sf9-III cells, expression of \u003cem\u003eNoboABCG1.3\u003c/em\u003e was detected early during infection and showed continuous increase from 1 to 6 dpi, with a decrease only at 4 dpi. This expression pattern may correlate with the formation of mature spores (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In the midgut of silkworms infected with \u003cem\u003eN. bombycis\u003c/em\u003e, \u003cem\u003eNoboABCG1.3\u003c/em\u003e expression was initially low during the pre-infection phase, but began to consistently increase after 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Transcriptional analysis reveals that NoboABCG1.3 dynamically participates in the proliferation process of \u003cem\u003eN. bombycis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing of\u003c/b\u003e \u003cb\u003eNoboABCG1.3\u003c/b\u003e \u003cb\u003einhibits the proliferation of\u003c/b\u003e \u003cb\u003eN. bombycis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter introducing dsRNA targeting \u003cem\u003eNoboABCG1.3\u003c/em\u003e into Sf9-III cells, samples were collected at 3 and 5 dpi following infection with \u003cem\u003eN. bombycis\u003c/em\u003e. RT-qPCR analysis showed that the transcript levels of \u003cem\u003eNoboABCG1.3\u003c/em\u003e were downregulated in infected cells by approximately 40% and 50% compared to control cells (dsEGFP groups), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The qPCR results showed that \u003cem\u003eN. bombycis\u003c/em\u003e can proliferate in both the dsABCG1.3 and dsEGFP groups. However, the pathogen load in cells transfected with dsABCG1.3 was significantly reduced at both 3 and 5 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). These findings indicate that NoboABCG1.3 exerts a pivotal role in the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e and represents a promising target for suppressing its growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the effect of the stabilizing interference system on the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e, the piggyBac plasmid \u003cem\u003eNoboABCG1.3\u003c/em\u003e-dsRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) was co-transfected with a helper plasmid into Sf9-III cells. After screening with G-418 for 4 months, a red fluorescent transgenic cell line was obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Then, the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e in NoboABCG1.3 interfering cell line and the control Sf9-III cell was assessed by qPCR. The results showed that the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e in the NoboABCG1.3 interfering cell line was significantly inhibited compared to Sf9-III cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). This result indicates that silencing \u003cem\u003eNoboABCG1.3\u003c/em\u003e inhibits the proliferation of \u003cem\u003eN. bombycis.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMicrosporidia lack mitochondria and numerous essential genes for independent energy metabolism, rendering them reliant on host nutrients for growth and development (Weidner et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Pan et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). To compensate for these deficiencies, many microsporidia have acquired transporter genes through horizontal gene transfer, facilitating nutrient acquisition and adaptation to parasitism (Nakjang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; He et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Among these transporter genes, ABC transporters are particularly significant. ABC transporters typically consist of TMDs and NBD domains (Beis \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this study, we cloned \u003cem\u003eNoboABCG1.3\u003c/em\u003e, which contains six TMDs and an NBD domain characteristic of ABC transporters. Previous research has shown that the NBD of NoboABCG1.1 protrudes into the host cytoplasm, likely facilitating ATP binding and hydrolysis for substrate transport (He et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Phylogenetic analysis indicates that NoboABCG1.3 shares high amino acid sequence similarity with NoboABCG1.1 and brancher similarly, suggesting that NoboABCG1.3 may play a comparable role in the growth and proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNoboABCG1.3\u003c/em\u003e exhibited consistent expression throughout the infection period, particularly during the proliferative cleavage phase, suggesting its potential significance in material transport. This study demonstrates that interference with \u003cem\u003eNoboABCG1.3\u003c/em\u003e significantly impairs the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e. Therefore, further investigation into the critical role of \u003cem\u003eN. bombycis\u003c/em\u003e ABCG in substrate transport is warranted. In \u003cem\u003eLeishmania\u003c/em\u003e species, drug resistance often arises due to changes in plasma membrane structure caused by gene amplification or point mutations in the ABCG protein family (Bigot et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, PfABCG in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e is located on the plasma membrane and plays a role in lipid transport (Edaye and Georges \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These findings offer valuable insights into substrate transport, drug resistance mechanisms, and energy metabolism in \u003cem\u003eN. bombycis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo date, stable gene editing techniques for \u003cem\u003eN. bombycis\u003c/em\u003e have remained elusive. However, a stable RNAi strategy has been established (Saleh et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Previous studies have successfully developed silkworms resistant to \u003cem\u003eB. mori\u003c/em\u003e nucleopolyhedrovirus using RNAi (Jiang et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this study, the Sf9-III cell line was induced to be resistant to \u003cem\u003eN. bombycis\u003c/em\u003e by RNAi targeting the \u003cem\u003eNoboABCG1.3\u003c/em\u003e gene. Nonetheless, the utility of transgenic cell lines for pathogen resistance is limited under high pathogen loads. Previous research has demonstrated that single-chain antibodies against \u003cem\u003eN. bombycis\u003c/em\u003e spore wall proteins, membrane proteins, and secretory proteins can significantly inhibits spore proliferation at the cellular level (Huang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, knockdown of key proteins using antibody-guided Trim21 or NSlmb degradation systems effectively inhibits spore growth in cells (Sun et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These studies suggest that future research should focus on generating single-chain antibodies against \u003cem\u003eNoboABCG1.3\u003c/em\u003e and expressing both dsRNA of \u003cem\u003eNoboABCG1.3\u003c/em\u003e and single-chain antibodies in individual silkworms using transgenic technology. This approach will enable exploration of NoboABCG1.3 function and promoter silkworm resistance to \u003cem\u003eN. bombycis\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this investigation, we characterized an ABC transporter within the microsporidian \u003cem\u003eN. bombycis\u003c/em\u003e. Following infection of Sf9-III cells and silkworms with \u003cem\u003eN. bombycis\u003c/em\u003e, \u003cem\u003eNoboABCG1.3\u003c/em\u003e exhibited heightened expression during the proliferative phase. Knockdown of \u003cem\u003eNoboABCG1.3\u003c/em\u003e in the Sf9-III cell line significantly impeded the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e. These observations underscore the pivotal role of NoboABCG1.3 in the growth dynamics of \u003cem\u003eN. bombycis\u003c/em\u003e and set the stage for developing transgenic silkworms resistant to this pathogen.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Yun Wang (Department of Cell Biology, College of Basic Medical Sciences, Army Medical University, China) for critical reading of the manuscript. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received approval from the Laboratory Animals Ethics Review Committee of Southwest University (Chongqing, China), ensuring adherence to the Guidelines for Ethical Review of Experimental Animal Welfare (GBT35892-2018) throughout all animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors confirm their participation in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to publication of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShaogang He, Data curation, Investigation, Methodology, Software, Validation, Visualization, Drafting the original manuscript, Reviewing and editing the manuscript.\u003c/p\u003e\n\u003cp\u003eShiyi Zheng, Data curation, Investigation, Methodology, Software, Validation, Visualization.\u003c/p\u003e\n\u003cp\u003eHonglin Zhu, Investigation, Methodology, Validation.\u003c/p\u003e\n\u003cp\u003eYuanke Hu, Conceptualization, Investigation, Methodology.\u003c/p\u003e\n\u003cp\u003eBin Yu, Conceptualization, Formal analysis, Drafting the original manuscript, Reviewing and editing the manuscript.\u003c/p\u003e\n\u003cp\u003eJunhong Wei, Conceptualization, Formal analysis, Reviewing and editing the manuscript.\u003c/p\u003e\n\u003cp\u003eGuoqing Pan, Conceptualization, Data curation, Funding acquisition, Project administration, Supervision.\u003c/p\u003e\n\u003cp\u003eZeyang Zhou, Conceptualization, Formal analysis, Funding acquisition, Project administration, Resources, Supervision.\u003c/p\u003e\n\u003cp\u003eChunfeng Li, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Visualization, Drafting the original manuscript, Reviewing and editing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Chongqing Modern Agricultural Industry Technology System (CQMAIT202311) and Natural Science Foundation of Chogqing, China (cstc2021jcyj-cxttX0005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe conclusions drawn in this article are substantiated by the integrated data within the text. Access to all data is available upon reasonable request without constraints. For data uploaded to the China National Center for Bioinformation, please visit https://www.cncb.ac.cn and log in to access.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBarletta A-BF, Smith JC, Burkart E, et al (2024) Mosquito midgut stem cell cellular defense response limits Plasmodium parasite infection. Nat Commun 15:1422. https://doi.org/10.1038/s41467-024-45550-2\u003c/li\u003e\n \u003cli\u003eBeis K (2015) Structural basis for the mechanism of ABC transporters. Biochem Soc Trans 43:889\u0026ndash;893. https://doi.org/10.1042/BST20150047\u003c/li\u003e\n \u003cli\u003eBhat S, Bashir I, Kamili A (2009) Microsporidiosis of silkworm, Bombyx mori L. (Lepidoptera bombycidae): A review. Afr J Agric Res 4:\u003c/li\u003e\n \u003cli\u003eBiemans-Oldehinkel E, Doeven MK, Poolman B (2006) ABC transporter architecture and regulatory roles of accessory domains. 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FEBS Lett 580:1183\u0026ndash;1191. https://doi.org/10.1016/j.febslet.2005.12.009\u003c/li\u003e\n \u003cli\u003eYu B, Zheng R, Bian M, et al (2023) A monoclonal antibody targeting spore wall protein 1 inhibits the proliferation of Nosema bombycis in Bombyx mori. Microbiol Spectr 11:e0068123. https://doi.org/10.1128/spectrum.00681-23\u003c/li\u003e\n \u003cli\u003eZheng S, Huang Y, Huang H, et al (2021) The role of NbTMP1, a surface protein of sporoplasm, in Nosema bombycis infection. Parasit Vectors 14:81. https://doi.org/10.1186/s13071-021-04595-8\u003c/li\u003e\n \u003cli\u003eZhu F, Xiao S, Qin X, et al (2022) Identification and subcellular localization of NbIAP in the microsporidian Nosema bombycis. J Invertebr Pathol 195:107846. https://doi.org/10.1016/j.jip.2022.107846\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Nosema bombycis, ATP-binding cassette (ABC) transporter, RNAi, resistance","lastPublishedDoi":"10.21203/rs.3.rs-4793566/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4793566/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eATP-binding cassette (ABC) transporter proteins, one of the largest families of membrane transport proteins, participate in almost all biological processes and widely exist in living organisms. Microsporidia are intracellular parasites, they can reduce crop yields and pose a threat to human health. The ABC proteins are also present in microsporidia and plays a critical role in their proliferation and energy transport. In this study, a novel ABC transporter protein of \u003cem\u003eNosema bombycis\u003c/em\u003e named NoboABCG1.3 was identified. The NoboABCG1.3 protein is comprised of 640 amino acids, which contains six transmembrane domains and one nucleotide-binding domain. After \u003cem\u003eN. bombycis\u003c/em\u003e infection of cells or tissues, quantitative reverse transcription polymerase chain reaction analysis revealed a progressive elevation in the transcript levels of \u003cem\u003eNoboABCG1.3\u003c/em\u003e. Downregulation of \u003cem\u003eNoboABCG1.3\u003c/em\u003e expression significantly inhibited \u003cem\u003eN. bombycis\u003c/em\u003e proliferation. Subsequently, a transgenic cell line stably expressing an interfering fragment of \u003cem\u003eNoboABCG1.3\u003c/em\u003e was established, which exhibited extreme inhibition on the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e. These findings indicate that NoboABCG1.3 plays a crucial role in the proliferation of \u003cem\u003eN. bombycis\u003c/em\u003e and holds promise as a target for developing \u003cem\u003eN. bombycis\u003c/em\u003e-resistant silkworms.\u003c/p\u003e","manuscriptTitle":"A novel ATP-binding cassette protein (NoboABCG1.3) p lays a vital role in the proliferation of Nosema bombycis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-26 05:02:38","doi":"10.21203/rs.3.rs-4793566/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-25T00:59:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-20T14:35:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-28T10:55:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-13T06:48:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"198138834464050709496687538756622640159","date":"2024-08-11T14:18:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172570433992794956089390226588230531135","date":"2024-08-08T17:47:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296700750731930765884848251665518330654","date":"2024-08-07T07:19:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-06T01:10:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-30T07:17:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-30T03:21:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasitology Research","date":"2024-07-24T08:20:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6301b738-cbcc-4c16-93e3-680fd764c47f","owner":[],"postedDate":"August 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:07:50+00:00","versionOfRecord":{"articleIdentity":"rs-4793566","link":"https://doi.org/10.1007/s00436-024-08440-6","journal":{"identity":"parasitology-research","isVorOnly":false,"title":"Parasitology Research"},"publishedOn":"2024-12-19 15:57:57","publishedOnDateReadable":"December 19th, 2024"},"versionCreatedAt":"2024-08-26 05:02:38","video":"","vorDoi":"10.1007/s00436-024-08440-6","vorDoiUrl":"https://doi.org/10.1007/s00436-024-08440-6","workflowStages":[]},"version":"v1","identity":"rs-4793566","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4793566","identity":"rs-4793566","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}