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Evolutionary and Functional Studies on Microsporidian ATP-Binding Infection, Genetics and Evolution 68 (2019) 136–144 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid Evolutionary and functional studies on microsporidian ATP-binding T cassettes: Insights into the adaptation of microsporidia to obligated intracellular parasitism Qiang Hea, Charles R. Vossbrinckc, Qiong Yangd, Xian-Zhi Menga, Jian Luoa, Guo-Qing Pana, ⁎ ⁎ Ze-Yang Zhoua,b, , Tian Lia, a State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, China b College of Life Science, Chongqing Normal University, Chongqing 400047, China c Department of Soil and Water, The Connecticut Agricultural Experiment Station, 123 Huntington Street, New Haven, CT 06511, USA d Sericulture and Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, China ARTICLE INFO ABSTRACT Keywords: ATP-binding cassette (ABC) transporters comprise the largest family of transmembrane proteins and are found in Microsporidian all domains of life. The ABCs are involved in a variety of biological processes and as exporters play important ABC transporter roles in multidrug resistance. However, the ABC transporters have not been addressed in microsporidia, which Genome are a very large group of obligate intracellular parasites that can infect nearly all animals, including humans. Evolution Here, a total of 234 ABC transporters were identified from 18 microsporidian genomes and classified intofive Nosema bombycis subfamilies, including 74 ABCBs, 2 ABCCs, 18 ABCEs, 15 ABCFs, 102 ABCGs and 23 uncategorized members. Two subfamilies, ABCA and ABCD, are found in most organisms, but lost in microsporidia. Phylogenetic analysis indicated that microsporidian ABCB and ABCG subfamilies expanded by recent gene duplications, which re- sulted in the two largest subfamilies in microsporidia. Functional analysis via qRT-PCR and Western blotting revealed that NoboABCG1.1, an ABCG member of Nosema bombycis, is expressed in mature spores and up- regulated from 1 dpi to 6 dpi in infected silkworm midgut. IFA and IEM analysis showed that NoboABCG1.1 is localized on the plasma membrane of the sporoplasm, meront and mature spore. The propagation of N. bombycis was significantly inhibited after the RNAi of NoboABCG1.1 expression, indicating that NoboABCG1.1 is im- portant to the pathogen proliferation. In conclusion, our study uncovered that the ABCs evolved during mi- crosporidia adaption to intracellular parasitism and play important roles in the pathogen development. 1. Introduction compensate for this lack of metabolites, microsporidia have transport systems to acquire nutrients from the host and export cytotoxic com- Microsporidia are a group of obligated intracellular parasites that pounds across the plasma membrane (Cuomo et al., 2012; Heinz et al., infect a broad range of animal phyla including vertebrates including 2014; Heinz et al., 2012). It has been suggested that the copying of the immunocompromised humans. Microsporidia are reported to be com- microsporidial genome may be a limiting step in the rate of reproduc- posed of at least 187 genera and 1500 species (Keeling, 2009; Vávra tion and that the this may be the reason for genome compression, in- and Lukeš, 2013). Microsporidial genomes are extremely compact and cluding the loss of genes in microsporidia (Xu et al., 2016). are missing genes for many metabolic pathways (Corradi et al., 2010; The ATP-binding cassette (ABC) transporters comprise one of the Katinka et al., 2001; Nakjang et al., 2013; Pombert et al., 2012), in- largest superfamilies of transmembrane proteins found in all living cluding the biosynthesis of nucleotides and amino acids, the tri- organisms (Jungwirth and Kuchler, 2006). ABC transporters are divided carboxylic acid cycle, beta-oxidation of fatty acids and the electron into two major subtypes; importers and exporters, although the im- transport chain (Bakowski et al., 2014; Chen et al., 2013; Corradi et al., porters seem to be found primarily in prokaryotes and plants (Rice 2009; Corradi et al., 2010; Heinz et al., 2012; Katinka et al., 2001; et al., 2014). Most of ABC transporters rely on the binding and hy- Keeling et al., 2010; Pan et al., 2013; Pombert et al., 2012). In order to drolysis of ATP to transport substrates, such as amino acids, lipids, ⁎ Correspondence authors: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, China. E-mail addresses: [email protected] (Z.-Y. Zhou), [email protected] (T. Li). https://doi.org/10.1016/j.meegid.2018.12.022 Received 7 October 2018; Received in revised form 10 December 2018; Accepted 17 December 2018 Available online 18 December 2018 1567-1348/ © 2018 Published by Elsevier B.V. Q. He et al. Infection, Genetics and Evolution 68 (2019) 136–144 sugars, peptides, ions, and drugs, from the cytosol to intracellular or 2.2. Identification of putative ABC transporter proteins extracellular regions (Dermauw and Van Leeuwen, 2014). Typical ABC full-length transporters contain two integral transmembrane domains To identify ABC transporter proteins in Microsporidia, all protein (TMDs) and two nucleotide binding domains (NBDs), while half-length sequences were searched against the TransportDB and Transporter transporters are composed of one TMD and one NBD domain (Beis, Classification Databases (TCDB) based on sequence similarity using the 2015; Hollenstein et al., 2007; Schneider and Hunke, 1998). According default parameters (Ren et al., 2007; Saier et al., 2016). All candidate to the homology of their NBDs, seven ABC subfamilies (ABCA - ABCG) ABC transporters were then searched against PFAM database to predict have been identified in humans (Dean et al., 2001). An additional ABC the nucleotide binding domain (NBD) using the Hidden Markov Model transporter, ABCH, was first discovered in Drosophila melanogaster, and (HMM) and confirmed using the NCBI CD-Search tool to predict con- has been found in other arthropods and zebrafish but not in mammals, served domains. The transmembrane helices were predicted using the plants or fungi (Dermauw and Van Leeuwen, 2014; Popovic et al., TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) (Krogh 2010). et al., 2001) web portal. Graphical representations of transmembrane Previous studies of parasite ABC transporters have shown their topology were created with TMRPres2D (http://bioinformatics.biol.u important role in exporting antibiotic compounds resulting in drug oa.gr/TMRPres2D). resistance. At present, 20 predicted genes encoding ABC transporters have been identified in the genome of Toxoplasma gondii (Sauvage et al., 2.3. Phylogenetic analysis 2006), 33 in Cryptosporidium parvum (Zapata et al., 2002), and 16 in Plasmodium falciparum (Koenderink et al., 2010). A recent study in T. Protein sequences were aligned using MUSCLE v3.8.31 and regions gondii has show that five of ABCG half transporters facilitate the export of uncertain alignment were eliminated with TrimAl using the default of cholesterol and phosphatidylserine when expressed in mammalian parameters (Capella-Gutiérrez et al., 2009; Edgar, 2004). The sub- COS cells (Ehrenman et al., 2010). However, another ABCG transporter, stitution model prediction option on MEGA (v6.06) (Tamura et al., TgABC107, localized in the parasite plasma membrane and para- 2011) was used to identify the best substitution models for the in- sitophorous vacuoles of T. gondii can import cholesterol from the host dividual protein homologue sets. Phylogenetic trees were constructed cell (Ehrenman et al., 2010). P. falciparum encodes a single ABCG using the Maximum Likelihood (ML) method in MEGA (Tamura et al., transporter (PfABCG) which is localized in the parasite plasma mem- 2011). The best substitution model was used and bootstrap support was brane and may be play a role in lipid transport consistent with human evaluated based on 100 replicates. The phylogenetic trees were visua- ABCG1 function (Edaye and Georges, 2015; Ehrenman et al., 2010). lized using Evolview (He et al., 2016). Meanwhile, Specific ABC transporters have been implicated in drug resistance in protozoan parasites such as TcABCG1 are naturally re- sistant to Benznidazole through over-expressed in Trypanosoma cruzi 2.4. Preparation of Nosema bombycis and cell cultivation (Klokouzas et al., 2003; Leprohon et al., 2006; Sauvage et al., 2009; Zingales et al., 2015). The first report of ABC transporters in micro- Mature spores of N. bombycis isolate CQ1, conserved in the China sporidia were EiABC1 and EiABC2 from Encephalitozoon intestinalis Veterinary Culture Collection Center (CVCC No. 102059) in Chongqing, (Bonafonte et al., 2001). Subsequently thirteen ABC transporter genes China, were propagated in and isolated from silkworm larvae as pre- were identified in the Encephalitozoon cuniculi and ten from Trachi- viously described (Wu et al., 2008). Spodoptera frugiperda ovary cell pleistophora hominis (Cornillot et al., 2002; Heinz et al., 2012). (Sf9) were purchased from Thermo Fisher Scientific and cultured in Sf- Here, we analyzed the ABC transporters from 18 microsporidian 900™ III SFM medium at 28 °C. genomes and characterized the subcellular localization and function of a G-subfamily member, NoboABCG1.1, in Nosema bombycis. 2.5. Expression of fusion proteins, antiserum production, and Western blotting 2. Materials and methods DNA was extracted as previously described (Pan et al., 2013). The outer membrane region of NoboABCG1.1 (GenBank: EOB15202.1) was 2.1.
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