Folgueira I, Lamas J, Sueiro R A, Leiro J M
Department of Microbiology and Parasitology, Institute of Research on Chemical and Biological Analysis (IAQBUS), University of Santiago de Compostela, 15782, Santiago de Compostela, Spain.
Department of Fundamental Biology, Institute of Aquaculture, University of Santiago de Compostela, 15782, Santiago de Compostela, Spain.
Sci Rep. 2021 Apr 19;11(1):8519. doi: 10.1038/s41598-021-88102-0.
Proton-translocating inorganic pyrophosphatases (H-PPases) are an ancient family of membrane bound enzymes that couple pyrophosphate (PPi) hydrolysis to H translocation across membranes. In this study, we conducted a molecular characterization of two isoenzymes (PdVP1 and PdVP2) located in respectively the alveolar sacs and in the membranes of the intracellular vacuoles of a scuticociliate parasite (Philasterides dicentrarchi) of farmed turbot. We analyzed the genetic expression of the isoenzymes after administration of antiparasitic drugs and after infection in the host. PdVP1 and PdVP2 are encoded by two genes of 2485 and 3069 bp, which respectively contain 3 and 11 exons and express proteins of 746 and 810 aa of molecular mass 78.9 and 87.6 kDa. Topological predictions from isoenzyme sequences indicate the formation of thirteen transmembrane regions (TMRs) for PdVP1 and seventeen TMRs for PdVP2. Protein structure modelling indicated that both isoenzymes are homodimeric, with three Mg binding sites and an additional K binding site in PdVP2. The levels of identity and similarity between the isoenzyme sequences are respectively 33.5 and 51.2%. The molecular weights of the native proteins are 158 kDa (PdVP1) and 178 kDa (PdVP2). The isoenzyme sequences are derived from paralogous genes that form a monophyletic grouping with other ciliate species. Genetic expression of the isoenzymes is closely related to the acidification of alveolar sacs (PdVP1) and intracellular vacuoles (PdVP2): antiparasitic drugs inhibit transcription, while infection increases transcription of both isoenzymes. The study findings show that P. dicentrarchi possesses two isoenzymes with H-PPase activity which are located in acidophilic cell compartment membranes and which are activated during infection in the host and are sensitive to antiparasitic drugs. The findings open the way to using molecular modelling to design drugs for the treatment of scuticociliatosis.
质子转运无机焦磷酸酶（H-PPases）是一类古老的膜结合酶家族，它们将焦磷酸（PPi）水解与质子跨膜转运偶联起来。在本研究中，我们对位于养殖大菱鲆的一种盾纤毛虫寄生虫（双盘藻体虫）肺泡囊和细胞内液泡膜中的两种同工酶（PdVP1和PdVP2）进行了分子特征分析。我们分析了在施用抗寄生虫药物后以及在宿主体内感染后这些同工酶的基因表达情况。PdVP1和PdVP2分别由两个长度为2485和3069 bp的基因编码，这两个基因分别包含3个和11个外显子，表达的蛋白质分别为746和810个氨基酸，分子量分别为78.9和87.6 kDa。根据同工酶序列进行的拓扑预测表明，PdVP1形成13个跨膜区（TMRs），PdVP2形成17个TMRs。蛋白质结构建模表明，这两种同工酶均为同二聚体，PdVP2有三个镁结合位点和一个额外的钾结合位点。同工酶序列之间的同一性和相似性水平分别为33.5%和51.2%。天然蛋白质的分子量分别为158 kDa（PdVP1）和178 kDa（PdVP2）。同工酶序列源自旁系同源基因，这些基因与其他纤毛虫物种形成一个单系类群。同工酶的基因表达与肺泡囊（PdVP1）和细胞内液泡（PdVP2）的酸化密切相关：抗寄生虫药物抑制转录，而感染则增加两种同工酶的转录。研究结果表明，双盘藻体虫拥有两种具有H-PPase活性的同工酶，它们位于嗜酸细胞区室膜中，在宿主体内感染期间被激活，并且对抗寄生虫药物敏感。这些发现为利用分子建模设计治疗盾纤毛虫病的药物开辟了道路。
