Asgari et al. Parasites Vectors (2020) 13:42 https://doi.org/10.1186/s13071-020-3889-6 Parasites & Vectors RESEARCH Open Access Molecular characterization of RNase III protein of Asaia sp. for developing a robust RNAi-based paratransgensis tool to afect the sexual life-cycle of Plasmodium or Anopheles ftness Majid Asgari1, Mahdokht Ilbeigikhamsehnejad1, Elham Rismani2, Navid Dinparast Djadid1* and Abbasali Raz1* Abstract Background: According to scientifc recommendations, paratransgenesis is one of the solutions for improving the efectiveness of the Global Malaria Eradication Programme. In paratransgenesis, symbiont microorganisms are used for distorting or blocking the parasite life-cycle, afecting the ftness and longevity of vectors or reducing the vecto- rial competence. It has been revealed recently that bacteria could be used as potent tools for double stranded RNA production and delivery to insects. Moreover, fndings showed that RNase III mutant bacteria are more competent for this aim. Asaia spp. have been introduced as potent paratransgenesis candidates for combating malaria and, based on their specifc features for this goal, could be considered as efective dsRNA production and delivery tools to Anopheles spp. Therefore, we decided to characterize the rnc gene and its related protein to provide the basic required informa- tion for creating an RNase III mutant Asaia bacterium. Methods: Asaia bacteria were isolated from feld-collected Anopheles stephensi mosquitoes. The rnc gene and its surrounding sequences were characterized by rapid amplifcation of genomic ends. RNase III recombinant protein was expressed in E. coli BL21 and biological activity of the purifed recombinant protein was assayed. Furthermore, Asaia RNaseIII amino acid sequence was analyzed by in silico approaches such as homology modeling and docking to determine its structural properties. Results: In this study, the structure of rnc gene and its related operon from Asaia sp. was determined. In addition, by performing superimposition and docking with specifc substrate, the structural features of Asaia RNaseIII protein such as critical residues which are involved and essential for proper folding of active site, binding of magnesium ions and double stranded RNA molecule to protein and cleaving of dsRNA molecules, were determined. Conclusions: In this study, the basic and essential data for creating an RNase III mutant Asaia sp. strain, which is the frst step of developing an efcient RNAi-based paratransgenesis tool, were acquired. Asaia sp. have been found in dif- ferent medically-important vectors and these data are potentially very helpful for researchers studying paratransgen- esis and vector-borne diseases and are interested in applying the RNAi technology in the feld. *Correspondence: [email protected]; [email protected] 1 Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Parasites Vectors (2020) 13:42 Page 2 of 19 Keywords: Vector-borne diseases, Paratransgenesis, Asaia, RNAi, RNase III Background these bacteria as appropriate candidates for paratrans- Vector-borne diseases (VBD) have enormous impacts on genic control of malaria and other VBD. Cultivation in human life [1–3]. Malaria is one of the most important cell-free media, colonization in diferent parts of the vec- protozoan infectious diseases, caused in humans by fve tor’s body (gut, salivary gland, reproductive organs, etc.), diferent members of the genus Plasmodium. Based on simple transformation capacity, transstadial and transo- the World Health Organization (WHO) annual report, varial transmission and cryo-preservation capability are there were approximately 219 million clinical malaria the main specifc features of the species of this genus that cases in 2017 which resulted in almost 0.5 million deaths discriminates it from the other symbiont microorganisms [4]. Te main at-risk groups for malaria disease are chil- [40, 41, 46, 47]. Recently, bacteria have been introduced dren under the age of fve years, but it also afects other and used as dsRNA producing agents and delivery vehi- risk groups such as pregnant women and non-immune cles to insects for triggering the RNAi pathway against adults [5]. the targeted genes [48–57]. Terefore, with regard to the After the failure of Global Malaria Eradication pro- unique features of Asaia spp., unsuitable environmental gram [6], the WHO has been updating the malaria policy conditions of malaria endemic regions, drawbacks of the instruments beginning with a consultative review process research-based dsRNA administration routes to insects involving the scientifc recommendations and fndings and the costs of in vitro dsRNA production, these bac- of scientists in 2007 [7]. Drug, vaccine, vector control, teria could be considered as valuable and cost-efective modeling, M&E and surveillance, integration strategies, candidates for applying the RNAi technology against health systems and operational research and diagnos- malaria and other VBD in endemic regions. tics and young investigators/basic research consultative Nevertheless, there is an important point that should groups were considered. Tree main subjects were rec- be considered for increasing the efectiveness of the ommended by the vector control consultative group: developed method based on symbiont bacteria and RNAi designing new and more efcient insecticides, developing technology. Studies have shown that the dsRNA-specifc of transgenic insects and applying paratransgenesis [8]. endonuclease (RNase III) mutant bacteria are more ef- Paratransgenesis is a kind of “Trojan horse” for con- cient for producing the dsRNA molecules in quantity in trolling and blocking the transmission of vector-borne comparison with their wild strains [49, 54, 57–60]. diseases [9]. Tis method is described as “the using of In fact, dsRNA substrates are subjected to cleavage diferent symbiotic microorganisms for delivering mole- by a specifc superfamily of dsRNA endonucleases that cules with anti-parasitic or anti-vector efects to the wild existed in most of prokaryotes and eukaryotes [61–65]. vector population” [2, 10–15]. Ribonuclease III (RNase III), an endoribonuclease, is Recently, diferent research studies have been per- encoded by the rnc gene, is an Mg 2+ dependent enzyme formed on symbiont microorganisms such as viruses, that is important for ribosomal RNA (rRNA) process- fungi, yeasts and bacteria as paratransgenesis agents [14, ing, cellular defense against the viral infections and 16–25]. Given the fact that bacteria are more available post-transcriptional gene expression control [61, 63–67]. and simpler than other microorganisms, they have been Hence, we decided to characterize the rnc locus and its at the focus of research for developing paratransgenesis related protein in a feld-isolated Asaia sp. by genomics, tools against VBD. Terefore, numerous studies have proteomics and in silico approaches to provide the basic been performed to reach this goal, and diferent bacte- and essential information for developing an rnc mutant ria have been introduced as suitable candidates for para- Asaia strain. Our fnal goal was to utilize the information transgenesis, especially for combating malaria [2, 9, 10, to create a strain which could be used as a pluripotent 12, 13, 16, 20, 24, 26–39]. Among the introduced candi- and robust dsRNA production tool and delivery vehicle dates, bacteria of the genus Asaia have special character- against target genes in Anopheles sp. (larvae and adults) istics that diferentiate them from the other introduced to control malaria vectors. bacteria [11, 40, 41]. Asaia spp. belong to the acid ace- tic alpha proteobacteria and are stably associated with Methods some mosquito species such as Anopheles spp., Culex Primer design spp. and Aedes spp. Tus, Asaia spp. have been the tar- 16S rRNA gene primers were used from our previous get of studies on vector microbiota [42–46]. Asaia spp. study [46, 67]. To characterize the rnc gene from the iso- have some features that persuade researchers to consider lated Asaia, full characterized sequences of the rnc gene Asgari et al. Parasites Vectors (2020) 13:42 Page 3 of 19 from diferent acetic acid bacteria species were aligned morphologically to species according to the Iranian using MEGA v.7.0 software [68]. After alignment, nine