Molecular characterization of RNase III protein of Asaia sp. for developing a robust RNAi-based paratransgensis tool to affect the sexual life-cycle of Plasmodium or Anopheles fitness

Abstract

Background: According to scientific recommendations, paratransgenesis is one of the solutions for improving the effectiveness of the Global Malaria Eradication Programme. In paratransgenesis, symbiont microorganisms are used for distorting or blocking the parasite life-cycle, affecting the fitness and longevity of vectors or reducing the vectorial competence. It has been revealed recently that bacteria could be used as potent tools for double stranded RNA production and delivery to insects. Moreover, findings 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 specific features for this goal, could be considered as effective 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 information for creating an RNase III mutant Asaia bacterium. Methods: Asaia bacteria were isolated from field-collected Anopheles stephensi mosquitoes. The rnc gene and its surrounding sequences were characterized by rapid amplification of genomic ends. RNase III recombinant protein was expressed in E. coli BL21 and biological activity of the purified 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 specific 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 first step of developing an efficient RNAi-based paratransgenesis tool, were acquired. Asaia sp. have been found in different medically-important vectors and these data are potentially very helpful for researchers studying paratransgenesis and vector-borne diseases and are interested in applying the RNAi technology in the field.

Culturable bacteria associated with Anopheles darlingi and their paratransgenesis potential

“…darlingi is anthropophilic, opportunistic, exophagic, and exophilic, long-lasting insecticidal nets and indoor residual spraying are not effective alone and novel vector control methods are needed to improve the control of malaria transmission by these mosquitoes [16][17][18]. For example, strategies deploying genetically-modified mosquitoes [19,20] and their endosymbionts (paratransgenesis) [21][22][23][24] have been proposed as vector-based tools for malaria control.…”

Paratransgenic manipulation of tsetsemiR275alters the physiological homeostasis of the fly’s midgut environment

“…Due to the lack of effective vaccines capable of preventing the majority of these diseases, and the increasing resistance of vector arthropods to pesticides, alternative approaches for disease control are urgently needed. Paratransgenic systems have been applied in efforts to reduce vector competence in mosquitoes (30,31,94,95), kissing bugs (96,97), sand flies (98) and tsetse flies (25)(26)(27)99). This technology has many benefits, including the absence of a reliance on inefficient germline modification procedures (88), and the fact that modified symbionts exert no fitness cost on their insect hosts (24) and can potentially spread through wild vector populations via vertical transmission (100).…”

“…'Paratransgenic' tsetse flies that house recSodalis have been successfully used to deliver antitrypanosomal nanobodies (25)(26)(27). Paratransgenesis has also been used to deliver dsRNA for gene silencing in kissing bugs (28,29) and in the malaria mosquito Anopheles gambiae (30,31).…”

Paratransgenic manipulation of a tsetse microRNA alters the physiological homeostasis of the fly’s midgut environment

“…Due to the lack of effective vaccines capable of preventing the majority of these diseases, and the increasing resistance of vector arthropods to pesticides, alternative approaches for disease control are urgently needed. Paratransgenic systems have been applied in efforts to reduce vector competence in mosquitoes [29,30,96,97], kissing bugs [98,99], sand flies [100] and tsetse flies [24][25][26]101]. This technology has many benefits, including the absence of a reliance on inefficient germline modification procedures [85], and the fact that modified symbionts exert no fitness cost on their insect hosts [23] and can potentially spread through wild vector populations via vertical transmission [102].…”

“…'Paratransgenic' tsetse flies that house recSodalis have been successfully used to deliver anti-trypanosomal nanobodies [24][25][26]. Paratransgenesis has also been used to deliver dsRNA for gene silencing in kissing bugs [27,28] and in the malaria mosquito Anopheles gambiae [29,30], and to express intronic miRNA (using a recombinant densovirus) in the mosquito Aedes albopictus [31]. However, paratransgenic expression of small RNA antagomirs to knockdown miRNA expression in tsetse fly has not been reported to date.…”