Molecular and Biochemical Analysis of Setaria digitata Novel Parasitic Nematode Specific Protein

Abstract

Setaria digitata is an ivory colored thread like animal parasitic nematode, which naturally resides in the peritoneum cavity of ungulates such as cattle and buffaloes. S. digitata share morphological, histological and drug responses similarities with Wucheraria bancrofti and Brugia malayi. Hence, it is used as a model organism to study W. bancrofti and B. malayi when specimens were not available. S. digitata novel protein (SDNP), is a single copy gene. It was found to be ubiquitously expressed in all the life stages of the nematode. It also shows a significant sequence resemblance with novel parasite specific genes of W. bancrofti, Loa, Brugia malayi and Onchocerca volvulus. In this study, the bioinformatics analysis, co-immunoprecipitation, RNA interference in-vitro (culture medium) with adults and in-vivo (inside the mosquito, Culex quinquefasciatus) with microfilariae were carried out to investigate the function of the SDNP. According to the bioinformatics analysis, the SDNP is a positively charged, basic protein with a better in-vivo half-life and thermostable tertiary structure. Due to the hydrophilic nature of the protein, it can be predicted that the protein is most likely to be in the cytosol than it to be in the membrane. The phylogenetic analysis of the protein showed that all of the taxonomic units in its cluster are animal parasitic nematodes and surprisingly this cluster is phylogentically related to a cluster having human parasites. To predict the three dimensional structure of the protein, CABS fold server was selected. The 3D structure obtained from the CABS fold server was refined using the Mod Refiner server and the model validation was carried out using ProSA and RAMPAGE tools. The outcomes of these analyses suggested that, a stable 3D structure had been predicted. The adult female S. digitata worm was subjected to RNAi by microinjecting the in-vivo synthesized SDNP siRNA and phenotypic and cellular changes associated with the interference were analyzed. The qPCR analysis of the SDNP transcript levels revealed a significant reduction of SDNP mRNA following siRNA microinjection into S. digitata adult worms. Similarly, immunohistochemical staining indicated a reduction of SDNP expression. Furthermore, worms treated with siRNA showed a significant reduction of microfilariae release together with embryonic lethality by arresting an early developmental stage compared to non-treated worms. A distinct motility reduction was also observed in treated worms compared to non-treated counterparts showing the amenability of S. digitata to the siRNA induced RNAi for the first time. The presence of interdomain linkers of muscle-specific twitchin kinase and calcium-dependent protein kinase isoform CDPK1, together with the results of foregoing and previous studies suggested that SDNP is most likely a protein involved in muscle movement, growth, and development of the nematode.