Identification And Interaction Of Associated Functional Molecules In Dermacentor Silvarum Midgut Involved In Anaplasma Ovis Infection

Anaplasma ovis(A.ovis),a tick-borne intra-erythrocytic gram negative bacterium,is the causative agent of ovine anaplasmosis.Ticks from the genus Dermacentor act as biological vector for the transmission of A.ovis.Msp1 a,AAAP,Vir B10 and Vir D4 are some of the important key molecules present in A.ovis genome which were used as bait proteins for screening Dermacentor silvarum(D.silvarum)midgut proteins via yeast-two-hybrid mating assay.For construction of yeast two-hybrid library,c DNA of midguts from D.silvarum was cloned into p GADT7-Sma I vector(Prey Plasmid)whereas the proteins of A.ovis were cloned into the p GBKT7 vector as bait plasmids after evaluating the auto-activation and toxicity assays in the yeast strain Y2 HGold.Mating of bait and prey plasmids into yeast strains Y2 HGold and Y187 respectively was carried out for yeast two-hybrid screening.The identified positive prey sequences were analyzed using BLAST,Uni Prot,STRING and SMART databases.Different vector proteins i.e.conserved hypothetical protein,ribosomal protein,large tegumental protein UL36,fatty acid binding protein,protein disulphide isomerase 3,coatomer subunit delta,cell envelope integrity inner membrane protein,flagellar biosynthesis regulator F1 h F,molecular chaperone,dosage compensation regulatory complex protein and uncharacterized protein LOC8036454 were identified as potential interacting proteins.Considering the role of ribosomal protein for structural integrity of ribosomes,in stringent response phenomenon,in biogenesis of macromolecules and in regulation of translation process,the recombinant plasmids p ET-32a-Vir D4 and p GEX-4T-1-RL12 were constructed for the in vitro Glutathione S-transferase(GST)pull-down assay.The proteins,GST-RL12 and HIS-Vir D4,successfully expressed in Western blot analysis via GST pull-down assay,demonstrated a real interaction between the two proteins.The study is the first of its kind reporting D.silvarum midgut protein interaction with Vir D4 from A.ovis.The functional annotations showed the protein is attributed with some important cellular processes,particularly in the stringent response and biogenesis whereas the results of study predict the possible involvement of Vir D4-RL12 interaction in the regulation of signaling pathways,which is a tool for understanding the pathogen-vector interaction.