Profiling The Proteins Cross-talking With The VirD4 And VirB10 Of Anaplasma Ovis In Salivary Gland Of Dermacentor Silvarum

Anaplasma ovis(A.ovis)is a gram-negative,tick-borne obligate intraerythrocytic pathogen,which causes ovine anaplasmosis in ruminants worldwide including areas of Asia,Europe,Africa,and North Amer ica.The transmission of Anaplasma ovis occurs mainly through ticks from the genera Dermacentor,Rhipicephalus and Hyalomma.The Type IV Secretion System(T4SS)of Anaplasma spp.has shown to be crucial for its cellular trafficking,survival,and pathogenicity.Both Vir D4 and Vir B10 are an integral component of the T4 SS and disease-causing bacteria use T4 SS to transfer virulence,DNAs and/or proteins from the bacteria,directly into the host cell.In this study,to better understand the interaction between Anaplasma ovis and Dermacentor silvarum,the Vir D4 and Vir B10 were used as bait plasmid to screen proteins interacting with the pathogen from a c DNA library of Dermacentor silvarum salivary gland by yeast two-hybrid(Y2H)system.The c DNA of Dermacentor silvarum salivary gland was cloned into pGADT7-SmaI vector(Prey Plasmid)for constructing the Y2 H c DNA library.The vird4 and virb10 genes were cloned into the pGBKT7 vector for bait plasmid construction excluding its auto-activation and toxicity in the yeast strain Y2 H Gold.The yeast two-hybrid screening was carried out via mating bait and prey plasmids into yeast str ains Y2 H Gold and Y187 respectively.Sequences of positive preys were analyzed us ing BLAST,Gene Ontology,UniProt,SMART,and STRING.In this study,4 Dermacentor silvarum salivary gland proteins: ribosomal protein L12,a conserved hypothetical protein(cadherin repeats),a conserved hypothetical protein(F lhF),and a tumor necrosis factor receptor-associated factor-like(TNF)protein were identif ied to interact with Vir D4.One large tegument protein,UL36 of Dermacentor silvarum salivary gland was identif ied to interact with Anaplasma ovis Vir B10.The results of the functional annotation of the identif ied interacting proteins showed their involvement in several important cellular functions such as cell adhesion,apoptosis regulation,cell proliferation,signaling,survival,late-stage replication,and ubiquitylation regulation.This study is the first to screen Dermacentor silvarum salivary gland proteins interacting with Anaplasma ovis Vir D4 and Vir B10.Five proteins: ribosomal protein L12,conserved hypothetical protein(cadherin repeats),conserved hypothetical protein(FlhF),tumor necrosis factor receptor-associated factor-(TNF)like protein and large tegument protein UL36 were identif ied using Y2 H technique.The findings help understand the interaction between Anaplasma ovis and Dermacentor silvarum.