Abstract
BackgroundAnaplasma ovis is a gram-negative, tick-borne obligate intraerythrocytic pathogen, which causes ovine anaplasmosis in small ruminants worldwide. VirB10 of A. ovis is an integral component of the Type IV Secretion System (T4SS). The T4SS is used by bacteria to transfer DNA and/or proteins undeviatingly into the host cell to increase their virulence. To more thoroughly understand the interaction between A. ovis and Dermacentor silvarum, a vector containing the virb10 gene of A. ovis was used as a bait plasmid to screen interacting proteins from the cDNA library of the D. silvarum salivary gland using the yeast two-hybrid system.MethodsThe cDNA of the D. silvarum salivary gland was cloned into the pGADT7-SmaI vector (prey plasmid) to construct the yeast two-hybrid cDNA library. The virb10 gene was cloned into the pGBKT7 vector to generate a bait plasmid. Any gene auto-activation or toxicity effects in the yeast strain Y2HGold were excluded. The screening was performed by combining the bait and prey plasmids in yeast strains to identify positive preys. The positive preys were then sequenced, and the obtained sequences were subjected to further analyses using Gene Ontology, UniProt, SMART, and STRING. Additionally, the interaction between the bait and the prey was evaluated using the glutathione S-transferase (GST) pull-down assay.ResultsA total of two clones were obtained from the cDNA library using the yeast two-hybrid system, and the sequence analysis showed that both clones encoded the same large tegument protein, UL36. Furthermore, the proteins GST-UL36 and His-VirB10 were successfully expressed in vitro and the interaction between the two proteins was successfully demonstrated by the GST pull-down assay.ConclusionsTo our knowledge, this study is the first to screen for D. silvarum salivary gland proteins that interact with A. ovis VirB10. The resulting candidate, UL36, is a multi-functional protein. Further investigations into the functionality of UL36 should be carried out, which might help in identifying novel prevention and treatment strategies for A. ovis infection. The present study provides a base for exploring and further understanding the interactions between A. ovis and D. silvarum.
Highlights
Anaplasma ovis is a gram-negative, tick-borne obligate intraerythrocytic pathogen, which causes ovine anaplasmosis in small ruminants worldwide
Construction of a yeast two‐hybrid complementary deoxyribonucleic acid (cDNA) library The quality of the cDNA library showed the cDNA library titer was 4.8 × 106 cfu (Fig. 1a), and the average size of inserted fragments was > 1000 bp, according to the polymerase chain reaction (PCR) amplification results of 24 random clones (Fig. 1b). These results indicated that the cDNA library could be used for yeast two-hybrid screening
The results of the bait plasmid auto-activation testing showed that the yeast transfected with a plasmid containing pGBKT7-VirB10 or empty pGBKT7 were white on SD/-Trp/X plates, and the positive control colonies were blue on DDO/X/A plates (Fig. 2c)
Summary
Anaplasma ovis is a gram-negative, tick-borne obligate intraerythrocytic pathogen, which causes ovine anaplasmosis in small ruminants worldwide. The economic impact of A. ovis is becoming increasingly serious in countries where sheep and goats are the most important livestock [4]. The transmission of this pathogen is primarily via tick bites [5]; the tick vector species are mainly species of the genera Dermacentor, Rhipicephalus and Hyalomma and play a crucial role in maintaining and spreading the pathogen [5,6,7]. It is known that A. ovis initially infects the midguts of ticks, and migrates to the salivary glands where the pathogens are transmitted to the new hosts during blood-feeding [8, 9]
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