Proteomic And MiRNAs Analysis Of Exosome-like Vesicles Isolated From Saliva Of Tick Haemaphysalis Longicornis

Extracellular vesicles originate from endosomal system or shed from the plasma membrane.These membranous structures have been identified in several biological fluids such as plasma,urine,saliva,semen,cerebrospinal fluid and amniotic fluid In recent years,their involvement in multiple physiological and biological processes has been verified These nano-sized structures are now considered as an additional mechanism of intercellular communication.It is now believed that cells can exchange proteins,lipids and genetic material by means of these cell-derived membranous structures.EVs have been classified into exosomes,microvesicles and apoptotic bodies.Excretory-secretory products of parasites have been known to play role in host parasite interactions.Therefore,these structures have been used to isolate nano-sized membrane vesicles.Discovery of EVs within excretory-secretory products of Fasciola hepatica,Echinostoma caproni,Heligmosomoides polygyrus,Dicrocoelium dendriticum,Schistosoma mansoni,Trichuris muris,Echinococcus granulosus,Leishmania amazonensis,Trichomonas vaginalis,Plasmodium vivax and Trypnosomacruzi has gained considerable interest.It is now believed that parasites can also use these membrane vesicles to communicate with their host environment.The tick Haemaphysalis longicornis is a kind of blood-sucking parasite and also is an important vector of human and animal disease-causing agents such as virus,bacteria and protozoan.Salivary glands of ticks play a role in blood feeding and pathogen transmission.Saliva is the carrier of interaction among tick,pathogen,and host.Is there exosome-like vesicles in tick saliva?What is its composition?Although,EVs have been identified within excretory products of parasites,ticks have not been studied so far.In a recent study,exosomes were isolated from the cell line of tick,Ixodes scapularis.Keeping in view the importance of these vesicles,this study was carried out to isolate vesicles directly from the saliva of tick,Haemaphysalislongicornis.Moreover,proteomics as well as transcriptomic analysis were also performed to get in touch with the proteins and RNA population within tick-derived EVs.In our first experiment,saliva was collected from partially engorged female tick of Haemaphysalis longicornis.Exosome-like vesicles were isolated from tick saliva by serial steps of ultracentrifugation and OptiPrepTM density gradient technique.Transmission electron microscopy confirms the presence of these vesicles having a size of 50 to 100 nm.Later on,LC-MS/MS was carried out to identify proteins within tickderived EVs.A total of 356 proteins were identified mainly corresponding to nuclear proteins(elongation factors and histones),cytoskeletal proteins(actin,tubulin)and stress-related proteins(HSPs).Proteome data of tick-derived EVs was validated by Western blot analysis.Immunodetection of Hsp70 and GAPDH proteins indicated that the proteomics data of tick-derived EVs were highly reliable.Bioinformatics analysis(Gene Ontology)indicated association of certain biological and molecular functions with proteins which may be helpful during tick development.Likewise,KEGG database revealed involvement of vesicular proteins in proton transport,detoxification,ECM-receptor interaction,ribosome,RNA transport,ABC transporters and oxidative.In second step,transcriptomic analysis of tick-derived EVs was carried out to get familiar with RNA population.For this purpose,total RNA was extracted from EVs and was analyzed by deep sequencing to determine miRNA profile.RNA-seq analysis showed that tick-derived EVs contained small non-coding RNA populations including miRNAs.The analysis of tick-derived EVs identified 36 known miRNAs,34 novel miRNAs and 842 novel Piwi-interacting RNAs(piRNA).Proteomics of tick-derived EVs identified nuclear proteins within them.These proteins(H2A,H2B,H3 and H4)have also been identified within vesicles of other parasites.However,there presence within tick EVs created some sort of confusion;tick specific or host specific.Keeping this in mind,comparative analysis was generated between fed and unfed ticks.Midguts and salivary glands of fed and unfed ticks were analyzed for the detection of H2A protein.Interestingly,it was observed that nuclear protein did not appear in midgut of unfed ticks as compared to fed ticks.Similarly,these proteins were not detected in salivary glands of unfed ticks.In previous studies,these proteins have also been identified in saliva of partially engorged and engorged ticks.Detection of proteins within tick saliva gave further confirmation about carrying of these proteins by tick from host These results indicate that nuclear proteins get entry into the tick during feeding.In addition,these proteins get entry into midgut,then to salivary glands by means of haemolymph and finally into the saliva.Therefore,we may here assume that nuclear proteins can be picked up by vesicles from tick saliva and then transfer them to host during feeding of ticks.In short,the results of current study provide evidence that exosome-like vesicles are being secreted by ticks in their saliva.In addition,presence of proteins and miRNAs within tick-derived EVs suggest their role in hostparasite relationship.However,better understanding of how EVs increase tick attachment to host skin as well as modulation of host immune responses will be helpful in pathogenesis and development of therapeutics and vaccine against ticks.