Profiling The Composition And Function Of Extracellular Vesicles From Eimeria Falciformis And Mice-infected With E. Falciformis

Extracellular vesicles（EVs）;,lipid bound vesicles secreted by organisms’cells under physiological and/or pathological conditions,can execute cellular communications,biomarkers,metabolic and/or immunological regulations.In parasitism,EVs are formed during parasite motility and development,and are also secreted as host responses to parasite infection.EVs have been characterized from host,parasitized host cells and parasites using differently modified culture mediums and physico-chemical stressors.However,host-parasite interaction as mechanism for EVs secretion has not been deeply investigated.During invasion process by Apicomplexan parasites,such as Plasmodium spp and Toxoplasma gondii,parasite secretomes usually include vesicle-free and EV-bound molecules but Eimeria falciformis EV and vesicle-free protein composition are yet to be reported.There is yet no empirical report of the roles of Eimeria-derived EVs on host cell inflammasome activation and regulation of non-coding RNAs（e.g.,lnc RNAs）which play crucial roles in intestinal epithelia cells（IECs）immunity in parasitic infections.Likewise,host-derived proteins conveyed in EVs during E.falciformis major developmental stages in mice have not been investigated.E.falciformis was utilized to study Eimeria-host interactions.EVs were isolated from caecum and bloodserum of E.falciformis-infected mice at time of oocyst ingestion,1^st generation merozont（68h）and 2^nd generation merozont（116h）stages,faecal oocyst shedding（7day）and time of host recovery（10day）post infection.Isolated EVs were purified,characterized,and profiled by tandem mass tag-based proteomics.In addition,E.falciformis sporozoites interacted with inactivated murine IECs（MIECs）in vitro.Secreted molecules were separated into EV and vesicle-free（VF）fractions by ultracentrifugation.Vesicle-free and EV fractions were purified and characterized as appropriate.The protein compositions of E.falciformis sporozoite EV and VF were analyzed by LC-MS/MS.Consequently,MIECs were stimulated with E.falciformis sporozoite EVs for differential regulations and expressions of cytokines,chemokines,inflammasomes and lnc RNAs.It was revealed that the subpopulation of EVs in E.falciformis-infected mice increased significantly as E.falciformis life stages progressed.Caecum-and serum-EVs were of different sizes with lipid bi-layer membranes.While caecum EVs contained CD9 and CD82 as EV markers,MHCs and Hsp70 were common marker in serum EVs.861 and 1,024 proteins were quantified in serum-and caecum-derived EVs respectively.Specifically,neutrophil gelatinase,interleukin 18,monocyte differentiation antigen,interferon inducible GTPase,macrophage colony stimulating factor,complements,immunoglobulins,and chemokine 8 were among differentially regulated immune-related serum-derived EV proteins.As well,superoxide dismutase,pyruvate kinase and zinc finger CCCH domain-containing protein 3 were among differentially regulated metabolic and/or ion exchange proteins in serum EVs.In contrast,differentially regulated,immune-associated caecum-derived EV proteins include signal peptide,inducible nitric oxide synthase（i NOS）,neutrophilic granule protein,TGFβ,STAT1,NLRP6,and CD47.Moreover,some common proteins involved in metabolic pathways were in the caecum-derived EVs,including NADH dehydrogenase[ubiquinone]I beta subcomplex subunit and cytochrome c oxidase in all of the collected stage samples,and sarcoplasmic/endoplasmic reticulum ATPase was found in EVs collected in the stage of 2nd merozont,7dpi and 10dpi.EVs from E.falciformis-infected mice caecum and serum were,at the least,enriched with immune/cell death associated proteins during first and second generation merozont stages of E.falciformis.Bioinformatic analyses indicated serum EV proteins were intricately linked to innate immunity and acute phase responses while caecum EV proteins were implicated in antigen processing and response to bacterium.In contrast to host-derived EVs,E.falciformis sporozoite-secreted EVs enclosed Hsp70,GAP45,refractile body associated aspartyl protease and eimepsin as well as aminopeptidases while E.falciformis sporozoite VF proteins include Hsp90,Vps,actin,and kinases among others.E.falciformis sporozoite EV and VF have EF1-αand Hsp70 as common proteins.In general,EV and VF proteins are crucial for sporozoite motility,pathogenesis,and survival.In addition,E.falciformis EVs down regulated the production of apoptotic cytokines but upregulated MIEC chemo-attractants and proinflammatory cytokines including IL-6,MCP1,IL-17,IL-1βand IL-18 at protein and m RNA levels in dose-and time-dependent treatments with sporozoite EVs.Also E.falciformis EVs upregulated pyroptotic-dependent NLRP6 and caspase 11 in E.falciformis sporozoite EV-stimulated MIECs with dose-and time-dependent LDH production.Furthermore,E.falciformis EVs differentially regulated MIEC m RNAs,lnc RNAs and cir RNA associated with TGF-β,MAPK and Fox O pathways.Identified lnc RNAs were significantly associated with MIEC homeostasis and cytokine expressions.In conclusion.E.falciformis infection co-opt cellular and humoral responses through EVs.IEC death during murine coccidiosis might involve diverse mechanisms as innate responses to the infection.E.falciformis and E.falciformis sporozoite EVs induced NLRP6-depedent cell death.Also,JAK-STAT pathway might play important roles in host response to E.faiciformis infection due to identification of caecum EV STAT1 and considerable number of lnc RNAs associated with JAK-STAT in E.faiciformis EV-stimulated MIECs.Some host-derived EV proteins are potential diagnostic and prognostic biomarkers just as differentially regulated lnc RNAs hold predictive values for coccidia infections.E.faiciformis sporozoite EVs contained immunogens and antigens as potential vaccines.Finally,EVs are important source of antigen discovery and immune regulatory motifs.