Molecular Cross Talk Between Apoptosis And Autophagy In The Degeneration Of Tick Salivary Gland

Ticks are the second largest pathogen vector after mosquitoes.The salivary glands of ticks play a key role in the blood feeding process and are the main transmission organs of tick-borne pathogens.Tick salivary glands undergo rapid degeneration post engorgement,but the mechanism is unknown.In this research,we focused on the mechanism of autophagy and apoptosis during salivary gland degeneration and the main findings are as follows:I.Dynamic detection of autophagy and apoptosis and the transcriptome analysis of tick salivary glands during the degeneration.Transmission electron microscopy?TEM?and TUNEL staining results showed that autophagosomes were observed throughout blood-sucking period.According to the results,unfed and engorged tick salivary glands were selected for transcriptome analysis to screen for key genes.The results showed that the transcription levels of autophagy-related genes?ATGs?in tick salivary glands did not change significantly,while post engorged the transcription levels of most apoptosis-related genes increased significantly,it is consistent with the results of TUNEL assay.2.Gene cloning,sequence analysis and recombinant protein expression of autophagy and apoptosis related genes.Based on the analysis of salivary gland transcriptomes at different blood-sucking times,we selected two of the ATGs and three Caspase like molecules for further research,and cloned the coding genes,respectively.According to the sequence alignments,the two ATGs were named RhATG5 and RhATG6.Three Caspase like molecules,named RhCaspases-7,-8,and-9,all of them have the conserved sequence characteristics of caspase.The recombinant protein was obtained by affinity chromatography purification.Recombinant proteins were prepared for antibody production.3.Interactions between RhCaspase-7,-8,-9 and their mechanism in tick salivary gland degenerationA qPCR assay demonstrated that the transcription of these three caspase genes increased significantly during the engorged periods of the tick developmental stages?engorged larval,nymph,and adult female ticks?.Transcriptional levels of RhCaspases-7,-8,and-9 in salivary glands increased more significantly than other tissues post engorgement.Co-transfection assays showed RhCaspase-7 is cleaved by RhCaspases-8 and-9,demonstrating that RhCaspases-8 and-9 are initiator caspases and RhCaspase-7 is an executioner caspase.RhCaspase-9-RNAi treatment significantly inhibited tick feeding.In contrast,knockdown of RhCaspase-7 and RhCaspase-8 had no influence on tick feeding.Compared to the control group,apoptosis levels were significantly reduced after interfering with RhCaspase-7,-8,and-9 expressions.4.ATG5 is instrumental in the transition from autophagy to apoptosis during the degeneration of tick salivary glandsDuring the fast feeding phase,RhATG5 was cleaved and both calcium concentration and the transcription of RhCalpains increased in the salivary glands.Recombinant RhATG5 was cleaved by?-Calpain only in the presence of calcium;the mutant RhATG5^191-199?was not cleaved.Treatment with20-hydroxyecdysone?20E?led to programmed cell death in the salivary glands of unfed ticks in vitro,autophagy was upregulated in ticks treated with low concentration of 20E.Conversely,autophagy decreased and apoptosis increased in ticks treated with a high concentration of 20E and transformed autophagy to apoptosis.High concentrations of 20E led to the cleavage of RhATG5.Calcium concentration and expression of RhCalpains were also upregulated in the tick salivary glands.RNA interference?RNAi?of RhATG5 in vitro inhibited both autophagy and apoptosis of the tick salivary glands.RNAi of RhATG5 in vivo significantly inhibited the normal feeding process.These results demonstrated that high concentrations of 20E led to the cleavage of RhATG5 by increasing the concentration of calcium and stimulated the transition from autophagy to apoptosis.5.Host-derived Beclin-1 regulates autophagy to apoptosis during the degeneration of tick salivary glands.It was found that Beclin-1 of host-derived Beclin-1 appeared in the tick salivary gland degeneration,and it appeared in tick hemolymph(5-7^th feeding day)earlier than the time point in tick salivary glands(1-2 ^th post engorgement).We proved that ATGs and apoptosis-related genes upregulated and the positive rates of TUNEL staining increased significantly in tick salivary glands after microinjected unfed ticks with Beclin-1 recombinant protein,consistent with the effect of 20E injection.After simultaneous injection of Beclin-1 and 20E,the salivary gland degeneration of ticks accelerated,and the situation improved when treatment with autophagy and apoptosis inhibitors.The cleavage of Beclin-1 mediated by Caspases is one of the pro-apoptotic mechanisms of Beclin-1.The results proved that the host-derived Beclin-1 was cleaved in tick salivary glands during the fast-blood feeding phase.We found that homo-Beclin-1 could be cleaved by three RhCaspases,but the cleavage did not appear in mutation homo-Beclin-1.Co-IP results showed that homo-Beclin-1 could bind to RhBCL-2 to form co-immunoprecipitation,which blocked the BH3 domain of RhBCL-2,and may lead to the anti-apoptotic effect of RhBCL-2 disappeared.In summary,this study indicates that the degeneration of tick salivary glands is dominated by apoptosis,and autophagy also involved in it.During the fast blood feeding phase,high concentrations of20E led to the cleavage of RhATG5 by increasing the concentration of calcium and stimulated the transition from autophagy to apoptosis;on the other hand,host-derived Beclin-1 accumulates in the salivary glands.We proved that host-derived Beclin-1 can be cleaved by tick-derived Caspases,or combined with tick-derived BCL-2 to further promote autophagy to apoptotic transformation and accelerate the degeneration process of tick salivary glands.Therefore,during the degeneration of tick salivary glands,the tick-derived ATG5 and host-derived Beclin-1 play a molecular link between autophagy and apoptosis.