Characterizaion Of Cysteine Proteases In Rhipicephalus Haemaphysaloides And Their Mechanisms Involved In Salivary Glands Apoptosis

Cysteine protease is a kind of ancestral and conservative protease, existing in virus, bacteria and various tissues and organs of mammals, which participate in embryogenesis, antigen presentation, apoptosis, maintain homeostasis and other essential physiological activities. As bloodfeeding arthropod ectoparasites, ticks are important vectors of human and animal diseases, transmitting virus, bacteria, fungi, protozoa and many other pathogens. The Salivary glands is an important target for tick‘s prevention and control as it is the passageway that host blood inflow while pathogens outflow. After its meal, the salivary glands become atrophied, which is associated with apoptosis induced by cysteine proteases, though the mechanism is unclear. This study focus on identification of cysteine proteases in tick salivary glands and the mechanism of cysteine proteases involved in apoptosis of salivary glands during blood feeding.Based on the sequence similarities to other species in databases, we analyzed the unfed and fed sialotranscriptome of R. haemaphysaloides. There are 1179 up-regulated genes and 574 down-regulated genes comparing with two sialotranscriptomes. From the sialotranscriptome, there are 25 cysteine protases EST in total(13 up-regulated EST and 1 down-regulated EST); Finally, six cysteine protease full-lengh sequences named Cathepsin B(CATB), Cathepsin L(CATL), Caspase-1(CASP1), Caspase-8(CASP8), Autophagy-related gene 4B and 4D(ATG4B and ATG4D) were cloned.We checked the transcription of 6 cysteine proteases in different developments and fed statuses by Real-time PCR, the results showed all the cysteine protases transcription were down-regulated significantly after feeding in larvas and nymphs, while up-regulated significantly in adults; and in salivary glands, their transcriptions were up-regulated significantly after feeding, especially CATB and CATL.We expressed and purificated 5 cysteine protases(without CASP8) in vitro, and got their specific antiserum in mouse. Analysis of recombination protein enzyme activity showed all the proteins were acticated in appropriate concentration and PH. The optimum PH of CASP1 is 5.5, and can achieve 60% activity. The activity of CATB and CATL was inhibited in high concentration. Whlie if cut off the 60-AA inhibition domain in CATL, its activity would be enhanced, suggesting the inhibition effect of CATL may be caused by its inhibitor and CATB may be associated with enzyme and substrate competition. ATG4 B and ATG4 D can achieve 40% activity with CASP1 specific substrate. ATG4 D also appeared inhibited effect in high concentration with CATB/L specific substrate. Western Blot showed natural protease forms of CATB and CATL in 35~40 kDa, ATG4 B in~36 kDa, ATG4 D in ~40 kDa, CASP1 in ~37 kDa and ~70 kDa. Immunohistochemical analysis showed the proteases mainly distributed in salivary glands and only a little ATG4 D in midgut.To find whether there is a relationship among theses cysteine protases, specifitic RNAi-dsRNA was transmitted into nymphs by electrotransformation. The results showed anyone of transcriptional inhibition in the cysteine protases would induce transcription changes of others. Expecially when one of CATB, CATL, ATG4 D and CASP8 was inhibited, the transcription of CASP1 was up-regulated significantly. What‘s more, when CASP1 was silenced by RNAi, the transcription of CATB, CATL, ATG4 D and CASP8 were almost silent, suggesting CASP1 may be the upstream regulator.Apoptosis involved in salivary glands during blood feeding was confirmed by dissection, TUNEL and Annexin Ⅴ. For the further mechenism, dsRNA-CASP1 was injected into the adult ticks and then the ticks were fed on the rabbits after 24 hours. We analysed the dynamic changes of cysteine proteases mRNA transcription and protein expression in salivary glands from unfed to engorged ticks and also checked the apoptosis by TUNEL and Annexin Ⅴto explore the roles of CASP1 in apoptosis of salivary glands. The results showed apoptosis was mainly occured after feeding 3~4 days, and then the salivary gland were necrosis. As effect caspases, the expression of CASP1 was increased from 5 to 7 days. The interference of CASP1 could delay the apoptosis of salivary gland, and affect its necrosis, and induce the enhancive expression of ATG4 D, indicating the closely relationship between ATG4 D and CASP1. Autophagy-related proteases and caspases may participate in the apoptosis of salivary gland together and regulate each other as compensatory mechanisms.In conclusion, 6 cysteine proteases in the salivary glands of tick R. haemaphysaloides were characterization, and their transcription were up-regulated after blood feeding. There was some dependence relationship among these protases. What‘more, autophagy-related proteases and caspases participate in the apoptosis of salivary gland together.