Structural and Evolutionary Adaptation of NOD-Like Receptors in Birds.

NOD-like receptors (NLRs) are intracellular sensors of the innate immune system that recognize intracellular pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Little information exists regarding the incidence of positive selection in the evolution of NLRs of birds or the structural differences between bird and mammal NLRs. Evidence of positive selection was identified in four avian NLRs (NOD1, NLRC3, NLRC5, and NLRP3) using the maximum likelihood approach. These NLRs are under different selection pressures which is indicative of different evolution patterns. Analysis of these NLRs showed a lower percentage of codons under positive selection in the LRR domain than seen in the studies of Toll-like receptors (TLRs), suggesting that the LRR domain evolves differently between NLRs and TLRs. Modeling of human, chicken, mammalian, and avian ancestral NLRs revealed the existence of variable evolution patterns in protein structure that may be adaptively driven.

Initiator and executioner caspases in salivary gland apoptosis of Rhipicephalus haemaphysaloides.

BACKGROUND: Apoptosis is fundamental in maintaining cell balance in multicellular organisms, and caspases play a crucial role in apoptosis pathways. It is reported that apoptosis plays an important role in tick salivary gland degeneration. Several different caspases have been found in ticks, but the interactions between them are currently unknown. Here, we report three new caspases, isolated from the salivary glands of the tick Rhipicephalus haemaphysaloides. METHODS: The full-length cDNA of the RhCaspases 7, 8 and 9 genes were obtained by transcriptome, and RhCaspases 7, 8 and 9 were expressed in E. coli; after protein purification and immunization in mice, specific polyclonal antibodies (PcAb) were created in response to the recombinant protein. Reverse-transcription quantitative PCR (RT-qPCR) and western blot were used to detect the existence of RhCaspases 7, 8 and 9 in ticks. TUNEL assays were used to determine the apoptosis level in salivary glands at different feeding times after gene silencing. The interaction between RhCaspases 7, 8 and 9 were identified by co-transfection assays. RESULTS: The transcription of apoptosis-related genes in R. haemaphysaloides salivary glands increased significantly after tick engorgement. Three caspase-like molecules containing conserved caspase domains were identified and named RhCaspases 7, 8 and 9. RhCaspase8 and RhCaspase9 contain a long pro-domain at their N-terminals. An RT-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. RhCaspase9-RNAi treatment significantly inhibited tick feeding. In contrast, knockdown of RhCaspase7 and RhCaspase8 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. Co-transfection assays showed RhCaspase7 was cleaved by RhCaspases 8 and 9, demonstrating that RhCaspases 8 and 9 are initiator caspases and RhCaspase7 is an executioner caspase. CONCLUSIONS: To the best of our knowledge, this is the first study to identify initiator and executioner caspases in ticks, confirm the interaction among them, and associate caspase activation with tick salivary gland degeneration.

Caspase-1 participates in apoptosis of salivary glands in Rhipicephalus haemaphysaloides.

BACKGROUND: Ticks are among the most harmful vectors worldwide. Their salivary glands play essential roles in blood-feeding and pathogen transmission and undergo apoptosis after feeding. Although it was previously reported that salivary degeneration in ixodid ticks is in response to hormonal stimulation, questions still exist with the underlying mechanisms of salivary gland apoptosis. METHODS: Salivary glands of Rhipicephalus haemaphysaloides were collected from 1 to 7 days after attachment to the host. TUNEL and Annexin V assays were used to check apoptosis during this time. To confirm the role of caspase-1, RNA interference was used to silence its expression, and the dynamic changes of associated cysteine proteases were also shown by quantitative real time PCR and western blot, while TUNEL and Annexin V assays were used to confirm apoptosis. RESULTS: In the present study, apoptosis of salivary glands in R. haemaphysaloides occurred 3 or 4 days after attachment to the host as determined by TUNEL and Annexin V assays. The expression of caspase-1 increased at 5-7 days. When the latter was silenced by RNA interference, apoptosis in the salivary glands was delayed. While there seemed to be another form of cell death in salivary glands of ticks, such occurrence may be caused by compensatory autophagy which involved autophagy-related gene 4D. CONCLUSIONS: This study describes the apoptosis of salivary glands in R. haemaphysaloides and the dynamic changes in cysteine proteases in this activity. Cysteine proteases were involved in this process, especially caspase-1. Caspase-1 participated in the apoptosis of salivary glands.

Differential sialotranscriptomes of unfed and fed Rhipicephalus haemaphysaloides, with particular regard to differentially expressed genes of cysteine proteases.

BACKGROUND: Rhipicephalus haemaphysaloides, a hard tick, is a common ectoparasite and can be found in many countries. It is recognized as the primary vector of bovine babesiosis in the south of China. During blood feeding, the tick's salivary glands secret numerous essential multifunctional proteins. In this study, a R. haemaphysaloides salivary gland transcriptome was described following the production and analysis of the transcripts from the two cDNA libraries of unfed and fed female ticks. The study focused on the differentially expressed genes and cysteine proteases, which play essential roles in the tick life cycle, that were detected most commonly in the up-regulation libraries. METHODS: The sialotranscriptome was assembled and analyzed though bioinformatic tools and the cysteine protease which is differentially expressed form sialotranscriptome were confirmed by Real-time PCR in salivary glands and different developments of ticks. RESULTS: On the basis of sequence similarities with other species in various databases, we analyzed the unfed and fed sialotranscriptome of R. haemaphysaloides to identify the differentially expressed proteins secreted from the salivary glands during blood feeding and to investigate their biological functions. There were 25,113 transcripts (35 % of the total assembled transcripts) that showed significant similarity to known proteins with high BLAST from other species annotated. In total, 88 % and 89 % of the sequencing reads could be mapped back to assembled sequences in the unfed and fed library, respectively. Comparison of the abundance of transcripts from similar contigs of the two salivary gland cDNA libraries allowed the identification of differentially expressed genes. In total, there were 1179 up-regulated genes and 574 down-regulated genes found by comparing the two libraries. Twenty-five predicted cysteine proteases were screened from the transcript databases, whereas only six protein molecules were confirmed by gene cloning and molecular expression in E.coli which all belonged to the cysteine protease family. Bioinformatic evolutionary analysis showed the relationship of cysteine proteases in ticks with those of other species, suggesting the origin and conservation of these genes. Analysis of sequences from different tick species indicated the further relationships among the proteases, suggesting the closely related function of these genes. Thus, we confirmed their changes in unfed, fed and engorged ticks and salivary glands. The dynamic changes revealed their important roles in the tick life cycle. CONCLUSIONS: Our survey provided an insight into the R. haemaphysaloides sialotranscriptome. The dynamic changes of cysteine proteases in ticks will assist further study of these proteases, which may contribute to the development of anti-tick vaccines or drugs, as well as improving understanding of the roles of cysteine proteases in the tick life cycle.

Characterization of a secreted cystatin from the tick Rhipicephalus haemaphysaloides.

A novel cystatin, designated RHcyst-2, was isolated from the tick Rhipicephalus haemaphysaloides. The full-length cDNA of RHcyst-2 is 773 bp, including an intact open reading frame encoding an expected protein of 139 amino acids and consisting of a 23 amino acids signal peptide. Predicted RHcyst-2 mature protein molecular weight is about 13 kDa, isoelectric point is 4.96. A sequence analysis showed that it has significant homology with the known type 2 cystatins. The recombinant protein of RHcyst-2 was expressed in a glutathione S-transferase-fused soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, C, H, and S, as well as papain, was identified by fluorogenic substrate analysis. The results showed that rRHcyst-2 can effectively inhibit the six cysteine proteases' enzyme activities. An investigation of the RHcyst-2 genes' expression profile by quantitative reverse transcription-PCR demonstrated that it was more richly transcribed in the embryo (egg) stage and mainly distributed in the mid-gut of adult ticks. Western blot analysis confirmed that RHcyst-2 was secreted into tick saliva.