Rhipicephalus microplus and Ixodes ovatus cystatins in tick blood digestion and evasion of host immune response

Abstract

Background: Cystatins are a group of cysteine protease inhibitors responsible for physiological proteolysis regulation and present in a wide range of organisms. Studies about this class of inhibitors in parasites have contributed to clarify their roles in important physiological processes, like blood digestion and modulation of host immune response during blood feeding. Thus, cystatins are a subject of research on the development of new parasite control methods. Additionally, the characterization of proteins shared by different parasite species represents a valuable strategy to find potential targets in multi-species control methods. However, cystatin functions in ticks remain undetermined, especially in Rhipicephalus microplus and Ixodes ovatus, two species that affect livestock and human health, respectively.

Methods: Here we report the inhibitory profile of two R. microplus (BrBmcys2b and BrBmcys2c) and one I. ovatus (JpIocys2a) cystatins to commercial cathepsins B, C, and L. The presence of native cystatins in R. microplus tissues was analyzed using sera against recombinant BrBmcys2b and BrBmcys2c. Also, a peptide from JpIocys2a was synthesized for rabbit immunization, and this serum was used to analyze the cross antigenicity between R. microplus and I. ovatus cystatins.

Results: Enzymatic inhibition profile of tick cystatins shows a distinct modulation for cathepsins related to tick blood digestion and evasion of host immune response. Furthermore, BrBmcys2b was detected in saliva and different tissues along tick stages, while BrBmcys2c was detected mainly in gut from partially engorged R. microplus females, demonstrating a distinct pattern of cystatin expression, secretion and traffic between tick tissues. Moreover, phylogenetic analysis suggests that JpIocys2a belongs to the group of tick gut secreted cystatins. Finally, cross-antigenicity assays revealed that antibodies against the JpIocys2a peptide recognize native and recombinant R. microplus cystatins.

Conclusion: The presence of these proteins in different tissues and their ability to differentially inhibit cathepsins suggest distinct roles for JpIocys2a, BrBmcys2b, and BrBmcys2c in blood digestion, egg and larvae development, and modulation of host immune response in tick physiology. The cross-antigenicity between native and recombinant cystatins supports further experiments using JpIocys2a, BrBmcys2b, and BrBmcys2c as vaccine antigens.

Figure 1

JpIocys2a nucleotide and predicted amino acid sequences. The predicted signal peptide (SignalP) is underlined. Cysteine residues are in gray and the conserved cystatin motifs PI (G), P II (QxVxG), and P III (PW) are boxed.

Figure 2

Conserved and antigenic tick cystatin regions for JpIocys2a peptide selection. Antigenic index plots for tick cystatins were predicted using the Jameson–Wolf algorithm. Graphic increased positivity shows predictive antigenic sites. Alignment shows conserved regions between JpIocys2a and R. microplus cystatins. Black boxes indicate conserved and antigenic amino acid region for each sequence. Asterisk indicate the selected region for peptide synthesis.

Figure 3

SDS-PAGE and Western blot of rJpIocys2a, rBrBmcys2b and rBrBmcys2c production. Western blot: purified rGST-JpIocys2a probed with anti-GST-Hl primary antibody and rabbit anti-IgG secondary antibody conjugate with alkaline phosphatase; purified rBrBmcys2b and rBrBmcys2c probed with anti-histidine tag primary antibody conjugate with alkaline phosphatase. Alkaline phosphatase revelations were performed with NBT and BCIP. SDS-PAGE: Recombinant cystatins resolved by 14% SDS-PAGE were stained with Coomassie blue G-250; purified rGST-JpIocys2a before and after thrombin cleavage (rGST and JpIocys2a); purified rJpIocys2a, rBrBmcys2b and rBrBmcys2c. MW: molecular weight.

Figure 4

Activity inhibition assay of cathepsins B, C, and L by rJpIocys2a, rBrBmcys2b, and rBrBmcys2c. Cathepsins B, C, and L were incubated with Z-Arg-Arg-pNA (0.125 mM), Gly-Phe-pNA (1.8 mM), or Z-Phe-Arg-MCA (0.02 mM), respectively, in the presence of different concentrations of rJpIocys2a, rBrBmcys2b, and rBrBmcys2c. The abscissa shows inhibitors concentration (nM, log₁₀); the ordinate shows percentage of remaining enzymatic activity. Incubation of cathepsins B, C, and L without rJpIocys2a, rBrBmcys2b, and rBrBmcys2c represents 100% of enzyme activity.

Figure 5

Cross-immunogenicity between native and recombinant tick cystatins. By Western blot, R. microplus and I. ovatus recombinant cystatins or R. microplus tissue extracts were analyzed using sera (1:50) against: A) rBrBmcys2b; B) rBrBmcys2c C) STQpep. SG, salivary glands; OV, ovary; FB, fatty body, S, saliva; H, hemolymph; L, larva; SGp, salivary glands from partially engorged female; SGt, salivary glands from fully engorged female; C2b, rBrBmcys2b; C2c, rBrBmcys2c; CIo, rJpIocys2a. MW: molecular weight. Anti-IgG alkaline phosphatase rabbit sera and peroxidase hamster sera conjugates were used as secondary antibodies. Alkaline phosphatase revelations were performed with NBT and BCIP. Peroxidase revelations were performed with DAB, H₂O₂ and CoCl₂.

Figure 6

Phylogenetic analysis of tick cystatins. R. microplus BrBmcys2b [GenBank: KC816580], R. microplus BrBmcys2c [GenBank: KC816581], R. microplus Rmcystatin-3 [GenBank: AIX97454]; I. ovatus JpIocys2a [GenBank: KP253747]; I. scapularis sialostatin 1 [GenBank: AF483724], I. scapularis sialostatin 2 [GenBank: DQ066048]; H. longicornis Hlcyst-2 [GenBank: DQ364159], H. longicornis Hlcyst-3 [GenBank: EU426545]. Bootstrap values of 1,000 simulations are shown at the branches.