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

Abstract

BackgroundCystatins 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.MethodsHere 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.ResultsEnzymatic 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.ConclusionThe 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.

Protease Inhibitors in Tick Saliva: The Role of Serpins and Cystatins in Tick-host-Pathogen Interaction

“…Their expression differs among various developmental stages and tissues, but since their presence has only been assessed by immunodetection methods, cross reactivity between antibodies is possible and has indeed been reported (Imamura et al, 2013 ). This study also examined the inhibitory specificity of two cystatins: while BrBmcys2b targeted cathepsins B, C, and L, BrBmcys2c only inhibited cathepsins C and L (Parizi et al, 2015 ). Antibodies raised against recombinant proteins detected BrBmcys2b in all tick tissues, while anti-BrBmcys2c serum only recognized the protein in the gut from partially engorged females and in the ovaries, salivary glands, and fat bodies from fully engorged females (Parizi et al, 2015 ).…”

“…This study also examined the inhibitory specificity of two cystatins: while BrBmcys2b targeted cathepsins B, C, and L, BrBmcys2c only inhibited cathepsins C and L (Parizi et al, 2015 ). Antibodies raised against recombinant proteins detected BrBmcys2b in all tick tissues, while anti-BrBmcys2c serum only recognized the protein in the gut from partially engorged females and in the ovaries, salivary glands, and fat bodies from fully engorged females (Parizi et al, 2015 ). The expression patterns suggest rather homeostatic function of these cystatins in ticks than immunomodulatory activity in the host (Imamura et al, 2013 ).…”

Strategies for new and improved vaccines against ticks and tick‐borne diseases

“…As OmC2 is expressed in both the salivary gland and gut tissues of O. moubata , the authors speculated about a possible ‘dual’ action that impairs both feeding (via interfering with modulation of host immune response) and blood digestion . More recently, identification and characterization of cystatins from other tick species, namely R. appendiculatus , R. microplus and Ixodes ovatus , were reported and the immunogenicity of corresponding recombinants tested as a first step towards their assessment as tick vaccine candidates .…”

Expression of cysteine proteinases and cystatins in parasites and use of cysteine proteinase inhibitors in parasitic diseases. Part II: Arthropods

“…As regards I. ovatus, a vector transmitting viral encephalitis in Japan, Parizi et al, (61) succeeded to identify CYS type II (five variants, JpIoCys2a-e). Their expression differs among various developmental stages and tissues (e.g.…”