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

Parizi LF, Sabadin GA, Alzugaray MF, Seixas A, Logullo C, Konnai S, Ohashi K, Masuda A, da Silva Vaz I - Parasit Vectors (2015)

Bottom Line: Finally, cross-antigenicity assays revealed that antibodies against the JpIocys2a peptide recognize native and recombinant R. microplus cystatins.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.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre, 91501-970, , RS, Brazil. luisfparizi@cbiot.ufrgs.br.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Fig5: 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.

Mentions: Sera against STQpep, rBrBmcys2b and rBrBmcys2c were used to determine the presence of cystatins in R. microplus tissues as well as the cross-antigenicity between peptide, native, and recombinant cystatins (Figure 5). Native cystatins present in saliva, larvae, ovary, gut, salivary glands, and fat body (apparent molecular mass of 12 kDa) were differentially recognized by these sera. Native cystatins were recognized by anti-rBrBmcys2b in all tissues (Figure 5A), whereas anti-rBrBmcys2c sera recognized cystatins in gut from partially engorged females and in ovary, salivary glands and fat body from fully engorged females (Figure 5B). rBrBmcys2b and native cystatins from partially and fully engorged female salivary glands were recognized by anti-STQpep sera (Figure 5C). The hosts sera inoculated with PBS did not recognize native cystatins in these tissues (data not shown). Furthermore, anti-rBrBmcys2b serum detected cystatin in hemolymph (Figure 5A), unlike anti-rBrBmcys2c and negative controls sera. Since these sera were raised against STQpep and recombinant cystatins, this recognition shows the cross-antigenicity between native and peptide/recombinant cystatins.Figure 5


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

Parizi LF, Sabadin GA, Alzugaray MF, Seixas A, Logullo C, Konnai S, Ohashi K, Masuda A, da Silva Vaz I - Parasit Vectors (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4340882&req=5

Fig5: 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.
Mentions: Sera against STQpep, rBrBmcys2b and rBrBmcys2c were used to determine the presence of cystatins in R. microplus tissues as well as the cross-antigenicity between peptide, native, and recombinant cystatins (Figure 5). Native cystatins present in saliva, larvae, ovary, gut, salivary glands, and fat body (apparent molecular mass of 12 kDa) were differentially recognized by these sera. Native cystatins were recognized by anti-rBrBmcys2b in all tissues (Figure 5A), whereas anti-rBrBmcys2c sera recognized cystatins in gut from partially engorged females and in ovary, salivary glands and fat body from fully engorged females (Figure 5B). rBrBmcys2b and native cystatins from partially and fully engorged female salivary glands were recognized by anti-STQpep sera (Figure 5C). The hosts sera inoculated with PBS did not recognize native cystatins in these tissues (data not shown). Furthermore, anti-rBrBmcys2b serum detected cystatin in hemolymph (Figure 5A), unlike anti-rBrBmcys2c and negative controls sera. Since these sera were raised against STQpep and recombinant cystatins, this recognition shows the cross-antigenicity between native and peptide/recombinant cystatins.Figure 5

Bottom Line: Finally, cross-antigenicity assays revealed that antibodies against the JpIocys2a peptide recognize native and recombinant R. microplus cystatins.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.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre, 91501-970, , RS, Brazil. luisfparizi@cbiot.ufrgs.br.

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.

No MeSH data available.


Related in: MedlinePlus