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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.


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.
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Fig3: 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.

Mentions: Soluble recombinant BrBmcys2b, BrBmcys2c, and GST-JpIocys2a were expressed in E. coli and purified by affinity chromatography. rGST-JpIocys2a was cleaved from GST-tag by thrombin and the purified rJpIocys2a was recovered (Figure 3). SDS-PAGE showed that the conjugated rGST-JpIocys2a protein weight was about 42 kDa (30 kDa from rGST plus 12 kDa from rJpIocys2a), while the weight of cystatins was approximately 12 kDa. These data are in accordance with in silico molecular weights estimation. rBrBmcys2b and rBrBmcys2c were recognized by anti-histidine tag antibodies, and rGST-JpIocys2a was recognized by anti-GST-Hl in Western blot assay. The purified recombinant cystatins were subsequently used in inhibitory and immunization assays.Figure 3


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)

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

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

Fig3: 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.
Mentions: Soluble recombinant BrBmcys2b, BrBmcys2c, and GST-JpIocys2a were expressed in E. coli and purified by affinity chromatography. rGST-JpIocys2a was cleaved from GST-tag by thrombin and the purified rJpIocys2a was recovered (Figure 3). SDS-PAGE showed that the conjugated rGST-JpIocys2a protein weight was about 42 kDa (30 kDa from rGST plus 12 kDa from rJpIocys2a), while the weight of cystatins was approximately 12 kDa. These data are in accordance with in silico molecular weights estimation. rBrBmcys2b and rBrBmcys2c were recognized by anti-histidine tag antibodies, and rGST-JpIocys2a was recognized by anti-GST-Hl in Western blot assay. The purified recombinant cystatins were subsequently used in inhibitory and immunization assays.Figure 3

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.