title: 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.
  
  
  CONCLUSIONS
  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.
authors.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.
authors.forename: Luís Fernando
authors.initials: LF
authors.lastname: Parizi
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre, 91501-970, , RS, Brazil. gabrielasabadin@hotmail.com.
authors.forename: Gabriela Alves
authors.initials: GA
authors.lastname: Sabadin
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre, 91501-970, , RS, Brazil. mafealga@gmail.com.|Departamento de Ciencias Microbiológicas, Laboratorio de Inmunología, Facultad de Veterinaria, UDELAR, Montevideo, Uruguay. mafealga@gmail.com.
authors.forename: María Fernanda
authors.initials: MF
authors.lastname: Alzugaray
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre, 91501-970, , RS, Brazil. adriseixas@gmail.com.|Departamento de Farmacociências, Universidade Federal de Ciências da Saúde de Porto Alegre, Rua Sarmento Leite, 245, Porto Alegre, 90050-170, , RS, Brazil. adriseixas@gmail.com.|Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil. adriseixas@gmail.com.
authors.forename: Adriana
authors.initials: A
authors.lastname: Seixas
authors.affiliation: Laboratório de Química e Função de Proteínas e Peptídeos-CBB-UENF and Unidade de Experimentação Animal, Avenida Alberto Lamego, 2000, Horto, Campos dos Goytacazes, 28015-620, , RJ, Brazil. carloslogullo@yahoo.com.br.|Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil. carloslogullo@yahoo.com.br.
authors.forename: Carlos
authors.initials: C
authors.lastname: Logullo
authors.affiliation: Department of Disease Control, Laboratory of Infectious Diseases, Graduate School of Veterinary Medicine, Hokkaido University, 060-0818, Sapporo, Hokkaido, Japan. konnai@vetmed.hokudai.ac.jp.
authors.forename: Satoru
authors.initials: S
authors.lastname: Konnai
authors.affiliation: Department of Disease Control, Laboratory of Infectious Diseases, Graduate School of Veterinary Medicine, Hokkaido University, 060-0818, Sapporo, Hokkaido, Japan. okazu@vetmed.hokudai.ac.jp.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ohashi
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre, 91501-970, , RS, Brazil. aoi.masuda@ufrgs.br.|Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil. aoi.masuda@ufrgs.br.
authors.forename: Aoi
authors.initials: A
authors.lastname: Masuda
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre, 91501-970, , RS, Brazil. itabajara.vaz@ufrgs.br.|Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9090, Porto Alegre, 91540-000, , RS, Brazil. itabajara.vaz@ufrgs.br.|Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil. itabajara.vaz@ufrgs.br.
authors.forename: Itabajara
authors.initials: I
authors.lastname: da Silva Vaz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001769:Blood; D006224:Cricetinae; D003429:Cross Reactions; D015891:Cystatins / Q000276:immunology*; D004063:Digestion; D005260:Female; D006790:Host-Parasite Interactions; D006801:Humans; D007114:Immunization / Q000662:veterinary*; D018884:Ixodes / Q000235:genetics / Q000276:immunology* / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D010802:Phylogeny; D011817:Rabbits; D011994:Recombinant Proteins; D048494:Rhipicephalus / Q000235:genetics / Q000276:immunology* / Q000502:physiology; D016415:Sequence Alignment
chemical_list: D015891:Cystatins; D011994:Recombinant Proteins