- Introduction
- Methods
- Experimental insects.
- Definition of the functional state of the complement system in blood ingested from T. infestans.
- Complementary DNA synthesis of TiCRT.
- TiCRT cloning.
- TiCRT subcloning.
- TiCRT expression.
- Sequence analysis and alignment of TiCRT.
- Cloning of the TiCRT S domain.
- Subcloning of the TiCRT S domain.
- Expression of the TiCRT S domain.
- Binding of human C1 to the TiCRT S domain.
- Inhibition of C4b deposit by the TiCRT S domain.
- Results
- No activation of the classical pathway of complement is detected in blood recovered from the anterior midgut of T. infestans.
- TiCRT DNA coding sequence identification.
- TiCRT shares important primary structure with insect and mammal CRTs, but not with TcCRT.
- Recombinant TiCRT S domain expression and purification.
- C1 binds to the TiCRT S domain.
- The TiCRT S domain inhibits the classical pathway of complement.
- Discussion
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Triatoma infestans Calreticulin: Gene Cloning and Expression of a Main Domain That Interacts with the Host Complement System
Triatoma infestans is an important hematophagous vector of Chagas disease, a neglected chronic illness affecting approximately 6 million people in Latin America. Hematophagous insects possess several molecules in their saliva that counteract host defensive responses. Calreticulin (CRT), a multifunctional protein secreted in saliva, contributes to the feeding process in some insects. Human CRT (HuCRT) and Trypanosoma cruzi CRT (TcCRT) inhibit the classical pathway of complement activation, mainly by interacting through their central S domain with complement component C1. In previous studies, we have detected CRT in salivary gland extracts from T. infestans. We have called this molecule TiCRT. Given that the S domain is responsible for C1 binding, we have tested its role in the classical pathway of complement activation in vertebrate blood. We have cloned and characterized the complete nucleotide sequence of CRT from T. infestans, and expressed its S domain. As expected, this S domain binds to human C1 and, as a consequence, it inhibits the classical pathway of complement, at its earliest stage of activation, namely the generation of C4b. Possibly, the presence of TiCRT in the salivary gland represents an evolutionary adaptation in hematophagous insects to control a potential activation of complement proteins, present in the massive blood meal that they ingest, with deleterious consequences at least on the anterior digestive tract of these insects.
Author Notes
Authors' addresses: Katherine Weinberger, Norberto Collazo, Juan Carlos Aguillón, María Carmen Molina, Carlos Rosas, Jaime Peña, Javier Pizarro, Ismael Maldonado, and Arturo Ferreira, Program of Immunology, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile, E-mails: kta.weinberger@gmail.com, norberto.collazo@gmail.com, jaguillo@med.uchile.cl, mcmolina@med.uchile.cl, carlosrosas@u.uchile.cl, jaime.pena.alvarez@gmail.com, jpizarrob@ug.uchile.cl, ismael_amf@yahoo.es, and aferreir@med.uchile.cl. Pedro E. Cattan, Departamento de Ciencias Biológicas Animales, Universidad de Chile, Santiago, Chile, E-mail: pcattan@u.uchile.cl. Werner Apt, Clinical Investigation, North Unit of Parasitology, Faculty of Medicine, University of Chile, Santiago, Chile, E-mail: wapt@med.uchile.cl.