A Kazal-type inhibitor is modulated by Trypanosoma cruzi to control microbiota inside the anterior midgut of Rhodnius prolixus
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2015-05-01
Autores
Soares, Tatiane S. [UNIFESP]
Buarque, Diego S. [UNIFESP]
Queiroz, Bruna R. [UNIFESP]
Gomes, Cicera M. [UNIFESP]
Braz, Gloria R. C.
Araujo, Ricardo N.
Pereira, Marcos H.
Guarneri, Alessandra A.
Tanaka, Aparecida S. [UNIFESP]
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The triatomine insect, Rhodnius prolixus, is a vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. the parasite must overcome immune response and microbiota to develop inside the midgut of triatomines. in this study, we expressed, purified and characterized a Kazal-type inhibitor from the midgut of Rhodnius prolixus, named RpTI, which may be involved in microbiota - Trypanosoma cruzi interactions. the qPCR showed that the RpTI transcript was primarily expressed in tissues from the intestinal tract and that it was upregulated in the anterior midgut after Trypanosoma cruzi infection. A 315-bp cDNA fragment encoding the mature protein was cloned into the pPIC9 vector and expressed in Pichia pastoris system. Recombinant RpTI (rRpTI) was purified on a trypsin-Sepharose column and had a molecular mass of 11.5 kDa as determined by SDS-PAGE analysis. This protein inhibited trypsin (K-i = 0.42 nM), whereas serine proteases from the coagulation cascade were not inhibited. Moreover, trypanocidal assays revealed that rRpTI did not interfere in the viability of Trypanosoma cruzi trypomastigotes. the RpTI transcript was also knocked down by RNA interference prior to infection of Rhodnius prolixus with Trypanosoma cruzi. the amount of Trypanosoma cruzi in the anterior midgut was significantly lower in RpTI knockdown insects compared to the non-silenced groups. We also verified that the bacterial load is higher in the anterior midgut of silenced and infected Rhodnius prolixus compared to non-silenced and infected insects. Our results suggest that Trypanosoma cruzi infection increases the expression of RpTI to mediate microbiota modulation and is important for parasite immediately after infection with Rhodnius prolixus. (C) 2015 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.
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Biochimie. Paris: Elsevier France-editions Scientifiques Medicales Elsevier, v. 112C, p. 41-48, 2015.