A Kazal-type inhibitor is modulated by Trypanosoma cruzi to control microbiota inside the anterior midgut of Rhodnius prolixus.
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 R. prolixus, named RpTI, which may be involved in microbiota - T. 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 T. 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 (Ki = 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 T. cruzi trypomastigotes. The RpTI transcript was also knocked down by RNA interference prior to infection of R. prolixus with T. cruzi. The amount of T. 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 R. prolixus compared to non-silenced and infected insects. Our results suggest that T. cruzi infection increases the expression of RpTI to mediate microbiota modulation and is important for parasite immediately after infection with R. prolixus.
Department of Biochemistry, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020 São Paulo, SP, Brazil. Electronic address: tatianesanches@yahoo.com.br.
Tatiane S
TS
Soares
Department of Biochemistry, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020 São Paulo, SP, Brazil. Electronic address: diego_bq@yahoo.com.br.
Diego S
DS
Buarque
Department of Biochemistry, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020 São Paulo, SP, Brazil. Electronic address: bruninharq@hotmail.com.
Bruna R
BR
Queiroz
Department of Biochemistry, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020 São Paulo, SP, Brazil. Electronic address: cicera_gomes@uol.com.br.
Cícera M
CM
Gomes
Department of Biochemistry, Instituto de Química, Universidade Federal do Rio de Janeiro, 21941-909 Rio de Janeiro, RJ, Brazil. Electronic address: grbraz@yahoo.com.br.
Glória R C
GR
Braz
Department of Parasitology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil. Electronic address: ricnaraujo@yahoo.com.
Ricardo N
RN
Araújo
Department of Parasitology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil. Electronic address: marcoshp@icb.ufmg.br.
Marcos H
MH
Pereira
Centro de Pesquisas René Rachou, FIOCRUZ, 30190-002 Belo Horizonte, MG, Brazil. Electronic address: guarneri@cpqrr.fiocruz.br.
Alessandra A
AA
Guarneri
Department of Biochemistry, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020 São Paulo, SP, Brazil. Electronic address: tanaka.bioq@epm.br.
Aparecida S
AS
Tanaka
D000818:Animals; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D007303:Insect Vectors / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D007422:Intestines / Q000382:microbiology*; D064307:Microbiota; D012239:Rhodnius / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D014349:Trypanosoma cruzi / Q000378:metabolism*; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000235:genetics / Q000378:metabolism
D019476:Insect Proteins; D014359:Trypsin Inhibitor, Kazal Pancreatic
Anterior midgut; Kazal family; Microbiota; Rhodnius prolixus; Trypanosoma cruzi