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Biochimie

A Kazal-type inhibitor is modulated by Trypanosoma cruzi to control microbiota inside the anterior midgut of Rhodnius prolixus.


PMID 25731714

Abstract

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.xa0prolixus, named RpTI, which may be involved in microbiota - T.xa0cruzi 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.xa0cruzi 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 11.5xa0kDa as determined by SDS-PAGE analysis. This protein inhibited trypsin (Kixa0=xa00.42xa0nM), whereas serine proteases from the coagulation cascade were not inhibited. Moreover, trypanocidal assays revealed that rRpTI did not interfere in the viability of T.xa0cruzi trypomastigotes. The RpTI transcript was also knocked down by RNA interference prior to infection of R.xa0prolixus with T.xa0cruzi. The amount of T.xa0cruzi 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.xa0prolixus compared to non-silenced and infected insects. Our results suggest that T.xa0cruzi infection increases the expression of RpTI to mediate microbiota modulation and is important for parasite immediately after infection with R.xa0prolixus.