title: Jaburetox affects gene expression and enzyme activities in Rhodnius prolixus, a Chagas' disease vector.
abstract: Jaburetox, a recombinant peptide of ∼11kDa derived from one of the Canavalia ensiformis (Jack Bean) urease isoforms, is toxic and lethal to insects belonging to different orders when administered orally or via injection. Previous findings indicated that Jaburetox acts on insects in a complex fashion, inhibiting diuresis and the transmembrane potential of Malpighian tubules, interfering with muscle contractility and affecting the immune system. In vitro, Jaburetox forms ionic channels and alters permeability of artificial lipid membranes. Moreover, recent data suggested that the central nervous system (CNS) is a target organ for ureases and Jaburetox. In this work, we employed biochemical, molecular and cellular approaches to explore the mode of action of Jaburetox using Rhodnius prolixus, one of the main Chagas' disease vectors, as experimental model. In vitro incubations with fluorescently labeled Jaburetox indicated a high affinity of the peptide for the CNS but not for salivary glands (SG). The in vitro treatment of CNS or SG homogenates with Jaburetox partially inhibited the activity of nitric oxide synthase (NOS), thus disrupting nitrinergic signaling. This inhibitory effect was also observed in vivo (by feeding) for CNS but not for SG, implying differential modulation of NOS in these organs. The inhibition of NOS activity did not correlate to a decrease in expression of its mRNA, as assessed by qPCR. UDP-N-acetylglucosamine pyrophosphorylase (UAP), a key enzyme in chitin synthesis and glycosylation pathways and a known target of Jaburetox in insect CNS, was also affected in SG, with activation of the enzyme seen after both in vivo or in vitro treatments with the peptide. Unexpectedly, incubation of Jaburetox with a recombinant R. prolixus UAP had no effect on its activity, implying that the enzyme's modulation by the peptide requires the participation of other factor(s) present in CNS or SG homogenates. Feeding Jaburetox to R. prolixus decreased the mRNA levels of UAP and chitin synthase, indicating a complex regulation exerted by the peptide on these enzymes. No changes were observed upon Jaburetox treatment in vivo and in vitro on the activity of the enzyme acid phosphatase, a possible link between UAP and NOS. Here we have demonstrated for the first time that the Jaburetox induces changes in gene expression and that SG are another target for the toxic action of the peptide. Taken together, these findings contribute to a better understanding of the mechanism of action of Jaburetox as well as to the knowledge on basic aspects of the biochemistry and neurophysiology of insects, and might help in the development of optimized strategies for insect control.
authors.affiliation: Brain Institute (Instituto do Cérebro-INSCER), Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6690, prédio 63, CEP 90610-000, Porto Alegre, Rio Grande do Sul, Brazil; Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, prédio 43431, CEP 91501-970, Porto Alegre, Rio Grande do Sul, Brazil. Electronic address: leofruttero@cbiot.ufrgs.br.
authors.forename: Leonardo L
authors.initials: LL
authors.lastname: Fruttero
authors.affiliation: Brain Institute (Instituto do Cérebro-INSCER), Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6690, prédio 63, CEP 90610-000, Porto Alegre, Rio Grande do Sul, Brazil. Electronic address: nataliarmoyetta@gmail.com.
authors.forename: Natalia R
authors.initials: NR
authors.lastname: Moyetta
authors.affiliation: Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, prédio 43431, CEP 91501-970, Porto Alegre, Rio Grande do Sul, Brazil. Electronic address: monie.krug@gmail.com.
authors.forename: Monique Siebra
authors.initials: MS
authors.lastname: Krug
authors.affiliation: Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, prédio 43431, CEP 91501-970, Porto Alegre, Rio Grande do Sul, Brazil. Electronic address: valbroll@ufrgs.br.
authors.forename: Valquiria
authors.initials: V
authors.lastname: Broll
authors.affiliation: Brain Institute (Instituto do Cérebro-INSCER), Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6690, prédio 63, CEP 90610-000, Porto Alegre, Rio Grande do Sul, Brazil. Electronic address: matheusgrahl@hotmail.com.
authors.forename: Matheus V Coste
authors.initials: MV
authors.lastname: Grahl
authors.affiliation: Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, prédio 43431, CEP 91501-970, Porto Alegre, Rio Grande do Sul, Brazil. Electronic address: rafael.guerra@ufrgs.br.
authors.forename: Rafael
authors.initials: R
authors.lastname: Real-Guerra
authors.affiliation: Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, prédio 43431, CEP 91501-970, Porto Alegre, Rio Grande do Sul, Brazil; Department of Molecular Biology and Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, prédio 43431, CEP 91501-970, Porto Alegre, Rio Grande do Sul, Brazil. Electronic address: fernanda.staniscuaski@ufgrs.br.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Stanisçuaski
authors.affiliation: Brain Institute (Instituto do Cérebro-INSCER), Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6690, prédio 63, CEP 90610-000, Porto Alegre, Rio Grande do Sul, Brazil; Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, prédio 43431, CEP 91501-970, Porto Alegre, Rio Grande do Sul, Brazil. Electronic address: celia.carlini@pucrs.br.
authors.forename: Celia R
authors.initials: CR
authors.lastname: Carlini
mesh_terms: D000818:Animals; D014355:Chagas Disease / Q000635:transmission; D002687:Chitin Synthase / Q000235:genetics; D004199:Disease Vectors; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007300:Insect Control / Q000379:methods*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins; D012239:Rhodnius / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D014510:Urease / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D010940:Plant Proteins; D019001:Nitric Oxide Synthase; D002687:Chitin Synthase; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D014510:Urease; C525847:jaburetox-2Ec, Canavalia ensiformis
keywords: Action mechanism; Enzymatic pathways; Jaburetox; Rhodnius prolixus; Ureases