High-Throughput Identification of the Rhodnius prolixus Midgut Proteome Unravels a Sophisticated Hematophagic Machinery

Abstract

Chagas disease is one of the most common parasitic infections in Latin America, which is transmitted by hematophagous triatomine bugs, of which Rhodnius prolixus is the vector prototype for the study of this disease. The protozoan parasite Trypanosoma cruzi, the etiologic agent of this disease, is transmitted by the vector to humans through the bite wound or mucosa. The passage of the parasite through the digestive tract of its vector constitutes a key step in its developmental cycle. Herewith, by a using high-throughput proteomic tool in order to characterize the midgut proteome of R. prolixus, we describe a set of functional groups of proteins, as well as the biological processes in which they are involved. This is the first proteomic analysis showing an elaborated hematophagy machinery involved in the digestion of blood, among which, several families of proteases have been characterized. The evaluation of the activity of cathepsin D proteases in the anterior part of the digestive tract of the insect suggested the existence of a proteolytic activity within this compartment, suggesting that digestion occurs early in this compartment. Moreover, several heat shock proteins, blood clotting inhibitors, and a powerful antioxidant enzyme machinery against reactive oxygen species (ROS) and cell detoxification have been identified. Highlighting the complexity and importance of the digestive physiology of insects could be a starting point for the selection of new targets for innovative control strategies of Chagas disease.

Data_Sheet_7.ZIP

“…In the context of the search for proteins possibly implicated in the triatomine immune system, we also detected three annexins (described initially in R. prolixus) in our proteome samples. After comparing these three annexins with sequences from Ribeiro et al (2014), Vieira et al (2015) and Ouali et al (2020) through Basic Local Alignment Search Tool (BLAST) analyses, we noted that these authors already found them confirming previous inferences from the complete genome sequence. Annexins are a family of proteins that are associated with many biological events, including calcium-binding, interaction with membranes, intracellular vesicle trafficking, arachidonic acid release, leukocyte migration, and that also affects several mediators involved in the inflammatory response including cyclo-oxygenase-2 (Cox-2) and inducible nitric oxide synthase (Minghetti et al, 1999;Ferlazzo et al, 2003;Rescher and Gerke, 2004;Gavins and Hickey, 2012).…”

“…To integrate the proteome of this study to previous knowledge, we gathered the sequences from our proteome samples with the datasets of R. prolixus from Ribeiro et al (2014), Vieira et al (2015) retrieved from the Bioinformatics Resource for Invertebrate Vectors of Human Pathogens (VectorBase, 4 ), and Ouali et al (2020) from ProteomeXchange Consortium. We retrieved the equivalent of UniprotKB accession considering an identity region (BLASTp) of 70% of the query sequence with a similarity ≥ 80% in the case of sequences from Ribeiro et al (2014) and Vieira et al (2015).…”

“…We retrieved the equivalent of UniprotKB accession considering an identity region (BLASTp) of 70% of the query sequence with a similarity ≥ 80% in the case of sequences from Ribeiro et al (2014) and Vieira et al (2015). Since the sequences from Ouali et al (2020) were already given with UniprotKB accessions, this exercise was not necessary. To sum up the gene onthologies (GO terms; Ashburner et al, 2000) of non-redundant protein sequences of TDT (Supplementary File 1) collected until now (n = 3,736; Supplementary File 1a) through the proteome analyses quoted above, we used ClueGO (v2.5.7;Bindea et al, 2009) whitin Cytoscape (v3.8.0;Shannon et al, 2003) with the default parameters (except for pV that was set to 0.5 given the limitation of ClueGO to analyze less than 1,473 vertices) and importing the R. prolixus dataset.…”

“…We also annotated the sequences of our samples and those of Ribeiro et al (2014), Vieira et al (2015) and Ouali et al (2020) for their involvement in the immune system by comparison (BLASTp) with the Insect Innate Immunity Database (IIID, http://bordensteinlab.vanderbilt.edu/IIID/test_immunity. php) (Brucker et al, 2012), transferring function from the subject to the query sequence following the same criteria as described above in the comparison of proteins with KEGG.…”

“…These studies show that the repertoire of genes in the immunological signaling pathway is substantially different in Hemiptera compared to Diptera. Since the methodology based on genomic sequences needs to be complemented by the direct analysis of gene products through functional genomics, a proteome analysis was, first, conducted by 2D gel electrophoresis by Vieira et al (2015) using the AM of R. prolixus and, later on, by Ouali et al (2020) using both AM and PM. These authors mainly found proteins involved in detoxification, amino acids, lipids and sugar degradation; they also confirmed the existence of these proteins by reference to the transcriptome and genome annotations by Ribeiro et al (2014) and Mesquita et al (2015).…”