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Genome Wide Mapping of Peptidases in Rhodnius Prolixus

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Genome Wide Mapping of Peptidases in Rhodnius Prolixus ORIGINAL RESEARCH published: 12 December 2017 doi: 10.3389/fphys.2017.01051 Genome Wide Mapping of Peptidases in Rhodnius prolixus: Identification of Protease Gene Duplications, Horizontally Transferred Proteases and Analysis of Peptidase A1 Structures, with Considerations on Their Role in the Evolution of Edited by: Xanthe Vafopoulou, Hematophagy in Triatominae York University, Canada Reviewed by: Bianca S. Henriques 1†, Bruno Gomes 1†, Samara G. da Costa 1, Caroline da Silva Moraes 1, Leonardo Luis Fruttero, Rafael D. Mesquita 2, 3, Viv M. Dillon 4, Eloi de Souza Garcia 1, 2, Patricia Azambuja 1, 2, Facultad de Ciencias Químicas, 5 1, 2 Universidad Nacional de Córdoba, Roderick J. Dillon and Fernando A. Genta * Argentina 1 Laboratory of Insect Physiology and Biochemistry, Oswaldo Cruz Institute – Oswaldo Cruz Foundation (IOC-FIOCRUZ), Rio Márcio Galvão Pavan, de Janeiro, Brazil, 2 National Institute of Science and Technology for Molecular Entomology (INCT-EM), Cidade Universitária, Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil, 3 Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, 4 Institute of Integrative Brazil Biology, University of Liverpool, Liverpool, United Kingdom, 5 Division of Biomedical and Life Sciences, Lancaster University, *Correspondence: Lancaster, United Kingdom Fernando A. Genta genta@ioc.fiocruz.br; [email protected] Triatominae is a subfamily of the order Hemiptera whose species are able to feed in the †These authors have contributed vertebrate blood (i.e., hematophagy). This feeding behavior presents a great physiological equally to this work. challenge to insects, especially in Hemipteran species with a digestion performed by lysosomal-like cathepsins instead of the more common trypsin-like enzymes. With the Specialty section: This article was submitted to aim of having a deeper understanding of protease involvement in the evolutionary Invertebrate Physiology, adaptation for hematophagy in Hemipterans, we screened peptidases in the Rhodnius a section of the journal Frontiers in Physiology prolixus genome and characterized them using common blast (NCBI) and conserved Received: 06 October 2017 domain analyses (HMMER/blast manager software, FAT, plus PFAM database). We Accepted: 30 November 2017 compared the results with available sequences from other hemipteran species and Published: 12 December 2017 with 18 arthropod genomes present in the MEROPS database. Rhodnius prolixus Citation: contains at least 433 protease coding genes, belonging to 71 protease families. Seven Henriques BS, Gomes B, Costa SG, Moraes CS, Mesquita RD, Dillon VM, peptidase families in R. prolixus presented higher gene numbers when compared to Garcia ES, Azambuja P, Dillon RJ and other arthropod genomes. Further analysis indicated that a gene expansion of the Genta FA (2017) Genome Wide Mapping of Peptidases in Rhodnius protease family A1 (Eukaryotic aspartyl protease, PF00026) might have played a major prolixus: Identification of Protease role in the adaptation to hematophagy since most of these peptidase genes seem to be Gene Duplications, Horizontally recently acquired, are expressed in the gut and present putative secretory pathway signal Transferred Proteases and Analysis of Peptidase A1 Structures, with peptides. Besides that, most R. prolixus A1 peptidases showed high frequencies of basic Considerations on Their Role in the residues at the protein surface, a typical structural signature of Cathepsin D-like proteins. Evolution of Hematophagy in Triatominae. Front. Physiol. 8:1051. Other peptidase families expanded in R. prolixus (i.e., C2 and M17) also presented doi: 10.3389/fphys.2017.01051 significant differences between hematophagous (higher number of peptidases) and Frontiers in Physiology | www.frontiersin.org 1 December 2017 | Volume 8 | Article 1051 Henriques et al. Rhodnius prolixus Peptidases non-hematophagous species. This study also provides evidence for gene acquisition from microorganisms in some peptidase families in R. prolixus: (1) family M74 (murein endopeptidase), (2) family S29 (Hepatitis C virus NS3 protease), and (3) family S24 (repressor LexA). This study revealed new targets for studying the adaptation of these insects for digestion of blood meals and their competence as vectors of Chagas disease. Keywords: Rhodnius prolixus, insect, genome, blood feeding, peptidase, gene transfer INTRODUCTION species are potential vectors of the Chagas disease parasite Trypanosoma cruzi (Beard, 2004). This neglected disease is The arthropod development requires several nutritional historically associated with countryside areas of Latin America components that are usually obtained through the digestive and currently affects up to 7 million people. The epidemiology function. In this process, enzymes are crucial to break down of Chagas disease has recently changed, with the increase complex compounds into simpler molecules that can be absorbed of its distribution to include several urban habitats (WHO, by the organism to be used for energy, growth, or reproduction. 2017). Rhodnius prolixus Stål (1859) is one of the main The molecular properties of these enzymes differ among vectors of Trypanosoma cruzi in South/Central America and arthropod taxa and are especially correlated to their phylogenetic one of the most used triatomine laboratory models to study background. However, other factors such as the adaptation to insect physiology and interaction with Trypanosoma spp. new feeding resources may have influenced the pre-established (Wigglesworth, 1972). composition of enzymes in closely related organisms (Terra and Haemoglobin and albumin are major components of Ferreira, 2005). vertebrate blood, and because of that the study of digestion Hematophagy (i.e., ability in feeding on vertebrate blood) in hematophagous insects has primarily focused in protein potentiates the ingestion of high amounts of proteins in degradation and absortion. In mosquitoes (Diptera), the main short time by arthropods. This feeding behavior has emerged proteases involved in blood digestion are serine proteases independently in several arthropod groups, particularly in as trypsin and chymotrypsin, plus carboxypeptidases and females that require higher amounts of proteins to produce aminopeptidases (Clements, 2000). In Triatomines like their offspring (Black and Kondratieff, 2004; Attardo et al., Rhodnius prolixus, belonging to the order Hemiptera, serine 2005; Grimaldi and Engel, 2005). However, hematophagy brings proteases (e.g., trypsin and chymotrypsin) activities are not several challenges to insects, due to an increase of contact major midgut activities. However, digestive Cathepsin D-like with vertebrates that may present predatory behavior or defense aspartic-proteinases, Cathepsin-L like cysteine-proteinases, plus mechanisms, and blood parasites that are potential pathogens aminopeptidases and carboxypeptidases have been described and (Devenport and Jacobs-Lorena, 2004; Balenghien et al., 2011). are involved in digestion of blood proteins and interaction with In addition to that, the heme molecule released by digestion of pathogens as T. cruzi (Garcia et al., 2010). Recent transcriptome hemoglobin in hematophagous is toxic and result in oxidative analysis of R. prolixus digestive tract described the main groups stress to the midgut cells (Graça-Souza et al., 2006). of proteins involved in the main functions in this tissue (e.g., In Hemiptera, Reduviidae, Polytecnidae and Cimicidae digestion, immune system), indicating possible gene expansion families present species that developed hematophagy. The in cysteine and aspartic proteinases (Ribeiro et al., 2014). Besides majority of the hemipteran species are phytophagous such as that, the genome of R. prolixus was recently sequenced (Mesquita aphids and cicadas, but the order also gathers other feeding et al., 2015) with the mapping of several proteinase families. behaviors such as predation of small vertebrates and insects Despite this evidence, there are still no detailed genomic studies (Beard, 2004). The adaptation to digesting vertebrate blood by supporting the hypothesis of proteinase family amplification in hemipteran species required different strategies when compared this hematophagous insect. to most studied hematophagous insects (mainly Diptera). In this study, we extended previous reports, screening the Hemipterans perform digestion in an acid gut by lysosomal-like R. prolixus genome to identify peptidases from each family cathepsins instead of the most common trypsin-like enzymes described in the MEROPS database (Rawlings et al., 2014). We secreted in an alkaline gut (Garcia and Garcia, 1977; Terra performed further analyses to determine and characterize genetic and Ferreira, 2005). It has been postulated that the recruitment expansion of peptidase families that may be linked to adaptations of lysosomal-like cathepsins in digestion in Hemipterans was for hematophagy in triatomines. We detected two main a secondary adaptation for protein digestion, after the loss of mechanisms of expansion in protease gene families: putative the peritrophic membrane, counter-current fluxes and serine gene duplication in ancestral gene families and horizontal endopeptidases that probably occurred in a sap-sucking ancestor gene transfer from microorganisms. These two mechanisms (Houseman and Downe, 1983; Terra, 2001). were probably necessary during evolution in triatomines as In Reduviidae, the Triatominae subfamily also known as they provided a more diverse
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