Isolation and molecular characterization of a major hemolymph serpin from the triatomine, Panstrongylus megistus

Abstract

BackgroundChagas disease kills 2.5 thousand people per year of 15 million persons infected in Latin America. The disease is caused by the protozoan, Trypanosome cruzi, and vectored by triatomine insects, including Panstrongylus megistus, an important vector in Brazil. Medicines treating Chagas disease have unpleasant side effects and may be ineffective, therefore, alternative control techniques are required. Knowledge of the T. cruzi interactions with the triatomine host needs extending and new targets/strategies for control identified. Serine and cysteine peptidases play vital roles in protozoan life cycles including invasion and entry of T. cruzi into host cells. Peptidase inhibitors are, therefore, promising targets for disease control.MethodsSDS PAGE and chromatograpy detected and isolated a P. megistus serpin which was peptide sequenced by mass spectrometry. A full amino acid sequence was obtained from the cDNA and compared with other insect serpins. Reverse transcription PCR analysis measured serpin transcripts of P. megistus tissues with and without T. cruzi infection. Serpin homology modeling used the Swiss Model and Swiss-PDB viewer programmes.ResultsThe P. megistus serpin (PMSRP1) has a ca. 40 kDa molecular mass with 404 amino acid residues. A reactive site loop contains a highly conserved hinge region but, based on sequence alignment, the normal cleavage site for serine proteases at P1-P1′ was translocated to the putative position P4′-P5′. A small peptide obtained corresponded to the C-terminal 40 amino acid region. The secondary structure of PMSRP1 indicated nine α-helices and three β-sheets, similar to other serpins. PMSRP1 transcripts occurred in all tested tissues but were highest in the fat body and hemocytes. Levels of mRNA encoding PMSRP1 were significantly modulated in the hemocytes and stomach by T. cruzi infection indicating a role for PMSRP1 in the parasite interactions with P. megistus.ConclusionsFor the first time, a constitutively expressed serpin has been characterized from the hemolymph of a triatomine. This opens up new research avenues into the roles of serine peptidases in the T. cruzi/P. megistus association. Initial experiments indicate a role for PMSRP1 in T. cruzi interactions with P. megistus and will lead to further functional studies of this molecule.

The study of plant protein accumulation in gut of insect using proteomics technique: Wheat–sunn pest interaction

“…Kansal et al (2008) showed that protease inhibitors when expressed in higher amounts in important crops are effective against phytophagous insects, also after denaturation by cooking, improvement of the quality occurs lead to release of essential amino acids. Extracted serpin from hemolymph of Panstrongylus megistus had ideal potential in control of Chagas disease was caused by protozoan, Trypanosome cruzi (Moreira et al, 2014).…”

Mosquito Surveillance for Prevention and Control of Emerging Mosquito-Borne Diseases in Portugal — 2008–2014

“…Some studies even indicate that the Flaviviridae family or otherwise, the genus Flavivirus , may suffer some reorganization in the near future, in a way to better assign this “incoming” species diversity [ 42 ]. Furthermore, the presence of DNA sequences similar to ISFs in mosquito genomes from the genera Aedes and Ochlerotatus also suggests a close evolution between these viruses and their mosquito hosts [ 43 , 44 , 45 , 46 ].…”

“…Never as today has the European region been faced with such concern regarding the introduction and dispersion of Aedes albopictus and Ae. aegypti among other invasive mosquitoes [ 1 , 2 ]. Reports of autochthonous transmission of Chikungunya and Dengue viruses in Italy, France and Croatia [ 3 , 4 , 5 , 6 ] and the 2012 Dengue outbreak in Madeira Island [ 7 , 8 ] make obvious the susceptibility to these diseases in areas where the primary mosquito vectors, Ae.…”

“…Moreover, some mosquito species are causing concern, both in posing a risk to public health and causing massive nuisance problems in some areas due to superabundance phenomena [ 9 , 10 ]. The re-emergence of pathogens through native mosquito populations such as West Nile virus (WNV) and malaria has been a major issue in several European regions [ 11 , 12 , 13 , 14 , 15 , 16 ]. In this context, the need to enhance knowledge about vector species, their distribution, abundance, and role as vectors of disease agents in Portugal, urged the establishment of a national vector surveillance programme—REVIVE ( RE de de VI gilância de VE ctores)—in 2008 under the custody of Portuguese Ministry of Health.…”

Serpin functions in host-pathogen interactions

“…The mechanism of serpin-inhibiting proteases relies on a reactive center loop (RCL) that interacts with the target enzyme. Structural studies reveal that serpins are characterized by a common core domain consisting of three -sheets and 8-9 -helices and a reactive center loop, as shown in Figure 1A (Moreira et al 2014;Patschull et al 2011;Pearce et al 2008). Although this core region is present in all serpins, sequence homology among members in the family could be as low as 25%.…”