Def-Aaa

An antimicrobial peptide belonging to the defensin family of small cationic peptides associated with innate immunity in insects was isolated from the hemolymph of Rhodnius prolixus, a vector of Chagas disease. This peptide, designated R. prolixus defensin A, was purified and sequenced. The active peptide contains 43 residues and aligns well with other insect defensins. However the pre-pro region of the sequence has little shared identity with other insect defensins. We have identified 3 isoforms of R. prolixus defensin from cDNA clones obtained from RNA isolated from the whole bodies of immune activated insects. Northern analysis and Real-Time Quantitative PCR indicate that there is a very low baseline transcription of this peptide in naïve insects, and that transcription increases significantly in the fat body of immune activated insects. In addition there is a delayed induction of transcription of this peptide in the intestine 24 h post activation suggesting that the midgut/intestine of this species is active in the immune response against pathogens.

Insects possess both cellular and humoral immune responses. The latter makes them capable to recognize and control invading pathogens after synthesis of a variety of small proteins, also known as antimicrobial peptides. Defensins, cysteine-rich cationic peptides with major activity against Gram-positive bacteria, are one ubiquitous class of antimicrobial peptides, widely distributed in different animal and plant taxa. Regarding triatomines in each of the so far analyzed species, various defensin gene isoforms have been identified. In the present study, these genes were sequenced and used as a molecular marker for phylogenetic analysis. Considering the vectors of Chagas disease the authors are reporting for the first time the presence of these genes in Triatoma sordida (Stål, 1859), Rhodnius nasutus (Stål, 1859), and Panstrongylus megistus (Burmeister, 1835). Members of the Triatoma brasiliensis species complex were included into the study to verify the genetic variability within these taxa. Mainly in their mature peptide, the deduced defensin amino acid sequences were highly conserved. In the dendrogram based on defensin encoding nucleotide, sequences the Triatoma Def3/4 genes were separated from the rest. In the dendrogram based on deduced amino acid sequences the Triatoma Def2/3/4 together with Rhodnius DefA/B pre-propeptides were separated from the rest. In the sub-branches of both the DNA and amino acid dendrograms, the genus Triatoma was separated from the genus Rhodnius as well as from P. megistus.

Chagas disease remains a serious health problem for countries where the most common mode of transmission is infection contracted from the feces of a Triatominae insect vector. In México, 32 species of Triatoma have been identified; amongst them, Triatoma (Meccus) pallidipennis is an endemic species reported to have high percentages of infection with T. cruzi Defensins, cysteine-rich cationic peptides, are a family of antimicrobial peptides (AMPs); the synthesis of these molecules is crucial for insect's immune defense. In the present study, the genes encoding defensins in T. pallidipennis were sequenced with the purpose of identifying the variability of these genes in a Mexican vector of T. cruzi We found 12 different genes encoding three mature peptides, all of which had the typical folding of a functional insect defensin. In this work two Defensins type 1 and one type 4 were identified. The pro-peptide domain was highly variable and the mature peptide was not. This is the first report focus on variability of defensins from an epidemiologically important Triatoma in Mexico.