The importance of antimicrobial peptides (AMPs) in relation to the survival of invertebrates is well known. The source and the mode of action on the insects' immune system of these molecules have been described from different perspectives. Insects produce their own AMPs as well as obtain these molecules from various sources, for example by absorption through the intestinal tract, as previously described for Boophilus microplus. Blood-sucking barber bug Triatoma infestans attracts social, economic and medical interest owing to its role in the transmission of Chagas disease. Despite new studies, descriptions of AMPs from this insect have remained elusive. Thus, the aims of this work were to characterize the antimicrobial potential of human fibrinopeptide A (FbPA) obtained from the T. infestans haemolymph and identify its natural source. Therefore, FbPA was isolated from the T. infestans haemolymph through liquid chromatography and identified by mass spectrometry. This peptide exhibited antimicrobial activity against Micrococcus luteus. Native FbPA from human blood and the synthetic FbPA also exhibited antimicrobial activity. The synthetic FbPA was conjugated with fluorescein isothiocyanate and offered to the insects. The haemolymph collected after 72 h exhibited fluorescence at the same wavelength as fluorescein isothiocyanate. Our experiments show that beyond intrinsic AMP production, T. infestans is able to co-opt molecules via internalization and may use them as AMPs for protection.

Antimicrobial peptides and proteins (AMPs) are molecules that can interact with microbial cells and lead to membrane disruption or intracellular molecule interactions and death. Several molecules with antimicrobial effects also present other biological activities. One such protein group representing the duplicity of activities is the tachykinin family. Tachykinins (TKs) form a family of neuropeptides in vertebrates with a consensus C-terminal region (F-X-G-Y-R-NH2). Invertebrate TKs and TK-related peptides (TKRPs) are subfamilies found in invertebrates that present high homology with TKs and have similar biological effects. Several of these molecules have already been described but reports of TKRP in Hemiptera species are limited. By analyzing the Triatoma infestans hemolymph by reversed-phase high-performance liquid chromatography, biological assays, and mass spectrometry, two antimicrobial molecules were isolated and identified as TKRPs, which we named as TRP1-TINF and TRP2-TINF (tachykinin-related peptides I and II from T. infestans). TRP1-TINF is a random secondary structure peptide with 9 amino acid residues. It is susceptible to aminopeptidases degradation and is active mainly against Micrococcus luteus (32 μM). TRP2-TINF is a 10-amino acid peptide with a 310 helix secondary structure and is susceptible to carboxypeptidases degradation. It has major antimicrobial activity against both Pseudomonas aeruginosa and Escherichia coli (45 μM). Neither molecule is toxic to human erythrocytes and both present minor toxicity toward Vero cells at a concentration of 1000 μM. As the first description of TKRPs with antimicrobial activity in T. infestans, this work contributes to the wider comprehension of the insects’ physiology and describes pharmacological relevant molecules.

Insects represent the oldest class of animals on the planet, and the present the wider geographic distribution, in a total of 81% of all catalogued species. This evolutive success is partially represented by their innate immune system (divided in cellular and humoral), that acts as their main defense mechanism. In highlight, there are the Antimicrobial Peptides or Proteins-AMPs. Triatoma infestans, member of the Triatominae subfamily, popularly known as kissing bugs, feeds with blood and has a major role as Chagas disease vector. Regarding new researches related to AMPs in triatomines, few molecules have been identified so far, and no research on this area have been developed with T. infestans, and due to the fact that this insect survives in a highly infectious habitat during its life cycle, we believe that it products antimicrobial molecules. On this study, four main AMPs have been identified and synthetized through the t-boc method. The samples nominated as Tin-TK-I and II were similar to Tachykinin-like proteins from insects. They were active against three bacteria and three fungi on the antimicrobial assay. They were not haemolytic, and showed different patterns of degradation, where Tin-TK-I were affected by aminopeptidases, as Tin-TK-II were affected by carboxipeptidases. On the CD, Tin-TK-I presents a random secondary structure, and Tin-TK-II has a helix 3 10 secondary structure, and both of them were able to disrupt negatively charged membranes. The sample nominated Triastina was similar to a glycine rich cuticular protein from T. infestans and was active against two bacteria and three fungi on the antimicrobial assay. It was not hemolytic and was affected by both carboxi and aminopeptidases. Triastina has a secondary structure of a helix 3 10 when analyzed by CD, and had the ability to generate pores that leads to intravesicular loss, and and then completely disrupts the membrane. The sample nominated Triatogen was similar to the human fibrinopeptide A. This peptide was active against three bacteria and six fungi on the antimicrobial assay. A analogue obtained during the SPPS of Triatomina (called Triato-A1) was also active against three bacteria and three fungi. They were not active against human eritrocites, and both were degenerated by aminopeptidases. They present a secondary structure of alfa-helix on the CD, and they were capable of generating pores on membranes. Our hypothesis is that beyond the intrinsic AMP production, T. infestans are able to absorb molecules from their feeding, using them as their own antimicrobial peptides. And, with these discoveries, new parts of its immune system start to be elucidated.

Hemoglobin is one of the most important molecules of the human body. Beyond its physiological activity, hemoglobins are able to inhibit the growth of several microorganisms. Since 1999, studies have reported that antimicrobial peptides can be produced by blood-feeding insects through hemoglobin digestion, and it has been reported that Triatoma infestans can generate an antimicrobial fragment from human fibrinopeptide. Thus T. infestans intestinal content was analyzed through Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), the eluted fractions were tested against Micrococcus luteus, Escherichia coli and Staphylococcus aureus, and the active fractions submitted to mass spectrometry. The data obtained were compared to hemoglobin databases to verify the presence of hemoglobin-derived fragments. Ten fractions eluted from chromatography presented antimicrobial activity, and when analyzed through mass spectrometry revealed the presence of 8 murine hemoglobin α-chain fragments and 24 fragments from murine hemoglobin β fragments. Through the compilation of the fragments is possible to obtain over 67% coverage of both sequences. Part of the amino acid sequences corresponds to the sequences already identified on other intestinal contents of arthropods, and are highly conserved between the blood of other wild animals that are the most common intermediate hosts of Chagas' disease in Brazil and some of the main natural blood source for triatomines.

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