Thus, it remains to be investigated whether FbPA internalized by comes from endogenous cleavage within the host or another process. Moreover, the role played by the peptide in the haemolymph requires further investigation. AMPs for protection. and (Cociancich et al., 1993), expression of two defensins (def3 and def4) in several tissues of the barber bug (Waniek et al., 2009), a description of trialysin expression in the salivary glands of (Assump??o et al., 2008) and two different types of digestive tract lysozymes (Kollien et al., 2003; Balczun et al., 2008; Flores-Villegas et al., 2015) provide evidence for the role of AMPs in triatomine immune defense mechanisms. Although there is evidence of AMP production by triatomines, there are no published descriptions of antimicrobial molecules isolated from haemolymph yet. Four AMPs were characterized among ten isolated from blood (Diniz, 2016unpublished data). The most relevant isolated finding was the presence of human fibrinopeptide A (FbPA) with antimicrobial activity. Regarding the relevance ENPP3 of the description of AMPs, elucidation of their role in the invertebrate immune system and, consequently, development of new AMP-dependent drugs, our aim was to identify and determine the origin of AMPs isolated from the Chagas disease-transmitting vector haemolymph. By combining mass spectrometry approaches with functional assays, our results provide evidence that is able to assimilate molecules through feeding and use them as part of their immune system, probably functioning as AMPs circulating in the haemolymph. Methods The experiments were performed under the exemption of the (CEUAIBComit de tica no uso de animais do Instituto Butantan) n I-1345/15. Bacterial strains The microorganisms (strain A270), (ATCC 29213), (Nalidixic resistant), (ATCC 10778), (ATCC 6633), (SBS363), -12, (ATCC 8750), (ATCC 4112), (ATCC 27853), (IOC 4564), (IOC 4558), (IOC 4560), sp. (bread isolated), (bread isolated), (bread isolated), (IBCB-215), and (ATCC 26362) were obtained from the Special Laboratory of Toxinology, Butantan Institute (S?o Paulo, Brazil). Animals were obtained from the Ecolyzer Group Entomology Laboratory and kept alive in the vivarium of the Special Laboratory of Toxinology, Butantan Institute (S?o Paulo, Brazil) at 37C and fed every 2 weeks with human blood from a healthy volunteer donor, in the presence of citrate buffer (150 mM, pH 7,4) (Martins et al., 2001). Bacteria inoculation and haemolymph collection One week after blood feeding, adult were injured with needles soaked in an and pool, both at logarithmic-phase growth. After 72 h, 300 L of haemolymph was collected by excising the metathoracic legs and pressing on the abdomen of the (Boman et al., 1974) in the presence of phenylthiourea (PTU), to avoid the activation of the phenoloxidase cascade, and stored at Zaleplon ?80C until use. Sample fractionation Acid and solid-phase extractions To release the contents of the haemocytes, the sample was incubated in acetic acid (2 M) for 5 min and centrifuged at 16.000 g for 30 min at 4C. The supernatant was injected into coupled Sep-Pack C18 cartridges (Waters Associates) equilibrated in Zaleplon 0.1% trifluoroacetic acid (TFA). The sample was eluted in three different acetonitrile (ACN) concentrations (5, 40, and 80%) and then concentrated and reconstituted in ultrapure water. Reverse-phase high-performance liquid chromatography (RP-HPLC) RP-HPLC separation was performed with a C18 column (Jupiter, 10 250 mm) equilibrated with 0.05% TFA. The elution gradient for the 5% ACN fraction was 2% to 20% (v/v) of solution B (0.10% (v/v) TFA in ACN) in solution A (0.05% (v/v) TFA in water). For the 40% ACN fraction, the gradient was 2C60% of solution B in solution A, and for the 80% ACN fraction, the gradient was 20C80% of solution B in solution A. RP-HPLC was performed for 60 min at a 1.5 mL/min flow rate. Effluent absorbance was monitored at 225 nm, and the fractions corresponding to absorbance peaks were hand-collected, concentrated under vacuum, and reconstituted in ultrapure water. When necessary, a second chromatographic step was performed on a VP-ODS analytic column (Shim-pack?), with a 1.0 mL/min flow rate for 60 min. This was performed to guarantee sample homogeneity. The gradients for these second chromatographic stages were determined by the target molecule’s retention time. Liquid growth inhibition assay The antimicrobial assay was performed against all the microorganisms listed previously in Methods section Bacterial Strains, using poor broth nutrient medium (PB: 1.0 g peptone in 100 mL of water containing 86 mM NaCl at pH 7.4; 217 mOsM) and Mller-Hinton medium (peptone 5.0 Zaleplon g/L; casein peptone 17.5 g/L; agar 15.0 g/L; Ca2+ 20.0C25.0 mg/L; Mg2+ 10.0C14.5 mg/L; pH 7.4) for bacteria and potato dextrose broth (1/2 PDB: 1.2 g potato dextrose.
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