Antifungal and antibacterial activities were detected in the hemolymph and gut contents of the cattle tick, Boophilus microplus. A peptide with antibacterial activity from the tick gut contents was purified to homogeneity by reversed-phase chromatography. The molecular mass of the purified peptide was 3,205.7 Da, measured by matrix-assisted laser desorption/ionization mass spectrometry. The amino acid sequence was obtained by Edman degradation and showed that the peptide was identical to a fragment of the bovine ␣-hemoglobin. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure.
The Araneae order is considered one of the most successful groups among venomous animals in the world. An important factor for this success is the production of venoms, a refined biological fluid rich in proteins, short peptides and cysteine-rich peptides (CRPs). These toxins may present pharmacologically relevant biological actions, as antimicrobial, antiviral and anticancer activities, for instance. Therefore, there is an increasing interest in the exploration of venom toxins for therapeutic reasons, such as drug development. However, the process of peptide sequencing and mainly the evaluation of potential biological activities of these peptides are laborious, considering the low yield of venom extraction and the high variability of toxins present in spider venoms. Here we show a robust methodology for identification, sequencing, and initial screening of potential bioactive peptides found in the venom of Acanthoscurria rondoniae. This methodology consists in a multiomics approach involving proteomics, peptidomics and transcriptomics analyses allied to in silico predictions of antibacterial, antifungal, antiviral, and anticancer activities. Through the application of this strategy, a total of 92,889 venom gland transcripts were assembled and 84 novel toxins were identified at the protein level, including seven short peptides and 10 fully sequenced CRPs (belonging to seven toxin families). In silico analysis suggests that seven CRPs families may have potential antimicrobial or antiviral activities, while two CRPs and four short peptides are potentially anticancer. Taken together, our results demonstrate an effective multiomics strategy for the discovery of new toxins and in silico screening of potential bioactivities. This strategy may be useful in toxin discovery, as well as in the screening of possible activities for the vast diversity of molecules produced by venomous animals.
Larval therapy (LT) is an alternative treatment for healing chronic wounds; its action is based on debridement, the removal of bacteria, and stimulating granulation tissue. The most important mechanism when using LT for combating infection depends on larval excretions and secretions (ES). Larvae are protected against infection by a spectrum of antimicrobial peptides (AMPs); special interest in AMPs has also risen regarding understanding their role in wound healing since they degrade necrotic tissue and kill different bacteria during LT. Sarconesiopsis magellanica (Diptera: Calliphoridae) is a promising medically-important necrophagous fly. This article reports a small AMP being isolated from S. magellanica ES products for the first time; these products were obtained from third-instar larvae taken from a previously-established colony. ES were fractionated by RP-HPLC using C18 columns for the first analysis; the products were then lyophilised and their antimicrobial activity was characterized by incubation with different bacterial strains. These fractions’ primary sequences were determined by mass spectrometry and de novo sequencing; five AMPs were obtained, the Sarconesin fraction was characterized and antibacterial activity was tested in different concentrations with minimum inhibitory concentrations starting at 1.2 μM. Potent inhibitory activity was shown against Gram-negative (Escherichia coli D31, E. coli DH5α, Salmonella enterica ATCC 13314, Pseudomonas aeruginosa 27853) and Gram-positive (Staphylococcus aureus ATCC 29213, S. epidermidis ATCC 12228, Micrococcus luteus A270) bacteria. Sarconesin has a significant similarity with Rho-family GTPases which are important in organelle development, cytoskeletal dynamics, cell movement, and wound repair. The data reported here indicated that Sarconesin could be an alternative candidate for use in therapeutics against Gram-negative and Gram-positive bacterial infections. Our study describes one peptide responsible for antibacterial activity when LT is being used. The results shown here support carrying out further experiments aimed at validating S. magellanica AMPs as novel resources for combating antibacterial resistance.
Antibiotic resistance is at dangerous levels and increasing worldwide. The search for new antimicrobial drugs to counteract this problem is a priority for health institutions and organizations, both globally and in individual countries. Sarconesiopsis magellanica blowfly larval excretions and secretions (ES) are an important source for isolating antimicrobial peptides (AMPs). This study aims to identify and characterize a new S. magellanica AMP. RP-HPLC was used to fractionate ES, using C18 columns, and their antimicrobial activity was evaluated. The peptide sequence of the fraction collected at 43.7 min was determined by mass spectrometry (MS). Fluorescence and electronic microscopy were used to evaluate the mechanism of action. Toxicity was tested on HeLa cells and human erythrocytes; physicochemical properties were evaluated. The molecule in the ES was characterized as sarconesin II and it showed activity against Gram-negative (Escherichia coli MG1655, Pseudomonas aeruginosa ATCC 27853, P. aeruginosa PA14) and Gram-positive (Staphylococcus aureus ATCC 29213, Micrococcus luteus A270) bacteria. The lowest minimum inhibitory concentration obtained was 1.9 μM for M. luteus A270; the AMP had no toxicity in any cells tested here and its action in bacterial membrane and DNA was confirmed. Sarconesin II was documented as a conserved domain of the ATP synthase protein belonging to the Fli-1 superfamily. The data reported here indicated that peptides could be alternative therapeutic candidates for use in infections against Gram-negative and Gram-positive bacteria and eventually as a new resource of compounds for combating multidrug-resistant bacteria.
Invasive Candida infections are an important growing medical concern and treatment options are limited to a few antifungal drug classes, with limited efficacies depending on the infecting organism. In this scenario, invasive infections caused by multiresistant Candida auris are emerging in several places around the world as important healthcare-associated infections. As antimicrobial peptides (AMPs) exert their activities primarily through mechanisms involving membrane disruption, they have a lower chance of inducing drug resistance than general chemical antimicrobials. Interestingly, we previously described the potent candicidal effect of a rattlesnake AMP, crotamine, against standard and treatment-resistant clinical isolates, with no hemolytic activity. We evaluated the antifungal susceptibility of several Candida spp. strains cultured from different patients by using the Clinical and Laboratory Standards Institute (CLSI) microdilution assay, and the antifungal activity of native crotamine was evaluated by a microbial growth inhibition microdilution assay. Although all Candida isolates evaluated here showed resistance to amphotericin B and fluconazole, crotamine (40–80 µM) exhibited in vitro activity against most isolates tested. We suggest that this native polypeptide from the South American rattlesnake Crotalus durissus terrificus has potential as a structural model for the generation of a new class of antimicrobial compounds with the power to fight against multiresistant Candida spp.
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