Larval excretion/secretion of dipters of Lucilia cuprina species induces death in promastigote and amastigote forms of Leishmania amazonensis.

Leishmaniasis is a neglected tropical disease that affects millions of people around the world. Larval excretion/secretion (ES) of the larvae of flies of the Calliphoridae family has microbicidal activity against Gram-positive and Gram-negative bacteria, in addition to some species of Leishmania. Our study aimed at assessing the in vitro efficacy of Lucilia cuprina larval ES against the promastigote and amastigote forms of Leishmania amazonensis, elucidating possible microbicidal mechanisms and routes of death involved. Larval ES was able to inhibit the viability of L. amazonensis at all concentrations, induce morphological and ultrastructural changes in the parasite, retraction of the cell body, roughness of the cytoplasmic membrane, leakage of intracellular content, ROS production increase, induction of membrane depolarization and mitochondrial swelling, the formation of cytoplasmic lipid droplets and phosphatidylserine exposure, thus indicating the possibility of apoptosis-like death. To verify the efficacy of larval ES on amastigote forms, we performed a phagocytic assay, measurement of total ROS and NO. Treatment using larval ES reduced the percentage of infection and the number of amastigotes per macrophage of lineage J774A.1 at all concentrations, increasing the production of ROS and TNF-α, thus indicating possible pro-inflammatory immunomodulation and oxidative damage. Therefore, treatment using larval ES is effective at inducing the death of promastigotes and amastigotes of L. amazonensis even at low concentrations.

Characterising four Sarconesiopsis magellanica (Diptera: Calliphoridae) larval fat body-derived antimicrobial peptides.

BACKGROUND: The inappropriate use of antibiotics has led to the accelerated growth of resistance to antibiotics. The search for new therapeutic strategies (i.e., antimicrobial peptides-AMPs) has thus become a pressing need. OBJECTIVE: Characterising and evaluating Sarconesiopsis magellanica larval fat body-derived AMPs. METHODS: Fat body extracts were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC); mass spectrometry was used for characterising the primary structure of the AMPs so found. ProtParam (Expasy) was used for analysing the AMPs' physico-chemical properties. Synthetic AMPs' antibacterial activity was evaluated. FINDINGS: Four new AMPs were obtained and called sarconesin III, IV, V and VI. Sarconesin III had an α-helix structure and sarconesins IV, V and VI had linear formations. Oligomer prediction highlighted peptide-peptide interactions, suggesting that sarconesins III, V and VI could form self-aggregations when in contact with the microbial membrane. AMPs synthesised from their native molecules' sequences had potent activity against Gram-positive bacteria and, to a lesser extent, against Gram-negative and drug-resistant bacteria. Sarconesin VI was the most efficient AMP. None of the four synthetic AMPs had a cytotoxic effect. MAIN CONCLUSIONS: S. magellanica larval fat body-derived antimicrobial peptides are an important source of AMPs and could be used in different antimicrobial therapies and overcoming bacterial resistance.

Characterising four Sarconesiopsis magellanica (Diptera: Calliphoridae) larval fat body-derived antimicrobial peptides

BACKGROUND The inappropriate use of antibiotics has led to the accelerated growth of resistance to antibiotics. The search for new therapeutic strategies (i.e., antimicrobial peptides-AMPs) has thus become a pressing need. OBJECTIVE Characterising and evaluating Sarconesiopsis magellanica larval fat body-derived AMPs. METHODS Fat body extracts were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC); mass spectrometry was used for characterising the primary structure of the AMPs so found. ProtParam (Expasy) was used for analysing the AMPs' physico-chemical properties. Synthetic AMPs' antibacterial activity was evaluated. FINDINGS Four new AMPs were obtained and called sarconesin III, IV, V and VI. Sarconesin III had an α-helix structure and sarconesins IV, V and VI had linear formations. Oligomer prediction highlighted peptide-peptide interactions, suggesting that sarconesins III, V and VI could form self-aggregations when in contact with the microbial membrane. AMPs synthesised from their native molecules' sequences had potent activity against Gram-positive bacteria and, to a lesser extent, against Gram-negative and drug-resistant bacteria. Sarconesin VI was the most efficient AMP. None of the four synthetic AMPs had a cytotoxic effect. MAIN CONCLUSIONS S. magellanica larval fat body-derived antimicrobial peptides are an important source of AMPs and could be used in different antimicrobial therapies and overcoming bacterial resistance.

Patagonin-CRISP: Antimicrobial Activity and Source of Antimicrobial Molecules in Duvernoy's Gland Secretion (Philodryas patagoniensis Snake).

Snake venom contains a variety of toxins with a range of biological activity, among these toxins cysteine-rich secreted proteins (CRISPs) can be found. The proteins of this family have masses of 20-30 kDa and display homologous amino acid sequences containing 16 cysteine residues, forming eight disulfide bonds. Some of these proteins have been explored, characterized, and described in terms of their activity; however, little is known about their range of activities. A search for new antimicrobial molecules is ongoing, as the number of microbial strains resistant to available antibiotics is increasing. We identified antimicrobial activity in the secretion of Duvernoy's gland of the rear-fanged Philodryas patagoniensis. Fractions of this venom were subjected to reverse-phase high performance liquid chromatography and analyzed to determine their antimicrobial activity with a liquid broth inhibition assay. One of the fractions presented activity against a Gram-negative bacterium and a filamentous fungus. This fraction was analyzed with LC-MS/MS, and a protein of 24,848.8 Da was identified. Database searches allowed us to identify it as a CRISP due to the presence of some unique fragments in the molecule. We called it patagonin-CRISP, as the same protein in the venom of P. patagoniensis had previously been characterized as having a different biological activity. Patagonin-CRISP presented activity at very low concentrations and showed no cytotoxic activity. This is the first time that antimicrobial activity has been identified for P. patagoniensis venom or for a CRISP family protein.

Sarconesin II, a New Antimicrobial Peptide Isolated from Sarconesiopsis magellanica Excretions and Secretions.

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.

Sarconesin: Sarconesiopsis magellanica Blowfly Larval Excretions and Secretions With Antibacterial Properties.

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.

The effect of Lucilia sericata- and Sarconesiopsis magellanica-derived larval therapy on Leishmania panamensis.

This study's main objective was to evaluate the action of larval therapy derived from Lucilia sericata and Sarconesiopsis magellanica (blowflies) regarding Leishmania panamensis using an in vivo model. Eighteen golden hamsters (Mesocricetus auratus) were used; they were divided into 6 groups. The first three groups consisted of 4 animals each; these, in turn, were internally distributed into subgroups consisting of 2 hamsters to be used separately in treatments derived from each blowfly species. Group 1 was used in treating leishmanial lesions with larval therapy (LT), whilst the other two groups were used for evaluating the used of larval excretions and secretions (ES) after the ulcers had formed (group 2) and before they appeared (group 3). The three remaining groups (4, 5 and 6), consisting of two animals, were used as controls in the experiments. Biopsies were taken for histopathological and molecular analysis before, during and after the treatments; biopsies and smears were taken for assessing parasite presence and bacterial co-infection. LT and larval ES proved effective in treating the ulcers caused by the parasite. There were no statistically significant differences between the blowfly species regarding the ulcer cicatrisation parameters. There were granulomas in samples taken from lesions at the end of the treatments. The antibacterial action of larval treatment regarding co-infection in lesions caused by the parasite was also verified. These results potentially validate effective LT treatment against cutaneous leishmaniasis aimed at using it with humans in the future.

Evaluating Sarconesiopsis magellanica blowfly-derived larval therapy and comparing it to Lucilia sericata-derived therapy in an animal model.

Larval therapy is used as alternative treatment for hard-to-heal chronic and infected wounds. Lucilia sericata is the most used blowfly species. However, it has been shown recently that Sarconesiopsis magellanica larval excretions and secretions have potent antibacterial activity; this blowfly belongs to the Calliphoridae family. The present work has dealt with evaluating larval therapy using S. magellanica on wounds induced in diabetic rabbits and its action was compared to the effect induced by L. sericata. Twelve New Zealand White rabbits (Oryctolagus cuniculus) were used; they were divided into 4 groups, the first two being treated with larval therapy derived from both aforementioned necrophagous blowflies, an antibiotic was used in the third and the fourth was used as control. All the animals were wounded on the back and infected with Pseudomonas aeruginosa and Staphylococcus aureus. Samples of the secretion from each animal's infected wound were taken and sown on blood agar. The colony forming units were then counted. The PUSH scale was used for the macroscopic evaluation of the wounds. Bacterial control was encountered 48 h post-treatment in the treatments involving larval therapy and to a lesser extent with the antibiotic. Likewise, wound debridement was quicker and more efficient with larval therapy compared to the antibiotic group; however, wound closing time was 23 days in all treatments. The group treated with S. magellanica larvae had relatively quicker evolution until the proliferation phase and the start of maturation, even though there were no significant differences between both blowfly species evaluated here regarding treatments by the end of the treatment period. The present study has validated the diabetic rabbit model for inducing chronic wounds regarding larval therapy and has likewise confirmed the effectiveness of S. magellanica-derived larval therapy as an alternative for curing and healing wounds.

Evaluating the effect of Sarconesiopsis magellanica (Diptera: Calliphoridae) larvae-derived haemolymph and fat body extracts on chronic wounds in diabetic rabbits.

We evaluated extracts taken from S. magellanica third instar larvae fat body and haemolymph using a diabetic rabbit model and compared this to the effect obtained with the same substances taken from Lucilia sericata larvae. Alloxan (a toxic glucose analogue) was used to induce experimental diabetes in twelve rabbits. Dorsal wounds were made in each animal and they were infected with Staphylococcus aureus and Pseudomonas aeruginosa. They were then treated with haemolymph and lyophilized extracts taken from the selected blowflies' larvae fat bodies. Each wound was then evaluated by using rating scales and histological analysis. More favourable scores were recorded on the PUSH and WBS scales for the wounds treated with fat body derived from the larvae of both species compared to that obtained with haemolymph; however, wounds treated with the substances taken from S. magellanica had better evolution. Histological analysis revealed that treatment led to tissue proliferation and more effective neovascularisation in less time with both species' fat body extracts compared to treatment with just haemolymph. The results suggest the effectiveness of the substances evaluated and validate them in the animal model being used here as topical agents in treating chronic wounds.

Evaluación de la actividad antibacterial de los extractos de cuerpos grasos y hemolinfa derivados de la mosca Sarconesiopsis magellanica (Diptera: Calliphoridae) / Assessment of antibacterial activity by fat bodies extracts and hemolymph derived from the blowfly Sarconesiopsis magellanica (Diptera: Calliphoridae)

Objetivo: Evaluar, en condiciones in vitro , la actividad antibacterial de los extractos de cuerpos grasos y de la hemolinfa de larvas de tercer estadio de Sarconesiopsis magellanica , la cual se comparó con los efectos obtenidos de las mismas sustancias derivadas de Lucilia sericata . S. magellanica (Diptera: Calliphoridae) es una mosca de importancia principalmente forense, utilizada en la determinación del intervalo post mortem . Por sus hábitos necrófagos, es considerada un modelo potencialmente útil en terapia larval. Material y métodos: Se extrajeron los cuerpos grasos de las larvas mediante la técnica de disección corporal y la hemolinfa se obtuvo mediante decapitación y centrifugación de los especímenes larvales. Las bacterias evaluadas fueron Staphylococcus aureus y Pseudomonas aeruginosa . Los métodos utilizados para evaluar la actividad antibacterial fueron difusión en agar y unidades formadoras de colonias (UFC/ml). Resultados: Después de la correspondiente incubación, los resultados generales mostraron que la actividad antibacterial de la hemolinfa y de los cuerpos grasos, tanto de L. sericata como de S. magellanica , fueron efectivos contra S. aureus y P. aeruginosa sin diferencias significativas entre las especies de moscas, aunque con algunas diferencias entre las cepas bacterianas. Conclusiones: Los resultados obtenidos sugieren que estas sustancias podrían tener un efecto similar en el tratamiento de heridas infectadas contra los microorganismos evaluados.

Objective: This study aimed to evaluate the in vitro antibacterial activity of fat body and hemolymph extracts from Sarconesiopsis magellanica (Diptera: Calliphoridae) third-instar larvae, compared to the effect obtained using the same extracts but derived from Lucilia sericata . S. magellanica blowflies are considered important in forensic sciences due to their usefulness in determining the post mortem interval. This blowfly could be useful in larval therapy due to its necrophagous habits. Materials and methods: Fat body from larvae was removed by dissection, and hemolymph via decapitation and centrifugation of larval specimens. The antibacterial effect was tested against Staphylococcus aureus and Pseudomonas aeruginosa using two methods: agar diffusion and colony forming units (CFU/mL). Results: Hemolymph and fat body extracts derived from both L. sericata and S. magellanica were effective against S. aureus and P. aeruginosa , with no significant differences between blowfly species, although with some differences between the bacterial strains. Conclusions: The results obtained suggest that S. magellanica and L. sericata fat body and hemolymph extracts might have a similar antimicrobial activity against these microorganisms when used to treat infected wounds.

Sarconesiopsis magellanica (Diptera: Calliphoridae) excretions and secretions have potent antibacterial activity.

The most important mechanism for combating infection using larval therapy depends on larval excretions and secretions (ES). The present work was aimed at evaluating Sarconesiopsis magellanica (Diptera: Calliphoridae) ES antibacterial activity in six bacterial strains (three Gram-positive and three Gram-negative) and comparing this to the effect of Lucilia sericata-derived ES. Antibacterial activity at 50µg/mL minimum inhibitory concentration (MIC) was observed for Staphylococcus epidermidis ATCC-12228 and Staphylococcus aureus ATCC-29213 strains, when the turbidimetry test involving S. magellanica ES was used; the rest of the bacterial strains (Staphylococcus aureus ATCC-6538, Pseudomonas aeruginosa ATCC-10145, Pseudomonas aeruginosa ATCC-9027 and Pseudomonas aeruginosa ATCC-27853) were inhibited at a 100µg/mL MIC. Twice the amount was required to inhibit the aforementioned bacteria with L. sericata-derived ES using this same technique; a similar trend was observed when the agar diffusion method was used instead. Furthermore, when the previously established MIC for each bacterial strain was used, their colonies became reduced following 1-6h incubation with S. magellanica derived ES, whilst the reduction occurred from 2 to 6hours with those from L. sericata. Although the MIC for each strain obtained with ciprofloxacin was lower than those established when using either blowfly derived-ES, the gradual reduction of the colonies occurred at a longer incubation time (6h or more). The results showed that S. magellanica ES antibacterial activity was more potent and effective, compared to that of L. sericata-derived ES.