RESUMO

Lactobacillus acidophilus are Gram-positive bacteria distributed in diverse environments, and as a component of the normal microbiota of gastrointestinal and urogenital tract, they are relevant for human beings. Classified as lactic acid bacteria, due to the production of lactic acid, Lactobacillus can also produce antimicrobial peptides (AMPs), which is a compound synthesized by all forms of life aiming for protecting themselves from threats and to increase their competitivity to survive in a specific environment. AMPs are molecules capable of inhibiting the growth of microorganisms and, due to the indiscriminate use of conventional antibiotics and the emergence of multi-resistant bacteria, they have become an alternative, not only for treating multi-resistant infections, but also for the identification of probiotic products and food conservation. Considering the rampant rise of bacterial resistance to classical antimicrobials, the present study aimed to isolate and characterize AMPs from L. acidophilus extracts. Lactobacillus acid extract was pre-fractionated on disposable cartridges, followed by a high-performance liquid chromatography (HPLC). The collected fractions were evaluated in a liquid growth inhibition assay allowing to identify eight fractions with antimicrobial activity, and one of them showed antimicrobial activity against Candida albicans and, for this reason, was further characterized by mass spectrometry (MS). A peptide with a molecular mass of 1788.01 Da, showing the primary sequence NEPTHLLKAFSKAGFQ, as determined by MS, was named as Doderlin. Interestingly, antimicrobial molecules isolated from L. acidophilus have already been described previously, but few reports describe AMPs effective against C. albicans as the one reported here. We show here that this newly discovered molecule has a biological property with potential to be used in pharmaceutical and food companies, in the fight against contamination and/or for treating infections caused by microorganisms, respectively.

RESUMO

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.

Assuntos

RESUMO

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.

Assuntos

RESUMO

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.

Assuntos

RESUMO

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.

RESUMO

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

RESUMO

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.

RESUMO

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.

RESUMO

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.

Assuntos

RESUMO

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.