RESUMO
Biofilm formation is a major health concern and studies have been pursued to find compounds able to prevent biofilm establishment and remove pre-existing biofilms. While biosurfactants (BS) have been well-known for possessing antibiofilm activities, bioemulsifiers (BE) are still scarcely explored for this purpose. The present study aimed to evaluate the bioemulsifying properties of cell-free supernatants produced by Bacillaceae and Vibrio strains isolated from marine sponges and investigate their antiadhesive and antibiofilm activities against different pathogenic Gram-positive and Gram-negative bacteria. The BE production by the marine strains was confirmed by the emulsion test, drop-collapsing, oil-displacement, cell hydrophobicity and hemolysis assays. Notably, Bacillus cereus 64BHI1101 displayed remarkable emulsifying activity and the ultrastructure analysis of its BE extract (BE64-1) revealed the presence of structures typically observed in macromolecules composed of polysaccharides and proteins. BE64-1 showed notable antiadhesive and antibiofilm activities against Staphylococcus aureus, with a reduction of adherence of up to 100 % and a dispersion of biofilm of 80 %, without affecting its growth. BE64-1 also showed inhibition of Staphylococcus epidermidis and Escherichia coli biofilm formation and adhesion. Thus, this study provides a starting point for exploring the antiadhesive and antibiofilm activities of BE from sponge-associated bacteria, which could serve as a valuable tool for future research to combat S. aureus biofilms.
Assuntos
Aderência Bacteriana , Biofilmes , Emulsificantes , Poríferos , Staphylococcus aureus , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Poríferos/microbiologia , Animais , Aderência Bacteriana/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Emulsificantes/farmacologia , Emulsificantes/química , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/fisiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Interações Hidrofóbicas e Hidrofílicas , Antibacterianos/farmacologia , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/fisiologia , Hemólise , Tensoativos/farmacologia , Tensoativos/metabolismo , Vibrio/efeitos dos fármacos , Vibrio/fisiologia , Vibrio/metabolismo , Testes de Sensibilidade Microbiana , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/fisiologiaRESUMO
The main goal of this work was to evaluate the therapeutic potential of green superparamagnetic iron oxide nanoparticles (SPIONs) produced with coconut water for treating cutaneous leishmaniasis caused by Leishmania amazonensis. Optical and electron microscopy techniques were used to evaluate the effects on cell proliferation, infectivity percentage, and ultrastructure. SPIONs were internalized by both parasite stages, randomly distributed in the cytosol and located mainly in membrane-bound compartments. The selectivity index for intracellular amastigotes was more than 240 times higher compared to current drugs used to treat the disease. The synthesized SPIONs showed promising activity against Leishmania and can be considered a strong candidate for a new therapeutic approach for treating leishmaniases.
RESUMO
Searching for new alternatives for treating leishmaniasis, we present the synthesis, characterization, and biological evaluation against Leishmania amazonensis of the new ZnCl2(H3)2 complex. H3 is 22-hydrazone-imidazoline-2-yl-chol-5-ene-3ß-ol, a well-known bioactive molecule functioning as a sterol Δ24-sterol methyl transferase (24-SMT) inhibitor. The ZnCl2(H3)2 complex was characterized by infrared, UV-vis, molar conductance measurements, elemental analysis, mass spectrometry, and NMR experiments. The biological results showed that the free ligand H3 and ZnCl2(H3)2 significantly inhibited the growth of promastigotes and intracellular amastigotes. The IC50 values found for H3 and ZnCl2(H3)2 were 5.2 µM and 2.5 µM for promastigotes, and 543 nM and 32 nM for intracellular amastigotes, respectively. Thus, the ZnCl2(H3)2 complex proved to be seventeen times more potent than the free ligand H3 against the intracellular amastigote, the clinically relevant stage. Furthermore, cytotoxicity assays and determination of selectivity index (SI) revealed that ZnCl2(H3)2 (CC50 = 5 µΜ, SI = 156) is more selective than H3 (CC50 = 10 µΜ, SI = 20). Furthermore, as H3 is a specific inhibitor of the 24-SMT, free sterol analysis was performed. The results showed that H3 was not only able to induce depletion of endogenous parasite sterols (episterol and 5-dehydroepisterol) and their replacement by 24-desalkyl sterols (cholesta-5,7,24-trien-3ß-ol and cholesta-7,24-dien-3ß-ol) but also its zinc derivative resulting in a loss of cell viability. Using electron microscopy, studies on the fine ultrastructure of the parasites showed significant differences between the control cells and parasites treated with H3 and ZnCl2(H3)2. The inhibitors induced membrane wrinkle, mitochondrial injury, and abnormal chromatin condensation changes that are more intense in the cells treated with ZnCl2(H3)2.
RESUMO
Snake venoms are complex cocktails of non-toxic and toxic molecules that work synergistically for the envenoming outcome. Alongside the immediate consequences, chronic manifestations and long-term sequelae can occur. Recently, extracellular vesicles (EVs) were found in snake venom. EVs mediate cellular communication through long distances, delivering proteins and nucleic acids that modulate the recipient cell's function. However, the biological roles of snake venom EVs, including possible cross-organism communication, are still unknown. This knowledge may expand the understanding of envenoming mechanisms. In the present study, we isolated and characterized the EVs from Bothrops jararaca venom (Bj-EVs), giving insights into their biological roles. Fresh venom was submitted to differential centrifugation, resulting in two EV populations with typical morphology and size range. Several conserved EV markers and a subset of venom related EV markers, represented mainly by processing enzymes, were identified by proteomic analysis. The most abundant protein family observed in Bj-EVs was 5'-nucleotidase, known to be immunosuppressive and a low abundant and ubiquitous toxin in snake venoms. Additionally, we demonstrated that mammalian cells efficiently internalize Bj-EVs. The commercial antibothropic antivenom partially recognizes Bj-EVs and inhibits cellular EV uptake. Based on the proteomic results and the in vitro interaction assays using macrophages and muscle cells, we propose that Bj-EVs may be involved not only in venom production and processing but also in host immune modulation and long-term effects of envenoming.
Assuntos
Bothrops , Venenos de Crotalídeos , Vesículas Extracelulares , Animais , Venenos de Crotalídeos/química , Proteômica , Proteínas , Venenos de Serpentes , MamíferosRESUMO
Leishmaniasis is a neglected disease caused by protozoan parasites of the Leishmania genus. Benzylamines are a class of compounds selectively designed to inhibit the squalene synthase (SQS) that catalyzes the first committed reaction on the sterol biosynthesis pathway. Herein, we studied seven new benzylamines (SBC 37-43) against Leishmania amazonensis. After the first screening of cell viability, two inhibitors (SBC 39 and SBC 40) were selected. Against intracellular amastigotes, SBC 39 and SBC 40 presented selectivity indexes of 117.7 and 180, respectively, indicating high selectivity. Analysis of the sterol composition revealed a depletion of endogenous 24-alkylated sterols such as episterol and 5-dehydroepisterol, with a concomitant accumulation of fecosterol, implying a disturbance in cellular lipid content. This result suggests a blockade of de novo sterol synthesis at the level of SQS and C-5 desaturase. Furthermore, physiological analysis and electron microscopy revealed three main alterations: (1) in the mitochondrion; (2) the presence of lipid bodies and autophagosomes; and (3) the appearance of projections in the plasma membrane. In conclusion, our results support the notion that benzylamines have a potent effect against Leishmania amazonensis and should be an exciting novel pharmaceutical lead for developing new chemotherapeutic alternatives to treat leishmaniasis.
Assuntos
Leishmania mexicana , Leishmania , Benzilaminas/farmacologia , Farnesil-Difosfato Farnesiltransferase/metabolismo , Estresse Oxidativo , Esteróis/metabolismoRESUMO
The new complexes Zn(ITZ)2Cl2 (1) and Zn(ITZ)2(OH)2 (2) were synthetized by a reaction of itraconazole with their respective zinc salts under reflux. These Zn-ITZ complexes were characterized by elemental analyses, molar conductivity, mass spectrometry, 1H and 13C{1H} nuclear magnetic resonance, and UV-vis and infrared spectroscopies. The antiparasitic and antifungal activity of Zn-ITZ complexes was evaluated against three protozoans of medical importance, namely, Leishmania amazonensis, Trypanosoma cruzi, and Toxoplasma gondii, and two fungi, namely, Sporothrix brasiliensis and Sporothrix schenckii The Zn-ITZ complexes exhibited a broad spectrum of action, with antiparasitic and antifungal activity in low concentrations. The strategy of combining zinc with ITZ was efficient to enhance ITZ activity since Zn-ITZ-complexes were more active than the azole alone. This study opens perspectives for future applications of these Zn-ITZ complexes in the treatment of parasitic diseases and sporotrichosis.
Assuntos
Antifúngicos/farmacologia , Antiparasitários/farmacologia , Itraconazol/farmacologia , Zinco/farmacologia , Leishmania/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Testes de Sensibilidade Parasitária , Sporothrix/efeitos dos fármacos , Toxoplasma/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Treatment of leishmaniasis involves the use of antimonials, miltefosine, amphotericin B or pentamidine. However, the side effects of these drugs and the reports of drug-resistant parasites demonstrate the need for new treatments that are safer and more efficacious. Histone deacetylase inhibitors are a new class of compounds with potential to treat leishmaniasis. Herein, we evaluated the effects of KH-TFMDI, a novel histone deacetylase inhibitor, on Leishmania amazonensis promastigotes and intracellular amastigotes. The IC50 values of this compound for promastigotes and intracellular amastigotes were 1.976 and 1.148 µM, respectively, after 72 h of treatment. Microscopic analyses revealed that promastigotes became elongated and thinner in response to KH-TFMDI, indicating changes in cytoskeleton organization. Immunofluorescence microscopy, western blotting and flow cytometry using an anti-acetylated tubulin antibody revealed an increase in the expression of acetylated tubulin. Furthermore, transmission electron microscopy revealed several ultrastructural changes, such as (a) mitochondrial swelling, followed by the formation of many vesicles inside the matrix; (b) presence of lipid bodies randomly distributed through the cytoplasm; (c) abnormal chromatin condensation; and (d) formation of blebs on the plasma membrane. Physiological studies for mitochondrial function, flow cytometry with propidium iodide and TUNEL assay confirmed the alterations in the mitochondrial metabolism, cell cycle, and DNA fragmentation, respectively, which could result to cell death by mechanisms related to apoptosis-like. All these together indicate that histone deacetylases are promising targets for the development of new drugs to treat Leishmania, and KH-TFMDI is a promising drug candidate that should be tested in vivo.
Assuntos
Compostos de Benzilideno/farmacologia , Morte Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Indóis/farmacologia , Leishmania/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Sirtuínas/antagonistas & inibidores , Animais , Antiparasitários/farmacologia , Antiparasitários/toxicidade , Apoptose/efeitos dos fármacos , Compostos de Benzilideno/toxicidade , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/metabolismo , Inibidores de Histona Desacetilases/toxicidade , Indóis/toxicidade , Concentração Inibidora 50 , Leishmania/citologia , Leishmania/crescimento & desenvolvimento , Leishmania/ultraestrutura , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Estresse Oxidativo/efeitos dos fármacosRESUMO
Phytomonas serpens are flagellates in the family Trypanosomatidae that parasitise the tomato plant (Solanum lycopersicum L.), which results in fruits with low commercial value. The tomato glycoalkaloid tomatine and its aglycone tomatidine inhibit the growth of P. serpens in axenic cultures. Tomatine, like many other saponins, induces permeabilisation of the cell membrane and a loss of cell content, including the cytosolic enzyme pyruvate kinase. In contrast, tomatidine does not cause permeabilisation of membranes, but instead provokes morphological changes, including vacuolisation. Phytomonas treated with tomatidine show an increased accumulation of labelled neutral lipids (BODYPY-palmitic), a notable decrease in the amount of C24-alkylated sterols and an increase in zymosterol content. These results are consistent with the inhibition of 24-sterol methyltransferase (SMT), which is an important enzyme that is responsible for the methylation of sterols at the 24 position. We propose that the main target of tomatidine is the sterols biosynthetic pathway, specifically, inhibition of the 24-SMT. Altogether, the results obtained in the present paper suggest a more general effect of alkaloids in trypanosomatids, which opens potential therapeutic possibilities for the treatment of the diseases caused by these pathogens.
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Tomatina/análogos & derivados , Tomatina/farmacologia , Trypanosomatina/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Colesterol/análise , Solanum lycopersicum/parasitologia , Metiltransferases/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Doenças das Plantas/parasitologia , Esteróis/análise , Esteróis/biossíntese , Trypanosomatina/metabolismo , Trypanosomatina/ultraestruturaRESUMO
Phytomonas serpens are flagellates in the family Trypanosomatidae that parasitise the tomato plant (Solanum lycopersicum L.), which results in fruits with low commercial value. The tomato glycoalkaloid tomatine and its aglycone tomatidine inhibit the growth of P. serpens in axenic cultures. Tomatine, like many other saponins, induces permeabilisation of the cell membrane and a loss of cell content, including the cytosolic enzyme pyruvate kinase. In contrast, tomatidine does not cause permeabilisation of membranes, but instead provokes morphological changes, including vacuolisation. Phytomonas treated with tomatidine show an increased accumulation of labelled neutral lipids (BODYPY-palmitic), a notable decrease in the amount of C24-alkylated sterols and an increase in zymosterol content. These results are consistent with the inhibition of 24-sterol methyltransferase (SMT), which is an important enzyme that is responsible for the methylation of sterols at the 24 position. We propose that the main target of tomatidine is the sterols biosynthetic pathway, specifically, inhibition of the 24-SMT. Altogether, the results obtained in the present paper suggest a more general effect of alkaloids in trypanosomatids, which opens potential therapeutic possibilities for the treatment of the diseases caused by these pathogens.
Assuntos
Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Cateterismo/métodos , Obstrução Intestinal/patologia , Obstrução Intestinal/terapia , Intestino Delgado/patologia , Biópsia , Constrição Patológica , Endoscopia do Sistema Digestório , Resultado do TratamentoRESUMO
Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical diseases. It is endemic in 98 countries, causing considerable morbidity and mortality. Pentavalent antimonials are the first line of treatment for leishmaniasis except in India. In resistant cases, miltefosine, amphotericin B and pentamidine are used. These treatments are unsatisfactory due to toxicity, limited efficacy, high cost and difficult administration. Thus, there is an urgent need to develop drugs that are efficacious, safe, and more accessible to patients. Trypanosomatids, including Leishmania spp. and Trypanosoma cruzi, have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. Inhibition of ergosterol biosynthesis is increasingly recognized as a promising target for the development of new chemotherapeutic agents. The aim of this work was to investigate the antiproliferative, physiological and ultrastructural effects against Leishmania amazonensis of itraconazole (ITZ) and posaconazole (POSA), two azole antifungal agents that inhibit sterol C14α-demethylase (CYP51). Antiproliferative studies demonstrated potent activity of POSA and ITZ: for promastigotes, the IC50 values were 2.74 µM and 0.44 µM for POSA and ITZ, respectively, and for intracellular amastigotes, the corresponding values were 1.63 µM and 0.08 µM, for both stages after 72 h of treatment. Physiological studies revealed that both inhibitors induced a collapse of the mitochondrial membrane potential (ΔΨm), which was consistent with ultrastructural alterations in the mitochondrion. Intense mitochondrial swelling, disorganization and rupture of mitochondrial membranes were observed by transmission electron microscopy. In addition, accumulation of lipid bodies, appearance of autophagosome-like structures and alterations in the kinetoplast were also observed. In conclusion, our results indicate that ITZ and POSA are potent inhibitors of L. amazonensis and suggest that these drugs could represent novel therapies for the treatment of leishmaniasis, either alone or in combination with other agents.