RESUMEN
Antimicrobial peptides (AMPs) are small molecules, which have a potential use as antibiotic or pharmacological tools. In chelicerate organisms, such as scorpions, these molecules constitute an alternative defense system against microorganisms. The aim of this work was to identify AMPs in the hemolymph of the Tityus serrulatus scorpion. Fractions of plasma and hemocytes were subjected to high-performance liquid chromatography (HPLC) and then analyzed to determine their activity in inhibiting microbial growth. One of the fractions from the hemocytes presents antimicrobial activity against microorganisms, such as Gram-negative and Gram-positive bacteria, fungi, and yeast. These fractions were analyzed by mass spectrometry, and a fragment of 3564 Da. was identified. The peptide was called serrulin, because it is derived from the species T. serrulatus. A comparison of the amino acid sequence of serrulin with databases shows that it has a similarity to the glycine-rich peptides described in Cupienius salai and Acanthoscurria gomesiana (spiders). Furthermore, serrulin has no hemolytic activity against human erythrocytes. While the presence of AMPs in T. serrulatus venom has been described in other works, this is the first work to characterize the presence of these molecules in the hemolymph (hemocytes) of this species and show its potential use as an alternative to conventional antibiotics against different species of microorganisms.
Asunto(s)
Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Adulto , Secuencia de Aminoácidos , Animales , Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/química , Aspergillus niger/efectos de los fármacos , Aspergillus niger/crecimiento & desarrollo , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Eritrocitos/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Glicina , Hemolinfa , Humanos , Micrococcus luteus/efectos de los fármacos , Micrococcus luteus/crecimiento & desarrollo , EscorpionesRESUMEN
The importance of antimicrobial peptides (AMPs) in relation to the survival of invertebrates is well known. The source and the mode of action on the insects' immune system of these molecules have been described from different perspectives. Insects produce their own AMPs as well as obtain these molecules from various sources, for example by absorption through the intestinal tract, as previously described for Boophilus microplus. Blood-sucking barber bug Triatoma infestans attracts social, economic and medical interest owing to its role in the transmission of Chagas disease. Despite new studies, descriptions of AMPs from this insect have remained elusive. Thus, the aims of this work were to characterize the antimicrobial potential of human fibrinopeptide A (FbPA) obtained from the T. infestans haemolymph and identify its natural source. Therefore, FbPA was isolated from the T. infestans haemolymph through liquid chromatography and identified by mass spectrometry. This peptide exhibited antimicrobial activity against Micrococcus luteus. Native FbPA from human blood and the synthetic FbPA also exhibited antimicrobial activity. The synthetic FbPA was conjugated with fluorescein isothiocyanate and offered to the insects. The haemolymph collected after 72 h exhibited fluorescence at the same wavelength as fluorescein isothiocyanate. Our experiments show that beyond intrinsic AMP production, T. infestans is able to co-opt molecules via internalization and may use them as AMPs for protection.
Asunto(s)
Antiinfecciosos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Fibrinopéptido A/aislamiento & purificación , Hemolinfa/química , Insectos Vectores/química , Triatoma/química , Animales , Cromatografía Liquida , Humanos , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Micrococcus luteus/efectos de los fármacos , Micrococcus luteus/crecimiento & desarrolloRESUMEN
CONTEXT: Lippia thymoides Mart. & Schauer (Verbenaceae) is used in folk medicine to treat wounds, fever, bronchitis, rheumatism, headaches, and weakness. OBJECTIVE: This study determinates the chemical composition of essential oils from L. thymoides, obtained at during each of the four seasons and correlates with pharmacological properties. MATERIALS AND METHODS: Essential oils were obtained by hydrodistillation and analyzed by gas chromatography coupled to mass spectroscopy (GC-MS). Antioxidant activity was determined by DPPH free radical scavenging and ß-carotene bleaching methods. The antimicrobial assays were performed by minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) methods. Isolated rat aorta and uterus, and guinea-pig trachea were utilized to evaluate relaxant potential in pre-contracted smooth muscle. RESULTS AND DISCUSSION: Essential oils from leaves of L. thymoides had the sesquiterpene ß-caryophyllene (17.22-26.27%) as the major constituent followed by borneol (4.45-7.36%), camphor (3.22-8.61%), camphene (2.64-5.66%), and germacrene D (4.72-6.18%). In vitro assays showed that these essential oils do not have antioxidant activity, have antimicrobial selectivity to Gram-positive bacteria Staphylococcus aureus (MIC = 0.004 mg/mL and MMC = 0.26-10.19 mg/mL) and Micrococcus luteus (MIC = 0.03 mg/mL and MMC = 8.43 mg/mL), relax isolated rat aorta (EC50 = 305-544 µg/mL, with endothelium; and EC50 = 150-283 µg/mL, without endothelium), and uterus (EC50 = 74-257 µg/mL), and minor potency, isolated guinea-pig trachea. CONCLUSIONS: Lippia thymoides is a source of natural products of pharmaceutical interest, being necessary additional studies to determine the substances involved in the biological activities.
Asunto(s)
Lippia/química , Aceites Volátiles/farmacología , Extractos Vegetales/farmacología , Aceites de Plantas/farmacología , Estaciones del Año , Resistencia de las Vías Respiratorias/efectos de los fármacos , Animales , Antiinfecciosos/química , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/farmacología , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Compuestos de Bifenilo/química , Relación Dosis-Respuesta a Droga , Femenino , Cromatografía de Gases y Espectrometría de Masas , Cobayas , Masculino , Pruebas de Sensibilidad Microbiana , Micrococcus luteus/efectos de los fármacos , Micrococcus luteus/crecimiento & desarrollo , Aceites Volátiles/química , Aceites Volátiles/aislamiento & purificación , Oxidación-Reducción , Fitoterapia , Picratos/química , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Hojas de la Planta , Aceites de Plantas/química , Aceites de Plantas/aislamiento & purificación , Plantas Medicinales , Ratas Wistar , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Tocolíticos/química , Tocolíticos/aislamiento & purificación , Tocolíticos/farmacología , Tráquea/efectos de los fármacos , Tráquea/fisiología , Contracción Uterina/efectos de los fármacos , Útero/efectos de los fármacos , Útero/fisiología , Vasodilatación/efectos de los fármacos , Vasodilatadores/química , Vasodilatadores/aislamiento & purificación , Vasodilatadores/farmacología , beta Caroteno/químicaRESUMEN
This study evaluated the antimicrobial and hemolytic activities and phytochemical constituents of hydroalcoholic extract and its fractions from Buchenavia tetraphylla leaves. Cyclohexane (BTCF), ethyl acetate (BTEF), and n-butanol-soluble (BTSBF) and non-soluble (BTNBF) fractions were obtained from a liquid-liquid partition of hydroalcoholic extract (BTHE) from B. tetraphylla leaves. The hemolytic activity of active fractions was checked. The BTHE inhibited the growth of Micrococcus luteus (MIC: 0.10 mg/mL), Pseudomonas aeruginosa (MIC: 0.20 mg/mL), Mycobacterium smegmatis (MIC: 0.39 mg/mL), Proteus vulgaris, and Staphylococcus aureus (MIC: 0.78 mg/mL for both). The more active fractions were BTCF and BTBSF. BTCF showed better potential to inhibit M. luteus (0.10 mg/mL), P. aeruginosa (0.20 mg/mL), S. enteritidis (0.39 mg/mL), and S. aureus (1.56 mg/mL). BTBSF showed the best results for M. luteus (0.10 mg/mL), M. smegmatis, B. subtilis (0.39 mg/mL for both), and P. vulgaris (0.10 mg/mL). The HC50 were greater than observed MIC: 20.30, 4.70 and 2.53 mg/mL, respectively, to BTBF, BTHE and BTCF, which. The phytochemical analysis detected the presence of flavanoids, triterpene, carbohydrate, and tannin. Our work showed for the first time the broad-spread antimicrobial activity of B. tetraphylla, which has nonhemolytic action, creating a new perspective on the interesting association of traditional and scientific knowledge.
Asunto(s)
Antibacterianos/farmacología , Combretaceae/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , 1-Butanol/química , Acetatos/química , Antibacterianos/aislamiento & purificación , Carbohidratos/análisis , Ciclohexanos/química , Relación Dosis-Respuesta a Droga , Flavonoides/análisis , Hemólisis/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Micrococcus luteus/efectos de los fármacos , Micrococcus luteus/crecimiento & desarrollo , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/crecimiento & desarrollo , Proteus vulgaris/efectos de los fármacos , Proteus vulgaris/crecimiento & desarrollo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Solubilidad , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Taninos/análisis , Triterpenos/análisisRESUMEN
OBJECTIVE: To verify the effect of scaling and root planing on the antimicrobial activity of intracanal medications on the external root surface of periodontally compromised teeth. METHOD AND MATERIALS: Forty single-rooted extracted teeth were randomly assigned for visible calculus removal or scaling and root planing. Samples were separated into four groups-chlorhexidine, tetracycline hydrochloride, calcium hydroxide, or saline solution-and immersed in semisolid media and seeded with Micrococcus luteus for 24 hours. The zones of inhibition were measured using a profile projector. RESULTS: Calcium hydroxide and saline solution did not show bacterial growth inhibition. Chlorhexidine and tetracycline showed the occurrence of zones of inhibition in 70% and 40% of specimens, respectively. CONCLUSION: Partial antimicrobial activity on the external root surface was found when chlorhexidine and tetracycline solutions were used as intracanal medications; however, root planing procedures may modify this condition.
Asunto(s)
Antiinfecciosos Locales/administración & dosificación , Periodontitis Crónica/terapia , Micrococcus luteus/efectos de los fármacos , Irrigantes del Conducto Radicular/administración & dosificación , Aplanamiento de la Raíz , Raíz del Diente/microbiología , Hidróxido de Calcio/administración & dosificación , Clorhexidina/administración & dosificación , Recuento de Colonia Microbiana , Preparaciones de Acción Retardada , Cálculos Dentales/terapia , Difusión , Pruebas Antimicrobianas de Difusión por Disco , Micrococcus luteus/crecimiento & desarrollo , Propiedades de Superficie , Tetraciclina/administración & dosificaciónRESUMEN
Free-living amoebae of the genus Acanthamoeba are widely distributed in soil and water collections, where trophozoites (vegetative, multiplicative stages) feed mainly by phagocytosis and thus control bacterial populations in the environment. Here, we examined the growth, encystment and survival of Acanthamoeba castellanii receiving different bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Bacillus subtilis, Bacillus megaterium, Micrococcus luteus, and Staphylococcus aureus) in nonnutrient saline. All bacteria assayed induced a dose-dependent proliferative response, in most cases maximized with a bacterial dose of 1 x 10(9) mL(-1); except for M. luteus, trophozoites grew better with viable than with heat-killed bacteria. In addition, Acanthamoeba growth was improved by adding bacteria on alternate days. Single-dose experiments indicated a temporal association between the growth of trophozoite and bacterial consumption, and higher consumption of M. luteus, E. coli and P. aeruginosa, bacterial species that allowed the highest trophozoite yields. Long-term Acanthamoeba-bacteria incubation revealed that encystment was significantly delayed by almost all the bacteria assayed (including S. aureus, which elicited a poor growth response) and that the presence of bacteria markedly increased cyst yield; final cyst recovery clearly depended on both the dose and the type of the bacterium given, being much higher with E. coli, M. luteus and P. aeruginosa.
Asunto(s)
Acanthamoeba castellanii/crecimiento & desarrollo , Acanthamoeba castellanii/fisiología , Bacterias/crecimiento & desarrollo , Conducta Alimentaria , Trofozoítos/crecimiento & desarrollo , Animales , Fenómenos Fisiológicos Bacterianos , Recuento de Colonia Microbiana , Escherichia coli K12/crecimiento & desarrollo , Escherichia coli K12/fisiología , Viabilidad Microbiana , Micrococcus luteus/crecimiento & desarrollo , Micrococcus luteus/fisiología , Fagocitosis , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/fisiologíaRESUMEN
The effect of anthocyanin- and proanthocyanidin-rich fractions isolated from cranberry juice was studied for their antibacterial activity against nine bacterial strains. Activity was assessed by the agar diffusion assay. Staphylococcus aureus ATCC 6538 was the only strain to exhibit some susceptibility to four out of 10 anthocyanin-rich fractions tested. A variable susceptibility of S. aureus, Enterococcus faecalis ATCC 10541, and Micrococcus luteus ATCC 9341 to proanthocyanidin- rich fractions was also observed. Streptococcus mutans strains as well as Escherichia coli ATCC 10538 and Pseudomonas aeruginosa ATCC 27853 were not susceptible to any of the cranberry juice samples or fractions at the tested concentrations. There was no clear correlation between Gram-positive or Gram-negative bacterial susceptibility to cranberry juice. In this work, the role of cranberry juice anthocyanic and proanthocyanic fractions upon bacterial viability is discussed.
Asunto(s)
Antocianinas/farmacología , Antibacterianos/farmacología , Extractos Vegetales/farmacología , Proantocianidinas/farmacología , Vaccinium macrocarpon/química , Recuento de Colonia Microbiana , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/crecimiento & desarrollo , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Inmunodifusión , Pruebas de Sensibilidad Microbiana , Micrococcus luteus/efectos de los fármacos , Micrococcus luteus/crecimiento & desarrollo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Streptococcus mutans/efectos de los fármacos , Streptococcus mutans/crecimiento & desarrolloRESUMEN
Hemoglobin is known to be a source of peptides involved in several functions. The peptide FLSFPTTKTYFPHFDLSHGSAQVKGHGAK (Hb33-61) is a proteolytic product of the bovine hemoglobin alpha-chain found in the gut content of the cattle tick, Boophilus microplus, and it possesses antimicrobial activity. Since in the past we showed that the amidated form of Hb33-61, Hb33-61a, is active against a few Gram-positive bacteria and fungi strains at micromolar concentration [Fogaca et al. (1999) J. Biol. Chem. 274, 25330-25334], we have been prompted to shed more light on its functional and structural features. Here we show that the peptide is able to disrupt the bacterial membrane ofMicrococcus luteus A270. As for its structure, it has a random conformation in water, and it does not interact with zwitterionic micelles. On the other hand, it binds to negatively charged micelles acquiring a finite structural organization. The 3D structure of Hb33-61a bound to SDS micelles exhibits a nonconventional conformation for an antimicrobial peptide. The backbone is characterized by the presence of a beta-turn in the N-terminus and by a beta-turn followed by a alpha-helical stretch in the C-terminus. A hinge, whose spatial organization is stabilized by side-chain-side-chain interactions, joins these two regions. Interestingly, it preserves structural features present in the corresponding segment of the bovine hemoglobin alpha-chain. Hb33-61a does not possess a well-defined amphipathic nature, and H/D exchange experiments show that while the C-terminal region is embedded in the SDS micelle, one face of the N-terminal half is partly exposed to the solvent.