RESUMEN
The synthesis, physico-chemical characterization and in vitro antiproliferative activity against the promastigote form of Leishmania amazonensis of two new cobalt(II) coordination compounds (i.e. [Co(HL1)Cl2]0.4,2H2O (1) and [Co(HL2)(Cl)(CH3OH)](ClO4).2H2O (2)) are reported, where HL1 = 4-{3-[bis(pyridin-2-ylmethyl)amino]-2-hydroxypropoxy}-2H-chromen-2-one and HL2 = 7-{3-[bis(pyridin-2-ylmethyl)amino]-2-hydroxypropoxy}-2H-chromen-2-one. X-ray diffraction studies were performed for complex (2) and the structure of complex (1) was built through Density Functional Theory (DFT) calculations. Complex (1) presented no cytotoxicity to LLC-MK2, but complex (2) was toxic. IC50 against promastigotes of L. amazonensis for complex (1) were 4.90 (24 h), 3.50 (48 h) and 3. 80 µmol L-1 (72 h), and for complex (2) were 2.09, 4.20 and 2.80 µmol L-1, respectively. Due to the high toxicity presented by complex (2) against LLC-MK2 host cells, mechanistic studies, to shed light on the probable mode of leishmanicidal activity, were carried out only for the non-cytotoxic complex. Complex (1) was able to elevate mitochondrial membrane potential of the parasites after treatment. Transmission electron microscopy revealed typical apoptotic condensation of chromatin, altered kinetoplast and mitochondria structures, suggesting that apoptosis-like cell death of the protozoa is probably mediated by an apoptotic mechanism associated with mitochondrial dysfunction (intrinsic pathway). Molecular docking studies with complex (1) upon protein tyrosine phosphatase (LmPRL-1) suggests a plausible positive complex anchoring mainly by hydrophobic and hydrogen bond forces close to the enzyme's catalytic site. These promising results for complex 1 will prompt future investigations against amastigote form of L. amazonensis.
Asunto(s)
Antiprotozoarios , Leishmania , Parásitos , Animales , Cobalto/farmacología , Simulación del Acoplamiento Molecular , Apoptosis , Mitocondrias , Antiprotozoarios/químicaRESUMEN
The necessity of drug combinations to treat leishmaniasis came to the surface mainly because of the toxicity of current treatments and the emergence of resistant strains. The calpain inhibitor MDL28170 has previously shown anti-Leishmania activity, therefore its use in association with standard drugs could provide a new alternative for the treatment strategy against leishmaniasis. In this study, we analyzed the potential of the combination of MDL28170 and the antileishmanial drug amphotericin B against Leishmania amazonensis and Leishmania chagasi. The compounds were tested in the combination of the ½ × IC50 value of MDL28170 plus the » × IC50 value of amphotericin B, which led to an increment in the anti-promastigote activity when compared to the single drug treatments. This drug association revealed several and severe morphophysiological changes on parasite cells, such as loss of plasma membrane integrity, reduced size of flagellum, and depolarization of mitochondrial membrane potential besides increased reactive oxygen species production. In addition, the combination of both drugs had a deleterious effect on the Leishmania-macrophage interaction, reflecting in a significant anti-amastigote action, which achieved a reduction of 50% in the association index. These results indicate that the combination treatment proposed here may represent a new alternative for leishmaniasis chemotherapy.
RESUMEN
Phialophora verrucosa causes several fungal human diseases, mainly chromoblastomycosis, which is extremely difficult to treat. Several studies have shown that human immunodeficiency virus peptidase inhibitors (HIV-PIs) are attractive candidates for antifungal therapies. This work focused on studying the action of HIV-PIs on peptidase activity secreted by P. verrucosa and their effects on fungal proliferation and macrophage interaction. We detected a peptidase activity from P. verrucosa able to cleave albumin, sensitive to pepstatin A and HIV-PIs, especially lopinavir, ritonavir and amprenavir, showing for the first time that this fungus secretes aspartic-type peptidase. Furthermore, lopinavir, ritonavir and nelfinavir reduced the fungal growth, causing remarkable ultrastructural alterations. Lopinavir and ritonavir also affected the conidia-macrophage adhesion and macrophage killing. Interestingly, P. verrucosa had its growth inhibited by ritonavir combined with either itraconazole or ketoconazole. Collectively, our results support the antifungal action of HIV-PIs and their relevance as a possible alternative therapy for fungal infections.
Asunto(s)
Antifúngicos/farmacología , Proteasas de Ácido Aspártico/antagonistas & inhibidores , Inhibidores de la Proteasa del VIH/farmacología , Macrófagos/efectos de los fármacos , Phialophora/efectos de los fármacos , Antifúngicos/síntesis química , Antifúngicos/química , Proteasas de Ácido Aspártico/metabolismo , Carbamatos/síntesis química , Carbamatos/química , Carbamatos/farmacología , Relación Dosis-Respuesta a Droga , Furanos , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/química , Humanos , Lopinavir/síntesis química , Lopinavir/química , Lopinavir/farmacología , Macrófagos/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Phialophora/enzimología , Phialophora/crecimiento & desarrollo , Ritonavir/síntesis química , Ritonavir/química , Ritonavir/farmacología , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
The emerging opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii [Ch], C. duobushaemulonii [Cd] and C. haemulonii var. vulnera[Chv]) are notable for their intrinsic antifungal resistance. Different clinical manifestations are associated with these fungal infections; however, little is known about their biology and potential virulence attributes. Herein, we evaluated some surface properties of 12 clinical isolates of Ch (n = 5), Cd (n = 4) and Chv (n = 3) as well as their virulence on murine macrophages and Galleria mellonella larvae. Scanning electron microscopy demonstrated the presence of homogeneous populations among the species of the C. haemulonii complex, represented by oval yeasts with surface irregularities able to form aggregates. Cell surface hydrophobicity was isolate-specific, exhibiting high (16.7%), moderate (25.0%) and low (58.3%) hydrophobicity. The isolates had negative surface charge, except for one. Mannose/glucose- and N-acetylglucosamine-containing glycoconjugates were evidenced in considerable amounts in all isolates; however, the surface expression of sialic acid was poorly detected. Cd isolates presented significantly higher amounts of chitin than Ch and Chv. Membrane sterol and lipid bodies, containing neutral lipids, were quite similar among all fungi studied. All isolates adhered to inert surfaces in the order: polystyrene > poly-L-lysine-coated glass > glass. Likewise, they interacted with murine macrophages in a quite similar way. Regarding in vivo virulence, the C. haemulonii species complex were able to kill at least 80% of the larvae after 120 hours. Our results evidenced the ability of C. haemulonii complex to produce potential surface-related virulence attributes, key components that actively participate in the infection process described in Candida spp.
Asunto(s)
Adhesividad/efectos de los fármacos , Antifúngicos/uso terapéutico , Candida/aislamiento & purificación , Candidiasis/tratamiento farmacológico , Candidiasis/fisiopatología , Farmacorresistencia Fúngica Múltiple/efectos de los fármacos , Virulencia/efectos de los fármacos , Arthrodermataceae/aislamiento & purificación , Brasil , Humanos , Macrófagos/efectos de los fármacos , Esporas Fúngicas/ultraestructuraRESUMEN
Candida parapsilosis sensu stricto (C. parapsilosis) has emerged as the second/third commonest Candida species isolated from hospitals worldwide. Candida spp. possess numerous virulence attributes, including peptidases that play multiple roles in both physiological and pathological events. So, fungal peptidases are valid targets for new drugs development. With this premise in mind, we have evaluated the effect of serine peptidase inhibitors (SPIs) on both cell biology and virulence aspects of C. parapsilosis. First, five different SPIs, phenylmethylsulfonyl fluoride, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, N-α-tosyl-L-lysine chloromethyl ketone hydrochloride, and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were tested, and TPCK showed the best efficacy to arrest fungal growth. Subsequently, the ability of TPCK to modulate physiopathological processes was investigated. Overall, TPCK was able to (i) inhibit the cell-associated serine peptidase activities, (ii) promote morphometric and ultrastructural alterations, (iii) induce an increase in the intracellular oxidation level, which culminates in a vigorous lipid peroxidation and accumulation of neutral lipids in cytoplasmic inclusions, (iv) modulate the expression/exposition of surface structures, such as mannose/glucose-rich glycoconjugates, N-acetylglucosamine-containing molecules, chitin, polypeptides and surface aspartic peptidases, (v) reduce the adhesion to either polystyrene or glass surfaces as well as to partially disarticulate the mature biofilm, (vi) block the fungal interaction with macrophages, and (vii) protect Galleria mellonella from fungal infection, enhancing larvae survivability. Altogether, these results demonstrated that TPCK induced several changes over fungal biology besides the interference with aspects associated to C. parapsilosis virulence and pathogenesis, which indicates that SPIs could be novel promising therapeutic agents in dealing with candidiasis.
Asunto(s)
Antifúngicos/farmacología , Candida parapsilosis/efectos de los fármacos , Candidiasis/prevención & control , Inhibidores de Serina Proteinasa/farmacología , Clorometilcetona de Tosilfenilalanila/farmacología , Animales , Antifúngicos/administración & dosificación , Candida parapsilosis/citología , Candida parapsilosis/crecimiento & desarrollo , Adhesión Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Larva/microbiología , Lepidópteros/microbiología , Estrés Oxidativo , Inhibidores de Serina Proteinasa/administración & dosificación , Análisis de Supervivencia , Clorometilcetona de Tosilfenilalanila/administración & dosificación , Resultado del Tratamiento , Virulencia/efectos de los fármacosRESUMEN
We have previously shown that metallocomplexes can control the growth of Toxoplasma gondii, the agent that causes toxoplasmosis. In order to develop new metallodrugs to treat this disease, we investigated the influence of the coordination of sulfadiazine (SDZ), a drug used to treat toxoplasmosis, on the biological activity of the iron(III) complex [Fe(HBPClNOL)Cl2]·H2O, 1, (H2BPClNOL=N-(2-hydroxybenzyl)-N-(2-pyridylmethyl)(3-chloro)(2-hydroxy)-propylamine). The new complex [(Cl)(SDZ)Fe(III)(µ-BPClNOL)2Fe(III)(SDZ)(Cl)]·2H2O, 2, which was obtained by the reaction between complex 1 and SDZ, was characterized using a range of physico-chemical techniques. The cytotoxic effect of the complexes and the ability of T. gondii to infect LLC-MK2 cells were assessed. It was found that both complexes reduced the growth of T. gondii while also causing low cytotoxicity in the host cells. After 48 h of treatment, complex 2 reduced the parasite's ability to proliferate by about 50% with an IC50 of 1.66 µmol/L. Meanwhile, complex 1 or SDZ alone caused a 40% reduction in proliferation, and SDZ displayed an IC50 of 5.3 µmol/L. In addition, complex 2 treatment induced distinct morphological and ultrastructural changes in the parasites and triggered the formation of cyst-like forms. These results show that the coordination of SDZ to the iron(III) complex is a good strategy for increasing the anti-toxoplasma activity of these compounds.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Hierro/farmacología , Sulfadiazina/farmacología , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis/tratamiento farmacológico , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Macaca mulatta , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Toxoplasma/efectos de los fármacos , Toxoplasmosis/parasitologíaRESUMEN
Acanthamoeba castellanii (Ac) are ubiquitously distributed in nature, and by contaminating medical devices such as heart valves and contact lenses, they cause a broad range of clinical presentations to humans. Although several molecules have been described to play a role in Ac pathogenesis, including parasite host-tissue invasion and escaping of host-defense, little information is available on their mechanisms of secretion. Herein, we describe the molecular components secreted by Ac, under different protein availability conditions to simulate host niches. Ac extracellular vesicles (EVs) were morphologically and biochemically characterized. Dynamic light scattering analysis of Ac EVs identified polydisperse populations, which correlated to electron microscopy measurements. High-performance thin liquid chromatography of Ac EVs identified phospholipids, steryl-esters, sterol and free-fatty acid, the last two also characterized by GC-MS. Secretome composition (EVs and EVs-free supernatants) was also determined and proteins biological functions classified. In peptone-yeast-glucose (PYG) medium, a total of 179 proteins were identified (21 common proteins, 89 exclusive of EVs and 69 in EVs-free supernatant). In glucose alone, 205 proteins were identified (134 in EVs, 14 common and 57 proteins in EVs-free supernatant). From those, stress response, oxidative and protein and amino acid metabolism proteins prevailed. Qualitative differences were observed on carbohydrate metabolism enzymes from Krebs cycle and pentose phosphate shunt. Serine proteases and metalloproteinases predominated. Analysis of the cytotoxicity of Ac EVs (upon uptake) and EVs-free supernatant to epithelial and glioblastoma cells revealed a dose-dependent effect. Therefore, the Ac secretome differs depending on nutrient conditions, and is also likely to vary during infection.
Asunto(s)
Acanthamoeba castellanii/metabolismo , Amebiasis/parasitología , Vesículas Extracelulares/metabolismo , Proteoma/metabolismo , Proteínas Protozoarias/metabolismo , Acanthamoeba castellanii/genética , Animales , Línea Celular , Vesículas Extracelulares/genética , Homeostasis , Humanos , Transporte de Proteínas , Proteoma/genética , Proteómica , Proteínas Protozoarias/genética , Vías SecretorasRESUMEN
Ethanol extracts obtained from Schinus terebinthifolius Raddi fruits and leaves were active against Escherichia coli with MIC of 78 µg mL-1 for both extracts. Phytochemical analyses revealed a major presence of phenolic acids, tannins, fatty acids and acid triterpenes in the leaves and phenolic acids, fatty acids, acid triterpenes and biflavonoids in the fruits. Major compounds isolated from the plant, such as the acid triterpene schinol, the phenolic acid derivative ethyl gallate and the biflavonoids agathisflavone and tetrahydroamentoflavone, showed very little activity against E. coli. Bioautography of the ethanol extracts on silica gel plate showed inhibition zones for E. coli. They were removed from the plate and the compounds identified as a mixture of myristic, pentadecanoic, palmitic, heptadecanoic, stearic, nonadecanoic, eicosanoic, heneicosanoic and behenic fatty acids.
Asunto(s)
Anacardiaceae/química , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Extractos Vegetales/farmacología , Antibacterianos/química , Biflavonoides/aislamiento & purificación , Biflavonoides/farmacología , Frutas/química , Ácido Gálico/análogos & derivados , Ácido Gálico/aislamiento & purificación , Ácido Gálico/farmacología , Pruebas de Sensibilidad Microbiana , Extractos Vegetales/análisis , Extractos Vegetales/química , Hojas de la Planta/química , Triterpenos/aislamiento & purificación , Triterpenos/farmacologíaRESUMEN
There is a general lack of effective and non-toxic chemotherapeutic agents against Chagas' disease despite more than a century of research. In this regard, we have verified the impact of human immunodeficiency virus aspartic peptidase inhibitors (HIV-PIs) on the viability and morphology of infective trypomastigote forms of Trypanosoma cruzi as well as on the aspartic peptidase and proteasome activities produced by this parasite. The effects of HIV-PIs on viability were assessed by counting motile parasites in a Neubauer chamber. Morphological alterations were detected by light microscopy of Giemsa-stained smears and scanning electron microscopy. Modulation of aspartic peptidase and proteasome activities by the HIV-PIs was measured by cleavage of fluorogenic peptide substrates. The majority of the HIV-PIs (6/9) were able to drastically decrease the viability of trypomastigotes after 4 h of treatment, with nelfinavir and lopinavir being the most effective compounds presenting LD50 values of 8.6 µM and 10.6 µM, respectively. Additionally, both HIV-PIs were demonstrated to be effective in a time- and cell density-dependent manner. Treatment with nelfinavir and lopinavir caused many morphological/ultrastructural alterations in trypomastigotes; parasites became round in shape, with reduced cell size and flagellar shortening. Nelfinavir and lopinavir were also capable of significantly inhibiting the aspartic peptidase and proteasome activities measured in trypomastigote extracts. These results strengthen the data on the positive effects of HIV-PIs on parasitic infections, possibly by targeting the parasite aspartic peptidase(s) and proteasome(s), opening a new possibility for the use of these clinically approved drugs as an alternative chemotherapy to treat Chagas' disease.
Asunto(s)
Antiprotozoarios/farmacología , Inhibidores de la Proteasa del VIH/farmacología , Trypanosoma cruzi/efectos de los fármacos , Tripanosomiasis/parasitología , Supervivencia Celular/efectos de los fármacos , Locomoción/efectos de los fármacos , Microscopía , Trypanosoma cruzi/citología , Trypanosoma cruzi/fisiología , Tripanosomiasis/tratamiento farmacológicoRESUMEN
Reported herein is the ability of Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans conidia to adhere, differentiate into hyphae and form biofilms on both polystyrene and lung epithelial cells. To different degrees, all of the fungi adhered to polystyrene after 4 h, with a predominance of those with germinated conidia. Prolonged fungi-polystyrene contact resulted in the formation of a monolayer of intertwined mycelia, which was identified as a typical biofilm structure due to the presence of a viable mycelial biomass, extracellular matrix and enhanced antifungal resistance. Ultrastructural details were revealed by SEM and CLSM, showing the dense compaction of the mycelial biomass and the presence of channels within the organized biofilm. A similar biofilm structure was observed following the co-culture of each fungus with A549 cells, revealing a mycelial trap covering all of the lung epithelial monolayer. Collectively, these results highlight the potential for biofilm formation by these clinically relevant fungal pathogens.
Asunto(s)
Ascomicetos/fisiología , Biopelículas/crecimiento & desarrollo , Células Epiteliales/microbiología , Hifa/crecimiento & desarrollo , Poliestirenos , Scedosporium/fisiología , Células A549 , Ascomicetos/ultraestructura , Adhesión Bacteriana , Biomasa , Humanos , Microscopía Confocal , Microscopía Electrónica de Rastreo , Poliestirenos/química , Scedosporium/ultraestructuraRESUMEN
Toxoplasma gondii infection can be severe during pregnancy and in immunocompromised patients. Current therapies for toxoplasmosis are restricted to tachyzoites and have little or no effect on bradyzoites, which are maintained in tissue cysts. Consequently, new therapeutic alternatives have been proposed as the use of atovaquone has demonstrated partial efficacy against tachyzoites and bradyzoites. This work studies the effect of 3-bromopyruvate (3-BrPA), a compound that is being tested against cancer cells, on the infection of LLC-MK2 cells with T. gondii tachyzoites, RH strain. No effect of 3-BrPA on host cell proliferation or viability was observed, but it inhibited the proliferation of T. gondii. The incubation of cultures with lectin Dolichos biflorus agglutinin (DBA) showed the development of cystogenesis, and an ultrastructural analysis of parasite intracellular development confirmed morphological characteristics commonly found in tissue cysts. Moreover, the presence of degraded parasites and the influence of 3-BrPA on endodyogeny were observed. Infected cultures were alternatively treated with a combination of this compound plus atovaquone. This resulted in a 73% reduction in intracellular parasites after 24 h of treatment and a 71% reduction after 48 h; cyst wall formation did not occur in these cultures. Therefore, we conclude that the use of 3-BrPA may serve as an important tool for the study of (i) in vitro cystogenesis; (ii) parasite metabolism, requiring a deeper understanding of the target of action of this compound on T. gondii; (iii) the alternative parasite metabolic pathways; and (iv) the molecular/cellular mechanisms that trigger parasite death.
Asunto(s)
Atovacuona/uso terapéutico , Piruvatos/uso terapéutico , Toxoplasma/patogenicidad , Toxoplasmosis Animal/tratamiento farmacológico , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Macaca mulatta , Ratones , Toxoplasma/efectos de los fármacosRESUMEN
BACKGROUND: Human cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC50 (15 µM) and two times the IC50 doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G0/G1 phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC50 dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis. CONCLUSIONS/SIGNIFICANCE: The data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the investigation of the potential clinical utility of calpain inhibitors in the chemotherapy of leishmaniases.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Fragmentación del ADN/efectos de los fármacos , ADN Protozoario/metabolismo , Dipéptidos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Protozoarias/biosíntesis , ADN Protozoario/genética , Fase G1/efectos de los fármacos , Humanos , Leishmania , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Cutánea/enzimología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Protozoarias/genética , Fase de Descanso del Ciclo Celular/efectos de los fármacosRESUMEN
The production of virulence attributes in three reference strains and 11 clinical isolates primarily identified as Candida parapsilosis was evaluated. Morphological and phenotypical tests were not able to discriminate among the three species of the C. parapsilosis complex; consequently, molecular methods were applied to solve this task. After employing polymerase chain reaction-based methods, nine clinical strains were identified as C. parapsilosis sensu stricto and two as C. orthopsilosis. Protease, catalase, and hemolysin were produced by all 14 strains, while 92.9% and 78.6% of strains secreted, respectively, esterase and phytase. No phospholipase producers were detected. Mannose/glucose, N-acetylglucosamine, and sialic acid residues were detected at the surface of all strains, respectively, in high, medium, and low levels. All strains presented elevated surface hydrophobicity and similar ability to form biofilm. However, the adhesion to inert substrates and mammalian cells was extremely diverse, showing typical intrastrain variations. Overall, the strains showed (1) predilection to adhere to plastic over glass and the number of pseudohyphae was more prominent than yeasts and (2) the interaction process was slightly enhanced in macrophages than fibroblasts, with the majority of fungal cells detected inside them. Positive/negative correlations were demonstrated among the production of these virulence traits in C. parapsilosis complex.
Asunto(s)
Candida/clasificación , Fenotipo , Biopelículas , Candida/fisiología , Candida/ultraestructura , Membrana Celular/química , Membrana Celular/metabolismo , Glicosilación , Humanos , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Tipificación Molecular , Filogenia , ARN de Hongos , ARN Ribosómico 28S , Virulencia/genéticaRESUMEN
BACKGROUND: Phosphatidylserine (PS) and surface carbohydrates (SC) are known as virulence factors that may contribute to the different clinical symptoms ranging from self-healing cutaneous leishmaniasis lesions to fatal visceral disease. Leishmania (Viannia) braziliensis causes localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). METHODS: We analyzed PS exposure and SC expression associated with 2 primary L. braziliensis isolates from patients with LCL or MCL. The role of PS exposure was also addressed during promastigotes phagocytosis by macrophages. RESULTS: We observed higher PS exposure on the surface of late stationary growth phase promastigotes from patients with LCL, compared with those from patients with MCL, and both strains were alive during PS display. Reduction in the infectivity index was observed during macrophage interaction with late stationary growth phase promastigotes in which PS was blocked by annexin V. The major surface carbohydrates detected on LCL and MCL promastigotes were α-Man, α-Glc, and α-Gal. However, α-ß-GalNAc, although observed on the surface of the LCL strain during the late stationary growth phase was highly expressed on the surface of early stationary growth phase promastigotes. CONCLUSIONS: Our results suggest that PS and SC can modulate interactions between Leishmania organisms and host cells and may be important for the outcome of the clinical course of diseases caused by L. braziliensis.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Leishmania braziliensis/metabolismo , Leishmaniasis Cutánea/metabolismo , Leishmaniasis Mucocutánea/metabolismo , Fosfatidilserinas/metabolismo , Pruebas de Aglutinación , Animales , Interacciones Huésped-Patógeno , Leishmania braziliensis/crecimiento & desarrollo , Leishmaniasis Cutánea/inmunología , Leishmaniasis Mucocutánea/inmunología , Macrófagos/inmunología , Macrófagos/parasitología , RatonesRESUMEN
Lonomia obliqua envenomation is characterized by intense local inflammatory reaction, which, dependent on the severity of the case, is followed by severe clinical manifestations related to hemorrhagic disorders that can lead to fatal outcome. These effects were imputed to several toxins present in L. obliqua venom, which are responsible for procoagulant, anticoagulant as well as antithrombotic activities, being also able to interfere with vascular cells functions. In this work, the intravital microscopy analysis show that after administration of low doses of L. obliqua venom (1-3 µg/ml) on hamster cheek pouch, there was no alterations neither on arterioles or venules caliber nor in the vascular permeability up to 30 min. However, after 10 min in contact with venom occurred a clear activation in the vascular bed, characterized by an increase in leukocyte rolling and adhesion on endothelium of hamster cheek pouch venules. A confocal analysis of vascular beds, confirmed these results showing an increase in endothelial E-selectin and VCAM-1 expression. The effects of L. obliqua venom on human endothelial cell (EC) in vitro were also investigated. The treatment of EC with venom (1-3 µg/ml) did not affect cell viability. However, at concentrations as low as 3 µg/ml of L. obliqua venom modifies actin cytoskeleton dynamics, and increases focal adhesion contacts, inducing stress fiber formation, focal adhesion kinase (FAK) phosphorylation and its subsequent association to actin. These effects are followed by the activation of NF-κB pathway, a critical signaling in several events associated to vascular inflammation. Accordingly, L. obliqua venom leads to a significant increase in COX-2, NOS-2, HO-1, MMP-2 and MMP-9 expression. Taken together the data show that, even at low concentrations, L. obliqua venom can activate endothelial cells, which assume a pro-inflammatory profile, contributing for local effects and probably also for systemic disturbances due to its ability to modulate the properties of the vascular system.
Asunto(s)
Células Endoteliales/metabolismo , Inflamación/inducido químicamente , Lepidópteros , Ponzoñas/toxicidad , Animales , Western Blotting , Cricetinae , Selectina E/metabolismo , Células Endoteliales/citología , Células Endoteliales/enzimología , Enzimas/metabolismo , Inmunoprecipitación , Fosforilación , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The flagellar pocket and the cytostome are surface domains of Trypanosoma cruzi epimastigote involved in acquisition of nutrients. The cytostome is physically connected to the flagellar complex. To investigate if this association plays a role in endocytosis in T. cruzi, the endocytic activity in wild type and gp72 null mutant (flagellum-cell body attachment region is absent) epimastigotes was compared. Both wild type and mutant cells were incubated with transferrin conjugated with Alexa 543 or gold particles over different time periods and thereafter qualitatively and quantitatively analyzed by flow cytometry and transmission electron microscopy. Flow cytometry analysis showed a reduction in transferrin uptake by null mutant after 30 min of incubation. In addition, at this time period, signals detected by fluorescence microscopy were slightly lower in null mutant cells. At lower incubation times, no differences between wild type and mutant epimastigotes could be observed. Quantitative data obtained by morphometric and flow cytometry analysis suggested that the speed of the endocytic process in the null mutant was similar to wild type cells, although null mutants were not able to bind cargo and therefore internalize as much as wild type epimastigotes. Our observations suggest that the physical association between cytostome and the flagellar complex plays a role in endocytosis efficiency by epimastigotes of T. cruzi.
Asunto(s)
Endocitosis/fisiología , Flagelos/metabolismo , Orgánulos/ultraestructura , Transferrina/metabolismo , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/ultraestructura , Animales , Flagelos/química , Flagelos/ultraestructura , Citometría de Flujo , Colorantes Fluorescentes/metabolismo , Oro/metabolismo , Cinética , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Mutación , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/genéticaRESUMEN
BACKGROUND: The pathogenic fungus Fonsecaea pedrosoi constitutively produces the pigment melanin, an important virulence factor in fungi. Melanin is incorporated in the cell wall structure and provides chemical and physical protection for the fungus.We evaluated the production of nitric oxide (NO) in macrophages, the oxidative burst and the inducible nitric oxide synthase (i-NOS) activity in interactions between activated murine macrophages and F. pedrosoi. Experiments were carried out with or without tricyclazole (TC) treatment, a selective inhibitor of the dihydroxynaphthalene (DHN)-melanin biosynthesis pathway in F. pedrosoi. The paramagnetisms of melanin and the TC-melanin were analysed by electron spin resonance. The fungal growth responses to H2O2 and to S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, were also evaluated. RESULTS: Melanised F. pedrosoi cells were more resistant to both H2O2 and NO. Nitrite was not detected in the supernatant of macrophages incubated with melanised fungal cells. However, i-NOS expression was unaffected by the presence of either untreated control F. pedrosoi or TC-treated F. pedrosoi. In addition, the inhibition of the DHN-melanin pathway by TC improved the oxidative burst capability of the macrophages. CONCLUSION: The NO-trapping ability of F. pedrosoi melanin is an important mechanism to escape the oxidative burst of macrophages.
Asunto(s)
Ascomicetos/metabolismo , Peróxido de Hidrógeno/metabolismo , Melaninas/metabolismo , Óxido Nítrico/metabolismo , Animales , Ascomicetos/química , Ascomicetos/crecimiento & desarrollo , Proliferación Celular/efectos de los fármacos , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/metabolismo , Interacciones Huésped-Patógeno , Peróxido de Hidrógeno/farmacología , Inmunohistoquímica , Macrófagos/citología , Macrófagos/microbiología , Melaninas/química , Ratones , Microscopía de Contraste de Fase , Microondas , Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/metabolismo , TiazolesRESUMEN
The present study analyses complement resistance, cell surface carbohydrates expression, lipidic composition and morphology in vivo and in vitro, of Leishmania (Viannia) shawi, a parasite identified in the Amazon region, Pará state, in 1989. We demonstrated that promastigotes in the stationary (STAT) growth phase are more resistant to complement lysis than in the logarithmic (LOG) growth phase. Ultrastructural analyses and imidazol technique showed accumulation of lipids in STAT growth phase promastigotes, which was confirmed by biochemical approach. Light and electron microscopy of skin lesion in hamster footpads caused by promastigotes in STAT growth phase, 90 days post inoculation, showed amastigotes inside of macrophage and free in the tissue surrounded by collagen fibers as well as extensive inflammatory reaction with tissue destruction. We also demonstrated, using lectins by agglutination assays and flow cytometry, the presence of fucose, mannose and/or glucose carbohydrate residues on the surface of LOG and STAT promastigotes. The results constitute the first characterization essay combining biochemical and morphological approaches dedicated to LOG and STAT growth phase promastigotes of L. (V) shawi contributing for a better knowledge of this poorly studied species of the New World.
Asunto(s)
Leishmania/clasificación , Leishmania/patogenicidad , Leishmaniasis Cutánea , Animales , Brasil , Carbohidratos/análisis , Carbohidratos/química , Proteínas del Sistema Complemento/inmunología , Cricetinae , Cobayas , Humanos , Lectinas/metabolismo , Leishmania/crecimiento & desarrollo , Leishmania/ultraestructura , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/patología , Mesocricetus , Microscopía/instrumentación , Microscopía ElectrónicaRESUMEN
In this paper, the lytic activity of two variants of Serratia marcescens against promastigotes of Leishmania braziliensis was studied. In vitro assays showed that S. marcescens variant SM365 lyses L. braziliensis promastigotes, while the variant DB11 did not. Scanning electron microscopy (SEM) revealed that S. marcescens SM365 adheres to all cellular body and flagellum of the parasite. Several filamentous structures were formed and identified as biofilms. After 120min incubation, they connect the protozoan to the developing bacterial clusters. SEM also demonstrated that bacteria, adhered onto L. braziliensis promastigote surface, formed small filamentous structures which apparently penetrates into the parasite membrane. d-mannose protects L. braziliensis against the S. marcescens SM365 lytic effect in a dose dependent manner. SM365 variant pre cultivated at 37 degrees C did not synthesize prodigiosin although the adherence and lysis of L. braziliensis were similar to the effect observed with bacteria cultivated at 28 degrees C, which produce high concentrations of prodigiosin. Thus, we suggest that prodigiosin is not involved in the lysis of promastigotes and that adherence promoted by bacterial mannose-sensitive (MS) fimbriae is a determinant factor in the lysis of L. braziliensis by S. marcescens SM365.
Asunto(s)
Fimbrias Bacterianas/metabolismo , Leishmania braziliensis/metabolismo , Prodigiosina/metabolismo , Serratia marcescens/fisiología , Animales , Adhesión Bacteriana , Carbohidratos/farmacología , Fimbrias Bacterianas/efectos de los fármacos , Cinética , Leishmania braziliensis/efectos de los fármacos , Leishmania braziliensis/ultraestructura , Manosa/farmacología , Microscopía Electrónica de Rastreo , Prodigiosina/aislamiento & purificación , Serratia marcescens/química , Serratia marcescens/ultraestructuraRESUMEN
Studies on the lysis of L. chagasi caused by the bacteria Serratia marcescens were carried out. In vitro experiments demonstrated that S. marcescens variant SM 365, a prodigiosin pigment producer, lysed this species of Leishmania but variant DB11, a nonpigmented bacteria, was unable to lyse the parasite. High concentrations of d-mannose were found to protect L. chagasi markedly diminishing the lysis by S. marcescens SM 365. Promastigotes of L. chagasi bound the lectin Concanavalin A conjugated with FITC, the fluorescence was intensely found at the base of the flagellum (flagellar pocket). Scanning electron microscopy revealed that the bacteria adherence occurred mainly in the flagellar pocket. S. marcescens SM 365 formed filamentous structures, identified as biofilms, which connect the protozoan to the developing bacterial clusters, in low concentrations of bacteria after 30 min incubation time. We suggest that bacterial mannose-sensitive (MS) fimbriae are relevant to S. marcescens SM 365 in the lysis of L. chagasi.