The protein map of the protozoan parasite Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum during growth phase transition and temperature stress.

Leishmania parasites cause a spectrum of diseases termed leishmaniasis, which manifests in two main clinical forms, cutaneous and visceral leishmaniasis. Leishmania promastigotes transit from proliferative exponential to quiescent stationary phases inside the insect vector, a relevant step that recapitulates early molecular events of metacyclogenesis. During the insect blood meal of the mammalian hosts, the released parasites interact initially with the skin, an event marked by temperature changes. Deep knowledge on the molecular events activated during Leishmania-host interactions in each step is crucial to develop better therapies and to understand the pathogenesis. In this study, the proteomes of Leishmania (Leishmania) amazonensis (La), Leishmania (Viannia) braziliensis (Lb), and Leishmania (Leishmania) infantum (syn L. L. chagasi) (Lc) were analyzed using quantitative proteomics to uncover the proteome modulation in three different conditions related to growth phases and temperature shifts: 1) exponential phase (Exp); 2) stationary phase (Sta25) and; 3) stationary phase subjected to heat stress (Sta34). Functional validations were performed using orthogonal techniques, focusing on α-tubulin, gp63 and heat shock proteins (HSPs). Species-specific and condition-specific modulation highlights the plasticity of the Leishmania proteome, showing that pathways related to metabolism and cytoskeleton are significantly modulated from exponential to stationary growth phases, while protein folding, unfolded protein binding, signaling and microtubule-based movement were differentially altered during temperature shifts. This study provides an in-depth proteome analysis of three Leishmania spp., and contributes compelling evidence of the molecular alterations of these parasites in conditions mimicking the interaction of the parasites with the insect vector and vertebrate hosts. SIGNIFICANCE: Leishmaniasis disease manifests in two main clinical forms according to the infecting Leishmania species and host immune responses, cutaneous and visceral leishmaniasis. In Brazil, cutaneous leishmaniasis (CL) is associated with L. braziliensis and L. amazonensis, while visceral leishmaniasis, also called kala-azar, is caused by L. infantum. Leishmania parasites remodel their proteomes during growth phase transition and changes in their mileu imposed by the host, including temperature. In this study, we performed a quantitative mass spectrometry-based proteomics to compare the proteome of three New world Leishmania species, L. amazonensis (La), L. braziliensis (Lb) and L. infantum (syn L. chagasi) (Lc) in three conditions: a) exponential phase at 25 °C (Exp); b) stationary phase at 25 °C (Sta25) and; c) stationary phase subjected to temperature stress at 34 °C (Sta34). This study provides an in-depth proteome analysis of three Leishmania spp. with varying pathophysiological outcomes, and contributes compelling evidence of the molecular alterations of these parasites in conditions mimicking the interaction of the parasites with the insect vector and vertebrate hosts.

Sugar-based colloidal nanocarriers for topical meglumine antimoniate application to cutaneous leishmaniasis treatment: Ex vivo cutaneous retention and in vivo evaluation.

The leishmaniases are a group of diseases caused by protozoan parasites from Leishmania species. Effectiveness therapies for cutaneous leishmaniasis (CL), the most common form, are still needed to be developed since the available drugs such as meglumine antimoniate (MA) present severe adverse reactions. Here, we develop and characterize maltodextrin polymeric colloidal nanocarriers containing MA (PCN-MA) for topical CL treatment. PCN-MA is composed of 5 to 8% maltodextrin, 0.3% NaCl, 1% MA in 21% of water as aqueous-internal phase, containing or no 3% Kolliphor® P-188, and 10% SF1540 dispersed in a silicone-based external phase. It formed a colloidal system dispersed in silicone with high encapsulation efficiency (87% to 92%) and composite spherical-shaped particles with the smooth and regular surface within the nanosized scale, which was confirmed by scanning electron microscopy (SEM) and dynamic light scattering (DLS) analysis. Ex vivo cutaneous retention studies using pig ears skin on Franz diffusion cells revealed that the MA cutaneous retention is improved when delivered by PCN. Topical PCN-MA evaluation in murine leishmaniasis model showed similar efficacy than the intraperitoneal injection of the reference medicine (Glucantime®) regarding parasite titer reduction and superior healing activity in terms of collagen area deposition. Our results suggest that this sugar-based PCN is a promising agent for topical delivery of meglumine antimoniate.

The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection

Trypanosoma cruzi, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the T. cruzi parasite, but their consumption produces an accumulation of NH4+ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS) plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene TcGS from T. cruzi encodes a functional glutamine synthetase; it can complement a defect in the GLN1 gene from Saccharomyces cerevisiae and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/ mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH4+ production is predictable. During host-cell invasion, TcGS is required for the parasite to escape from the parasitophorous vacuole, a process sine qua non for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection.

The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection

Trypanosoma cruzi, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the T. cruzi parasite, but their consumption produces an accumulation of NH4+ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS) plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene TcGS from T. cruzi encodes a functional glutamine synthetase; it can complement a defect in the GLN1 gene from Saccharomyces cerevisiae and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/ mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH4+ production is predictable. During host-cell invasion, TcGS is required for the parasite to escape from the parasitophorous vacuole, a process sine qua non for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection.

Chemical composition and amoebicidal activity of Croton pallidulus, Croton ericoides, and Croton isabelli (Euphorbiaceae) essential oils.

Acanthamoeba is a free-living amoebae genus that causes amoebic keratitis which is a painful sight-threatening disease of the eyes. Its treatment is difficult, and the search for new drugs is very important. Here, essential oils obtained from the aerial parts of Croton pallidulus, Croton isabelli, and Croton ericoides (Euphorbiaceae), native plants of Southern Brazil, were tested against Acanthamoeba polyphaga and analyzed by gas chromatography and gas chromatography-mass spectrometry. The essential oils of C. pallidulus and C. isabelli were characterized by the presence of sesquiterpenes: germacrene D (15.5 %), terpinen-4-ol (13.2 %), and ß-caryophyllene (13.1 %) in C. pallidulus and bicyclogermacrene (48.9 %) in C. isabelli. The essential oil of C. ericoides presented mainly monoterpenes, ß-pinene (39.0 %) being the main component. Laboratory tests were carried out to determine the effect of the essential oils against A. polyphaga trophozoites. The essential oil of C. ericoides was the most active, killing 87 % of trophozoites at the concentration of 0.5 mg/mL. The essential oil of C. pallidulus killed only 29 % of the trophozoites at the same concentration. The essential oil of C. isabelli presented the lowest activity, killing only 4 % of the trophozoites at the concentration of 10 mg/mL. The essential oils of the three species showed cytotoxic effect by the methyl thiazolyl tetrazolium (MTT) method in Vero cells. The oil of C. ericoides, which showed the highest amoebicidal activity, was the most cytotoxic on these mammalian cells.

Amoebicidal activity and chemical composition of Pterocaulon polystachyum (Asteraceae) essential oil.

Acanthamoeba species are free-living amoebae that constitute an etiological agent of Acanthamoeba keratitis, an illness that may cause severe ocular inflammation and blindness and has a very difficult treatment. These molecules that are found in plants may be an alternative for the development of new drugs. Plants of the genus Pterocaulon (Asteraceae) are used in folk medicine as an antiseptic and antifungal agent. In this work, we analyzed Pterocaulon polystachyum essential oil and assessed its amoebicidal activity against Acanthamoeba polyphaga. The leaves of the fresh plant submitted to hydrodistillation yielded 0.15% (w/v) of essential oil that was analyzed by gas chromatography-mass spectrometry being E-sesquilavandulyl acetate as the major component, representing 43.8% of the oil. For the assessment of the amoebicidal activity, concentrations of 20, 10, 5, 2.5, and 1.25 mg/mL of essential oil were tested, being lethal to 100% of the A. polyphaga trophozoites at the concentrations of 10 and 20 mg/mL in 24 and 48 h. The cytotoxic effect of essential oil was also tested in mammalian cells using MTT assay. Amoebicidal activity results are in accordance with previous work in which the lipophilic compounds from this plant were active against Acanthamoeba castellanii. However, further studies with the major component of the essential oil will be carried out.

Lipoteichoic acid from Staphylococcus aureus increases matrix metalloproteinase 9 expression in RAW 264.7 macrophages: modulation by A2A and A2B adenosine receptors.

Peptidoglycan (PEG) and lipoteichoic acid (LTA) are the main constituents of Gram-positive bacteria cell wall and are described to modulate immune functions. Increased levels of matrix metalloproteinases (MMPs) were described in endotoxemia, suggesting that they participate to tecidual damage, multiple organs failure and vascular disfunction. Staphylococcus aureus PEG is described to increase MMPs 2 and 9 levels in plasma from rat and MMP 9 secretion by human neutrophils, however, the effect of LTA on MMPs is unknown. In this work, was evaluated the modulation of MMPs 2 and 9 expression and secretion in RAW 264.7 macrophages by LTA from S. aureus. The role of A2A and A2B adenosine receptors was also investigated. LTA increased MMP 9 expression and secretion at 12h of treatment. The modulation of MMP 9 secretion was dose dependent, with maximal effect above 1microg/ml. The inhibitor of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway (U0126, 10microM) prevented LTA stimulation of MMP 9 secretion; however, the inhibitors of p38 (SB203580, 10microM) and Jun N-terminal kinase (JNK; SP600125, 10microM) presented any effect. A2A and A2B adenosine receptors pharmacological blockade or gene knockdown resulted in exacerbated MMP 9 secretion, while an adenosine receptors agonist inhibited LTA-stimulated MMP 9 secretion. These results suggest that LTA increased MMP 9 secretion in macrophages could be involved in complications associated to S. aureus infections. Moreover, LTA modulation of MMP 9 is dependent on MEK/ERK pathway and is regulated by A2A and A2B adenosine receptors.

High glucose increases RAW 264.7 macrophages activation by lipoteichoic acid from Staphylococcus aureus.

BACKGROUND: Type 2 diabetes mellitus is associated with an increased risk of cardiovascular diseases and accelerated atherosclerosis, which has been associated to hyperglycemia and chronic inflammation. Activated macrophages are described to participate in atherosclerosis due to foam cell formation and pro-inflammatory mediators production. Bacterial infections are described to accelerate atherosclerosis, moreover, gram-positive and negative bacterial DNA was described in atherosclerotic plaques. METHODS: We studied the glucose modulation of RAW 264.7 macrophages activation by the gram-positive bacterial antigen lipoteichoic acid (LTA), evaluating nitrite production, tumor necrosis factor alpha secretion and matrix metalloproteinase 9 activity. RESULTS: High glucose increased macrophages activation by LTA, evidenced by exacerbated nitric oxide and tumor necrosis factor alpha production, as well matrix metalloproteinase 9 secretion. CONCLUSIONS: These effects could contribute to atherosclerotic risk parameters, like atherome plaque instability, and participate in chronic inflammation present in type 2 diabetes.