Unsaturation of serine lipids modulating the interaction of a cytosporone with models of the external leaflet of tumorigenic cell membranes.

Cytosporone-B was isolated from fungi and incorporated in models of tumorigenic cell membranes using palmitoyloleoylglycerophosphoserine (POPS) and dipalmitoyl glycerophosphoserine (DPPS) lipids. While for DPPS, the compound condensed the monolayer and decreased the surface compressional modulus, it expanded and kept the compressional modulus for POPS. Hysteresis for compression-expansion cycles was more sensitive for POPS than for DPPS, while a high degree of destabilization was observed for POPS. As observed with infrared spectroscopy and Brewster angle microscopy, specific changes were selective regarding molecular organization and morphology. Atomic force microscopy for transferred monolayers as Langmuir-Blodgett films also confirmed such specificities. We believe these data can help understand the mechanism of action of bioactive drugs in lipid interfaces at the molecular level.

Host autophagy is exploited by the intracellular parasite Toxoplasma gondii to enhance amino acids levels.

Toxoplasma gondii, a widespread parasite, has the ability to infect nearly any nucleated cell in warm-blooded vertebrates. It is estimated that around 2 billion people globally have been infected by this pathogen. Although most healthy individuals can effectively control parasite replication, certain parasites may evade the immune response, establishing cysts in the brain that are refractory to the immune system and resistance to available drugs. For its chronic persistence in the brain, the parasite relies on host cells' nutrients, particularly amino acids and lipids. Therefore, understanding how latent parasites persist in the brain is crucial for identifying potential drug targets against chronic forms. While shielded within parasitophorous vacuoles (PVs) or cysts, Toxoplasma exploits the host endoplasmic reticulum (ER) metabolism to sustains its persistence in the brain, resulting in host neurological alterations. In this study, we demonstrate that T. gondii disrupts the host ER homeostasis, resulting in accumulation of unfolded protein with the host ER. The host counters this stress by initiating an autophagic pathway known as ER-phagy, which breaks down unfolded proteins into amino acids, promoting their recycling. Remarkably, the persistence of latent forms in cell culture as well as behavioral changes in mice caused by the latent infection could be successfully reversed by restricting the availability of various amino acids during T. gondi infection. Our findings unveil the underlying mechanisms employed by T. gondii to exploit host ER and lysosomal pathways, enhancing nutrient levels during infection. These insights provide new strategies for the treatment of toxoplasmosis. Importance: Intracellular parasites employ several mechanisms to manipulate the cellular environment, enabling them to persist in the host. Toxoplasma gondii , a single-celled parasite, possesses the ability to infect virtually any nucleated cell of warm-blooded vertebrates, including nearly 2 billion people worldwide. Unfortunately, existing treatments and immune responses are not entirely effective in eliminating the chronic persisting forms of the parasite. This study reveals that T. gondii induces the host's autophagic pathway to boost amino acid levels in infected cells. The depletion of amino acids, in turn, influences the persistence of the parasite's chronic forms, resulting in a reduction of neurological alterations caused by chronic infection in mice. Significantly, our investigation establishes the crucial role of host ER-phagy in the parasite's persistence within the host during latent infection.

Coxiella burnetii actively blocks IL-17-induced oxidative stress in macrophages.

Coxiella burnetii is a highly infectious pathogen that causes Q fever, a leading cause of culture-negative endocarditis. Coxiella first targets alveolar macrophages and forms a phagolysosome-like compartment called the Coxiella-Containing Vacuole (CCV). Successful host cell infection requires the Type 4B Secretion System (T4BSS), which translocates bacterial effector proteins across the CCV membrane into the host cytoplasm, where they manipulate numerous cell processes. Our prior transcriptional studies revealed that Coxiella T4BSS blocks IL-17 signaling in macrophages. Given that IL-17 is known to protect against pulmonary pathogens, we hypothesize that C. burnetii T4BSS downregulates intracellular IL-17 signaling to evade the host immune response and promote bacterial pathogenesis. Using a stable IL-17 promoter reporter cell line, we confirmed that Coxiella T4BSS blocks IL-17 transcription activation. Assessment of the phosphorylation state of NF-κB, MAPK, and JNK revealed that Coxiella downregulates IL-17 activation of these proteins. Using ACT1 knockdown and IL-17RA or TRAF6 knockout cells, we next determined that IL17RA-ACT1-TRAF6 pathway is essential for the IL-17 bactericidal effect in macrophages. In addition, macrophages stimulated with IL-17 generate higher levels of reactive oxygen species, which is likely connected to the bactericidal effect of IL-17. However, C. burnetii T4SS effector proteins block the IL-17-mediated oxidative stress, suggesting that Coxiella blocks IL-17 signaling to avoid direct killing by the macrophages.

Coxiella burnetii Sterol-Modifying Protein Stmp1 Regulates Cholesterol in the Intracellular Niche.

Coxiella burnetii replicates in a phagolysosome-like vacuole called the Coxiella-containing vacuole (CCV). While host cholesterol readily traffics to the CCV, cholesterol accumulation leads to CCV acidification and bacterial death. Thus, bacterial regulation of CCV cholesterol content is essential for Coxiella pathogenesis. Coxiella expresses a sterol-modifying protein, Stmp1, that may function to lower CCV cholesterol through enzymatic modification. Using an Stmp1 knockout (Δstmp1), we determined that Stmp1 is not essential for axenic growth. Inside host cells, however, Δstmp1 mutant bacteria form smaller CCVs which accumulate cholesterol, preferentially fuse with lysosomes, and become more acidic, correlating with a significant growth defect. However, in cholesterol-free cells, Δstmp1 mutant bacteria grow similarly to wild-type bacteria but are hypersensitive to cholesterol supplementation. To better understand the underlying mechanism behind the Δstmp1 mutant phenotype, we performed sterol profiling. Surprisingly, we found that Δstmp1 mutant-infected macrophages accumulated the potent cholesterol homeostasis regulator 25-hydroxycholesterol (25-HC). We next determined whether dysregulated 25-HC alters Coxiella infection by treating wild-type Coxiella-infected cells with 25-HC. Similar to the Δstmp1 mutant phenotype, 25-HC increased CCV proteolytic activity and inhibited bacterial growth. Collectively, these data indicate that Stmp1 alters host cholesterol metabolism and is essential to establish a mature CCV which supports Coxiella growth. IMPORTANCE Coxiella burnetii is the causative agent of human Q fever, an emerging infectious disease and significant cause of culture-negative endocarditis. Acute infections are often undiagnosed, there are no licensed vaccines in the United States, and chronic Q fever requires a prolonged antibiotic treatment. Therefore, new treatment and preventive options are critically needed. Coxiella is an obligate intracellular bacterium that replicates within a large acidic phagolysosome-like compartment, the Coxiella-containing vacuole (CCV). We previously discovered that cholesterol accumulation in the CCV increases its acidification, leading to bacterial death. Therefore, in order to survive in this harsh environment, Coxiella likely regulates CCV cholesterol levels. Here, we found that Coxiella sterol modifying protein (Stmp1) facilitates bacterial growth by reducing CCV cholesterol and host cell 25-hydroxycholesterol (25-HC) levels, which prevents excessive CCV fusion with host lysosomes and CCV acidification. This study establishes that Stmp1-mediated regulation of host cholesterol homeostasis is essential for Coxiella intracellular survival.

TgIF2K-B Is an eIF2α Kinase in Toxoplasma gondii That Responds to Oxidative Stress and Optimizes Pathogenicity.

Toxoplasma gondii is an obligate intracellular parasite that persists in its vertebrate hosts in the form of dormant tissue cysts, which facilitate transmission through predation. The parasite must strike a balance that allows it to disseminate throughout its host without killing it, which requires the ability to properly counter host cell defenses. For example, oxidative stress encountered by Toxoplasma is suggested to impair parasite replication and dissemination. However, the strategies by which Toxoplasma mitigates oxidative stress are not yet clear. Among eukaryotes, environmental stresses induce the integrated stress response via phosphorylation of a translation initiation factor, eukaryotic initiation factor 2 (eIF2). Here, we show that the Toxoplasma eIF2 kinase TgIF2K-B is activated in response to oxidative stress and affords protection. Knockout of the TgIF2K-B gene, Δtgif2k-b, disrupted parasite responses to oxidative stresses and enhanced replication, diminishing the ability of the parasite to differentiate into tissue cysts. In addition, parasites lacking TgIF2K-B exhibited resistance to activated macrophages and showed greater virulence in an in vivo model of infection. Our results establish that TgIF2K-B is essential for Toxoplasma responses to oxidative stress, which are important for the parasite's ability to establish persistent infection in its host.IMPORTANCEToxoplasma gondii is a single-celled parasite that infects nucleated cells of warm-blooded vertebrates, including one-third of the human population. The parasites are not cleared by the immune response and persist in the host by converting into a latent tissue cyst form. Development of tissue cysts can be triggered by cellular stresses, which activate a family of TgIF2 kinases to phosphorylate the eukaryotic translation initiation factor TgIF2α. Here, we establish that the TgIF2 kinase TgIF2K-B is activated by oxidative stress and is critical for maintaining oxidative balance in the parasite. Depletion of TgIF2K-B alters gene expression, leading to accelerated growth and a diminished ability to convert into tissue cysts. This study establishes that TgIF2K-B is essential for the parasite's oxidative stress response and its ability to persist in the host as a latent infection.

Host sensing and signal transduction during Toxoplasma stage conversion.

The intracellular parasite Toxoplasma gondii infects nucleated cells in virtually all warm-blooded vertebrates, including one-third of the human population. While immunocompetent hosts do not typically show symptoms of acute infection, parasites are retained in latent tissue cysts that can be reactivated upon immune suppression, potentially damaging key organ systems. Toxoplasma has a multistage life cycle that is intimately linked to environmental stresses and host signals. As this protozoan pathogen is transmitted between multiple hosts and tissues, it evaluates these external signals to appropriately differentiate into distinct life cycle stages, such as the transition from its replicative stage (tachyzoite) to the latent stage (bradyzoite) that persists as tissue cysts. Additionally, in the gut of its definitive host, felines, Toxoplasma converts into gametocytes that produce infectious oocysts (sporozoites) that are expelled into the environment. In this review, we highlight recent advances that have illuminated the interfaces between Toxoplasma and host and how these interactions control parasite stage conversion. Mechanisms underlying these stage transitions are important targets for therapeutic intervention aimed at thwarting parasite transmission and pathogenesis.

Toxoplasma gondii Co-opts the Unfolded Protein Response To Enhance Migration and Dissemination of Infected Host Cells.

Toxoplasma gondii is an intracellular parasite that reconfigures its host cell to promote pathogenesis. One consequence of Toxoplasma parasitism is increased migratory activity of host cells, which facilitates dissemination. Here, we show that Toxoplasma triggers the unfolded protein response (UPR) in host cells through calcium release from the endoplasmic reticulum (ER). We further identify a novel role for the host ER stress sensor protein IRE1 in Toxoplasma pathogenesis. Upon infection, Toxoplasma activates IRE1, engaging its noncanonical role in actin remodeling through the binding of filamin A. By inducing cytoskeletal remodeling via IRE1 oligomerization in host cells, Toxoplasma enhances host cell migration in vitro and dissemination of the parasite to host organs in vivo Our study has identified novel mechanisms used by Toxoplasma to induce dissemination of infected cells, providing new insights into strategies for treatment of toxoplasmosis.IMPORTANCE Cells that are infected with the parasite Toxoplasma gondii exhibit heightened migratory activity, which facilitates dissemination of the infection throughout the body. In this report, we identify a new mechanism used by Toxoplasma to hijack its host cell and increase its mobility. We further show that the ability of Toxoplasma to increase host cell migration involves not the enzymatic activity of IRE1 but rather IRE1 engagement with actin cytoskeletal remodeling. Depletion of IRE1 from infected host cells reduces their migration in vitro and significantly hinders dissemination of Toxoplasma in vivo Our findings reveal a new mechanism underlying host-pathogen interactions, demonstrating how host cells are co-opted to spread a persistent infection around the body.

Regulation of arginine transport by GCN2 eIF2 kinase is important for replication of the intracellular parasite Toxoplasma gondii.

Toxoplasma gondii is a prevalent protozoan parasite that can infect any nucleated cell but cannot replicate outside of its host cell. Toxoplasma is auxotrophic for several nutrients including arginine, tryptophan, and purines, which it must acquire from its host cell. The demands of parasite replication rapidly deplete the host cell of these essential nutrients, yet Toxoplasma successfully manages to proliferate until it lyses the host cell. In eukaryotic cells, nutrient starvation can induce the integrated stress response (ISR) through phosphorylation of an essential translation factor eIF2. Phosphorylation of eIF2 lowers global protein synthesis coincident with preferential translation of gene transcripts involved in stress adaptation, such as that encoding the transcription factor ATF4 (CREB2), which activates genes that modulate amino acid metabolism and uptake. Here, we discovered that the ISR is induced in host cells infected with Toxoplasma. Our results show that as Toxoplasma depletes host cell arginine, the host cell phosphorylates eIF2 via protein kinase GCN2 (EIF2AK4), leading to induced ATF4. Increased ATF4 then enhances expression of the cationic amino acid transporter CAT1 (SLC7A1), resulting in increased uptake of arginine in Toxoplasma-infected cells. Deletion of host GCN2, or its downstream effectors ATF4 and CAT1, lowers arginine levels in the host, impairing proliferation of the parasite. Our findings establish that Toxoplasma usurps the host cell ISR to help secure nutrients that it needs for parasite replication.

Guanabenz Reverses a Key Behavioral Change Caused by Latent Toxoplasmosis in Mice by Reducing Neuroinflammation.

Toxoplasma gondii is an intracellular parasite that has infected one-third of humans. The infection is permanent because the replicative form (tachyzoite) converts into a latent tissue cyst form (bradyzoite) that evades host immunity and is impervious to current drugs. The continued presence of these parasitic cysts hinders treatment and leads to chronic infection that has been linked to behavioral changes in rodents and neurological disease in humans. How these behavioral changes occur, and whether they are due to parasite manipulation or the host response to infection, remains an outstanding question. We previously showed that guanabenz possesses antiparasitic activity; here, we show that guanabenz reproducibly lowers brain cyst burden up to 80% in chronically infected male and female BALB/cJ mice when given intraperitoneally but not when administered by gavage or in food. Regardless of the administration route, guanabenz reverses Toxoplasma-induced hyperactivity in latently infected mice. In contrast, guanabenz increases cyst burden when given to chronically infected C57BL/6J mice yet still reverses Toxoplasma-induced hyperactivity. Examination of the brains from chronically infected BALB/cJ and C57BL/6J mice shows that guanabenz decreases inflammation and perivascular cuffing in each strain. Our study establishes a robust model for cyst reduction in BALB/cJ mice and shows for the first time that it is possible to reverse a key behavioral change associated with latent toxoplasmosis. The rescue from parasite-induced hyperactivity correlates with a decrease in neuroinflammation rather than reduced cyst counts, suggesting that some behavioral changes arise from host responses to infection.IMPORTANCEToxoplasma gondii is a common parasite of animals, including up to one-third of humans. The single-celled parasite persists within hosts for the duration of their life as tissue cysts, giving rise to chronic infection. Latent toxoplasmosis is correlated with neurological dysfunction in humans and results in dramatic behavioral changes in rodents. When infected, mice and rats adapt behaviors that make them more likely to be devoured by cats, the only host that supports the sexual stage of the parasite. In this study, we establish a new mouse model of tissue cyst depletion using a drug called guanabenz and show that it is possible to reverse a key behavior change back to normal in infected animals. We also show that the mechanism appears to have nothing to do with parasite cyst burden but rather the degree of neuroinflammation produced by chronic infection.

Effects of PERK eIF2α Kinase Inhibitor against Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that has infected one-third of the population. Upon infection of warm-blooded vertebrates, the replicating form of the parasite (tachyzoite) converts into a latent form (bradyzoite) present in tissue cysts. During immune deficiency, bradyzoites can reconvert into tachyzoites and cause life-threatening toxoplasmosis. We previously reported that translational control through phosphorylation of the α subunit of T. gondii eukaryotic initiation factor 2 (eIF2α) (TgIF2α) is a critical component of the parasite stress response. Diverse stresses can induce the conversion of tachyzoites to bradyzoites, including those disrupting the parasite's endoplasmic reticulum (ER) (ER stress). Toxoplasma possesses four eIF2α kinases, one of which (TgIF2K-A) localizes to the parasite ER analogously to protein kinase R-like endoplasmic reticulum kinase (PERK), the eIF2α kinase that responds to ER stress in mammalian cells. Here, we investigated the effects of a PERK inhibitor (PERKi) on Toxoplasma Our results show that the PERKi GSK2606414 blocks the enzymatic activity of TgIF2K-A and reduces TgIF2α phosphorylation specifically in response to ER stress. PERKi also significantly impeded multiple steps of the tachyzoite lytic cycle and sharply lowered the frequency of bradyzoite differentiation in vitro Pretreatment of host cells with PERKi prior to infection did not affect parasite infectivity, and PERKi still impaired parasite replication in host cells lacking PERK. In mice, PERKi conferred modest protection from a lethal dose of Toxoplasma Our findings represent the first pharmacological evidence supporting TgIF2K-A as an attractive new target for the treatment of toxoplasmosis.

Translational Control in the Latency of Apicomplexan Parasites.

Apicomplexan parasites Toxoplasma gondii and Plasmodium spp. use latent stages to persist in the host, facilitate transmission, and thwart treatment of infected patients. Therefore, it is important to understand the processes driving parasite differentiation to and from quiescent stages. Here, we discuss how a family of protein kinases that phosphorylate the eukaryotic initiation factor-2 (eIF2) function in translational control and drive differentiation. This translational control culminates in reprogramming of the transcriptome to facilitate parasite transition towards latency. We also discuss how eIF2 phosphorylation contributes to the maintenance of latency and provides a crucial role in the timing of reactivation of latent parasites towards proliferative stages.

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria.

BACKGROUND: Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. METHODS: Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). RESULTS: The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 µg/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 µg/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. CONCLUSIONS: The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.

Vascular function in asthmatic children and adolescents.

BACKGROUND: Epidemiological studies have demonstrated an increased incidence of cardiovascular events in patients with bronchial asthma, but little is known about the relationship between asthma and vascular function. The purpose of this study was to evaluate endothelial function and arterial stiffness in children and adolescents with asthma. METHODS: A cross-sectional controlled study was designed. Measurements of endothelial function and arterial stiffness in asthmatic (13.6 ± 0.6 years) and control groups (14.9 ± 0.7 years) were taken by the non-invasive peripheral arterial tonometry (EndoPAT2000) determined by using the natural logarithm of the reactive hyperemia index (LnRHI) and the augmentation index (AIx@75%), respectively. Patients with asthma were also administered two questionnaires to evaluate asthma control and quality of life. Exercise functional capacity was evaluated using the Shuttle Walking Test (SWT). Only male participants were included in the present study. RESULTS: LnRHI and the walked distance during the SWT were similar between groups (p = 0.23 and p = 0.50, respectively). AIx@75% was significantly higher in the asthmatic group (-7.75 ± 1.7) compared to the control group (-15.25 ± 1.8), p < 0.04. In the control group, the LnRHI correlated positively with baseline systolic blood pressure (r = 0.53, p = 0.02) and mean arterial pressure (r = 0.50, p = 0.03), age (r = 0.61, p = 0.007), weight (r = 0.63, p = 0.004) and height (r = 0.56, p = 0.015). Besides that LnRHI correlated with FVC (r = 0.69, p = 0.002), FEV1, (r = 0.53, p = 0.03) and negatively with Tiffeneau index (FEV1/FVC%, r = -0.49 p = 0.04). The LnRHI of the asthmatic group did not correlate with the different variables evaluated. CONCLUSION: The increased AIx@75% without changes in LnRHI in asthmatic patients could mean that an early detection of vascular impairment may precede endothelial dysfunction, and that different mechanisms may contribute to the pathogenesis and progression of cardiovascular events in this population. A large prospective and randomized controlled study should be done to evaluate the physiopathological mechanisms underlying the association between arterial stiffness and asthma.

Ergosterol isolated from the basidiomycete pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

Background: Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. Methods: Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). Results: The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 mu g/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 mu g/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. Conclusions: The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

Abstract Background Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. Methods Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). Results The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 g/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 g/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. Conclusions The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

Background Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. Methods Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). Results The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 μg/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 μg/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. Conclusions The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.(AU)

Expression of non-acetylatable lysines 10 and 14 of histone H4 impairs transcription and replication in Trypanosoma cruzi.

The histone H4 from Trypanosomatids diverged from other eukaryotes in the N-terminus, a region that undergoes post-translation modifications involved in the control of gene expression, DNA replication, and chromatin assembly. Nonetheless, the N-terminus of Trypanosoma cruzi histone H4 is mainly acetylated at lysine 4. The lysines 10 and 14 are also acetylated, although at less extent, increasing during the S-phase or after DNA damage, which suggests a regulatory function. Here, we investigated the roles of these acetylations by expressing non-acetylated forms of histone H4 in T. cruzi. We found that histone H4 containing arginines at positions 10 or 14, to prevent acetylation were transported to the nucleus and inserted into the chromatin. However, their presence, even at low levels, interfered with DNA replication and transcription, causing a significant growth arrest of the cells. The absence of acetylation also increased the amount of soluble endogenous histones H3 and H4 and affected the interaction with Asf1, a histone chaperone. Therefore, acetylation of lysines 10 and 14 of the histone H4 in trypanosomes could be required for chromatin assembly and/or remodeling required for transcription and replication.

Ikaros could be a key factor in the maintenance of "B-side" of B-1 cells.

Ikaros, a zinc finger transcription factor, is an important regulator of the hematopoietic system. Several studies have suggested the role of Ikaros in the development, maturation, activation and differentiation of lymphocytes. To elucidate this mechanism, it is important to understand how this transcription factor works in the dichotomy of the hematopoietic system, a topic that remains uncertain. Herein, we investigated the role of Ikaros in the control of the lymphomyeloid phenotype of B-1 lymphocytes. We found that Ikaros, as well as its target genes, are expressed in B-1 cells,. Moreover, Ikaros positively regulates the expression of Flt3, Gfi and Il7r, while it down-regulates PU.1. During the induction of differentiation of B-1 cells toward phagocytes, Ikaros transcription was reduced. Taken together, these data pointed to the relevance of Ikaros in the maintenance of the promiscuous gene profile of B-1 cells. It could be suggested that Ikaros functions as a guardian of B-1 lymphoid pattern, and that its absence directs the differentiation of B-1 cells into phagocytes.

Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease.

Acetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD(+)-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation of Trypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion by T. cruzi at concentrations that did not affect host cell viability. In addition, in vivo infection was partially controlled upon administration of salermide. There are two sirtuins in T. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed in Escherichia coli with a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 µM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

Comparison between whole-body vibration, light-emitting diode, and cycling warm-up on high-intensity physical performance during sprint bicycle exercise.

The purpose of this study was to compare the effects of light-emitting diode (LED) irradiation and whole-body vibration (WBV) delivered either in isolation or combination (LED + WBV), warm-up (WU), and a control (C) treatment on performance during a sprint bicycle exercise. Ten cyclists performed a 30-second sprint cycle test under these conditions. The LED light was applied at 4 points bilaterally. Whole-body vibration consisted of 5 minutes of squats associated with WBV. LED + WBV consisted of WBV followed by LED therapy. Warm-up consisted of 17 minutes of moderate-intensity bicycle exercise. Control consisted of 10 minutes at rest. Blood lactate (BL) and ammonia (BA) levels and skin temperature (ST) were determined. Peak power (842 ± 117 vs. 800 ± 106 vs. 809 ± 128 W [p = 0.02 and p = 0.01]), relative power (12.1 ± 1.0 vs. 11.5 ± 0.9 vs. 11.6 ± 1.0 W·kg [p = 0.02 and p = 0.02]), and relative work (277 ± 23 vs. 263 ± 24 vs. 260 ± 23 J·kg [p = 0.02 and p = 0.003]) were higher in the WU group compared with the control and LED groups. In the LED + WBV group, peak (833 ± 115 vs. 800 ± 106 W [p = 0.02]) and relative (11.9 ± 0.9 vs. 11.5 ± 0.9 W·kg [p = 0.02]) power were higher than those in the control group, and relative work (272 ± 22 vs. 260 ± 23 J·kg [p = 0.02]) were improved compared with the LED group. There were no differences for BL, BA, and ST. The findings of this study confirmed the effectiveness of a warm-up as a preparatory activity and demonstrated that LED + WBV and WBV were as effective as WU in improving cyclist performance during a sprint bicycle exercise.