A polyphenol, pyrogallol changes the acidic pH of the digestive vacuole of Plasmodium falciparum

Pyrogallol has a capability of generating free radicals like other antimalarial drugs such as artemisinin, which is thought to inhibit the proton pump located in the membrane of the Plasmodium falciparum digestive vacuole, thus alkalinising this acidic organelle. This study aimed to determine pH changes of the malaria parasite’s digestive vacuole following treatment with pyrogallol. The antimalarial activity of this compound was evaluated by a malarial SYBR Green 1 fluorescence-based assay to determine the 50% inhibitory concentration (IC50). Based on the IC50 value, different concentrations of pyrogallol were selected to ensure changes of the digestive vacuole pH were not due to parasite death. This was measured by flow cytometry after 4-hour pyrogallol treatment on the fluorescein isothiocyanate-dextran-accumulated digestive vacuole of the mid-trophozoite stage parasites. Pyrogallol showed a moderate antimalarial activity with the IC50 of 2.84 ± 9.40 µM. The treatment of 1.42, 2.84 and 5.67 µM pyrogallol increased 2.9, 3.0 and 3.1 units of the digestive vacuole pH, respectively as compared with the untreated parasite (pH 5.6 ± 0.78). The proton pump, V-type H+-ATPase might be inhibited by pyrogallol, hence causing the digestive vacuole pH alteration, which is similar with the result shown by a standard V-type H+-ATPase inhibitor, concanamycin A. This study provides a fundamental understanding on the antimalarial activity and mechanism of action of pyrogallol that has a potential to be the antimalarial drug candidate.

Split luciferase-based assay to detect botulinum neurotoxins using human motor neurons derived from induced pluripotent stem cell.

Botulinum neurotoxins (BoNTs) have been widely used clinically as a muscle relaxant. These toxins target motor neurons and cleave proteins essential for neurotransmitter release like Synaptosomal-associated protein of 25 kDa (SNAP-25). Most in vitro assays for BoNT testing use rodent cells or immortalized cell lines, which showed limitations in accuracy and physiological relevance. Here, we report a cell-based assay for detecting SNAP25-cleaving BoNTs by combining human induced Pluripotent Stem Cells (hiPSC)-derived motor neurons and a luminescent detection system based on split nanoluc luciferase. This assay is convenient, rapid, free-of-specialized antibodies, and can discriminate the potency of different BoNTs, with a detection sensitivity of femtomolar concentrations of toxin and can be used to study the different steps of BoNT intoxication. Abreviations: BoNT, Botulinum neurotoxin, SNAP-25, Synaptosomal-associated protein of 25 kDa, hiPSC, human induced Pluripotent Stem Cells, SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor, SV2, synaptic vesicle proteins, MLB, mouse lethality bioassay, LD50, toxin’s dose lethal for half of the animal injected, CB-assay, cell-based assays, FRET, Förster resonance energy transfer, Concanamycin A, EC50, Half maximal effective concentration, MNs, motor neurons.

Lipidome Remodeling and Autophagic Respose in the Arachidonic-Acid-Rich Microalga Lobosphaera incisa Under Nitrogen and Phosphorous Deprivation

The green microalga Lobosphaera incisa accumulates triacylglycerols (TAGs) with exceptionally high levels of long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid (ARA) under nitrogen (N) deprivation. Phosphorous (P) deprivation induces milder changes in fatty acid composition, cell ultrastructure, and growth performance. We hypothesized that the resource-demanding biosynthesis and sequestration of ARA-rich TAG in lipid droplets (LDs) are associated with the enhancement of catabolic processes, including membrane lipid turnover and autophagic activity. Although this work focuses mainly on N deprivation, a comparative analysis of N and P deprivation responses is included. The results of lipidomic profiling showed a differential impact of N and P deprivation on the reorganization of glycerolipids. The formation of TAG under N deprivation was associated with the enhanced breakdown of chloroplast glycerolipids and the formation of lyso-lipids. N-deprived cells displayed a profound reorganization of cell ultrastructure, including internalization of cellular material into autophagic vacuoles, concomitant with the formation of LDs, while P-deprived cells showed better cellular ultrastructural integrity. The expression of the hallmark autophagy protein ATG8 and the major lipid droplet protein (MLDP) genes were coordinately upregulated, but to different extents under either N or P deprivation. The expression of the Δ5-desaturase gene, involved in the final step of ARA biosynthesis, was coordinated with ATG8 and MLDP, exclusively under N deprivation. Concanamycin A, the inhibitor of vacuolar proteolysis and autophagic flux, suppressed growth and enhanced levels of ATG8 and TAG in N-replete cells. The proportions of ARA in TAG decreased with a concomitant increase in oleic acid under both N-replete and N-deprived conditions. The photosynthetic apparatus’s recovery from N deprivation was impaired in the presence of the inhibitor, along with the delayed LD degradation. The GFP-ATG8 processing assay showed the release of free GFP in N-replete and N-deprived cells, supporting the existence of autophagic flux. This study provides the first insight into the homeostatic role of autophagy in L. incisa and points to a possible metabolic link between autophagy and ARA-rich TAG biosynthesis.

Concanamycin A counteracts HIV-1 Nef to enhance immune clearance of infected primary cells by cytotoxic T lymphocytes

Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.

Lysosomal Activation Is Required for Priming of Quiescent Hematopoietic Stem Cells

Despite their immense in vivo repopulating capacity, hematopoietic stem cells (HSCs) are largely quiescent at the steady-state. However, mechanisms that regulate HSC quiescence/cycling remain incompletely understood. Using mitochondrial membrane potential (MMP) to dissect the heterogeneity of HSCs (LSKCD150+CD48-), we find that HSCs within 25% lowest MMP (MMP-low) fractions are almost entirely (~95% ±2.65) in G0 as measured by Pyronin Y/Hoechst staining (p<0.05, n=3). In contrast, HSCs within 25% highest MMP (MMP-high HSCs) are in majority in cycling (see abstract 129099). To elucidate mechanisms implicated in the regulation of HSC cycling at the single cell level in quiescent MMP-low versus primed MMP-high HSCs we used single-cell RNA-Seq (scRNA-Seq) analysis. Cycling analysis in silico in each cell by CYCLONE further confirmed that over 80% of MMP-low HSCs are within G0/G1, as compared to less than 40% of MMP-high HSCs that are mostly in the S/G2/M phase. Notably, GO enrichment analysis related to protein degradation through lysosomal- and proteasomal-mediated pathways were significantly enriched in MMP-low HSCs (p=0.002). Strikingly, and in agreement with our scRNA-seq analysis, a greater abundance of lysosomes was observed in MMP-low relative to -high HSCs (p=0.002). Higher expression of lysosomal genes was further confirmed by qRT-PCR in MMP-low relative to -high HSCs. Analysis of lysosomal content by immunofluorescence staining showed that while the lysosomal specific marker LAMP2 was barely detectable in MMP-high HSCs, LAMP2 was readily found in MMP-low HSCs, results further confirmed by additional markers LAMP1 and LysoTracker Green. Lysosomes are, among others, a major component of organelle degradation through autophagy, which is required for the maintenance of HSCs however, whether lysosomes are implicated in regulating HSC beyond autophagy is unknown. To address this we examined the effect of the suppression (and not activation that is required for autophagy) of lysosomal activation on in vitro HSC maintenance. Treatment with concanamycin-A (ConA), a specific inhibitor of lysosomal acidification via inhibition of the vacuolar H+ -adenosine triphosphatase ATPase (v-ATPase) led to 3 fold improved frequency of phenotypically defined HSCs from optimally cultured lineage-negative cells in 24 hours (p<0.05, n=4). This was associated with 4-fold greater retention of the MMP-low HSC fraction (p<0.05, n=4). Cell divisions of single MMP-low and -high GFP+ HSCs treated with ConA or vehicle control was tracked up to 60 hours in culture. Over 70% of control treated MMP-low GFP+ HSCs did not divide during this time, whereas the majority (>85%) of MMP-high GFP+ HSCs divided at least once (p=0.001, n=5). While ConA treatment had only a slight effect on non-dividing MMP-low HSCs in culture, it significantly increased the frequency of non-dividing MMP-high GFP+ HSCs (p=0.007). Priming of MMP-low to -high HSCs was associated with lysosomal recruitment, and activation of mTOR signaling in MMP-high HSCs (p=0.001, n=5). Importantly, ConA-treatment led to the repression of mTOR expression and activity in MMP-high HSCs (p<0.001). In addition, a 48-hours ConA treatment led to enhanced frequency of LTC-ICs recovered in limiting dilution analysis of both MMP-low (p=0.023) and -high (p=0.004) HSCs ex vivo. To further investigate the role of suppression of lysosomal activation in vivo, FACS-purified MMP-low and -high HSCs were treated with vehicle control or ConA ex vivo for 4 days before 50 ConA- or control-treated MMP-low or -high HSCs were mixed with CD45.2 (2x105) competitor cells and injected into lethally irradiated mice (n=7) in a competitive repopulation assay. Reconstitution levels were consistently more robust in ConA-treated populations of MMP-low (p= 0.001) and -high (p=0.001) HSCs after 18 weeks as compared to control. Importantly, HSC-derived lineage output was balanced in its composition up to 18 weeks in recipients of MMP-low HSC regardless of ConA treatment as well as in ConA-treated MMP-high HSCs, while control MMP-high HSC was myeloid-biased. Overall our results, based on HSC mitochondrial heterogeneity, suggest that lysosomal -content and activity participate in the maintenance of HSC quiescence. Based on these findings, we propose a model that stipulates that lysosomal activation primes HSCs (G0⇒G1) while lysosomal suppression maintains HSC quiescence. Disclosures Ghaffari: Rubius Therapeutics: Consultancy.

Vacuolar processing enzyme translocates to the vacuole through the autophagy pathway to induce programmed cell death

The caspase-like vacuolar processing enzyme (VPE) is a key factor in programmed cell death (PCD) associated with plant stress responses. Growth medium lacking a carbon source and dark conditions caused punctate labeling of 35S::VPE1-GFP (StVPE1-GFP) in potato leaves. Carbon starvation of BY-2 cells induced higher VPE activity and PCD symptoms. Growing VPE-RNAi BY-2 cells without sucrose reduced VPE activity and prevented PCD symptoms. During extended exposure to carbon starvation, VPE expression and activity levels peaked, with a gradual increase in BY-2 cell death. Histological analysis of StVPE1-GFP in BY-2 cells showed that carbon starvation induces its translocation from the endoplasmic reticulum to the central vacuole, through tonoplast engulfment. Exposure of BY-2 culture to the autophagy inhibitor concanamycin A caused autophagic bodies accumulation in the cell vacuole. Such accumulation did not occur in the presence of 3-methyladenine, an inhibitor of early-stage autophagy. BY-2 cells constitutively expressing StATG8IL-RFP, an autophagosome marker, showed colocalization with the StVPE1-GFP protein in the cytoplasm and vacuole. RNAi silencing of the core autophagy component ATG4 in BY-2 cells reduced VPE activity and cell death. These results are the first to suggest that VPE translocates to the cell vacuole through the autophagy pathway, leading to PCD.One sentence summaryCarbon starvation induced programmed cell death by trafficking vacuolar processing enzyme through the autophagy pathway to the vacuole.

Down-regulation of microRNA-138 improves immunologic function via negatively targeting p53 by regulating liver macrophage in mice with acute liver failure

MicroRNAs (miRNAs) have been frequently identified as key mediators in almost all developmental and pathological processes, including those in the liver. The present study was conducted with aims of investigating the role of microRNA-138 (miR-138) in acute liver failure (ALF) via a mechanism involving p53 and liver macrophage in a mouse model. The ALF mouse model was established using C57BL/6 male mice via tail vein injection of Concanamycin A (Con A) solution. The relationship between miR-138 and p53 was tested. The mononuclear macrophages were infected with mimic and inhibitor of miR-138 in order to identify roles of miR-138 in p53 and levels of inflammatory factors. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot analysis and ELISA were conducted in order to determine the levels of miR-138, inflammatory factors, and p53 during ALF. The results showed an increase in the levels of miR-138 and inflammatory factors in ALF mice induced by the ConA as time progressed and reached the peak at 12 h following treatment with ConA, while it was on the contrary when it came to the level of p53. Dual-luciferase reporter gene assay revealed that p53 was a target gene of miR-138. Furthermore, the results from the in vitro transfection experiments in primary macrophages of ALF mouse showed that miR-138 down-regulated p53 and enhanced levels of inflammatory factors; thus, improving immune function in ALF mice. In conclusion, by negatively targeting p53, the decreased miR-138 improves immunologic function by regulating liver macrophage in mouse models of ALF.

Intramelanocytic Acidification Plays a Role in the Antimelanogenic and Antioxidative Properties of Vitamin C and Its Derivatives

Although vitamin C (VC, L-ascorbic acid) has been widely used as a skin lightening agent for a long time, the mechanism by which it inhibits melanogenesis remains poorly understood. It is well-documented that the intramelanocytic pH is an important factor in regulating tyrosinase function and melanosome maturation. The activity of tyrosinase, the rate-limiting enzyme required for melanin synthesis, is generally minimal in an acidic environment. Given that VC is an acidic compound, we might speculate that the intracellular acidification of melanocytes induced by VC likely reduces melanin content through the suppression of tyrosinase activity. The results of this study reveal that treatment of melanocytes with VC or its derivatives, magnesium ascorbyl phosphate (MAP) and 3-O-ethyl-L-ascorbic acid (AAE), resulted in significant decreases in the tyrosinase activity and melanin content and in the levels of intracellular reactive oxygen species (ROS), indicating that VC and its derivatives possess antimelanogenic and antioxidative activities. Western blotting analysis indicated that VC, MAP, and AAE exert their antimelanogenic activity by inhibiting the tyrosinase activity rather than by downregulating the expression of melanogenic proteins such as tyrosinase, premelanosome protein 17 (Pmel17) and microphthalmia-associated transcription factor (MITF). Further, we found that the reduced tyrosinase activity of melanocytes treated with VC or its derivatives could be reactivated following intracellular neutralization induced by ammonium chloride (NH4Cl) or concanamycin A (Con A). Finally, we examined the expression of sodium-dependent VC transporter-2 (SVCT-2) using western blotting and qPCR, which revealed that there was a significant increase in the expression of SVCT-2 in melanocytes following treatment with VC. VC-mediated intracellular acidification was neutralized by phloretin (a putative SVCT-2 inhibitor) in a dose-dependent manner. Taken together, these data show that VC and its derivatives suppress tyrosinase activity through cytoplasmic acidification that potentially results from enhanced VC transmembrane transport via the VC transporter SVCT-2.

Niclosamide Triggers Non-Canonical LC3 Lipidation

Autophagy is a highly- evolutionarily-conserved catabolic pathway activated by various cellular stresses. Recently, non-canonical autophagy (NCA), which does not require all of the ATG proteins to form autophagosome or autophagosome-like structures, has been found in various conditions. Moreover, mounting evidence has indicated that non-canonical LC3 lipidation (NCLL) may reflect NCA. We and others have reported that niclosamide (Nic), an anti-helminthic drug approved by the Food and Drug Administration, could induce canonical autophagy via a feedback downregulation of mTOR complex 1. In this study, we found that Nic could also induce NCLL, which is independent of the ULK1 complex and Beclin 1 complex, but dependent on ubiquitin-like conjugation systems. Although bafilomycin A1 and concanamycin A, two known V-ATPase inhibitors, significantly inhibited Nic-induced NCLL, Nic-induced NCLL was demonstrated to be independent of V-ATPase. In addition, the Golgi complex and vimentin were involved in Nic-induced NCLL, which might be a platform or membrane source for Nic-induced LC3-positive structures. These results would be helpful to broaden our understanding of the working mechanisms of Nic and evaluate its pharmacological activities in diseases.

Inhibition of Amebic Lysosomal Acidification Blocks Amebic Trogocytosis and Cell Killing

ABSTRACTEntamoeba histolyticaingests fragments of live host cells in a nibbling-like process termed amebic trogocytosis. Amebic trogocytosis is required for cell killing and contributes to tissue invasion, which is a hallmark of invasive amebic colitis. Work done prior to the discovery of amebic trogocytosis showed that acid vesicles are required for amebic cytotoxicity. In the present study, we show that acidified lysosomes are required for amebic trogocytosis and cell killing. Interference with lysosome acidification using ammonium chloride, a weak base, or concanamycin A, a vacuolar H+ATPase inhibitor, decreased amebic trogocytosis and amebic cytotoxicity. Our data suggest that the inhibitors do not impair the ingestion of an initial fragment but rather block continued trogocytosis and the ingestion of multiple fragments. The acidification inhibitors also decreased phagocytosis, but not fluid-phase endocytosis. These data suggest that amebic lysosomes play a crucial role in amebic trogocytosis, phagocytosis, and cell killing.IMPORTANCEE. histolyticais a protozoan parasite that is prevalent in low-income countries, where it causes potentially fatal diarrhea, dysentery, and liver abscesses. Tissue destruction is a hallmark of invasiveE. histolyticainfection. The parasite is highly cytotoxic to a wide range of human cells, and parasite cytotoxic activity is likely to drive tissue destruction.E. histolyticais able to kill human cells through amebic trogocytosis. This process also contributes to tissue invasion. Trogocytosis has been observed in other organisms; however, little is known about the mechanism in any system. We show that interference with lysosomal acidification impairs amebic trogocytosis, phagocytosis, and cell killing, indicating that amebic lysosomes are critically important for these processes.