Double-membrane Vacuoles Research Articles

Torsional Twist of the SARS-CoV and SARS-CoV-2 SUD-N and SUD-M domains.

Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vacuole that houses the replication-transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated, but rather, have significant rotational flexibility. Phylogenetic analysis supports that the disulfide bond cysteines are also absent in pangolin-SARS and closely related viruses, consistent with pangolins being the presumed intermediate host in the emergence of SARS-CoV-2. The absence of these cysteines does not impact viral replication or protein translation.

Pushing boundaries: mechanisms enabling bacterial pathogens to spread between cells.

For multiple intracellular bacterial pathogens, the ability to spread directly into adjacent epithelial cells is an essential step for disease in humans. For pathogens such as Shigella, Listeria, Rickettsia, and Burkholderia, this intercellular movement frequently requires the pathogens to manipulate the host actin cytoskeleton and deform the plasma membrane into structures known as protrusions, which extend into neighboring cells. The protrusion is then typically resolved into a double-membrane vacuole (DMV) from which the pathogen quickly escapes into the cytosol, where additional rounds of intercellular spread occur. Significant progress over the last few years has begun to define the mechanisms by which intracellular bacterial pathogens spread. This review highlights the interactions of bacterial and host factors that drive mechanisms required for intercellular spread with a focus on how protrusion structures form and resolve.

Shenlian extract decreases mitochondrial autophagy to regulate mitochondrial function in microvascular to alleviate coronary artery no-reflow.

Shenlian (SL) extract has been proven to be effective in the prevention and treatment of atherosclerosis and myocardial ischemia. However, the function and molecular mechanisms of SL on coronary artery no-reflow have not been fully elucidated. This study was designed to investigate the contribution of SL extract in repressing excessive mitochondrial autophagy to protect the mitochondrial function and prevent coronary artery no-reflow. The improvement of SL on coronary artery no-reflow was observed in vivo experiments and the molecular mechanisms were further explored through vitro experiments. First, a coronary artery no-reflow rat model was built by ligating the left anterior descending coronary artery for 2hr of ischemia, followed by 24 hr of reperfusion. Thioflavin S (6%, 1ml/kg) was injected into the inferior vena cava to mark the no-reflow area. Transmission electron microscopy was performed to observe the cellular structure, mitochondrial structure, and mitochondrial autophagy of the endothelial cells. Immunofluorescence was used to observe the microvascular barrier function and microvascular inflammation. Cardiac microvascular endothelial cells (CMECs) were isolated from rats. The CMECs were deprived of oxygen-glucose deprivation (OGD) for 2hr and reoxygenated for 4hr to mimic the Myocardial ischemia-reperfusion (MI/R) injury-induced coronary artery no-reflow in vitro. Mitochondrial membrane potential was assessed using JC-1 dye. Intracellular adenosine triphosphate (ATP) levels were determined using an ATP assay kit. The cell total reactive oxygen species (ROS) levels and cell apoptosis rate were analyzed by flow cytometry. Colocalization of mitochondria and lysosomes indirectly indicated mitophagy. The representative ultrastructural morphologies of the autophagosomes and autolysosomes were also observed under transmission electron microscopy. The mitochondrial autophagy-related proteins (LC3II/I, P62, PINK, and Parkin) were analyzed using Western blot analysis. In vivo, results showed that, compared with the model group, SL could reduce the no-reflow area from 37.04 ± 9.67% to 18.31 ± 4.01% (1.08 g·kg-1 SL), 13.79 ± 4.77% (2.16 g·kg-1 SL), and 12.67 ± 2.47% (4.32 g·kg-1 SL). The extract also significantly increased the left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS) (p < 0.05 or p < 0.01). The fluorescence intensities of VE-cadherin, which is a junctional protein that preserves the microvascular barrier function, decreased to ~74.05% of the baseline levels in the no-reflow rats and increased to 89.87%(1.08 g·kg-1 SL), 82.23% (2.16 g·kg-1 SL), and 89.69% (4.32 g·kg-1 SL) of the baseline levels by SL treatment. SL administration repressed the neutrophil migration into the myocardium. The oxygen-glucose deprivation/reoxygenation (OGD/R) model was induced in vitro to mimic microvascular ischemia-reperfusion injury. The impaired mitochondrial function after OGD/R injury led to decreased ATP production, calcium overload, the excessive opening of the Mitochondrial Permeability Transition Pore, decreased mitochondrial membrane potential, and reduced ROS scavenging ability (p < 0.05 or p < 0.01). The normal autophagosomes (double-membrane vacuoles with autophagic content) in the sham group were rarely found. The large morphology and autophagosomes were frequently observed in the model group. By contrast, SL inhibited the excessive activation of mitochondrial autophagy. The mitochondrial autophagy regulated by the PINK/Parkin pathway was excessively activated. However, administration of SL prevented the activation of the PINK/Parkin pathway and inhibited excessive mitochondrial autophagy to regulate mitochondrial dysfunction. Results also demonstrated that mitochondrial dysfunction stimulated endothelial cell barrier dysfunction, but Evans blue transmission was significantly decreased and transmembrane resistance was increased significantly by SL treatment (p < 0.05 or p < 0.01). Carbonylcyanide-3-chlorophenylhydrazone (CCCP) could activate the PINK/Parkin pathway. CCCP reversed the regulation of SL on mitochondrial autophagy and mitochondrial function. SL could alleviate coronary artery no-reflow by protecting the microvasculature by regulating mitochondrial function. The underlying mechanism was related to decreased mitochondrial autophagy by the PINK/Parkin pathway.

Autophagy in the Lifetime of Plants: From Seed to Seed.

Autophagy is a highly conserved self-degradation mechanism in eukaryotes. Excess or harmful intracellular content can be encapsulated by double-membrane autophagic vacuoles and transferred to vacuoles for degradation in plants. Current research shows three types of autophagy in plants, with macroautophagy being the most important autophagic degradation pathway. Until now, more than 40 autophagy-related (ATG) proteins have been identified in plants that are involved in macroautophagy, and these proteins play an important role in plant growth regulation and stress responses. In this review, we mainly introduce the research progress of autophagy in plant vegetative growth (roots and leaves), reproductive growth (pollen), and resistance to biotic (viruses, bacteria, and fungi) and abiotic stresses (nutrients, drought, salt, cold, and heat stress), and we discuss the application direction of plant autophagy in the future.

Preclinical investigation of artesunate as a therapeutic agent for hepatocellular carcinoma via impairment of glucosylceramidase-mediated autophagic degradation

Artesunate (ART) has been indicated as a candidate drug for hepatocellular carcinoma (HCC). Glucosylceramidase (GBA) is required for autophagic degradation. Whether ART regulates autophagic flux by targeting GBA in HCC remains to be defined. Herein, our data demonstrated that the dramatic overexpression of GBA was significantly associated with aggressive progression and short overall survival times in HCC. Subsequent experiments revealed an association between autophagic activity and GBA expression in clinical HCC samples, tumor tissues from a rat model of inflammation-induced HCC and an orthotopic mouse model, and human HCC cell lines. Interestingly, probe labeling identified GBA as an ART target, which was further verified by both a glutathione-S-transferase pulldown assay and surface plasmon resonance analysis. The elevated protein expression of LC3B, the increased numbers of GFP-LC3B puncta and double-membrane vacuoles, and the enhanced expression of SQSTM1/p62 indicated that the degradation of autophagosomes in HCC cells was inhibited by ART treatment. Both the in vitro and in vivo data revealed that autophagosome accumulation through targeting of GBA was responsible for the anti-HCC effects of ART. In summary, this preclinical study identified GBA as one of the direct targets of ART, which may have promising potential to inhibit lysosomal autophagy for HCC therapy.

The type three secretion system effector protein IpgB1 promotes Shigella flexneri cell-to-cell spread through double-membrane vacuole escape.

S. flexneri is an important human pathogen that causes bacillary dysentery. During infection, S. flexneri invades colonic epithelial cells, hijacks the host cell cytoskeleton to move in the cytosol of infected cells, and spreads from cell to cell through formation of membrane protrusions that project into adjacent cells and resolve into double membrane vacuoles (DMVs). S. flexneri cell-to-cell spread requires the integrity of the bacterial type three secretion system (T3SS). However, the exact role of the T3SS effector proteins in the dissemination process remains poorly understood. Here, we investigated the role of the T3SS effector protein IpgB1 in S. flexneri dissemination. IpgB1 was previously characterized as a guanine nucleotide exchange factor (GEF) that contributes to invasion. In addition to the invasion defect, we showed that the ipgB1 mutant formed smaller infection foci in HT-29 cells. Complementation of this phenotype required the GEF activity of IpgB1. Using live confocal microscopy, we showed that the ipgB1 mutant is specifically impaired in DMV escape. Depletion of Rac1, the host cell target of IpgB1 during invasion, as well as pharmacological inhibition of Rac1 signaling, reduced cell-to-cell spread and DMV escape. In a targeted siRNA screen, we uncovered that RhoA depletion restored ipgB1 cell-to-cell spread and DMV escape, revealing a critical role for the IpgB1-Rac1 axis in antagonizing RhoA-mediated restriction of DMV escape. Using an infant rabbit model of shigellosis, we showed that the ipgB1 mutant formed fewer and smaller infection foci in the colon of infected animals, which correlated with attenuated symptoms of disease, including epithelial fenestration and bloody diarrhea. Our results demonstrate that, in addition to its role during invasion, IpgB1 modulates Rho family small GTPase signaling to promote cell-to-cell spread, DMV escape, and S. flexneri pathogenesis.

The type 3 secretion effector IpgD promotes S. flexneri dissemination.

The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery worldwide every year, resulting in more than 200,000 deaths. S. flexneri pathogenic properties rely on its ability to invade epithelial cells and spread from cell to cell within the colonic epithelium. This dissemination process relies on actin-based motility in the cytosol of infected cells and formation of membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the pathogen escapes, thereby achieving cell-to-cell spread. S. flexneri dissemination is facilitated by the type 3 secretion system (T3SS) through poorly understood mechanisms. Here, we show that the T3SS effector IpgD facilitates the resolution of membrane protrusions into DMVs during S. flexneri dissemination. The phosphatidylinositol 4-phosphatase activity of IpgD decreases PtdIns(4,5)P2 levels in membrane protrusions, thereby counteracting de novo cortical actin formation in protrusions, a process that restricts the resolution of protrusions into DMVs. Finally, using an infant rabbit model of shigellosis, we show that IpgD is required for efficient cell-to-cell spread in vivo and contributes to the severity of dysentery.

Presumed SARS-CoV-2 Viral Particles in the Human Retina of Patients With COVID-19

The presence of the SARS-CoV-2 virus in the retina of deceased patients with COVID-19 has been suggested through real-time reverse polymerase chain reaction and immunological methods to detect its main proteins. The eye has shown abnormalities associated with COVID-19 infection, and retinal changes were presumed to be associated with secondary microvascular and immunological changes. To demonstrate the presence of presumed SARS-CoV-2 viral particles and its relevant proteins in the eyes of patients with COVID-19. The retina from enucleated eyes of patients with confirmed COVID-19 infection were submitted to immunofluorescence and transmission electron microscopy processing at a hospital in São Paulo, Brazil, from June 23 to July 2, 2020. After obtaining written consent from the patients' families, enucleation was performed in patients deceased with confirmed SARS-CoV-2 infection. All patients were in the intensive care unit, received mechanical ventilation, and had severe pulmonary involvement by COVID-19. Presence of presumed SARS-CoV-2 viral particles by immunofluorescence and transmission electron microscopy processing. Three patients who died of COVID-19 were analyzed. Two patients were men, and 1 was a woman. The age at death ranged from 69 to 78 years. Presumed S and N COVID-19 proteins were seen by immunofluorescence microscopy within endothelial cells close to the capillary flame and cells of the inner and the outer nuclear layers. At the perinuclear region of these cells, it was possible to observe by transmission electron microscopy double-membrane vacuoles that are consistent with the virus, presumably containing COVID-19 viral particles. The present observations show presumed SARS-CoV-2 viral particles in various layers of the human retina, suggesting that they may be involved in some of the infection's ocular clinical manifestations.

Activation of Autophagy in Models of SF3B1 Mutant MDS

Background:SF3B1 is a key pre-mRNA splicing factor that is abundantly expressed in normal erythroid cells and whose mutation has been linked with high frequency to myelodysplastic syndromes with ringed sideroblasts (MDS with RS) implying that this gene may be an important driver of the pathogenesis of this subtype. Given that MDS with RS is characterized by ineffective erythropoiesis, we previously investigated the link between SF3B1 aberrations and the regulation of pathways that control erythroid maturation and iron homeostasis. Indeed, knockdown of SF3B1 led to significant impairment of the growth of erythroid cells and delayed erythroid differentiation. MDS erythroid cells were unable to properly process iron, resulting in its accumulation in mitochondria. Iron build-up inhibited the proliferation of erythroid progenitors and induced mitochondrial DNA-damage. During erythroid maturation, autophagy plays critical roles in the clearance of damaged mitochondria and maintaining iron homeostasis. Knockout of the essential autophagy gene, Atg7 leads to anemia due to a delayed clearance of mitochondria with a consequent increase in iron levels, ultimately resulting in increased reactive oxygen species formation. As such, we set out to determine if autophagy upregulation alleviates oxidative stress and thereby increases erythropoiesis in MDS with RS.Methods: Transmission electron microscopy (TEM) was performed to examine the cytoplasmic vacuolization, autophagosome content, and morphology of vacuoles and mitochondria in SF3B1 mutant (SF3B1MUT) vs. wild-type (SF3B1WT) cells. Immunoblotting was used to detect microtubule-associated protein 1 light chain 3 (LC3B), p62, and cathepsin D (marker of autophagic degradation). Whole exome sequencing (WES) was used to detect somatic alterations in autophagy genes and RNA-Sequencing and qPCR were conducted to evaluate differences in mRNA expression levels of autophagy genes. A missense mutation in SF3B1 resulting in K700E amino-acid substitution was introduced using EGE-CRISPR/Cas9-stimulated homology-mediated repair to generate heterozygous K562 cells expressing K700E.Transgenic heterozygous SF3B1 mice were used as an in vivo model. Temsirolimus (CCI-779), an mTOR inhibitor with autophagy-inducing properties, was tested in vitro and in vivo.Results: We identified numerous autophagosomes, double-membraned autophagic vacuoles, and single-membrane cytoplasmic structures in SF3B1MUTvs. SF3B1WT cells. LC3B protein was higher in primary SF3B1MUTvs. SF3B1WT cells. WES did not detect any somatic mutations in autophagy genes in the patients analyzed by TEM. RNA-Sequencing and qPCR showed that primary SF3B1MUT MDS with RS cells displayed increased expression levels of key genes of the ATG-autophagy pathway: [ATG2A/B, ATG3, ATG5 (FC=2), ATG9, (FC=5); P=.05], serine/threonine kinases [ULK1 (FC=2), ULK3 (FC=4); P=.05], and cathepsins [CTSB/D (FC=2), CTSE (FC=6); P=.01]. CRISPR/Cas9 K700E cells showed high autophagosome counts, increased basal expression of LC3B protein, and a 2-fold increase in autophagy genes (ATG5, ATG7, ULK1) compared to parental cells. Treatment with temsirolimus (10-100 nM) for 24 hours induced growth inhibition (IC50, 50 nM), enhanced the amount of autophagosomes containing degraded organelles, increased the percentage of dividing CD71+ cells and induced a 2-fold increase in LC3B in CRISPR/Cas9 K700E cells compared to parental cells. Temsirolimus (10 mg/kg i.p. 5d/wk) administered for two weeks to 6-8-month-old transgenic SF3B1 heterozygous mice (N=20) induced autophagy by increasing the number of autophagosomes and autophagolysosomes containing entrapped mitochondria, increased hemoglobin (7.5 vs. 6.6 g/dL; P=.05), and doubled the percentage of CD71+ erythroid cells (5-10%) in temsirolimus-treated vs. untreated SF3B1 mice.Conclusions: Basal autophagy is significantly upregulated in SF3B1MUT cells. Further stimulation of autophagic activity using FDA approved pathway-activating drugs may improve rates of erythrocyte production by SF3B1MUT clones and thus improve control of anemia in MDS with RS cases. DisclosuresAdvani:Takeda/ Millenium: Research Funding; Pfizer: Consultancy. Kelly:Pharmacyclics: Honoraria; Amgen: Honoraria; Abbvie: Honoraria; Jannsen: Honoraria. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Carew:Millenium/ Takeda: Research Funding.

Outer membrane protein a in Acinetobacter baumannii induces autophagy through mTOR signalling pathways in the lung of SD rats

Outer membrane protein A (OmpA) of Acinetobacter baumannii (A. baumannii) is associated with autophagy, which plays an important role in its pathogenicity. However, its exact pathophysiological role in the process of lung tissue cell autophagy remains unclear. In this study, animal and cell infection models were established by wild A. baumannii strain and An OmpA knockout mutant (OmpA-/- A. baumannii) strain. The expression levels of markers autophagy, histological change, cell viability and protein expression levels of inflammatory cytokines were examined. OmpA-/-A. baumannii was successfully constructed. The capacities of bacterial adhesion and invasion to host cells increased more obviously in the AB group and the AB + Rapa group than in the OmpA-/- AB group and AB + CQ group. The AB group and AB + Rapa group could produce double membrane vacuoles, endoplasmic reticulum dilation, mitochondrial ridge rupture, and mitochondrial vacuoles. OmpA could lead to increased LC3, AMPK, and PAMPK protein release, and decreased levels of P62, mTOR and pmTOR proteins in vivo and in vitro. OmpA caused lung pathology and the release of inflammatory cytokines. A. baumannii OmpA promotes autophagy in lung cells through the mTOR signalling pathway, which increases the bacterial colonization ability in the double-layer membrane autophagosome formed by the autophagy reaction to escape the clearance of bacteria by the host, promote the release of inflammatory mediators and aggravate the damage to the host.

Characteristics of in vitro infection of human monocytes, by Rickettsia helvetica

Eighteen species of rickettsiae are reported to cause infections in humans. One of these is Rickettsia helvetica, which is endemic in European and Asian countries and transmitted by the tick Ixodes ricinus. Besides fever, it has been demonstrated to cause meningitis and is also associated with perimyocarditis. One of the initial targets for rickettsiae after inoculation by ticks is the macrophage/monocyte. How rickettsiae remain in the macrophages/monocytes before establishing their infection in vascular endothelial cells remains poorly understood. The main aim of the present study was to investigate the impact on and survival of R. helvetica in a human leukemic monocytic cell line, THP-1. Our results show that R. helvetica survives and propagates in the THP-1 cells. The infection in monocytes was followed for seven days by qPCR and for 30 days by TEM, where invasion of the nucleus was also observed as well as double membrane vacuoles containing rickettsiae, a finding suggesting that R. helvetica might induce autophagy at the early stage of infection. Infected monocytes induced TNF-α which may be important in host defence against rickettsial infections and promote cell survival and inhibiting cell death by apoptosis. The present findings illustrate the importance of monocytes to the pathogenesis of rickettsial disease.

The propagation of HSV-1 in high autophagic activity

Autophagy is an intracellular process involving double-membrane vacuoles that ultimately merge with the lysosomes and play a key role in the inhibition of herpessimplex virus 1 (HSV-1) proliferation. This virus is an agent of some lethal neuronal diseases like encephalitis. HSV-1 requires the expression of its latent proteins, such as ICP34.5, to promote cell infection, which disrupts the autophagy process. In this study, we aimed to evaluate the effect of autophagy induction on HSV-1 replication in host cells at the early and late stages of its replication. Furthermore, we explored the consequences of autophagy induction before and after cell infection.Cells were transfected through Beclin-1-expressing plasmids. Autophagy induction was performed with microtubule-associated protein 1 light chain 3 (LC3-II) as an autophagosome formation marker by using flow cytometry. In the first stage, HSV-1 was inoculated into transfected cells 18 hours post-transfection. Next, viral DNA was extracted 18 and 48 hours post-infection, and eventually viral copies per milliliter were calculated through real-time polymerase chain reaction (PCR). For the second stage, the plasmid containing Beclin-1 was transfected to the cells following virus inoculation to examine the influence of autophagy induction after cell infection.Study results have shown that in neuroblastoma cells autophagy activation reduces virus yield from 4×10 5 copies/ml (control sample) to 9×10 4 copies/ml at 24 h postinfection and viral load after 48 h declines up to 1×10 6 copies/ml, which is less than that of the control sample about 5 logs. However, in HeLa cells, we observed a significant reduction in autophagy induction with reducing HSV-1 propagation. Despite these results, HSV-1 proliferation in both cell types increased and these viruses were able to maintain their ability to propagate even in high autophagic activity. Hyperactivation of autophagy can only slow the rate of virus replication. This study may provide new insight into the effect of autophagy on HSV-1 replication.

Mechanistic and compositional studies of the autophagy-inducing areca nut ingredient

Background/purposeWe previously found that the partially purified 30–100 kDa fraction of areca-nut-extract (ANE 30–100K) induces autophagy in different types of cells including oral carcinoma OECM-1 cells. This study was to analyze the composition and possible mechanisms of ANE 30-100K-induced autophagy (AIA). Materials and methodsPhenol-sulfuric acid method and high performance anion exchange chromatography were utilized to analyze the composition of ANE 30–100K. OECM-1 and esophageal CE81T/VGH cells were taken as the experimental models. Microscope and transmission electron microscope were used to observe morphological changes. Cell viability and specific proteins were respectively measured by XTT and Western bot assay. shRNA and chemical inhibitors were applied to assess the involvement of Atg5, caveolin, and proteasome in AIA. ResultsANE 30–100K contains ∼67% carbohydrate, which is composed of fucose (5.938%), arabinose (24.631%), glucosamine (8.066%), galactose (26.820%), glucose (21.388%), and mannose (13.157%). After ANE 30–100K stimulation, CE81T/VGH cells showed intracellular vacuoles, acidic vesicles, double-membrane vacuoles, and elevated LC3-II level. ANE 30-100K-induced cytotoxicity and LC3-II accumulation were significantly inhibited by Atg5 knockdown. Furthermore, the endocytosis inhibitor (methyl-β-cyclodextrin) and two caveolin shRNAs, as well as two proteasome inhibitors (lactacystin and epoxomicin), were shown to significantly attenuate ANE 30-100K-induced cytotoxicity and LC3-II accumulation in both OECM-1 and CE81T/VGH cells. ConclusionThe major components of ANE 30–100K are carbohydrates. CE81T/VGH also exhibited autophagic responses to ANE 30–100K. Caveolin-mediated endocytosis and proteasome are involved in AIA. This study may have provided new knowledges of the action mechanisms and compositions of ANE 30–100K.

Microautophagy upregulation in cutaneous lymph nodes of dogs naturally infected by Leishmania infantum.

This is the first study showing an in vivo microautophagy upregulation by Leishmania infantum in dogs. Both Leishmania amastigotes and promastigotes were detected in the cytoplasm of many professional and nonprofessional phagocytic cells of popliteal lymph node of three dogs suffering from chronic cutaneous leishmaniasis. Ultrastructurally, parasites appeared to be wrapped by lysosomes and/or multivesicular bodies. Neither phagophores nor double-membraned vacuoles consistent with autophagosomes were observed. Transcription factor EB (TFEB), a key factor involved in lysosome biogenesis, showed a statistically significant increase in the total component when examined by western blot in samples from leishmaniotic dogs compared with samples from healthy dogs. Instead, phosphorylated TFEB showed unmodified expression levels both in leishmaniotic and healthy dogs. Furthermore, Hsc70 and endosomal sorting complex required for transport (ESCRT)-I, which are known to play a role in microautophagy, showed no variation in expression levels both in diseased and healthy animals. Vps4A/B, an evolutionary conserved ATPase responsible for ESCRT-I complex disassembly and MVB maturation, was statistically significantly overexpressed in lymph nodal samples from leishmaniotic dogs. Bag3 was downregulated in diseased dogs whereas CHIP, p62, and LC3-II did not show any variation in expression levels. The altered expression profile of Bag3 suggested an altered interaction of Bag3 with Hsc70 and CHIP, which usually form a molecular complex involved in autophagosome-lysosome pathways. Ultrastructural and molecular findings suggested that the microautophagy pathway is upregulated in lymph nodes of dogs suffering from a chronic natural infection by Leishmania infantum.

Autophagic cell death in viral infection: Do TAM receptors play a role?

Containment and clearance of invading pathogens, such as viruses, by suppression of viral replication through antiviral mechanisms (e.g. CRISPR, interferon response or programmed cell death) provide examples of evolutionary developed responses by hosts to limit the establishment of infection. Degradation of the cytoplasm en masse provides an ideal cellular response against intruding pathogens. Degradation of such scale is achieved by a process called (macro)autophagy, where double membrane vacuoles, autophagosomes, engulf cytoplasm and organelles for lysosomal degradation. However, chronic and unrestrained autophagy poses catastrophic consequences to a cell especially when vital organelles (e.g. mitochondria or nucleus) are engulfed and destroyed. Recent findings in the field of autophagy and cell death regulation describe mechanisms that distinguish whether autophagy takes a moderate or excess route. This review aims to present new perspectives and re-examines current assumptions related to cell death regulation by autophagy. The emerging role of TAM receptors in the modulation of autophagy (i.e. both homeostatic and lethal) in the context of virus infections is also discussed in addition to chemical strategies for studying autophagy.

De Novo Organelle Biogenesis in the Cyanobacterium TDX16 Released from the Green Alga &amp;lt;i&amp;gt;Haematococcus pluvialis&amp;lt;/i&amp;gt;

It is believed that eukaryotes arise from prokaryotes, which means that organelles can form de novo in prokaryotes. Such events, however, had not been observed previously. Here, we report the biogenesis of organelles in the endosymbiotic cyanobacterium TDX16 (prokaryote) that was released from its senescent/necrotic host cell of green alga Haematococcus pluvialis (eukaryote). Microscopic observations showed that organelle biogenesis in TDX16 initiated with cytoplasm compartmentalization, followed by de-compartmentalization, DNA allocation, and re-compartmentalization, as such two composite organelles-the primitive chloroplast and primitive nucleus sequestering minor and major fractions of cellular DNA respectively were formed. Thereafter, the eukaryotic cytoplasmic matrix was built up from the matrix extruded from the primitive nucleus; mitochondria were assembled in and segregated from the primitive chloroplast, whereby the primitive nucleus and primitive chloroplast matured into the nucleus and chloroplast respectively. While mitochondria subsequently turned into double-membraned vacuoles after matrix degradation. Results of pigment analyses, 16S rRNA and genome sequencing revealed that TDX16 is a phycocyanin-containing cyanobacterium resembling Chroococcidiopsis thermalis, which had acquired 9,017,401 bp DNAs with 10,301 genes from its host. Accordingly, we conclude that organelle biogenesis in TDX16 is achieved by hybridizing the acquired eukaryotic DNAs with its own one and expressing the hybrid genome. The formation of organelles in cyanobacterium TDX16 is the first case of organelle biogenesis in prokaryotes observed so far, which sheds an unprecedented light on eukaryotes and their connections with prokaryotes, and thus has broad implications on biology.

Autophagic flux disruption contributes to Ganoderma lucidum polysaccharide-induced apoptosis in human colorectal cancer cells via MAPK/ERK activation

Targeting autophagy may serve as a promising strategy for cancer therapy. Ganoderma lucidum polysaccharide (GLP) has been shown to exert promising anti-cancer effects. However, the underlying mechanisms remain elusive. Whether GLP regulates autophagy in cancer has never been reported. In this study, GLP induced the initiation of autophagy in colorectal cancer (CRC) HT-29 and HCT116 cells, as evidenced by enhanced level of LC3-II protein, GFP-LC3 puncta, and increased formation of double membrane vacuoles. However, GLP treatment caused marked increase of p62 expression. Addition of late stage autophagy inhibitor, chloroquine (CQ), further enhanced LC3-II and p62 level, as well as increased autophagosome accumulation, suggesting a blockage of autophagic flux by GLP in CRC cells. We then found GLP blocked autophagosome and lysosome fusion as determined by mRFP-GFP-LC3 colocalization analysis. Mechanistic study revealed that GLP-induced disruption of autophagosome-lysosome fusion is due to reduced lysosome acidification and lysosomal cathepsin activities. Cell viability and flow cytometry assays revealed that GLP-induced autophagosome accumulation is responsible for GLP-induced apoptosis in CRC cells. In line with this, inhibition of autophagy initiation by 3-methyladenine (3-MA), an early stage autophagy inhibitor, attenuated GLP-induced apoptosis. In contrast, suppression of autophagy at late stage by CQ enhanced the anti-cancer effect of GLP. Furthermore, we demonstrated that GLP-induced autophagosome accumulation and apoptosis is mediated via MAPK/ERK activation. Finally, GLP inhibited tumor growth and also inhibited autophagic flux in vivo. These results unveil new molecular mechanism underlying anti-cancer effects of GLP, suggesting that GLP is a potent autophagy inhibitor and might be useful in anticancer therapy.

Spatial, Temporal, and Functional Assessment of LC3-Dependent Autophagy in Shigella flexneri Dissemination.

Shigella flexneri disseminates within the colonic mucosa by displaying actin-based motility in the cytosol of epithelial cells. Motile bacteria form membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the bacteria escape, thereby achieving cell-to-cell spread. During dissemination, S. flexneri is targeted by LC3-dependent autophagy, a host cell defense mechanism against intracellular pathogens. The S. flexneri type III secretion system effector protein IcsB was initially proposed to counteract the recruitment of the LC3-dependent autophagy machinery to cytosolic bacteria. However, a recent study proposed that LC3 was recruited to bacteria in DMVs formed during cell-to-cell spread. To resolve the controversy and clarify the role of autophagy in S. flexneri infection, we tracked dissemination using live confocal microscopy and determined the spatial and temporal recruitment of LC3 to bacteria. This approach demonstrated that (i) LC3 was exclusively recruited to wild-type or icsB bacteria located in DMVs and (ii) the icsB mutant was defective in cell-to-cell spread due to failure to escape LC3-positive as well as LC3-negative DMVs. Failure of S. flexneri to escape DMVs correlated with late LC3 recruitment, suggesting that LC3 recruitment is the consequence and not the cause of DMV escape failure. Inhibition of autophagy had no positive impact on the spreading of wild-type or icsB mutant bacteria. Our results unambiguously demonstrate that IcsB is required for DMV escape during cell-to-cell spread, regardless of LC3 recruitment, and do not support the previously proposed notion that autophagy counters S. flexneri dissemination.

Tamoxifen overrides autophagy inhibition in Beclin-1-deficient glioma cells and their resistance to adenovirus-mediated oncolysis via upregulation of PUMA and BAX.

Autophagy is an evolutionarily conserved process regulating cellular homeostasis via digestion of dysfunctional proteins and whole cellular organelles by mechanisms, involving their enclosure into double-membrane vacuoles that are subsequently fused to lysosomes. Glioma stem cells utilize autophagy as a main mechanism of cell survival and stress response. Most recently, we and others demonstrated induction of autophagy in gliomas in response to treatment with chemical drugs, such as temozolomide (TMZ) or oncolytic adenoviruses (Ads). As autophagy has been implicated in the mechanism of Ad-mediated cell killing, autophagy deficiency in some glioma tumors could be the reason for their resistance to oncolysis. Despite the observed connection, the exact relationship between autophagy-activating cell signaling and adenoviral infection remains unclear. Here, we report that inhibition of autophagy in target glioma cells induces their resistance to killing by oncolytic agent CRAd-S-5/3. Furthermore, we found that downregulation of autophagy inducer Beclin-1 inhibits replication-competent Ad-induced oncolysis of human glioma by suppressing cell proliferation and inducing premature senescence. To overcome the autophagy-deficient state of such glioma cells and restore their susceptibility to oncolytic Ad infection, we propose treating glioma tumors with an anticancer drug tamoxifen (TAM) as a means to induce apoptosis in Ad-targeted cancer cells via upregulation of BAX/PUMA genes. In agreement with the above hypothesis, our data suggest that TAM improves susceptibility of Beclin-1-deficient glioma cells to CRAd-S-5/3 oncolysis by means of activating autophagy and pro-apoptotic signaling pathways in the target cancer cells.

Autophagic-related cell death of Trypanosoma brucei induced by bacteriocin AS-48

The parasitic protozoan Trypanosoma brucei is the causative agent of human African trypanosomiasis (sleeping sickness) and nagana. Current drug therapies have limited efficacy, high toxicity and/or are continually hampered by the appearance of resistance. Antimicrobial peptides have recently attracted attention as potential parasiticidal compounds. Here, we explore circular bacteriocin AS-48's ability to kill clinically relevant bloodstream forms of T. brucei gambiense, T. brucei rhodesiense and T. brucei brucei. AS-48 exhibited excellent anti-trypanosomal activity in vitro (EC50 = 1–3 nM) against the three T. brucei subspecies, but it was innocuous to human cells at 104-fold higher concentrations. In contrast to its antibacterial action, AS-48 does not kill the parasite through plasma membrane permeabilization but by targeting intracellular compartments. This was evidenced by the fact that vital dye internalization-prohibiting concentrations of AS-48 could kill the parasite at 37 °C but not at 4 °C. Furthermore, AS-48 interacted with the surface of the parasite, at least in part via VSG, its uptake was temperature-dependent and clathrin-depleted cells were less permissive to the action of AS-48. The bacteriocin also caused the appearance of myelin-like structures and double-membrane autophagic vacuoles. These changes in the parasite's ultrastructure were confirmed by fluorescence microscopy as AS-48 induced the production of EGFP-ATG8.2-labeled autophagosomes. Collectively, these results indicate AS-48 kills the parasite through a mechanism involving clathrin-mediated endocytosis of VSG-bound AS-48 and the induction of autophagic-like cell death. As AS-48 has greater in vitro activity than the drugs currently used to treat T. brucei infection and does not present any signs of toxicity in mammalian cells, it could be an attractive lead compound for the treatment of sleeping sickness and nagana.