Francisella tularensis Harvests Nutrients Derived via ATG5-Independent Autophagy to Support Intracellular Growth

Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.     

COST1 balances plant growth and stress tolerance via attenuation of autophagy

ABSTRACT

In plants, macroautophagy/autophagy has been reported to function in various biotic and abiotic stress-response pathways, but few direct regulators linking stress and autophagy have yet been identified. Other than the conserved nutrient sensing kinase TOR (Target of Rapamycin), negative regulators that can directly modulate plant autophagy are unknown. We recently identified a mutant, termed cost1 (Constitutively Stressed 1), which has strong drought tolerance with constitutive induction of autophagy and broad expression of normally stress-responsive genes. The COST1 protein negatively regulates autophagy by direct interaction with the key autophagy adaptor ATG8E, thus directly linking autophagy and drought tolerance. Moreover, plant growth and development in a cost1 mutant is greatly retarded, suggesting that COST1 controls the tradeoff between growth and stress tolerance.     

Multifunction of autophagy-related genes in filamentous fungi

Autophagy (macroautophagy), a highly conserved eukaryotic mechanism, is a non-selective degradation process, helping to maintain a balance between the synthesis, degradation and subsequent recycling of macromolecules to overcome various stress conditions. The term autophagy denotes any cellular process which involves the delivery of cytoplasmic material to the lysosome for degradation. Autophagy, in filamentous fungi plays a critical role during cellular development and pathogenicity. Autophagy, like the mitogen-activated protein (MAP) kinase cascade and nutrient-sensing cyclic AMP (cAMP) pathway, is also an important process for appressorium turgor accumulation in order to penetrate the leaf surface of host plant and destroy the plant defense. Yeast, an autophagy model, has been used to compare the multi-valued functions of ATG (autophagy-related genes) in different filamentous fungi. The autophagy machinery in both yeast and filamentous fungi is controlled by Tor kinase and both contain two distinct phosphatidylinositol 3-kinase complexes. In this review, we focus on the functions of ATG genes during pathogenic development in filamentous fungi.     

自噬在病原真菌生殖中的作用

自噬是真核生物中重要且高度保守的蛋白降解过程。在此过程中,细胞中的细胞器、长寿蛋白及其他大分子物质被双层膜的自噬体包裹并运送至降解细胞器中进行降解并重新利用。自噬在病原真菌诸如细胞分化、营养动态平衡以及致病性等各种细胞过程中起重要作用。在本综述中,我们简要介绍了自噬过程,并以人体病原真菌新生隐球菌为例介绍了病原真菌的有性生殖过程;同时我们也总结了目前模式病原真菌中自噬相关基因的研究情况以及自噬调控病原真菌无性和有性生殖的可能机理;最后我们总结全文并讨论了未来自噬调控真菌有性生殖机理研究的工作方向。     

Autophagosomes and human diseases

The autophagosome is a double-membrane bound compartment that initiates macroautophagy, a degradative pathway for cytoplasmic material terminating in the lysosomal compartment. The discovery of ATG genes involved in the formation of autophagosomes has greatly increased our understanding of the molecular basis of macroautophagy, and its role in cell function. Macroautophagy plays a pivotal role in cell fitness by removing obsolete organelles and protein aggregates. Its stimulation is an adaptive response to stressful situations, such as nutrient deprivation, intended to maintain a level of ATP compatible with cell survival. Macroautophagy is central for organ homeostasis, embryonic development, and longevity. Malfunctioning autophagy is observed in many human diseases including cancer, neurodegenerative diseases, cardiac and muscular diseases, infectious and inflammatory diseases, diabetes, and obesity. Discovering potential drug therapies that can be used to modulate macroautophagy is a major challenge, and likely to enhance the therapeutic arsenal against many human diseases.     

Autophagy regulates programmed cell death during the plant innate immune response

The plant innate immune response includes the hypersensitive response (HR), a form of programmed cell death (PCD). PCD must be restricted to infection sites to prevent the HR from playing a pathologic rather than protective role. Here we show that plant BECLIN 1, an ortholog of the yeast and mammalian autophagy gene ATG6/VPS30/beclin 1, functions to restrict HR PCD to infection sites. Initiation of HR PCD is normal in BECLIN 1-deficient plants, but remarkably, healthy uninfected tissue adjacent to HR lesions and leaves distal to the inoculated leaf undergo unrestricted PCD. In the HR PCD response, autophagy is induced in both pathogen-infected cells and distal uninfected cells; this is reduced in BECLIN 1-deficient plants. The restriction of HR PCD also requires orthologs of other autophagy-related genes including PI3K/VPS34, ATG3, and ATG7. Thus, the evolutionarily conserved autophagy pathway plays an essential role in plant innate immunity and negatively regulates PCD.     

Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy

Microtubules, the major components of cytoskeleton, are involved in various fundamental biological processes in plants. Recent studies in mammalian cells have revealed the importance of microtubule cytoskeleton in autophagy. However, little is known about the roles of microtubules in plant autophagy. Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana. Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy. Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts. Our findings reveal that an intact microtubule network is important for efficient macroautophagy and leaf starch degradation.     

Autophagy Is Involved in the Reduction of Myelinating Schwann Cell Cytoplasm during Myelin Maturation of the Peripheral Nerve

Peripheral nerve myelination involves dynamic changes in Schwann cell morphology and membrane structure. Recent studies have demonstrated that autophagy regulates organelle biogenesis and plasma membrane dynamics. In the present study, we investigated the role of autophagy in the development and differentiation of myelinating Schwann cells during sciatic nerve myelination. Electron microscopy and biochemical assays have shown that Schwann cells remove excess cytoplasmic organelles during myelination through macroautophagy. Inhibition of autophagy via Schwann cell-specific removal of ATG7, an essential molecule for macroautophagy, using a conditional knockout strategy, resulted in abnormally enlarged abaxonal cytoplasm in myelinating Schwann cells that contained a large number of ribosomes and an atypically expanded endoplasmic reticulum. Small fiber hypermyelination and minor anomalous peripheral nerve functions are observed in this mutant. Rapamycin-induced suppression of mTOR activity during the early postnatal period enhanced not only autophagy but also developmental reduction of myelinating Schwann cells cytoplasm in vivo. Together, our findings suggest that autophagy is a regulatory mechanism of Schwann cells structural plasticity during myelination.     

Autophagy Negatively Regulates Cell Death by Controlling NPR1-Dependent Salicylic Acid Signaling during Senescence and the Innate Immune Response in Arabidopsis

Autophagy is an evolutionarily conserved intracellular process for vacuolar degradation of cytoplasmic components. In higher plants, autophagy defects result in early senescence and excessive immunity-related programmed cell death (PCD) irrespective of nutrient conditions; however, the mechanisms by which cells die in the absence of autophagy have been unclear. Here, we demonstrate a conserved requirement for salicylic acid (SA) signaling for these phenomena in autophagy-defective mutants (atg mutants). The atg mutant phenotypes of accelerated PCD in senescence and immunity are SA signaling dependent but do not require intact jasmonic acid or ethylene signaling pathways. Application of an SA agonist induces the senescence/cell death phenotype in SA-deficient atg mutants but not in atg npr1 plants, suggesting that the cell death phenotypes in the atg mutants are dependent on the SA signal transducer NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. We also show that autophagy is induced by the SA agonist. These findings imply that plant autophagy operates a novel negative feedback loop modulating SA signaling to negatively regulate senescence and immunity-related PCD.     

Glucose-starved Cells Do Not Engage in Prosurvival Autophagy

In response to nutrient shortage or organelle damage, cells undergo macroautophagy. Starvation of glucose, an essential nutrient, is thought to promote autophagy in mammalian cells. We thus aimed to determine the role of autophagy in cell death induced by glucose deprivation. Glucose withdrawal induces cell death that can occur by apoptosis (in Bax, Bak-deficient mouse embryonic fibroblasts or HeLa cells) or by necrosis (in Rh4 rhabdomyosarcoma cells). Inhibition of autophagy by chemical or genetic means by using 3-methyladenine, chloroquine, a dominant negative form of ATG4B or silencing Beclin-1, Atg7, or p62 indicated that macroautophagy does not protect cells undergoing necrosis or apoptosis upon glucose deprivation. Moreover, glucose deprivation did not induce autophagic flux in any of the four cell lines analyzed, even though mTOR was inhibited. Indeed, glucose deprivation inhibited basal autophagic flux. In contrast, the glycolytic inhibitor 2-deoxyglucose induced prosurvival autophagy. Further analyses indicated that in the absence of glucose, autophagic flux induced by other stimuli is inhibited. These data suggest that the role of autophagy in response to nutrient starvation should be reconsidered.     

Dicot-specific ATG8-interacting ATI3 proteins interact with conserved UBAC2 proteins and play critical roles in plant stress responses

Selective macroautophagy/autophagy targets specific cargo by autophagy receptors through interaction with ATG8 (autophagy-related protein 8)/MAP1LC3 (microtubule associated protein 1 light chain 3) for degradation in the vacuole. Here, we report the identification and characterization of 3 related ATG8-interacting proteins (AT1G17780/ATI3A, AT2G16575/ATI3B and AT1G73130/ATI3C) from Arabidopsis. ATI3 proteins contain a WxxL LC3-interacting region (LIR) motif at the C terminus required for interaction with ATG8. ATI3 homologs are found only in dicots but not in other organisms including monocots. Disruption of ATI3A does not alter plant growth or development but compromises both plant heat tolerance and resistance to the necrotrophic fungal pathogen Botrytis cinerea. The critical role of ATI3A in plant stress tolerance and disease resistance is dependent on its interaction with ATG8. Disruption of ATI3B and ATI3C also significantly compromises plant heat tolerance. ATI3A interacts with AT3G56740/UBAC2A and AT2G41160/UBAC2B (Ubiquitin-associated [UBA] protein 2a/b), 2 conserved proteins implicated in endoplasmic reticulum (ER)-associated degradation. Disruption of UBAC2A and UBAC2B also compromised heat tolerance and resistance to B. cinerea. Overexpression of UBAC2 induces formation of ATG8- and ATI3-labeled punctate structures under normal conditions, likely reflecting increased formation of phagophores or autophagosomes. The ati3 and ubac2 mutants are significantly compromised in sensitivity to tunicamycin, an ER stress-inducing agent, but are fully competent in autophagy-dependent ER degradation under conditions of ER stress when using an ER lumenal marker for detection. We propose that ATI3 and UBAC2 play an important role in plant stress responses by mediating selective autophagy of specific unknown ER components.     

Autophagy in Drosophila ovaries is induced by starvation and is required for oogenesis

Autophagy, an evolutionarily conserved lysosome-mediated degradation, promotes cell survival under starvation and is controlled by insulin/target of rapamycin (TOR) signaling. In Drosophila, nutrient depletion induces autophagy in the fat body. Interestingly, nutrient availability and insulin/TOR signaling also influence the size and structure of Drosophila ovaries, however, the role of nutrient signaling and autophagy during this process remains to be elucidated. Here, we show that starvation induces autophagy in germline cells (GCs) and in follicle cells (FCs) in Drosophila ovaries. This process is mediated by the ATG machinery and involves the upregulation of Atg genes. We further demonstrate that insulin/TOR signaling controls autophagy in FCs and GCs. The analysis of chimeric females reveals that autophagy in FCs, but not in GCs, is required for egg development. Strikingly, when animals lack Atg gene function in both cell types, ovaries develop normally, suggesting that the incompatibility between autophagy-competent GCs and autophagy-deficient FCs leads to defective egg development. As egg morphogenesis depends on a tightly linked signaling between FCs and GCs, we propose a model in which autophagy is required for the communication between these two cell types. Our data establish an important function for autophagy during oogenesis and contributes to the understanding of the role of autophagy in animal development.     

Genetic aberrations in macroautophagy genes leading to diseases

The catabolic process of macroautophagy, through the rapid degradation of unwanted cellular components, is involved in a multitude of cellular and organismal functions that are essential to maintain homeostasis. Those functions include adaptation to starvation, cell development and differentiation, innate and adaptive immunity, tumor suppression, autophagic cell death, and maintenance of stem cell stemness. Not surprisingly, an impairment or block of macroautophagy can lead to severe pathologies. A still increasing number of reports, in particular, have revealed that mutations in the autophagy-related (ATG) genes, encoding the key players of macroautophagy, are either the cause or represent a risk factor for the development of several illnesses. The aim of this review is to provide a comprehensive overview of the diseases and disorders currently known that are or could be caused by mutations in core ATG proteins but also in the so-called autophagy receptors, which provide specificity to the process of macroautophagy. Our compendium underlines the medical relevance of this pathway and underscores the importance of the eventual development of therapeutic approaches aimed at modulating macroautophagy.     

Macroautophagy occurs in distal TMV-uninfected root tip tissue of tomato taking place systemic PCD

Autophagy is an important mechanism for recycling cell materials upon encountering stress conditions. Our previous studies had shown that TMV infection could lead to systemic PCD in the distal uninfected tissues, including root tip and shoot tip tissues. But it is not clear whether there is autophagy in the distal apical meristem of TMV-induced plants. To better understand the autophagy process during systemic PCD, here we investigated the formation and type of autophagy in the root meristem cells occurring PCD. Transmission electron microscopy assay revealed that the autophagic structures formed by the fusion of vesicles, containing the sequestered cytoplasm, multilamellar bodies, and degraded mitochondria. In the PCD progress, many mitochondria appeared degradation with blurred inner membrane structure. And the endoplasmic reticulum was broke into small fragments. Finally, the damaged mitochodria were engulfed and degraded by the autophagosomes. These results indicated that during the systemic PCD process of root tip cells, the classical macroautophagy occurred, and the cell contents and damaged organelles (mitochondria) would be self-digested by autophagy.     

Nutrient deprivation induces autophagy as well as apoptosis in Chinese hamster ovary cell culture

During Chinese hamster ovary (CHO) cell culture for foreign protein production, cells are subjected to programmed cell death (PCD). A rapid death at the end of batch culture is accelerated by nutrient starvation. In this study, type II PCD, autophagy, as well as type I PCD, apoptosis, was found to take place in two antibody-producing CHO cell lines, Ab1 and Ab2, toward the end of batch culture when glucose and glutamine were limiting. The evidence of autophagy was observed from the accumulation of a common autophagic marker, a 16 kDa form of LC3-II during batch culture. Moreover, a significant percentage of the total cells (80% of Ab1 cells and 86% of Ab2 cells) showed autophagic vacuoles containing cytoplasmic material by transmission electron microscopy. An increased level of PARP cleavage and chromosomal DNA fragmentation supported that starvation-induced apoptosis also occurred simultaneously with autophagy.     

Autophagosome formation is initiated at phosphatidylinositol synthase‐enriched ER subdomains

The autophagosome, a double‐membrane structure mediating degradation of cytoplasmic materials by macroautophagy, is formed in close proximity to the endoplasmic reticulum (ER). However, how the ER membrane is involved in autophagy initiation and to which membrane structures the autophagy‐initiation complex is localized have not been fully characterized. Here, we were able to biochemically analyze autophagic intermediate membranes and show that the autophagy‐initiation complex containing ULK and FIP200 first associates with the ER membrane. To further characterize the ER subdomain, we screened phospholipid biosynthetic enzymes and found that the autophagy‐initiation complex localizes to phosphatidylinositol synthase (PIS)‐enriched ER subdomains. Then, the initiation complex translocates to the ATG9A‐positive autophagosome precursors in a PI3P‐dependent manner. Depletion of phosphatidylinositol (PI) by targeting bacterial PI‐specific phospholipase C to the PIS domain impairs recruitment of downstream autophagy factors and autophagosome formation. These findings suggest that the autophagy‐initiation complex, the PIS‐enriched ER subdomain, and ATG9A vesicles together initiate autophagosome formation.     

Molecular characterization of proprotein convertase subtilisin/kexin type 9-mediated degradation of the LDLR

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that promotes degradation of cell surface LDL receptors (LDLRs) in selected cell types. Here we used genetic and pharmacological inhibitors to define the pathways involved in PCSK9-mediated LDLR degradation. Inactivating mutations in autosomal recessive hypercholesterolemia (ARH), an endocytic adaptor, blocked PCSK9-mediated LDLR degradation in lymphocytes but not in fibroblasts. Thus, ARH is not specifically required for PCSK9-mediated LDLR degradation. Knockdown of clathrin heavy chain with siRNAs prevented LDLR degradation. In contrast, prevention of ubiquitination of the LDLR cytoplasmic tail, inhibition of proteasomal activity, or disruption of proteins required for lysosomal targeting via macroautophagy (autophagy related 5 and 7) or the endosomal sorting complex required for trafficking (ESCRT) pathway (hepatocyte growth factor-regulated Tyr-kinase substrate and tumor suppressor gene 101) failed to block PCSK9-mediated LDLR degradation. These findings are consistent with a model in which the LDLR-PCSK9 complex is internalized via clathrin-mediated endocytosis and then routed to lysosomes via a mechanism that does not require ubiquitination and is distinct from the autophagy and proteosomal degradation pathways. Finally, the PCSK9-LDLR complex appears not to be transported by the canonical ESCRT pathway.     

Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. Thus far, plant autophagy has been studied primarily using morphological analyses. A recent genome-wide search revealed significant conservation among autophagy genes (ATGs) in yeast and plants. It has not been proved, however, that Arabidopsis thaliana ATG genes are required for plant autophagy. To evaluate this requirement, we examined the ubiquitination-like Atg8 lipidation system, whose component genes are all found in the Arabidopsis genome. In Arabidopsis, all nine ATG8 genes and two ATG4 genes were expressed ubiquitously and were induced further by nitrogen starvation. To establish a system monitoring autophagy in whole plants, we generated transgenic Arabidopsis expressing each green fluorescent protein-ATG8 fusion (GFP-ATG8). In wild-type plants, GFP-ATG8s were observed as ring shapes in the cytoplasm and were delivered to vacuolar lumens under nitrogen-starved conditions. By contrast, in a T-DNA insertion double mutant of the ATG4s (atg4a4b-1), autophagosomes were not observed, and the GFP-ATG8s were not delivered to the vacuole under nitrogen-starved conditions. In addition, we detected autophagic bodies in the vacuoles of wild-type roots but not in those of atg4a4b-1 in the presence of concanamycin A, a V-ATPase inhibitor. Biochemical analyses also provided evidence that autophagy in higher plants requires ATG proteins. The phenotypic analysis of atg4a4b-1 indicated that plant autophagy contributes to the development of a root system under conditions of nutrient limitation.     

EDS1-mediated activation of autophagy regulates <Emphasis Type="Italic">Pst</Emphasis> DC3000 (<Emphasis Type="Italic">AvrRps4</Emphasis>)-induced programmed cell death in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Autophagy is a conserved intracellular process through which cytoplasmic components are degraded and recycled under stress conditions. In the innate immunity of higher plants, autophagy has either pro-survival or pro-death functions in pathogen-induced programmed cell death (PCD). In aged leaves, autophagy negatively regulates PCD by eliminating redundant salicylic acid. However, in young leaves, the specific pro-death mechanisms of autophagy and signaling pathways related to the autophagic process have not been elucidated. Here, we demonstrate that enhanced disease susceptibility 1 (EDS1) mediated the activation of autophagy and played a key role in the pro-death mechanism of autophagy during avirulent Pst DC3000 (AvrRps4) infection. The path through which autophagosomes enter the vacuole was blocked. Additionally, formation of the ATG12–ATG5 complex and the level of enzymatic activity associated with ATG8 cleavage decreased in eds1 mutants. The expression of EDS1 in atg5 mutants was also much lower than that in wild-type plants during pathogen-triggered PCD. These findings implied that EDS1 may regulate autophagy by affecting the activities of the two ubiquitin-like protein-conjugating pathways. Moreover, autophagy may regulate immunity-related PCD by affecting the expression of EDS1 in young plants. Our results provide important insights into the mechanisms of EDS1 in autophagy during infection with avirulent Pst DC3000 (AvrRps4) in Arabidopsis.