Bacterial toxins can inhibit host cell autophagy through cAMP generation

Autophagy plays a significant role in innate and adaptive immune responses to microbial infection. Some pathogenic bacteria have developed strategies to evade killing by host autophagy. These include the use of ‘camouflage’ proteins to block targeting to the autophagy pathway and the use of pore-forming toxins to block autophagosome maturation. However, general inhibition of host autophagy by bacterial pathogens has not been observed to date. Here we demonstrate that bacterial cAMP-elevating toxins from B. anthracis and V. cholera can inhibit host anti-microbial autophagy, including autophagic targeting of S. Typhimurium and latex bead phagosomes. Autophagy inhibition required the cAMP effector protein kinase A. Formation of autophagosomes in response to rapamycin and the endogenous turnover of peroxisomes was also inhibited by cAMP-elevating toxins. These findings demonstrate that cAMP-elevating toxins, representing a large group of bacterial virulence factors, can inhibit host autophagy to suppress immune responses and modulate host cell physiology.     

Interactions of pathogenic bacteria with autophagy systems

Autophagy is a conserved cellular degradative pathway that is now established to be a vital part of the host immune response to microbial infection. Autophagy can directly eliminate intracellular pathogens by mediating their delivery to lysosomes. Canonical autophagy is characterized by the formation of a double-membrane autophagosome and the involvement of over 35 autophagy-related proteins (Atgs), including a commonly used autophagosome marker in mammalian cells, LC3. Recent studies have shown that a subset of autophagy components can lead to LC3 conjugation onto phagosomes. This process of LC3-associated phagocytosis (LAP) results in the degradation of the cargo by promoting phagosome fusion with lysosomes. Other components of the autophagy machinery also play roles in immunity that are distinct from the canonical autophagy and LAP pathways. This minireview highlights the complicated relationship between autophagy components and intracellular bacteria, including bacterial targeting mechanisms and the interaction between autophagy and effectors/toxins secreted by bacteria.     

Mechanisms and consequences of bacterial targeting by the autophagy pathway

Autophagy is a key component of our immune response to invading pathogens. Autophagic targeting of intracellular bacteria within vacuolar compartments or the cytosol helps to control bacterial replication in the host cell. The mechanism by which these invading pathogens are selectively targeted for degradation is of particular interest. Recently, several signaling factors have been shown to play roles in the specific targeting of bacteria by the autophagy pathway including: pattern recognition receptors, reactive oxygen species, ubiquitin and diacylglycerol. Here, we discuss these signaling factors and the consequences of bacterial targeting by autophagy during infection of host cells.     

细菌与自噬的抗争——死亡或重生

自噬是一种高度保守的细胞内成分的降解过程,不仅维持细胞的代谢稳定,还与机体对抗各种病原菌感染有着密切关系。自噬能协助机体清除病原体,但有些细菌进化出多种策略干扰自噬信号通路或抑制自噬体与溶酶体融合形成自噬溶酶体来逃避自噬的降解,甚至利用自噬来促进其生长增殖。文中从自噬的分子机制出发,讨论多种致病菌与宿主细胞自噬关系的最新进展,以及自噬与病原菌感染的作用和意义,以期为病原菌感染导致的自噬研究提供参考。     

Induction of autophagy via innate bacterial recognition

Macroautophagy (referred to hereafter as autophagy) functions not only in self-digestion, but also in the killing and degradation of infectious pathogens in in vitro-cultured cells. Based on genetic manipulations of both the host, Drosophila and pathogen, Listeria monocytogenes, we recently reported that L. monocytogenes-induced autophagy is dependent on the recognition of the pathogen by the Drosophila pattern recognition protein, PGRP-LE. Autophagy and PGRP-LE are crucial for inhibition of the intracellular growth of bacteria in hemocytes, the target cells of L. monocytogenes infection in vivo. The importance of autophagy in the resistance of Drosophila against L. monocytogenes is further indicated in in vivo survival experiments. The signaling pathway(s) that induces autophagy by PGRP-LE is independent of the known immune signaling pathways, suggesting that another unidentified pathway(s) is involved. The results of the present study demonstrate that the induction of autophagy, as an innate immune response targeting intracellular pathogens, is activated by intracellular sensors through unidentified pathways.     

Ferritinophagy drives uropathogenic Escherichia coli persistence in bladder epithelial cells

Autophagy is a cellular recycling pathway, which in many cases, protects host cells from infections by degrading pathogens. However, uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections (UTIs), persist within the urinary tract epithelium (urothelium) by forming reservoirs within autophagosomes. Iron is a critical nutrient for both host and pathogen, and regulation of iron availability is a key host defense against pathogens. Iron homeostasis depends on the shuttling of iron-bound ferritin to the lysosome for recycling, a process termed ferritinophagy (a form of selective autophagy). Here, we demonstrate for the first time that UPEC shuttles with ferritin-bound iron into the autophagosomal and lysosomal compartments within the urothelium. Iron overload in urothelial cells induces ferritinophagy in an NCOA4-dependent manner causing increased iron availability for UPEC, triggering bacterial overproliferation and host cell death. Addition of even moderate levels of iron is sufficient to increase and prolong bacterial burden. Furthermore, we show that lysosomal damage due to iron overload is the specific mechanism causing host cell death. Significantly, we demonstrate that host cell death and bacterial burden can be reversed by inhibition of autophagy or inhibition of iron-regulatory proteins, or chelation of iron. Together, our findings suggest that UPEC persist in host cells by taking advantage of ferritinophagy. Thus, modulation of iron levels in the bladder may provide a therapeutic avenue to controlling UPEC persistence, epithelial cell death, and recurrent UTIs.     

Pharmacological Inhibition of ULK1 Kinase Blocks Mammalian Target of Rapamycin (mTOR)-dependent Autophagy

Autophagy is a cell-protective and degradative process that recycles damaged and long-lived cellular components. Cancer cells are thought to take advantage of autophagy to help them to cope with the stress of tumorigenesis; thus targeting autophagy is an attractive therapeutic approach. However, there are currently no specific inhibitors of autophagy. ULK1, a serine/threonine protein kinase, is essential for the initial stages of autophagy, and here we report that two compounds, MRT67307 and MRT68921, potently inhibit ULK1 and ULK2 in vitro and block autophagy in cells. Using a drug-resistant ULK1 mutant, we show that the autophagy-inhibiting capacity of the compounds is specifically through ULK1. ULK1 inhibition results in accumulation of stalled early autophagosomal structures, indicating a role for ULK1 in the maturation of autophagosomes as well as initiation.     

RhoGTPases and inflammasomes: Guardians of effector-triggered immunity

Pathogens have evolved smart strategies to invade hosts and hijack their immune responses. One such strategy is the targeting of the host RhoGTPases by toxins or virulence factors to hijack the cytoskeleton dynamic and immune processes. In response to this microbial attack, the host has evolved an elegant strategy to monitor the function of virulence factors and toxins by sensing the abnormal activity of RhoGTPases. This innate immune strategy of sensing bacterial effector targeting RhoGTPase appears to be a bona fide example of effector-triggered immunity (ETI). Here, we review recently discovered mechanisms by which the host can sense the activity of these toxins through NOD and NOD-like receptors (NLRs).     

Autophagy Enhances Bacterial Clearance during P. aeruginosa Lung Infection

Pseudomonas aeruginosa is an opportunistic bacterial pathogen which is the leading cause of morbidity and mortality among cystic fibrosis patients. Although P. aeruginosa is primarily considered an extacellular pathogen, recent reports have demonstrated that throughout the course of infection the bacterium acquires the ability to enter and reside within host cells. Normally intracellular pathogens are cleared through a process called autophagy which sequesters and degrades portions of the cytosol, including invading bacteria. However the role of autophagy in host defense against P. aeruginosa in vivo remains unknown. Understanding the role of autophagy during P. aeruginosa infection is of particular importance as mutations leading to cystic fibrosis have recently been shown to cause a blockade in the autophagy pathway, which could increase susceptibility to infection. Here we demonstrate that P. aeruginosa induces autophagy in mast cells, which have been recognized as sentinels in the host defense against bacterial infection. We further demonstrate that inhibition of autophagy through pharmacological means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance. Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung. Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infection.     

Inhibition of HIV-1 replication with stable RNAi-mediated knockdown of autophagy factors

Autophagy is a cellular process leading to the degradation of cytoplasmic components such as organelles and intracellular pathogens. It has been shown that HIV-1 relies on several components of the autophagy pathway for its replication, but the virus also blocks late steps of autophagy to prevent its degradation. We generated stable knockdown T cell lines for 12 autophagy factors and analyzed the impact on HIV-1 replication. RNAi-mediated knockdown of 5 autophagy factors resulted in inhibition of HIV-1 replication. Autophagy analysis confirmed a specific defect in the autophagy pathway for 4 of these 5 factors. We also scored the impact on cell viability, but no gross effects were observed. Upon simultaneous knockdown of 2 autophagy factors (Atg16 and Atg5), an additive inhibitory effect was scored on HIV-1 replication. Stable knockdown of several autophagy factors inhibit HIV-1 replication without any apparent cytotoxicity. We therefore propose that targeting of the autophagy pathway can be a novel therapeutic approach against HIV-1.     

Old antibiotics target TB with a new trick

Autophagy is now recognized as a cellular defense mechanism that can restrict the growth of Mycobacterium tuberculosis (Mtb). In this issue of Cell Host & Microbe, Kim et?al. (2012) demonstrate that antibiotics routinely used to treat Mtb infection elicit a host autophagy response critical for bacterial clearance.     

Avoiding death by autophagy: interactions of Listeria monocytogenes with the macrophage autophagy system

Autophagy restricts the growth of a variety of intracellular pathogens. However, cytosol-adapted pathogens have evolved ways to evade restriction by this innate immune mechanism. Listeria monocytogenes is a Gram-positive bacterial pathogen that utilizes a cholesterol-dependent pore-forming toxin, listeriolysin O (LLO), to escape from the phagosome. Autophagy targets L. monocytogenes in LLO-damaged phagosomes and also in the cytosol under some experimental conditions. However, this bacterium has evolved multiple mechanisms to evade restriction by autophagy, including actin-based motility in the cytosol and an as yet undefined mechanism mediated by bacterial phospholipases C (PLCs). A population of L. monocytogenes with inefficient LLO activity forms Spacious Listeria-containing Phagosomes (SLAPs), which are autophagosome-like compartments that do not mature, allowing slow bacterial growth within enlarged vesicles. SLAPs may represent a stalemate between bacterial LLO action and the host autophagy system, resulting in persistent infection.     

p62 and NDP52 proteins target intracytosolic Shigella and Listeria to different autophagy pathways

Autophagy is an important mechanism of innate immune defense. We have recently shown that autophagy components are recruited with septins, a new and increasingly characterized cytoskeleton component, to intracytosolic Shigella that have started to polymerize actin. On the other hand, intracytosolic Listeria avoids autophagy recognition by expressing ActA, a bacterial effector required for actin polymerization. Here, we exploit Shigella and Listeria as intracytosolic tools to characterize different pathways of selective autophagy. We show that the ubiquitin-binding adaptor proteins p62 and NDP52 target Shigella to an autophagy pathway dependent upon septin and actin. In contrast, p62 or NDP52 targets the Listeria ActA mutant to an autophagy pathway independent of septin or actin. TNF-α, a host cytokine produced upon bacterial infection, stimulates p62-mediated autophagic activity and restricts the survival of Shigella and the Listeria ActA mutant. These data provide a new molecular framework to understand the emerging complexity of autophagy and its ability to achieve specific clearance of intracytosolic bacteria.     

Autophagy in the intestinal epithelium regulates Citrobacter rodentium infection

Autophagy, a ubiquitous degradation pathway, is important for the survival and homeostasis of cells. Previous studies have demonstrated the role of autophagy in host defense against bacterial infection, but the importance of autophagy in the intestinal epithelium for the regulation of bacterial infection has not been fully elucidated. In this study, we showed that the essential autophagy protein Atg7 is required for resistance to Citrobacter rodentium infection in the intestinal epithelium. Infected mice in which Atg7 had been conditionally deleted from the intestinal epithelium exhibited greater clinical evidence of disease and higher expression levels of pro-inflammatory cytokine mRNA in the large intestine. Moreover, C. rodentium clearance was reduced in the Atg7 conditional knockout mice. These results demonstrate that autophagy in intestinal epithelial cells plays an important role in host defense against C. rodentium infection and the regulation of C. rodentium infectious colitis.     

Apoptosis and autophagy as mechanisms of dinoflagellate symbiont release during cnidarian bleaching: every which way you lose

Cnidarian bleaching results from the breakdown in the symbiosis between the host cnidarian and its dinoflagellate symbiont. Coral bleaching in recent years has increasingly caused degradation and mortality of coral reefs on a global scale. Although much is understood about the environmental causes of bleaching, the underlying cellular mechanisms of symbiont release that drive the process are just beginning to be described. In this study, we investigated the roles of two cellular pathways, host cell apoptosis and autophagy, in the bleaching process of the symbiotic anemone Aiptasia pallida. Host cell apoptosis was experimentally manipulated using gene knockdown of an anemone caspase by RNA interference, chemical inhibition of caspase using ZVAD-fmk and an apoptosis-inducer wortmannin. Autophagy was manipulated by chemical inhibition using wortmannin or induction using rapamycin. The applications of multiple single treatments resulted in some increased bleaching in anemones under control conditions but no significant drop in bleaching in individuals subjected to a hyperthermic stress. These results indicated that no single pathway is responsible for symbiont release during bleaching. However, when multiple inhibitors were applied simultaneously to block both apoptosis and autophagy, there was a significant reduction in bleaching in heat-stressed anemones. Our results allow us to formulate a model for cellular processes involved in the control of cnidarian bleaching where apoptosis and autophagy act together in a see-saw mechanism such that if one is inhibited the other is induced. Similar interconnectivity between apoptosis and autophagy has previously been shown in vertebrates including involvement in an innate immune response to pathogens and parasites. This suggests that the bleaching response could be a modified immune response that recognizes and removes dysfunctional symbionts.     

Autophagy: a new player in hepatic stellate cell activation

Hepatic stellate cell (HSC) activation, the transition from a resident quiescent HSC to a myofibroblastic collagen-producing HSC, is a fundamental feature of liver fibrosis. Autophagy has been implicated in major liver pathologies, such as HCV infection and hepatocarcinoma. However, its role in HSC biology is largely unknown. Recently, we were able to demonstrate that HSC activation is followed by an increased autophagic flux and that its inhibition can partially inhibit the HSC myofibroblastic transition. These results point to autophagy as a possible target in the prevention of HSC activation.     

Manipulation of autophagy by bacteria for their own benefit

Autophagy is the host innate immune system's first line of defense against microbial intruders. When the innate defense system recognizes invading bacterial pathogens and their infection processes, autophagic proteins act as cytosolic sensors that allow the autophagic pathway to be rapidly activated. However, many intracellular bacterial pathogens deploy highly evolved mechanisms to evade autophagic recognition, manipulate the autophagic pathway, and remodel the autophagosomal compartment for their own benefit. Here current topics regarding the recognition of invasive bacteria by the cytosolic innate immune system are highlighted, including autophagy and the mechanisms that enable bacteria to evade autophagy. Also highlighted are some selective examples of bacterial activities that manipulate the autophagic pathways for their own benefit.     

The ubiquitin-binding adaptor proteins p62/SQSTM1 and NDP52 are recruited independently to bacteria-associated microdomains to target Salmonella to the autophagy pathway

Autophagy is an innate immune defense against bacterial invasion. Recent studies show that two adaptor proteins, p62 and NDP52, are required for autophagy of the bacterial pathogen Salmonella enterica serovar Typhimurium (S. typhimurium). However, it is not known why two different adaptors are required to target the same bacterial cargo to autophagy. Here we show that both adaptors are recruited to bacteria with similar kinetics, that they are recruited to bacteria independently of each other, and that depletion of either adaptor leads to impairment of antibacterial autophagy. Depletion of both adaptors does not synergistically impair autophagy, indicating they act in the same pathway. Remarkably, we observed that these adaptors do not colocalize, but rather form non-overlapping microdomains surrounding bacteria. We conclude that p62 and NDP52 act cooperatively to drive efficient antibacterial autophagy by targeting the protein complexes they coordinate to distinct micro-domains associated with bacteria.     

KIM-1-/TIM-1-mediated phagocytosis links ATG5-/ULK1-dependent clearance of apoptotic cells to antigen presentation

Phagocytosis of apoptotic cells by both professional and semi-professional phagocytes is required for resolution of organ damage and maintenance of immune tolerance. KIM-1/TIM-1 is a phosphatidylserine receptor that is expressed on epithelial cells and can transform the cells into phagocytes. Here, we demonstrate that KIM-1 phosphorylation and association with p85 results in encapsulation of phagosomes by lipidated LC3 in multi-membrane organelles. KIM-1-mediated phagocytosis is not associated with increased ROS production, and NOX inhibition does not block LC3 lipidation. Autophagy gene expression is required for efficient clearance of apoptotic cells and phagosome maturation. KIM-1-mediated phagocytosis leads to pro-tolerogenic antigen presentation, which suppresses CD4 T-cell proliferation and increases the percentage of regulatory T cells in an autophagy gene-dependent manner. Taken together, these data reveal a novel mechanism of epithelial biology linking phagocytosis, autophagy and antigen presentation to regulation of the inflammatory response.     

Analysis of the role of autophagy inhibition by two complementary human cytomegalovirus BECN1/Beclin 1-binding proteins

Autophagy is activated early after human cytomegalovirus (HCMV) infection but, later on, the virus blocks autophagy. Here we characterized 2 HCMV proteins, TRS1 and IRS1, which inhibit autophagy during infection. Expression of either TRS1 or IRS1 was able to block autophagy in different cell lines, independently of the EIF2S1 kinase, EIF2AK2/PKR. Instead, TRS1 and IRS1 interacted with the autophagy protein BECN1/Beclin 1. We mapped the BECN1-binding domain (BBD) of IRS1 and TRS1 and found it to be essential for autophagy inhibition. Mutant viruses that express only IRS1 or TRS1 partially controlled autophagy, whereas a double mutant virus expressing neither protein stimulated autophagy. A mutant virus that did not express IRS1 and expressed a truncated form of TRS1 in which the BBD was deleted, failed to control autophagy. However, this mutant virus had similar replication kinetics as wild-type virus, suggesting that autophagy inhibition is not critical for viral replication. In fact, using pharmacological modulators of autophagy and inhibition of autophagy by shRNA knockdown, we discovered that stimulating autophagy enhanced viral replication. Conversely, inhibiting autophagy decreased HCMV infection. Thus, our results demonstrate a new proviral role of autophagy for a DNA virus.