Shigella and autophagy

Bacterial invasion of eukaryotic cells, and host recognition and elimination of the invading bacteria, determines the fate of bacterial infection. Once inside mammalian cells, many pathogenic bacteria enter the host cytosol to escape from the lytic compartment and gain a replicative niche. Recent studies indicate that autophagy also recognizes intracellular bacteria. Although autophagy is a conserved membrane trafficking pathway in eukaryotic cells that sequesters undesirable or recyclable cytoplasmic components or organelles and delivers them to lysosomes, autophagy has recently been described as playing a pivotal role as an intracellular surveillance system for recognition and eradication of the pathogens that have invaded the cytoplasm. Indeed, unless they are able to circumvent entrapping by autophagosomes, bacteria ultimately undergo degradation by delivery into autolysosomes. In this review we discuss recent discoveries regarding Shigella strategies for infecting mammalian cells, and then focus on recent studies of an elegant bacterial survival strategy against autophagic degradation.     

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.     

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。     

Bacterial pathogens and the autophagic response

The host cell recognition and removal of invading pathogens are crucial for the control of microbial infections. However, several microorganisms have developed mechanisms that allow them to survive and replicate intracellularly. Autophagy is an ubiquitous physiological pathway in eukaryotic cells, which maintains the cellular homeostasis and acts as a cell quality control mechanism to eliminate aged organelles and unnecessary structures. In addition, autophagy has an important role as a housekeeper since cells that have to get rid of invading pathogens use this pathway to assist this eradication. In this review we will summarize some strategies employed by bacterial pathogens to modulate autophagy to their own benefit and, on the other hand, the role of autophagy as a protective process of the host cell. In addition, we will discuss here recent studies that show the association of LC3 to a pathogen-containing compartment without a classical autophagic sequestering process (i.e. formation of a double membrane structure).     

Eating the strangers within: host control of intracellular bacteria via xenophagy

Many bacterial pathogens rely on an intracellular cycle to ensure their proliferation within infected hosts, through their ability to avoid or circumvent host bactericidal pathways. Recent evidence supports an increasingly important role for the autophagy pathway in innate immune defences against intracellular pathogens, as a mechanism of capture of either cytosol-adapted or vacuolar bacteria that redirect them to the lysosomal compartment for killing. Antibacterial autophagy, also referred to as xenophagy, involves selective recognition of intracellular bacteria and their targeting to the autophagic machinery for degradation. Here we review recent advances in our molecular understanding of these processes, and in how bacteria have adapted to avoid xenophagy or even take advantage of this innate immune process.     

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.     

Stimulation of autophagy suppresses the intracellular survival of Burkholderia pseudomallei in mammalian cell lines

Burkholderia pseudomallei is the causative agent of melioidosis, a tropical infection of humans and other animals. The bacterium is an intracellular pathogen that can escape from endosomes into the host cytoplasm, where it replicates and infects adjacent cells. We investigated the role played by autophagy in the intracellular survival of B. pseudomallei in phagocytic and non-phagocytic cell lines. Autophagy was induced in response to B. pseudomallei invasion of murine macrophage (RAW 264.7) cells and a proportion of the bacteria co-localized with the autophagy effector protein LC3, a marker for autophagosome formation. Pharmacological stimulation of autophagy in RAW 264.7 and murine embryonic fibroblast (MEF) cell lines resulted in increased co-localization of B. pseudomallei with LC3 while basal levels of co-localization could be abrogated using inhibitors of the autophagic pathway. Furthermore, induction of autophagy decreased the intracellular survival of B. pseudomallei in these cell lines, but bacterial survival was not affected in MEF cell lines deficient in autophagy. Treatment of infected macrophages with chloramphenicol increased the proportion of bacteria within autophagosomes indicating that autophagic evasion is an active process relying on bacterial protein synthesis. Consistent with this hypothesis, we identified a B. pseudomallei type III secreted protein, BopA, which plays a role in mediating bacterial evasion of autophagy. We conclude that the autophagic pathway is a component of the innate defense system against invading B. pseudomallei, but which the bacteria can actively evade. However, when autophagy is pharmacologically induced using rapamycin, bacteria are actively sequestered in autophagosomes, ultimately decreasing their survival.     

Stimulation of autophagy suppresses the intracellular survival of Burkholderia pseudomallei in mammalian cell lines

Burkholderia pseudomallei is the causative agent of melioidosis, a tropical infection of humans and other animals. The bacterium is an intracellular pathogen that can escape from endosomes into the host cytoplasm, where it replicates and infects adjacent cells. We investigated the role played by autophagy in the intracellular survival of B. pseudomallei in phagocytic and non-phagocytic cell lines. Autophagy was induced in response to B. pseudomallei invasion of murine macrophage (RAW 264.7) cells and a proportion of the bacteria co-localized with the autophagy effector protein LC3, a marker for autophagosome formation. Pharmacological stimulation of autophagy in RAW 264.7 and murine embryonic fibroblast (MEF) cell lines resulted in increased co-localization of B. pseudomallei with LC3 while basal levels of co-localization could be abrogated using inhibitors of the autophagic pathway. Furthermore, induction of autophagy decreased the intracellular survival of B. pseudomallei in these cell lines, but bacterial survival was not affected in MEF cell lines deficient in autophagy. Treatment of infected macrophages with chloramphenicol increased the proportion of bacteria within autophagosomes indicating that autophagic evasion is an active process relying on bacterial protein synthesis. Consistent with this hypothesis, we identified a B. pseudomallei type III secreted protein, BopA, which plays a role in mediating bacterial evasion of autophagy. We conclude that the autophagic pathway is a component of the innate defense system against invading B. pseudomallei, but which the bacteria can actively evade. However, when autophagy is pharmacologically induced using rapamycin, bacteria are actively sequestered in autophagosomes, ultimately decreasing their survival.     

Autophagy limits Listeria monocytogenes intracellular growth in the early phase of primary infection

Autophagy has been recently proposed to be a component of the innate cellular immune response against several types of intracellular microorganisms. However, other intracellular bacteria including Listeria monocytogenes have been thought to evade the autophagic cellular surveillance. Here, we show that cellular infection by L. monocytogenes induces an autophagic response, which inhibits the growth of both the wild-type and a DeltaactA mutant strain, impaired in cell-to-cell spreading. The onset of early intracellular growth is accelerated in autophagy-deficient cells, but the growth rate once bacteria begin to multiply in the cytosol does not change. Moreover, a significant fraction of the intracellular bacteria colocalize with autophagosomes at the early time-points after infection. Thus, autophagy targets L. monocytogenes during primary infection by limiting the onset of early bacterial growth. The bacterial expression of listeriolysin O but not phospholipases is necessary for the induction of autophagy, suggesting a possible role for permeabilization of the vacuole in the induction of autophagy. Interestingly, the growth of a DeltaplcA/B L. monocytogenes strain deficient for bacterial phospholipases is impaired in wild-type cells, but restored in the absence of autophagy, suggesting that bacterial phospholipases may facilitate the escape of bacteria from autophagic degradation. We conclude that L. monocytogenes are targeted for degradation by autophagy during the primary infection, in the early phase of the intracellular cycle, following listeriolysin O-dependent vacuole perforation but preceding active multiplication in the cytosol, and that expression of bacterial phospholipases is necessary for the evasion of autophagy.     

The Role of Autophagy during Group B Streptococcus Infection of Blood-Brain Barrier Endothelium

Bacterial meningitis occurs when bloodborne pathogens invade and penetrate the blood-brain barrier (BBB), provoking inflammation and disease. Group B Streptococcus (GBS), the leading cause of neonatal meningitis, can enter human brain microvascular endothelial cells (hBMECs), but the host response to intracellular GBS has not been characterized. Here we sought to determine whether antibacterial autophagy, which involves selective recognition of intracellular organisms and their targeting to autophagosomes for degradation, is activated in BBB endothelium during bacterial infection. GBS infection resulted in increased punctate distribution of GFP-microtubule-associated protein 1 light chain 3 (LC3) and increased levels of endogenous LC3-II and p62 turnover, two hallmark indicators of active autophagic flux. Infection with GBS mutants revealed that bacterial invasion and the GBS pore-forming β-hemolysin/cytolysin (β-h/c) trigger autophagic activation. Cell-free bacterial extracts containing β-h/c activity induced LC3-II conversion, identifying this toxin as a principal provocative factor for autophagy activation. These results were confirmed in vivo using a mouse model of GBS meningitis as infection with WT GBS induced autophagy in brain tissue more frequently than a β-h/c-deficient mutant. Elimination of autophagy using Atg5-deficient fibroblasts or siRNA-mediated impairment of autophagy in hBMECs led to increased recovery of intracellular GBS. However, electron microscopy revealed that GBS was rarely found within double membrane autophagic structures even though we observed GBS-LC3 co-localization. These results suggest that although autophagy may act as a BBB cellular defense mechanism in response to invading and toxin-producing bacteria, GBS may actively thwart the autophagic pathway.     

Selective autophagy mediated by autophagic adapter proteins

Mounting evidence suggests that autophagy is a more selective process than originally anticipated. The discovery and characterization of autophagic adapters, like p62 and NBR1, has provided mechanistic insight into this process. p62 and NBR1 are both selectively degraded by autophagy and able to act as cargo receptors for degradation of ubiquitinated substrates. A direct interaction between these autophagic adapters and the autophagosomal marker protein LC3, mediated by a so-called LIR (LC3-interacting region) motif, their inherent ability to polymerize or aggregate as well as their ability to specifically recognize substrates are required for efficient selective autophagy. These three required features of autophagic cargo receptors are evolutionarily conserved and also employed in the yeast cytoplasm-to-vacuole targeting (Cvt) pathway and in the degradation of P granules in C. elegans. Here, we review the mechanistic basis of selective autophagy in mammalian cells discussing the degradation of misfolded proteins, p62 bodies, aggresomes, mitochondria and invading bacteria. The emerging picture of selective autophagy affecting the regulation of cell signaling with consequences for oxidative stress responses, tumorigenesis and innate immunity is also addressed.     

Selective autophagy mediated by autophagic adapter proteins

Mounting evidence suggests that autophagy is a more selective process than originally anticipated. The discovery and characterization of autophagic adapters, like p62 and NBR1, has provided mechanistic insight into this process. p62 and NBR1 are both selectively degraded by autophagy and able to act as cargo receptors for degradation of ubiquitinated subtstrates. A direct interaction between these autophagic adapters and the autophagosomal marker protein LC3, mediated by a so-called LIR (LC3-interacting region) motif, their inherent ability to polymerize or aggregate as well as their ability to specifically recognize substrates are required for efficient selective autophagy. These three required features of autophagic cargo receptors are evolutionarily conserved and also employed in the yeast cytoplasm-to-vacuole targeting (Cvt) pathway and in the degradation of P granules in C. elegans. Here, we review the mechanistic basis of selective autophagy in mammalian cells discussing the degradation of misfolded proteins, p62 bodies, aggresomes, mitochondria and invading bacteria. The emerging picture of selective autophagy affecting the regulation of cell signaling with consequences for oxidative stress responses, tumorigenesis and innate immunity is also addressed.     

Cytoplasmic bacteria can be targets for autophagy

Autophagy is an important constitutive cellular process involved in size regulation, protein turnover and the removal of malformed or superfluous subcellular components. The process involves the sequestration of cytoplasm and organelles into double-membrane autophagic vacuoles for subsequent breakdown within lysosomes. In this work, we demonstrate that the intracellular pathogen Listeria monocytogenes can also be a target for autophagy. If infected macrophages are treated with chloramphenicol after phagosome lysis, the bacteria are internalized from the cell cytoplasm into autophagic vacuoles. The autophagic vacuoles appear to form by fusion of small cytoplasmic vesicles around the bacteria. These vesicular structures immunolabel with antibodies to protein disulphide isomerase, a marker for the rough ER. Internalization of metabolically arrested cytoplasmic L. monocytogenes represents an autophagic process as the vacuoles have double membranes and the process can be inhibited by the autophagy inhibitors 3-methyladenine and wortmannin. Additionally, the rate of internalization can be accelerated under starvation conditions and the vacuoles fuse with the endocytic pathway. Metabolic inhibition of cytoplasmic bacteria prevents them from adapting to the intracellular niche and reveals a host mechanism utilizing the autophagic pathway as a defence against invading pathogens by providing a route for their removal from the cytoplasm and subsequent delivery to the endocytic pathway for degradation.     

Autophagy in innate immunity against intracellular bacteria

Many pathogenic bacteria can invade phagocytic and non-phagocytic cells and colonize them intracellularly, then become disseminated to other cells. The endocytic degradation pathway is thought to be the only prevention against such intracellular pathogens. Autophagy, a fundamental cellular homeostasis pathway that operates with the intracellular degradation/recycling system, causes the turnover of cellular components by delivering portions of the cytoplasm and organelles to lysosomes. Recently, we reported that autophagic degradation is a previously unrecognized effector of host innate immunity. Streptococcus pyogenes (Group A Streptococcus; GAS) successfully enters human epithelial cells via endocytosis. GAS immediately escapes from the endosomes to the cytoplasm and gains a replicative niche, after which GAS in the cytoplasm is trapped in autophagosome-like compartments and degraded upon fusion with lysosomes. This process indicates that autophagy plays a protective role in infectious diseases. We also found that autophagic degradation was induced against Staphylococcus aureus, while methicillin-resistant S. aureus were resistant to autophagic degradation. The present review focuses on the protective function of autophagy against bacterial invasion of cells.     

Autophagy Limits Listeria monocytogenes Intracellular Growth in the Early Phase of Primary Infection

Autophagy has been recently proposed to be a component of the innate cellular immune response against several types of intracellular microorganisms. However, other intracellular bacteria including Listeria monocytogenes have been thought to evade the autophagic cellular surveillance. Here, we show that cellular infection by L. monocytogenes induces an autophagic response, which inhibits the growth of both the wild-type and a delta actA mutant strain, the latter being impaired in cell-to-cell spreading. The onset of early intracellular growth is accelerated in autophagy-deficient cells, but the growth rate once bacteria begin to multiply in the cytosol does not change. Moreover, a significant fraction of the intracellular bacteria co-localize with autophagosomes at the early time-points after infection. Thus, autophagy targets L. monocytogenes during primary infection by limiting the onset of early bacterial growth. The bacterial expression of listeriolysin O but not phospholipases is necessary for the induction of autophagy, suggesting a possible role for permeabilization of the vacuole in the induction of autophagy. Interestingly, the growth of a delta plcA/B L. monocytogenes strain deficient for bacterial phospholipases is impaired in wild-type cells, but restored in the absence of autophagy, suggesting that bacterial phospholipases may facilitate the escape of bacteria from autophagic degradation. We conclude that L. monocytogenes are targeted for degradation by autophagy during the primary infection, in the early phase of the intracellular cycle, following listeriolysin O-dependent vacuole perforation but preceding active multiplication in the cytosol, and that expression of bacterial phospholipases is necessary for the evasion of autophagy.     

The Salmonella Deubiquitinase SseL Inhibits Selective Autophagy of Cytosolic Aggregates

Cell stress and infection promote the formation of ubiquitinated aggregates in both non-immune and immune cells. These structures are recognised by the autophagy receptor p62/sequestosome 1 and are substrates for selective autophagy. The intracellular growth of Salmonella enterica occurs in a membranous compartment, the Salmonella-containing vacuole (SCV), and is dependent on effectors translocated to the host cytoplasm by the Salmonella pathogenicity island-2 (SPI-2) encoded type III secretion system (T3SS). Here, we show that bacterial replication is accompanied by the formation of ubiquitinated structures in infected cells. Analysis of bacterial strains carrying mutations in genes encoding SPI-2 T3SS effectors revealed that in epithelial cells, formation of these ubiquitinated structures is dependent on SPI-2 T3SS effector translocation, but is counteracted by the SPI-2 T3SS deubiquitinase SseL. In macrophages, both SPI-2 T3SS-dependent aggregates and aggresome-like induced structures (ALIS) are deubiquitinated by SseL. In the absence of SseL activity, ubiquitinated structures are recognized by the autophagy receptor p62, which recruits LC3 and targets them for autophagic degradation. We found that SseL activity lowers autophagic flux and favours intracellular Salmonella replication. Our data therefore show that there is a host selective autophagy response to intracellular Salmonella infection, which is counteracted by the deubiquitinase SseL.     

Autophagosome formation in response to intracellular bacterial invasion

Autophagy is an intracellular bulk degradation system in which double‐membrane vesicles, called autophagosomes, engulf cytoplasmic components and later fuse with lysosomes to degrade the autophagosome content. Although autophagy was initially thought a non‐selective process, recent studies have clarified that it can selectively target intracellular bacteria and function as an intracellular innate immune system that suppresses bacterial survival. A key mechanism for the recognition of cytosol‐invading bacteria is ubiquitination, and the recognition of the ubiquitinated target by the autophagy machinery can be accomplished multiple ways. In this review, we discuss recent findings regarding the induction of autophagosome formation in response to intracellular bacterial invasion.     

Autophagy recognizes intracellular Salmonella enterica serovar Typhimurium in damaged vacuoles

Autophagy is responsible for the degradation of cytosolic components within eukaryotic cells. Interestingly, autophagy also appears to play a role in recognizing invading intracellular pathogens. Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that normally resides and replicates within the Salmonella-containing vacuole (SCV). However, during in vitro infection a population of S. Typhimurium damage and escape from the SCV to enter the cytosol. We have observed that some intracellular S. Typhimurium are recognized by autophagy under in vitro infection conditions. Immunofluorescence studies revealed that autophagy recognizes the population of S. Typhimurium within damaged SCVs early after infection. The consequences of autophagic recognition of S. Typhimurium are still being elucidated, though a restrictive effect on intracellular bacterial replication has been demonstrated. Results of our in vitro infection studies are consistent with autophagy playing a role in cellular defense against S. Typhimurium that become exposed to the cytosol.     

Autophagy Recognizes Intracellular Salmonella enterica serovar Typhimurium in Damaged Vacuoles

Autophagy is responsible for the degradation of cytosolic components within eukaryotic cells. Interestingly, autophagy also appears to play a role in recognizing invading intracellular pathogens. Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that normally resides and replicates within the Salmonella-containing vacuole (SCV). However, during in vitro infection a population of S. Typhimurium damage and escape from the SCV to enter the cytosol. We have observed that some intracellular S. Typhimurium are recognized by autophagy under in vitro infection conditions. Immunofluorescence studies revealed that autophagy recognizes the population of S.Typhimurium within damaged SCVs early after infection. The consequences of autophagic recognition of S. Typhimurium are still being elucidated, though a restrictive effect on intracellular bacterial replication has been demonstrated. Results of our in vitro infection studies are consistent with autophagy playing a role in cellular defense against S. Typhimurium that become exposed to the cytosol.