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.     

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.     

How cells deploy ubiquitin and autophagy to defend their cytosol from bacterial invasion

Autophagy serves as a cell-autonomous effector mechanism of innate immunity in the cytosol. Autophagy restricts bacterial proliferation by separating bacteria from the nutrient-rich cytosol and delivering them into bactericidal autolysosomes. Autophagy also restricts inflammation by enclosing the membrane remnants of vacuoles from which bacteria have escaped. In contrast to starvation-induced autophagy, which engulfs cytosol nonspecifically, antibacterial autophagy is receptor-mediated and selective. Several distinct pathways of antibacterial autophagy have been identified recently, which can be triggered by either bacterial PAMPs, host-mediated modifications of bacteria-containing vacuoles, or cytosolic bacteria that have become decorated with ubiquitin. Ubiquitin-coated bacteria are sensed by p62, a promiscuous autophagy receptor required for the uptake of a variety of ubiquitin-marked autophagy substrates, and by NDP52, an autophagy receptor that, by associating with the immunoregulatory kinase TBK1, may serve a dedicated function in cytosolic immunity.     

How cells deploy ubiquitin and autophagy to defend their cytosol from bacterial invasion

Autophagy serves as a cell-autonomous effector mechanism of innate immunity in the cytosol. Autophagy restricts bacterial proliferation by separating bacteria from the nutrient-rich cytosol and delivering them into bactericidal autolysosomes. Autophagy also restricts inflammation by enclosing the membrane remnants of vacuoles from which bacteria have escaped. In contrast to starvation-induced autophagy, which engulfs cytosol nonspecifically, antibacterial autophagy is receptor-mediated and selective. Several distinct pathways of antibacterial autophagy have been identified recently, which can be triggered by either bacterial PAMPs, host-mediated modifications of bacteria-containing vacuoles, or cytosolic bacteria that have become decorated with ubiquitin. Ubiquitin-coated bacteria are sensed by p62, a promiscuous autophagy receptor required for the uptake of a variety of ubiquitin-marked autophagy substrates, and by NDP52, an autophagy receptor that, by associating with the immuno-regulatory kinase TBK1, may serve a dedicated function in cytosolic immunity.     

NDP52, a novel autophagy receptor for ubiquitin-decorated cytosolic bacteria

Autophagy functions as a cell-autonomous effector mechanism of innate immunity by separating bacteria from cytosolic resources and delivering them for lysosomal destruction. How cytosolic bacteria are targeted for autophagy is incompletely understood. We recently discovered that Salmonella enterica serotype Typhimurium and Streptococcus pyogenes are detected by NDP52 (nuclear dot protein 52kDa), after these bacteria enter the cytosol of human cells and become decorated with poly-ubiquitinated proteins. NDP52 binds the bacterial ubiquitin coat as well as ATG8/LC3 and delivers cytosolic bacteria into autophagosomes. In the absence of NDP52 ubiquitin-coated bacteria accumulate outside ATG8/LC3⁺ autophagosomes. Cells lacking NDP52 fail to restrict bacterial proliferation, as do cells depleted of TBK1, an IKK family kinase colocalizing with NDP52 at the bacterial surface. Our findings demonstrate the existence of a receptor for the selective autophagy of cytosolic bacteria, suggesting that cells are able to differentiate between anti-bacterial and other forms of autophagy.     

An essential role for the ATG8 ortholog LC3C in antibacterial autophagy

Autophagy defends the mammalian cytosol against bacterial invasion. Efficient bacterial engulfment by autophagy requires cargo receptors that bind (a) homolog(s) of the ubiquitin-like protein Atg8 on the phagophore membrane. The existence of multiple ATG8 orthologs in higher eukaryotes suggests that they may perform distinct functions. However, no specific role has been assigned to any mammalian ATG8 ortholog. We recently discovered that the autophagy receptor CALCOCO2/NDP52, which detects cytosol-invading Salmonella enterica serovar Typhimurium (S. Typhimurium), preferentially binds LC3C. The CALCOCO2/NDP52-LC3C interaction is essential for cell-autonomous immunity against cytosol-exposed S. Typhimurium, because cells lacking either protein fail to target bacteria into the autophagy pathway. The selectivity of CALCOCO2/NDP52 for LC3C is determined by a novel LC3C interacting region (CLIR), in which the lack of the key aromatic residue of canonical LIRs is compensated by LC3C-specific interactions. Our findings provide a new layer of regulation to selective autophagy, suggesting that specific interactions between autophagy receptors and the ATG8 orthologs are of biological importance.     

自噬蛋白NDP52与细菌感染的研究进展

自噬（autophagy）是哺乳动物清除入侵细菌的主要途径,可保卫宿主细胞免受细菌的损伤。核点蛋白52（nuclear dot protein 52,NDP52）——核点家族成员之一,是除p62/SQSTM1和NBR1等之外最新发现的自噬关键蛋白。它连接自噬体表面的微管相关蛋白1轻链3（microtubule associ-ated protein 1 light chain 3,LC3）,将披上＂泛素大衣＂的病原菌（如沙门氏菌和化脓性链球菌）递送至自噬体内加以清除。这一发现有助于人们深入了解自噬抵抗病原微生物感染的具体分子机制,为预防和治疗细菌感染提供了新靶点。     

Intracellular Salmonella induces aggrephagy of host endomembranes in persistent infections

Xenophagy has been studied in epithelial cells infected with Salmonella enterica serovar Typhimurium (S. Typhimurium). Distinct autophagy receptors target this pathogen to degradation after interacting with ubiquitin on the surface of cytosolic bacteria, and the phagophore- and autophagosome-associated protein MAP1LC3/LC3. Glycans exposed in damaged phagosomal membranes and diacylglycerol accumulation in the phagosomal membrane also trigger S. Typhimurium xenophagy. How these responses control intraphagosomal and cytosolic bacteria remains poorly understood. Here, we examined S. Typhimurium interaction with autophagy in fibroblasts, in which the pathogen displays limited growth and does not escape into the cytosol. Live-cell imaging microscopy revealed that S. Typhimurium recruits late endosomal or lysosomal compartments that evolve into a membranous aggregate connected to the phagosome. Active dynamics and integrity of the phagosomal membrane are requisite to induce such aggregates. This membranous structure increases over time to become an aggresome that engages autophagy machinery at late infection times (> 6 h postentry). The newly formed autophagosome harbors LC3 and the autophagy receptor SQSTM1/p62 but is devoid of ubiquitin and the receptor CALCOCO2/NDP52. Live-cell imaging showed that this autophagosome captures and digests within the same vacuole the aggresome and some apposed intraphagosomal bacteria. Other phagosomes move away from the aggresome and avoid destruction. Thus, host endomembrane accumulation resulting from activity of intracellular S. Typhimurium stimulates a novel type of aggrephagy that acts independently of ubiquitin and CALCOCO2, and destroys only a few bacteria. Such selective degradation might allow the pathogen to reduce its progeny and, as a consequence, to establish persistent infections.     

Burkholderia cenocepacia J2315 escapes to the cytosol and actively subverts autophagy in human macrophages

Selective autophagy functions to specifically degrade cellular cargo tagged by ubiquitination, including bacteria. Strains of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that cause life‐threatening infections in patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD). While there is evidence that defective macrophage autophagy in a mouse model of CF can influence B. cenocepacia susceptibility, there have been no comprehensive studies on how this bacterium is sensed and targeted by the host autophagy response in human macrophages. Here, we describe the intracellular life cycle of B. cenocepacia J2315 and its interaction with the autophagy pathway in human cells. Electron and confocal microscopy analyses demonstrate that the invading bacteria interact transiently with the endocytic pathway before escaping to the cytosol. This escape triggers theselective autophagy pathway, and the recruitment of ubiquitin, the ubiquitin‐binding adaptors p62 and NDP52 and the autophagosome membrane‐associated protein LC3B, to the bacterial vicinity. However, despite recruitment of these key autophagy pathway effectors, B. cenocepacia blocks autophagosome completion and replicates in the host cytosol. We find that a pre‐infection increase in cellular autophagy flux can significantly inhibit B. cenocepacia replication and that lower autophagy flux in macrophages from immunocompromised CGD patients could contribute to increased B. cenocepacia susceptibility, identifying autophagy manipulation as a potential therapeutic approach to reduce bacterial burden in B. cenocepacia infections.     

A role for diacylglycerol in antibacterial autophagy

Antibacterial autophagy is understood to be a key cellular immune response to invading microbes. However, the mechanism(s) by which bacteria are selected as targets of autophagy remain unclear. We recently identified diacylglycerol as a novel signaling molecule that targets bacteria to the autophagy pathway, and show that it acts via protein kinase C activation. We also found that Pkc1 is required for autophagy in yeast, indicating that this kinase plays a conserved role in autophagy regulation.Key words: bacteria, Salmonella, innate immunity, adaptor, lipid second messenger, diacylglycerol, ubiquitin, NDP52, p62, SQSTM1The mechanism by which bacteria and other subcellular targets are identified and degraded by the autophagy pathway is an area of intense research. Ubiquitin has been recently found to act as an essential signal required for the autophagy of bacteria and proteins. We have previously observed ubiquitin on autophagy-targeted Salmonella enterica serovar Typhimurium (S. Typhimurium) but were surprised to see that only 50% of these bacteria were positive for ubiquitin. This indicated the possibility that an alternate signal was required for efficient autophagic targeting of the nonubiquitinated population of these bacteria.We initially performed a screen quantifying the colocalization of different lipid second messengers (diacylglycerol (DAG), PtdIns(3)P, PtdIns(4,5)P₂, PtdIns(3,4) P₂, and PtdIns(3,4,5)P₃) with autophagytargeted (i.e., LC3⁺) S. Typhimurium. We observed that DAG preferentially localizes with LC3⁺ bacteria. A kinetic analysis revealed that maximal DAG colocalization with bacteria (45 min post-infection) precedes maximal autophagy of the bacteria (60 min post-infection). Using pharmacological agents, siRNA and dominant negative constructs we were able to determine that DAG localization to the bacteria requires the action of phospholipase D (PLD; phosphatidylcholine to phosphatidic acid conversion) and phosphatidic acid phosphatase (PAP; phosphatidic acid to DAG conversion). We observed that inhibition of these pathways significantly reduces DAG localization to bacteria as well as concomitant autophagy of the bacteria, indicating a role for this lipid second messenger in the regulation of this process.Having determined that DAG is necessary for autophagy of bacteria we subsequently wanted to identify the effector through which it was signaling. Conventional and novel isoforms of the protein kinase C (PKC) family contain DAG-binding C1 domains. Accordingly, we targeted PKC isoforms using pharmacological agents, siRNA and knockout cell lines and were able to determine that DAG is signaling through the δ isoform of PKC. Inhibition of this serine/threonine kinase results in significant inhibition of antibacterial autophagy. Furthermore, bacterial replication in PKCδ knockout mouse embryonic fibroblasts is significantly higher compared to control fibroblasts, consistent with previous observations demonstrating that autophagy impairs intracellular replication of S. Typhimurium (Birmingham et al. 2006).We addressed the possibility that DAG and ubiquitin are functioning in a cooperative manner to target Salmonella for degradation by autophagy. We simultaneously inhibited both pathways using siRNA or pharmacological agents and observed additive inhibitory effects on autophagy of the bacteria. While this is indicative of two independent pathways, we cannot discount the possibility that there is still cooperation between the two pathways, especially as we did observe a small population of bacteria that were positive for both DAG and ubiquitin (Fig. 1). There are also a number of technical limitations in the methods we used, such as detection levels of the probes and antibodies that warrant caution in concluding that the two pathways are completely independent. Nonetheless, our studies clearly demonstrate a role for both DAG (Shahnazari et al. 2010) and ubiquitin (Zheng et al. 2009) in autophagy of S. Typhimurium. Future studies are required to further examine how these signals contribute to regulation of antibacterial autophagy.Open in a separate windowFigure 1Autophagic targeting of Salmonella Typhimurium. Invading S. Typhimurium can be targeted to the autophagy pathway by two independent signaling mechanisms. The first requires ubiquitin and the autophagy adaptors p62 and NDP52. The second requires DAG generation and PKCδ function. DAG generation on the SCV may occur through interaction of the SCV with DAG-positive endocytic vesicles (pathway 1) or through direct DAG production on the SCV (pathway 2). SCV, Salmonella-containing vacuole; PA, phosphatidic acid; DAG, diacylglycerol; PAP, phosphatidic acid phosphatase; PKCδ, protein kinase C delta; Ub, ubiquitin.Having characterized this pathway in antibacterial autophagy we were interested in determining whether these components were required for general autophagy. We therefore tested whether DAG localizes with rapamycin-induced autophagosomes. We observed DAG on these compartments and also found a requirement for PAP and PKCδ in this process. Other PKC isoforms are involved in alternate types of autophagy including ER stress-induced autophagy (Sakaki et al. 2008) as well as hypoxia-induced autophagy (Chen et al. 2009). As a result, we were interested in determining whether PKC function in autophagy was evolutionarily conserved. We therefore tested a role for the yeast ortholog, Pkc1, in this process and observed that it is required for starvation-induced autophagy in Saccharomyces cerevisiae.Having identified and characterized a novel signal and effector for antibacterial autophagy, further work still remains to be done in order to obtain a complete picture of this process. This includes additional study of the mechanism by which DAG is generated and the subcellular localization of PLD and PAP during this process. It is possible that DAG⁺ endocytic vesicles fuse with the Salmonella-containing vacuole (SCV) coating this compartment with DAG (pathway 1, see Fig. 1). It is also possible that both PLD and PAP function directly on the SCV, converting phosphatidylcholine to DAG via the phosphatidic acid intermediate (pathway 2, Fig. 1).More work also needs to be done to dissect DAG and ubiquitin signaling contributions to this pathway. Questions to be answered include the identification of the ubiquitinated protein(s) on the SCV, which may be host or bacterial proteins. Additionally, while we know that DAG is present on the SCV we do not yet know the signal that induces its generation. One intriguing possibility is that DAG generation occurs in response to bacterial-induced damage to the SCV during invasion. To date, PKC has been implicated in at least three different types of autophagy, and the possibility exists that other PKC isoforms (DAG responsive or not) are also involved in this process.     

Autophagy and p62/sequestosome 1 generate neo-antimicrobial peptides (cryptides) from cytosolic proteins

In a manifestation of the immunological autophagy termed xenophagy, autophagic adapter proteins such as p62 and NDP52 directly capture microbes for delivery to autophagosomal organelles where they are eliminated. In a mirror image phenomenon, which is also an immunological variant of the process termed decryption, p62 and autophagy contribute to the elimination of Mycobacterium tuberculosis. During decryption, p62 sequesters cytosolic proteins into autophagosomes where they are proteolytically converted into peptides termed cryptides. A subset of cryptides possesses antimicrobial peptide properties exhibited upon their delivery to parasitophorous vacuoles where they kill intracellular microbes.     

Species‐specific impact of the autophagy machinery on Chikungunya virus infection

Chikungunya virus (CHIKV) is a recently re‐emerged arbovirus that triggers autophagy. Here, we show that CHIKV interacts with components of the autophagy machinery during its replication cycle, inducing a cytoprotective effect. The autophagy receptor p62 protects cells from death by binding ubiquitinated capsid and targeting it to autophagolysosomes. By contrast, the human autophagy receptor NDP52—but not its mouse orthologue—interacts with the non‐structural protein nsP2, thereby promoting viral replication. These results highlight the distinct roles of p62 and NDP52 in viral infection, and identify NDP52 as a cellular factor that accounts for CHIKV species specificity.     

LAMTOR2/LAMTOR1 complex is required for TAX1BP1‐mediated xenophagy

Xenophagy, also known as antibacterial autophagy, plays a role in host defence against invading pathogens such as Group A Streptococcus (GAS) and Salmonella. In xenophagy, autophagy receptors are used in the recognition of invading pathogens and in autophagosome maturation and autolysosome formation. However, the mechanism by which autophagy receptors are regulated during bacterial infection remains poorly elucidated. In this study, we identified LAMTOR2 and LAMTOR1, also named p14 and p18, respectively, as previously unrecognised xenophagy regulators that modulate the autophagy receptor TAX1BP1 in response to GAS and Salmonella invasion. LAMTOR1 was localized to bacterium‐containing endosomes, and LAMTOR2 was recruited to bacterium‐containing damaged endosomes in a LAMTOR1‐dependent manner. LAMTOR2 was dispensable for the formation of autophagosomes targeting damaged membrane debris surrounding cytosolic bacteria, but it was critical for autolysosome formation, and LAMTOR2 interacted with the autophagy receptors NBR1, TAX1BP1, and p62 and was necessary for TAX1BP1 recruitment to pathogen‐containing autophagosomes. Notably, knockout of TAX1BP1 caused a reduction in autolysosome formation and subsequent bacterial degradation. Collectively, our findings demonstrated that the LAMTOR1/2 complex is required for recruiting TAX1BP1 to autophagosomes and thereby facilitating autolysosome formation during bacterial infection.     

NDP52 associates with phosphorylated tau in brains of an Alzheimer disease mouse model

We previously showed that NDP52 (also known as calcoco2) plays a role as an autophagic receptor for phosphorylated tau facilitating its clearance via autophagy. Here, we examined the expression and association of NDP52 with autophagy-regulated gene (ATG) proteins including LC3, as well as phosphorylated tau and amyloid-beta (Aβ) in brains of an AD mouse model. NDP52 was expressed not only in neurons, but also in microglia and astrocytes. NDP52 co-localized with ATGs and phosphorylated tau as expected since it functions as an autophagy receptor for phosphorylated tau in brain. Compared to wild-type mice, the number of autophagic vesicles (AVs) containing NDP52 in both cortex and hippocampal regions was significantly greater in AD model mice. Moreover, the protein levels of NDP52 and phosphorylated tau together with LC3-II were also significantly increased in AD model mice, reflecting autophagy impairment in the AD mouse model. By contrast, a significant change in p62/SQSTM1 level was not observed in this AD mouse model. NDP52 was also associated with intracellular Aβ, but not with the extracellular Aβ of amyloid plaques. We conclude that NDP52 is a key autophagy receptor for phosphorylated tau in brain. Further our data provide clear evidence for autophagy impairment in brains of AD mouse model, and thus strategies that result in enhancement of autophagic flux in AD are likely to be beneficial.     

Autophagy and p62/sequestosome 1 generate neo-antimicrobial peptides (cryptides) from cytosolic proteins

In a manifestation of the immunological autophagy termed xenophagy, autophagic adapter proteins such as p62 and NDP52 directly capture microbes for delivery to autophagosomal organelles where they are eliminated. In a mirror image phenomenon, which is also an immunological variant of the process termed decryption, p62 and autophagy contribute to the elimination of Mycobacterium tuberculosis. During decryption, p62 sequesters cytosolic proteins into autophagosomes where they are proteolytically converted into peptides termed cryptides. A subset of cryptides possesses antimicrobial peptide properties exhibited upon their delivery to parasitophorous vacuoles where they kill intracellular microbes.Key words: autophagy, tuberculosis, ribosome, ubiquitin, antimicrobial peptidesAutophagy is an evolutionarily conserved cytoplasm-homeostatic process with a multitude of functions supporting, for the most part, cellular viability. During autophagy, cytoplasmic targets ranging from protein aggregates to whole organelles such as mitochondria and intracellular microbes are sequestered into a double-membrane bound organelle called the autophagosome. Autophagosomes mature into autolysosomes through fusion with lysosomes or their transport intermediates, bringing about acidification and acquisition of hydrolases leading to the digestion of the captured substrates. It is generally assumed that autophagy produces terminal degradative products such as free amino acids that are then used by the cell or the body as nutrients at times of starvation. Recently, we have discovered that autophagy generates, by proteolysis of captured cytosolic proteins, a mixture of peptides conferring potential cryptic biological functions, termed “cryptides.” Some of the cryptides with thus far assigned biological functions are the neo-antimicrobial peptides liberated from innocuous cytoplasmic proteins such as the ribosomal protein precursor FAU and ubiquitin.Our study was motivated by the search for factors or ingredients that make autophagic organelles particularly mycobactericidal, as Mycobacterium tuberculosis can survive the environment of the conventional phagolysosome. This was shown in the 1970s by the classical work of Armstrong and D''Arcy Hart at the same time when these authors established the more broadly appreciated and well-ingrained reputation of the tubercle bacillus as inhibiting the conventional phagosome-lysosome fusion. The approach to identifying such hypothetical ingredients was to first examine the steps of the autophagic pathway that are necessary for the mycobactericidal nature of macrophages induced for autophagy by, for example, starvation. We have found that not only are all stages of autophagy (initiation, elongation/closure and maturation) required for full mycobactericidal potency, but that p62, the first autophagic adapter characterized by the Johansen group, and also known as sequestosome 1, is absolutely required for autophagic elimination of M. tuberculosis. Sequestosome 1/p62 recognizes ubiquitinated protein aggregates and possibly ubiquitinated depolarized mitochondria and other targets, and delivers them to nascent autophagosomes; p62 also binds to the mammalian Atg8 paralog LC3 via its LC3-interaction region (LIR), thus conveniently bridging the targets with forming phagophores.At first blush, it may seem that mycobacteria follow the same fate demonstrated for several other bacteria, whereby p62 or another autophagic adapter, NDP52, capture cytosolic microbes and deliver them to autophagosomes. For example, the fraction of Salmonellae that are no longer retained within phagosomes and are free in the cytosol, or Shigella and Listeria that actively escape into the cytosol, are associated with ubiquitinated material or become otherwise recognized by p62 or NDP52, and end up being sequestered into autophagosomes. However, we found no evidence for p62 acting directly to transfer intraphagosomal mycobacteria into autophagic vacuoles. Instead, we observed p62-positive organelles as periodically fusing with mycobacterial phagosomes. At the same time, we found by imaging and biochemical means that proteins recruited by p62 from the cytosol into conventional autophagic organelles are subsequently transferred to model (latex bead phagosomes formed upon feeding 1 µm beads to macrophages) or mycobacterial phagosomes, as they gradually acquire autolysosomal characteristics. Next, we established that p62-captured cytosolic proteins (ribosomal protein rpS30 precursor FAU and ubiquitin) are proteolytically degraded into smaller peptides, and that specific peptides from these complex mixtures show antimycobacterial activity. Thus, the emerging model posits that autophagy captures cytosolic proteins and converts them into neo-antimycobacterial peptides that can then kill M. tuberculosis upon delivery to mycobacteria-containing phagosomes, which in turn gradually acquire autolysosomal properties (Fig. 1).Open in a separate windowFigure 1Elimination of M. tuberculosis by autophagy and p62. Mycobacteria are phagocytosed by macrophages and at least for some time reside within phagosomes. Upon induction of autophagy, p62, as a bifunctional agent interacting with autophagic substrates and with LC3, recruits into autophagosomes pre-antimicrobicidal cytosolic substrates. Autophagosome maturation including acquisition of lysosomal hydrolases leads to the proteolytic cleavage of p62 substrates and their conversion into peptides (cryptides) that can act as antimicrobial peptides.In contrast to the direct mechanism of capturing bacteria employed in some instances described above, in the case of M. tuberculosis, an organism that resides within the phagosomes, the adapter molecule p62 exerts its anti-microbial action through an indirect, but rather sophisticated mechanism. By sequestering into autophagosomes the initially harmless cytosolic components and by proteolytically processing them within maturing autophagosomes, p62 and autophagy liberate antimicrobial peptides from the otherwise innocuous substrates. This amounts to a resourceful utilization by the cell of otherwise spent or to-be-discarded cytoplasmic proteins and gives them an after-function upon completion of their “day jobs” that they performed as whole proteins.Our studies have uncovered a previously unappreciated function for autophagy in generating neo-antimicrobial peptides, and perhaps also opened the prospect for other biological functions potentially engendered by the products of autophagic proteolysis. Given that autophagy has the capacity to capture en masse and subject to digestion large sections of the cytoplasm, most cellular proteins are undergoing, or can undergo, processing into peptides or peptide intermediates within autophagic organelles. We postulate that the antimicrobial peptide production revealed in our studies thus far is only one manifestation of a spectrum of potential biological functions of cryptides generated by autophagy.     

Rab35 GTPase recruits NDP52 to autophagy targets

Autophagy targets intracellular molecules, damaged organelles, and invading pathogens for degradation in lysosomes. Recent studies have identified autophagy receptors that facilitate this process by binding to ubiquitinated targets, including NDP52. Here, we demonstrate that the small guanosine triphosphatase Rab35 directs NDP52 to the corresponding targets of multiple forms of autophagy. The active GTP‐bound form of Rab35 accumulates on bacteria‐containing endosomes, and Rab35 directly binds and recruits NDP52 to internalized bacteria. Additionally, Rab35 promotes interaction of NDP52 with ubiquitin. This process is inhibited by TBC1D10A, a GAP that inactivates Rab35, but stimulated by autophagic activation via TBK1 kinase, which associates with NDP52. Rab35, TBC1D10A, and TBK1 regulate NDP52 recruitment to damaged mitochondria and to autophagosomes to promote mitophagy and maturation of autophagosomes, respectively. We propose that Rab35‐GTP is a critical regulator of autophagy through recruiting autophagy receptor NDP52.     

The Autophagy Receptor TAX1BP1 and the Molecular Motor Myosin VI Are Required for Clearance of Salmonella Typhimurium by Autophagy

Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of myosin VI motor activity as a potential therapeutic target in cellular immunity.     

Host and Bacterial Proteins That Repress Recruitment of LC3 to Shigella Early during Infection

Shigella spp. are intracytosolic gram-negative pathogens that cause disease by invasion and spread through the colonic mucosa, utilizing host cytoskeletal components to form propulsive actin tails. We have previously identified the host factor Toca-1 as being recruited to intracellular S. flexneri and being required for efficient bacterial actin tail formation. We show that at early times during infection (40 min.), the type three-secreted effector protein IcsB recruits Toca-1 to intracellular bacteria and that recruitment of Toca-1 is associated with repression of recruitment of LC3, as well as with repression of recruitment of the autophagy marker NDP52, around these intracellular bacteria. LC3 is best characterized as a marker of autophagosomes, but also marks phagosomal membranes in the process LC3-associated phagocytosis. IcsB has previously been demonstrated to be required for S. flexneri evasion of autophagy at late times during infection (4–6 hr) by inhibiting binding of the autophagy protein Atg5 to the Shigella surface protein IcsA (VirG). Our results suggest that IcsB and Toca-1 modulation of LC3 recruitment restricts LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants. Together with published results, our findings suggest that IcsB inhibits innate immune responses in two distinct ways, first, by inhibiting LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants early during infection, and second, by inhibiting autophagy late during infection.     

Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes disease in a variety of hosts. S. Typhimurium actively invade host cells and typically reside within a membrane-bound compartment called the Salmonella-containing vacuole (SCV). The bacteria modify the fate of the SCV using two independent type III secretion systems (TTSS). TTSS are known to damage eukaryotic cell membranes and S. Typhimurium has been suggested to damage the SCV using its Salmonella pathogenicity island (SPI)-1 encoded TTSS. Here we show that this damage gives rise to an intracellular bacterial population targeted by the autophagy system during in vitro infection. Approximately 20% of intracellular S. Typhimurium colocalized with the autophagy marker GFP-LC3 at 1 h postinfection. Autophagy of S. Typhimurium was dependent upon the SPI-1 TTSS and bacterial protein synthesis. Bacteria targeted by the autophagy system were often associated with ubiquitinated proteins, indicating their exposure to the cytosol. Surprisingly, these bacteria also colocalized with SCV markers. Autophagy-deficient (atg5-/-) cells were more permissive for intracellular growth by S. Typhimurium than normal cells, allowing increased bacterial growth in the cytosol. We propose a model in which the host autophagy system targets bacteria in SCVs damaged by the SPI-1 TTSS. This serves to retain intracellular S. Typhimurium within vacuoles early after infection to protect the cytosol from bacterial colonization. Our findings support a role for autophagy in innate immunity and demonstrate that Salmonella infection is a powerful model to study the autophagy process.     

Toll‐like receptor signalling cross‐activates the autophagic pathway to restrict Salmonella Typhimurium growth in macrophages

It has been long recognised that activation of toll‐like receptors (TLRs) induces autophagy to restrict intracellular bacterial growth. However, the mechanisms of TLR‐induced autophagy are incompletely understood. Salmonella Typhimurium is an intracellular pathogen that causes food poisoning and gastroenteritis in humans. Whether TLR activation contributes to S. Typhimurium‐induced autophagy has not been investigated. Here, we report that S. Typhimurium and TLRs shared a common pathway to induce autophagy in macrophages. We first showed that S. Typhimurium‐induced autophagy in a RAW264.7 murine macrophage cell line was mediated by the AMP‐activated protein kinase (AMPK) through activation of the TGF‐β‐activated kinase (TAK1), a kinase activated by multiple TLRs. AMPK activation led to increased phosphorylation of Unc‐51‐like autophagy activating kinase (ULK1) at S317 and S555. ULK1 phosphorylation at these two sites in S. Typhimurium‐infected macrophages overrode the inhibitory effect of mTOR on ULK1 activity due to mTOR‐mediated ULK1 phosphorylation at S757. Lipopolysaccharide (LPS), flagellin, and CpG oligodeoxynucleotide, which activate TLR4, TLR5, and TLR9, respectively, increased TAK1 and AMPK phosphorylation and induced autophagy in RAW264.7 cells and in bone marrow‐derived macrophages. However, LPS was unable to induce TAK1 and AMPK phosphorylation and autophagy in TLR4‐deficient macrophages. TAK1 and AMPK‐specific inhibitors blocked S. Typhimurium‐induced autophagy and xenophagy and increased the bacterial growth in RAW264.7 cells. These observations collectively suggest that activation of the TAK1–AMPK axis through TLRs is essential for S. Typhimurium‐induced autophagy and that TLR signalling cross‐activates the autophagic pathway to clear intracellular bacteria.