Unconventional autophagy mediated by the WD40 domain of ATG16L1 is derailed by the T300A Crohn disease risk polymorphism

A coding polymorphism of the critical autophagic effector ATG16L1 (T300A) increases the risk of Crohn disease, but how this mutation influences the function of ATG16L1 has remained unclear. In a recent report, we showed that the A300 allele alters the ability of the C-terminal WD40 domain of ATG16L1 to interact with proteins containing a specific amino acid motif able to recognize this region. This defect impairs the capacity of the motif-containing transmembrane molecule TMEM59 to induce the unconventional autophagic labeling of the same single-membrane vesicles where this protein is located. Such alteration derails the intracellular trafficking of TMEM59 and the xenophagic response against bacterial infection. In contrast, canonical autophagy remains unaffected in the presence of ATG16L1^T300A. These data argue that the T300A polymorphism impairs the unconventional autophagic activities carried out by the WD40 domain, a region of ATG16L1 whose function has remained poorly understood.     

NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets

Autophagy is an evolutionary conserved cell survival process for degradation of long-lived proteins, damaged organelles and protein aggregates. The mammalian proteins p62 and NBR1 are selectively degraded by autophagy and can act as cargo receptors or adaptors for the autophagic degradation of ubiquitinated substrates. Despite differing in size and primary sequence, both proteins share a similar domain architecture containing an N-terminal PB1 domain, a LIR motif interacting with ATG8 family proteins, and a C-terminal UBA domain interacting with ubiquitin. The LIR motif is essential for their autophagic degradation, indicating that ATG8 family proteins are responsible for the docking of p62 and NBR1 to nucleating autophagosomes. p62 and NBR1 co-operate in the sequestration of misfolded and ubiquitinated proteins in p62 bodies and are both required for their degradation by autophagy. Here we discuss the role of p62 and NBR1 in degradation of ubiquitinated cargoes and the putative role of LIR as a general motif for docking of proteins to ATG8 family proteins.     

TMEM41B is a novel regulator of autophagy and lipid mobilization

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well‐characterized autophagy‐related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2‐ and DFCP1‐positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and β‐oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER‐localized regulator of autophagosome biogenesis and lipid mobilization.     

Structure of the WD40‐domain of human ATG16L1

Autophagy‐related protein ATG16L1 is a component of the mammalian ATG12～ATG5/ATG16L1 complex, which acts as E3‐ligase to catalyze lipidation of LC3 during autophagosome biogenesis. The N‐terminal part of ATG16L1 comprises the ATG5‐binding site and coiled‐coil dimerization domain, both also present in yeast ATG16 and essential for bulk and starvation induced autophagy. While absent in yeast ATG16, mammalian ATG16L1 further contains a predicted C‐terminal WD40‐domain, which has been shown to be involved in mediating interaction with diverse factors in the context of alternative functions of autophagy, such as inflammatory control and xenophagy. In this work, we provide detailed information on the domain boundaries of the WD40‐domain of human ATG16L1 and present its crystal structure at a resolution of 1.55 Å.     

The WD40 domain of ATG16L1 is required for its non‐canonical role in lipidation of LC3 at single membranes

A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double‐membrane phagophore, which is driven by the ATG16L1/ATG5‐ATG12 complex. In contrast, non‐canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3‐associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non‐canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat‐containing C‐terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non‐canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non‐canonical autophagy specifically, we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD. Further, we demonstrate activation of non‐canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non‐canonical use of autophagy machinery.     

Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure

Sphingomyelin is an essential cellular lipid that traffics between plasma membrane and intracellular organelles until directed to lysosomes for SMPD1 (sphingomyelin phosphodiesterase 1)-mediated degradation. Inactivating mutations in the SMPD1 gene result in Niemann-Pick diseases type A and B characterized by sphingomyelin accumulation and severely disturbed tissue homeostasis. Here, we report that sphingomyelin overload disturbs the maturation and closure of autophagic membranes. Niemann-Pick type A patient fibroblasts and SMPD1-depleted cancer cells accumulate elongated and unclosed autophagic membranes as well as abnormally swollen autophagosomes in the absence of normal autophagosomes and autolysosomes. The immature autophagic membranes are rich in WIPI2, ATG16L1 and MAP1LC3B but display reduced association with ATG9A. Contrary to its normal trafficking between plasma membrane, intracellular organelles and autophagic membranes, ATG9A concentrates in transferrin receptor-positive juxtanuclear recycling endosomes in SMPD1-deficient cells. Supporting a causative role for ATG9A mistrafficking in the autophagy defect observed in SMPD1-deficient cells, ectopic ATG9A effectively reverts this phenotype. Exogenous C12-sphingomyelin induces a similar juxtanuclear accumulation of ATG9A and subsequent defect in the maturation of autophagic membranes in healthy cells while the main sphingomyelin metabolite, ceramide, fails to revert the autophagy defective phenotype in SMPD1-deficient cells. Juxtanuclear accumulation of ATG9A and defective autophagy are also evident in tissues of smpd1-deficient mice with a subsequent inability to cope with kidney ischemia-reperfusion stress. These data reveal sphingomyelin as an important regulator of ATG9A trafficking and maturation of early autophagic membranes.     

Expression of a ULK1/2 binding-deficient ATG13 variant can partially restore autophagic activity in ATG13-deficient cells

Autophagy describes an intracellular process responsible for the lysosome-dependent degradation of cytosolic components. The ULK1/2 complex comprising the kinase ULK1/2 and the accessory proteins ATG13, RB1CC1, and ATG101 has been identified as a central player in the autophagy network, and it represents the main entry point for autophagy-regulating kinases such as MTOR and AMPK. It is generally accepted that the ULK1 complex is constitutively assembled independent of nutrient supply. Here we report the characterization of the ATG13 region required for the binding of ULK1/2. This binding site is established by an extremely short peptide motif at the C terminus of ATG13. This motif is mandatory for the recruitment of ULK1 into the autophagy-initiating high-molecular mass complex. Expression of a ULK1/2 binding-deficient ATG13 variant in ATG13-deficient cells resulted in diminished but not completely abolished autophagic activity. Collectively, we propose that autophagy can be executed by mechanisms that are dependent or independent of the ULK1/2-ATG13 interaction.     

Human autophagy gene ATG16L1 is post-transcriptionally regulated by MIR142-3p

Multiple genetic studies have implicated the autophagy-related gene, ATG16L1, in the pathogenesis of Crohn disease (CD). While CD-related research on ATG16L1 has focused on the functional significance of ATG16L1 genetic variations, the mechanisms underlying the regulation of ATG16L1 expression are unclear. Our laboratory has described that microRNAs (miRNAs), key regulators of gene expression, are dysregulated in CD. Here, we report miRNA-mediated regulation of ATG16L1 in colonic epithelial cells as well as Jurkat T cells. Dual luciferase reporter assays following the transfection of vectors containing the ATG16L1 3′-untranslated region (3′UTR) or truncated 3′UTR fragments suggest that the first half of ATG16L1 3′UTR in the 5′ end is more functional for miRNA targeting. Of 5 tested miRNAs with putative binding sites within the region, MIR142-3p, upon transient overexpression in the cells, resulted in decreased ATG16L1 mRNA and protein levels. Further observation demonstrated that the luciferase reporter vector with a mutant MIR142-3p binding sequence in the 3′UTR was unresponsive to the inhibitory effect of MIR142-3p, suggesting ATG16L1 is a gene target of MIR142-3p. Moreover, the regulation of ATG16L1 expression by a MIR142-3p mimic blunted starvation- and L18-MDP-induced autophagic activity in HCT116 cells. Additionally, we found that a MIR142-3p inhibitor enhanced starvation-induced autophagy in Jurkat T cells. Our study reveals MIR142-3p as a new autophagy-regulating small molecule by targeting ATG16L1, implying a role of this miRNA in intestinal inflammation and CD.     

Poliovirus induces autophagic signaling independent of the ULK1 complex

Poliovirus (PV), like many positive-strand RNA viruses, subverts the macroautophagy/autophagy pathway to promote its own replication. Here, we investigate whether the virus uses the canonical autophagic signaling complex, consisting of the ULK1/2 kinases, ATG13, RB1CC1, and ATG101, to activate autophagy. We find that the virus sends autophagic signals independent of the ULK1 complex, and that the members of the autophagic complex are not required for normal levels of viral replication. We also show that the SQSTM1/p62 receptor protein is not degraded in a conventional manner during infection, but is likely cleaved in a manner similar to that shown for coxsackievirus B3. This means that SQSTM1, normally used to monitor autophagic degradation, cannot be used to accurately monitor degradation during poliovirus infection. In fact, autophagic degradation may be affected by the loss of SQSTM1 at the same time as autophagic signals are being sent. Finally, we demonstrate that ULK1 and ULK2 protein levels are greatly reduced during PV infection, and ATG13, RB1CC1, and ATG101 protein levels are reduced as well. Surprisingly, autophagic signaling appears to increase as ULK1 levels decrease. Overexpression of wild-type or dominant-negative ULK1 constructs does not affect virus replication, indicating that ULK1 degradation may be a side effect of the ULK1-independent signaling mechanism used by PV, inducing complex instability. This demonstration of ULK1-independent autophagic signaling is novel and leads to a model by which the virus is signaling to generate autophagosomes downstream of ULK1, while at the same time, cleaving cargo receptors, which may affect cargo loading and autophagic degradative flux. Our data suggest that PV has a finely-tuned relationship with the autophagic machinery, generating autophagosomes without using the primary autophagy signaling pathway.

Abbreviations: ACTB - actin beta; ATG13 - autophagy related 13; ATG14 - autophagy related 14; ATG101 - autophagy related 101; BECN1 - beclin 1; CVB3 - coxsackievirus B3; DMV - double-membraned vesicles; EM - electron microscopy; EMCV - encephalomyocarditis virus; EV-71 - enterovirus 71; FMDV - foot and mouth disease virus; GFP - green fluorescent protein; MAP1LC3B/LC3B - microtubule associated protein 1 light chain 3 beta; MOI - multiplicity of infection; MTOR - mechanistic target of rapamycin kinase; PIK3C3 - phosphatidylinositol 3-kinase catalytic subunit type 3; PRKAA2 - protein kinase AMP-activated catalytic subunit alpha 2; PSMG1 - proteasome assembly chaperone 1; PSMG2 - proteasome assembly chaperone 2PV - poliovirus; RB1CC1 - RB1 inducible coiled-coil 1; SQSTM1 - sequestosome 1; ULK1 - unc-51 like autophagy activating kinase 1; ULK2 - unc-51 like autophagy activating kinase 2; WIPI1 - WD repeat domain, phosphoinositide interacting 1     

Transiently expressed ATG16L1 inhibits autophagosome biogenesis and aberrantly targets RAB11-positive recycling endosomes

The membrane source for autophagosome biogenesis is an unsolved mystery in the study of autophagy. ATG16L1 forms a complex with ATG12–ATG5 (the ATG16L1 complex). The ATG16L1 complex is recruited to autophagic membranes to convert MAP1LC3B-I to MAP1LC3B-II. The ATG16L1 complex dissociates from the phagophore before autophagosome membrane closure. Thus, ATG16L1 can be used as an early event marker for the study of autophagosome biogenesis. We found that among 3 proteins in the ATG16L1 complex, only ATG16L1 formed puncta-like structures when transiently overexpressed. ATG16L1⁺ puncta formed by transient expression could represent autophagic membrane structures. We thoroughly characterized the transiently expressed ATG16L1 in several mammalian cell lines. We found that transient expression of ATG16L1 not only inhibited autophagosome biogenesis, but also aberrantly targeted RAB11-positive recycling endosomes, resulting in recycling endosome aggregates. We conclude that transient expression of ATG16L1 is not a physiological model for the study of autophagy. Caution is warranted when reviewing findings derived from a transient expression model of ATG16L1.     

TRIM-directed selective autophagy regulates immune activation

Selectivity of autophagy is achieved by target recognition; however, the number of autophagy receptors identified so far is limited. In this study we demonstrate that a subset of tripartite motif (TRIM) proteins mediate selective autophagy of key regulators of inflammatory signaling. MEFV/TRIM20, and TRIM21 act as autophagic receptors recognizing their cognate targets and delivering them for autophagic degradation. MEFV recognizes the inflammasome components NLRP3, CASP1 and NLRP1, whereas TRIM21 specifically recognizes the activated, dimeric from of IRF3 inducing type I interferon gene expression. MEFV and TRIM21 have a second activity, whereby they act not only as receptors but also recruit and organize key components of autophagic machinery consisting of ULK1, BECN1, ATG16L1, and mammalian homologs of Atg8, with a preference for GABARAP. MEFV capacity to organize the autophagy apparatus is affected by common mutations causing familial Mediterranean fever. These findings reveal a general mode of action of TRIMs as autophagic receptor-regulators performing a highly-selective type of autophagy (precision autophagy), with MEFV specializing in the suppression of inflammasome and CASP1 activation engendering IL1B/interleukin-1β production and implicated in the form of cell death termed pyroptosis, whereas TRIM21 dampens type I interferon responses.     

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.     

Ubiquilin4 is an adaptor protein that recruits Ubiquilin1 to the autophagy machinery

Ubiquilins (Ubqlns)—a family of ubiquitin‐binding proteins—are involved in several protein degradation pathways and have been implicated in various neurodegenerative diseases. Ubqln1 regulates autophagosome maturation during autophagy‐mediated degradation. We now show that Ubqln4 mediates the interaction between Ubqln1 and the autophagy machinery by recruiting Ubqln1 to LC3. This targeting of Ubqln1 to autophagosomes requires the Ubqln4 UBL domain and the Ubqln1 UBA domain. This study identifies a new role for Ubqln4, expanding the role for Ubqlns in protein degradation.     

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.     

NBR1 co-operates with p62 in selective autophagy of ubiquitinated targets

Selective degradation of intracellular targets, such as misfolded proteins and damaged organelles, is an important homeostatic function that autophagy has acquired in addition to its more general role in restoring the nutrient balance during stress and starvation. Although the exact mechanism underlying selection of autophagic substrates is not known, ubiquitination is a candidate signal for autophagic degradation of misfolded and aggregated proteins. p62/SQSTM1 was the first protein shown to bind both target-associated ubiquitin (Ub) and LC3 conjugated to the phagophore membrane, thereby effectively acting as an autophagic receptor for ubiquitinated targets. Importantly, p62 not only mediates selective degradation but also promotes aggregation of ubiquitinated proteins that can be harmful in some cell types. Is p62 the only autophagic receptor for selective autophagy? Looking for proteins that interact with ATG8 family proteins, we identified NBR1 (neighbor of BRCA1 gene 1) as an additional LC3- and Ub-binding protein. NBR1 is degraded by autophagy depending on its LC3-interacting region (LIR) but does not strictly require p62 for this process. Like p62, NBR1 accumulates and aggregates when autophagy is inhibited and is a part of pathological inclusions. We propose that NBR1 together with p62 promotes autophagic degradation of ubiquitinated targets and simultaneously regulates their aggregation when autophagy becomes limited.     

Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy

ABSTRACT

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.

Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2     

Subversion of cellular autophagy machinery by hepatitis B virus for viral envelopment

Autophagy is a conserved eukaryotic mechanism that mediates the removal of long-lived cytoplasmic macromolecules and damaged organelles via a lysosomal degradative pathway. Recently, a multitude of studies have reported that viral infections may have complex interconnections with the autophagic process. The findings reported here demonstrate that hepatitis B virus (HBV) can enhance the autophagic process in hepatoma cells without promoting protein degradation by the lysosome. Mutation analysis showed that HBV small surface protein (SHBs) was required for HBV to induce autophagy. The overexpression of SHBs was sufficient to induce autophagy. Furthermore, SHBs could trigger unfolded protein responses (UPR), and the blockage of UPR signaling pathways abrogated the SHB-induced lipidation of LC3-I. Meanwhile, the role of the autophagosome in HBV replication was examined. The inhibition of autophagosome formation by the autophagy inhibitor 3-methyladenine (3-MA) or small interfering RNA duplexes targeting the genes critical for autophagosome formation (Beclin1 and ATG5 genes) markedly inhibited HBV production, and the induction of autophagy by rapamycin or starvation greatly contributed to HBV production. Furthermore, evidence was provided to suggest that the autophagy machinery was required for HBV envelopment but not for the efficiency of HBV release. Finally, SHBs partially colocalized and interacted with autophagy protein LC3. Taken together, these results suggest that the host's autophagy machinery is activated during HBV infection to enhance HBV replication.     

Actin‐based motility allows Listeria monocytogenes to avoid autophagy in the macrophage cytosol

Listeria monocytogenes grows in the host cytosol and uses the surface protein ActA to promote actin polymerisation and mediate actin‐based motility. ActA, along with two secreted bacterial phospholipases C, also mediates avoidance from autophagy, a degradative process that targets intracellular microbes. Although it is known that ActA prevents autophagic recognition of L. monocytogenes in epithelial cells by masking the bacterial surface with host factors, the relative roles of actin polymerisation and actin‐based motility in autophagy avoidance are unclear in macrophages. Using pharmacological inhibition of actin polymerisation and a collection of actA mutants, we found that actin polymerisation prevented the colocalisation of L. monocytogenes with polyubiquitin, the autophagy receptor p62, and the autophagy protein LC3 during macrophage infection. In addition, the ability of L. monocytogenes to stimulate actin polymerisation promoted autophagy avoidance and growth in macrophages in the absence of phospholipases C. Time‐lapse microscopy using green fluorescent protein‐LC3 macrophages and a probe for filamentous actin showed that bacteria undergoing actin‐based motility moved away from LC3‐positive membranes. Collectively, these results suggested that although actin polymerisation protects the bacterial surface from autophagic recognition, actin‐based motility allows escape of L. monocytogenes from autophagic membranes in the macrophage cytosol.     

Impaired autophagy of an intracellular pathogen induced by a Crohn's disease associated ATG16L1 variant

The genetic risk factors predisposing individuals to the development of inflammatory bowel disease are beginning to be deciphered by genome-wide association studies. Surprisingly, these new data point towards a critical role of autophagy in the pathogenesis of Crohn's disease. A single common coding variant in the autophagy protein ATG16L1 predisposes individuals to the development of Crohn's disease: while ATG16L1 encoding threonine at amino acid position 300 (ATG16L1*300T) confers protection, ATG16L1 encoding for alanine instead of threonine (ATG16L1*300A, also known as T300A) mediates risk towards the development of Crohn's disease. Here we report that, in human epithelial cells, the Crohn's disease-associated ATG16L1 coding variant shows impairment in the capture of internalized Salmonella within autophagosomes. Thus, we propose that the association of ATG16L1*300A with increased risk of Crohn's disease is due to impaired bacterial handling and lowered rates of bacterial capture by autophagy.