Ju Huang

Activation of antibacterial autophagy by NADPH oxidases

Autophagy plays an important role in immunity to microbial pathogens. The autophagy system can target bacteria in phagosomes, promoting phagosome maturation and preventing pathogen escape into the cytosol. Recently, Toll-like receptor (TLR) signaling from phagosomes was found to initiate their targeting by the autophagy system, but the mechanism by which TLR signaling activates autophagy is unclear. Here we show that autophagy targeting of phagosomes is not exclusive to those containing TLR ligands. Engagement of either TLRs or the Fcγ receptors (FcγRs) during phagocytosis induced recruitment of the autophagy protein LC3 to phagosomes with similar kinetics. Both receptors are known to activate the NOX2 NADPH oxidase, which plays a central role in microbial killing by phagocytes through the generation of reactive oxygen species (ROS). We found that NOX2-generated ROS are necessary for LC3 recruitment to phagosomes. Antibacterial autophagy in human epithelial cells, which do not express NOX2, was also dependent on ROS generation. These data reveal a coupling of oxidative and nonoxidative killing activities of the NOX2 NADPH oxidase in phagocytes through autophagy. Furthermore, our results suggest a general role for members of the NOX family in regulating autophagy.

Autophagy and Human Disease

As a conserved cellular degradative pathway in eukaryotes, autophagy relieves cells from various types of stress. There are different forms of autophagy, and the ongoing studies of the molecular mechanisms and cellular functions of these processes are unraveling their significant roles in human health. Currently, the best-studied of these pathways is macroautophagy, which is linked to a range of human disease. For example, as part of the host immune defense mechanism, macroautophagy is activated to eliminate invasive pathogenic bacteria; however, in some cases bacteria subvert this process for their own replication. Autophagy also contributes to endogenous major histocompatibility complex class II antigen presentation, reflecting its role in adaptive immunity. In certain neurodegenerative diseases, which are associated with aggregation-prone proteins, macroautophagy plays a protective role in preventing or reducing cytotoxicity by clearance of the toxic proteins; however, the autophagy-dependent processing of some components correlates with the pathogenesis of certain myopathies. Finally, autophagy acts as a mechanism for tumor suppression, although some cancer cells use it as a cytoprotective mechanism. Thus, a fundamental paradox of autophagy is that it can act to promote both cell survival and cell death, depending on the specific conditions.

Bacteria–autophagy interplay: a battle for survival

Autophagy is a cellular process that targets proteins, lipids and organelles to lysosomes for degradation, but it has also been shown to combat infection with various pathogenic bacteria. In turn, bacteria have developed diverse strategies to avoid autophagy by interfering with autophagy signalling or the autophagy machinery and, in some cases, they even exploit autophagy for their growth. In this Review, we discuss canonical and non-canonical autophagy pathways and our current knowledge of antibacterial autophagy, with a focus on the interplay between bacterial factors and autophagy components.

Trs85 directs a Ypt1 GEF, TRAPPIII, to the phagophore to promote autophagy

Macroautophagy (hereafter autophagy) is a ubiquitous process in eukaryotic cells that is integrally involved in various aspects of cellular and organismal physiology. The morphological hallmark of autophagy is the formation of double-membrane cytosolic vesicles, autophagosomes, which sequester cytoplasmic cargo and deliver it to the lysosome or vacuole. Thus, autophagy involves dynamic membrane mobilization, yet the source of the lipid that forms the autophagosomes and the mechanism of membrane delivery are poorly characterized. The TRAPP complexes are multimeric guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1, which is required for secretion. Here we describe another form of this complex (TRAPPIII) that acts as an autophagy-specific GEF for Ypt1. The Trs85 subunit of the TRAPPIII complex directs this Ypt1 GEF to the phagophore assembly site (PAS) that is involved in autophagosome formation. Consistent with the observation that a Ypt1 GEF is directed to the PAS, we find that Ypt1 is essential for autophagy. This is an example of a Rab GEF that is specifically targeted for canonical autophagosome formation.

Autophagy signaling through reactive oxygen species.

Autophagy is a degradative pathway that involves delivery of cytoplasmic components, including proteins, organelles, and invaded microbes to the lysosome for digestion. Autophagy is implicated in the pathology of various human diseases. The association of autophagy to inflammatory bowel diseases is consistent with recent discoveries of its role in immunity. A complex of signaling pathways control the induction of autophagy in different cellular contexts. Reactive oxygen species (ROS) are highly reactive oxygen free radicals or non-radical molecules that are generated by multiple mechanisms in cells, with the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and mitochondria as major cellular sources. These ROS are important signaling molecules that regulate many signal-transduction pathways and play critical roles in cell survival, death, and immune defenses. ROS were recently shown to activate starvation-induced autophagy, antibacterial autophagy, and autophagic cell death. Current findings implicate ROS in the regulation of autophagy through distinct mechanisms, depending on cell types and stimulation conditions. Conversely, autophagy can also suppress ROS production. Understanding the mechanisms behind ROS-induced autophagy will provide significant therapeutic implications for related diseases.

Antibacterial autophagy occurs at PI(3)P-enriched domains of the endoplasmic reticulum and requires Rab1 GTPase.

Autophagy mediates the degradation of cytoplasmic components in eukaryotic cells and plays a key role in immunity. The mechanism of autophagosome formation is not clear. Here we examined two potential membrane sources for antibacterial autophagy: the ER and mitochondria. DFCP1, a marker of specialized ER domains known as ‘omegasomes,’ associated with Salmonella-containing autophagosomes via its PtdIns(3)P and ER-binding domains, while a mitochondrial marker (cytochrome b5-GFP) did not. Rab1 also localized to autophagosomes, and its activity was required for autophagosome formation, clearance of protein aggregates and peroxisomes, and autophagy of Salmonella. Overexpression of Rab1 enhanced antibacterial autophagy. The role of Rab1 in antibacterial autophagy was independent of its role in ER-to-Golgi transport. Our data suggest that antibacterial autophagy occurs at omegasomes and reveal that the Rab1 GTPase plays a crucial role in mammalian autophagy.

Trs85 is Required for Macroautophagy, Pexophagy and Cytoplasm to Vacuole Targeting in Yarrowia lipolytica and Saccharomyces cerevisiae

Yarrowia lipolytica was recently introduced as a new model organism to study peroxisome degradation in yeasts. Transfer of Y. lipolytica cells from oleate/ethylamine to glucose/ammonium chloride medium leads to selective macroautophagy of peroxisomes. To decipher the molecular mechanisms of macropexophagy we made use of Y. lipolytica tagged mutants affected in the inactivation of peroxisomal enzymes under pexophagy conditions, Ain16 and Ain19. Both strains appeared to be disrupted at two different sites of the same gene, YlTRS85, the ortholog of Saccharomyces cerevisiae TRS85 that encodes 85 kDa subunit of transport protein particle (TRAPP). Y. lipolytica trs85 mutants had multiple defects of protein transport to external medium, cell wall and vacuoles, indicating that YlTrs85 is indeed the ScTrs85 functional homologue, required early in the classical secretory pathway. Interestingly, peroxisomes were not able to reach vacuoles under pexophagy conditions in both Ain16 and Ain19 strains. Therefore, the essential role of the early secretory flow in selective macroautophagy of peroxisomes is suggested.

Hepatic autophagy mediates endoplasmic reticulum stress–induced degradation of misfolded apolipoprotein B

Induction of endoplasmic reticulum (ER) stress was previously shown to impair hepatic apolipoprotein B100 (apoB) production by enhancing cotranslational and posttranslational degradation of newly synthesized apoB. Here, we report the involvement of autophagy in ER stress–induced degradation of apoB and provide evidence for a significant role of autophagy in regulating apoB biogenesis in primary hepatocyte systems. Induction of ER stress following short‐term glucosamine treatment of McA‐RH7777 cells resulted in significantly increased colocalization of apoB with green fluorescent protein–microtubule‐associated protein 1 light chain 3 (GFP‐LC3), referred to as apoB‐GFP‐LC3 puncta, in a dose‐dependent manner. Colocalization with this autophagic marker correlated positively with the reduction in newly synthesized apoB100. Treatment of McA‐RH7777 cells with 4‐phenyl butyric acid, a chemical ER stress inhibitor, prevented glucosamine‐ and tunicamycin‐induced increases in GRP78 and phosphorylated eIF2α, rescued newly synthesized [35S]‐labeled apoB100, and substantially blocked the colocalization of apoB with GFP‐LC3. Autophagic apoB degradation was also observed in primary rat and hamster hepatocytes at basal conditions as well as upon the induction of ER stress. In contrast, this pathway was inactive in HepG2 cells under ER stress conditions, unless proteasomal degradation was blocked with N‐acetyl‐leucinyl‐leucinyl‐norleucinal and the medium was supplemented with oleate. Transient transfection of McA‐RH7777 cells with a wild‐type protein kinase R–like ER kinase (PERK) complementary DNA resulted in dramatic induction of apoB autophagy. In contrast, transfection with a kinase inactive mutant PERK gave rise to reduced apoB autophagy, suggesting that apoB autophagy may occur via a PERK signaling–dependent mechanism. Conclusion: Taken together, these data suggest that induction of ER stress leads to markedly enhanced apoB autophagy in a PERK‐dependent pathway, which can be blocked with the chemical chaperone 4‐phenyl butyric acid. ApoB autophagy rather than proteasomal degradation may be a more pertinent physiological mechanism regulating hepatic lipoprotein production in primary hepatocytes. (HEPATOLOGY 2011;)

NADPH oxidases contribute to autophagy regulation

Reactive oxygen species (ROS) are emerging as regulators of autophagy in various cellular contexts. There are many cellular sources of ROS in eukaryotic cells. In phagocytes, the critical immune cells for host defense, the Nox2 NADPH oxidase generates ROS during phagocytosis and plays a central role in microbial killing. Toll-like receptors (TLRs) are important membrane microbial sensing receptors, which can activate Nox2,1 and were recently demonstrated to signal autophagy targeting of phagosomes to promote their maturation.2 Our recent study reveals that Nox2 activity and its generated ROS are key signals that induce TLR-activated autophagy of phagosomes. Our results provide the first evidence that ROS from the Nox2 NADPH oxidase can contribute to regulating autophagy in host defense against bacteria. The association of TLR, Nox2 and autophagy with inflammatory bowel disease (IBD) suggests a significant role of this antibacterial pathway in these diseases.

Salmonella exploits Arl8B-directed kinesin activity to promote endosome tubulation and cell-to-cell transfer.

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium establishes a replicative niche, the Salmonella‐containing vacuole (SCV), in host cells. Here we demonstrate that these bacteria exploit the function of Arl8B, an Arf family GTPase, during infection. Following infection, Arl8B localized to SCVs and to tubulated endosomes that extended along microtubules in the host cell cytoplasm. Arl8B+ tubules partially colocalized with LAMP1 and SCAMP3. Formation of LAMP1+ tubules (the Salmonella‐induced filaments phenotype; SIFs) required Arl8B expression. SIFs formation is known to require the activity of kinesin‐1. Here we find that Arl8B is required for kinesin‐1 recruitment to SCVs. We have previously shown that SCVs undergo centrifugal movement to the cell periphery at 24 h post infection and undergo cell‐to‐cell transfer to infect neighbouring cells, and that both phenotypes require kinesin‐1 activity. Here we demonstrate that Arl8B is required for migration of the SCV to the cell periphery 24 h after infection and for cell‐to‐cell transfer of bacteria to neighbouring cells. These results reveal a novel host factor co‐opted by S. Typhimurium to manipulate the host endocytic pathway and to promote the spread of infection within a host.