INAUGURAL ARTICLE by a Recently Elected Academy Member:Autophagosomes induced by a bacterial Beclin 1 binding protein facilitate obligatory intracellular infection

Autophagy is an essential and highly regulated eukaryotic cellular homeostatic process that sequesters and digests intracellular components to recycle (1). Autophagy begins with the nucleation of an isolation membrane that elongates to envelop autophagic targets into double-membrane vacuole autophagosome, which expands and eventually fuses with lysosomes to become degradative autolysosomes (1). Autophagy plays important roles in immunological processes, including direct pathogen elimination, pathogen-associated molecular pattern processing for pattern recognition receptor, inflammasome regulation and autosecretion of alarmins, and cytosolic antigen processing for MHC-II presentation (2). Against intracellular pathogens, such as virus, bacteria, and protozoa, autophagy is one of the primary innate defense mechanisms (1). Intracellular invading bacteria, including Streptococcus pyogenes, Listeria monocytogenes, Salmonella enterica, and Shigella flexneri, have been shown to be ubiquitin-coated for autophagic degradation (1). To fight back, some intracellular pathogens were shown to have evolved strategies to either inhibit or avoid autophagy (1). For example, Shigella and Salmonella cause membrane damage at an early stage of cellular invasion, which inhibits the mammalian target of rapamycin (mTOR) activation of an amino acid starvation-induced autophagy pathway (3). Although Shigella escapes from phagosomes, Salmonella rapidly repairs the membrane damage to block autophagosome development (3). Shigella VirG, an outer membrane protein required for its intracellular actin-based motility, also induces autophagy by binding to Atg5 (4). To escape this autophagy, Shigella secretes a type III secretion effector IcsB, which competitively binds to VirG to evade Atg5 recognition (4). By binding to Beclin 1 (human homolog of yeast Atg6), Herpes simplex virus 1 protein ICP34.5 and γ-herpes virus-encoded viral Bcl-2-like proteins inhibit autophagosome initiation (1), whereas HIV accessory protein Nef or the influenza virus matrix protein 2 blocks autophagosome maturation (1). However, some intracellular bacterial pathogens benefit from autophagy activation, such as Coxiella (5), and Brucella (6), although the detailed mechanisms are not well-understood. Autophagy has been also observed in obligatory intracellular bacterial infection, including Rickettsia conorii (7) and Orientia (former R.) tsutsugamushi (8). We have been studying the role of autophagy in Anaplasma phagocytophilum (Ap) infection. Ap is a Gram-negative obligatory intracellular bacterium that belongs to the order Rickettsiales (9). Ap infects granulocytes and endothelial cells of various mammalian species (9). In humans, Ap causes an emerging and major tick-borne disease called human granulocytic anaplasmosis, an acute febrile disease that is potentially fatal, especially in elderly or immunocompromised individuals (10). Ap replicates inside the membrane-bound vacuole (inclusion) that is secluded from the endosome–lysosome pathway (11). We previously showed that the Ap inclusion resembles the early autophagosome based on the presence of double-lipid bilayer membranes, endogenous Beclin 1, and GFP-tagged LC3 (human ortholog of yeast Atg8) and the absence of the late endosome–lysosome marker (12). Autophagy becomes evident during the early bacterial growth stage with clumps of Beclin 1 and LC3 and their colocalization with the Ap inclusion (12). However, contrary to the dogma, this autophagy does not seem to be an innate immune response against Ap infection, because inhibition of the autophagy with the class III PI3K (PI3KC3) inhibitor 3-methyladenine (3-MA) does not enhance Ap infection; rather, it arrests its growth (12). Furthermore, although rapamycin induction of autophagy clears Salmonella infection (13), it significantly promotes Ap replication (12). This finding led us to suggest that Ap actively induces inclusion-targeted autophagosome formation to aid its replication. The type IV secretion (T4S) system is one of the important bacterial secretion systems that transfers bacterial proteins and/or DNA into the eukaryotic host in an ATP-dependent manner to alter critical host cell functions in favor of bacterial growth (14). Ap has genes encoding the T4S machinery, all of which are expressed by Ap in human leukocytes (15). So far, two T4S effectors, an ankyrin repeat-rich protein A (AnkA) and Ap translocated substrate 1 (Ats-1), have been identified experimentally. Secreted AnkA localizes in the host cell cytoplasm and nucleus (16). AnkA is essential for Ap infection, and it binds Abi-1 and the major tyrosine-phosphorylated protein in the infected cells (16). Unlike AnkA, Ats-1 lacks a tyrosine phosphorylation site, but it is imported into the mitochondrial matrix and delays mitochondria-mediated host cell apoptosis to benefit Ap (17). Ats-1 also localizes to Ap inclusions (17). Here, we report that Ats-1 is a unique example of a bacterial Beclin 1 binding protein that hijacks the Beclin 1-Atg14L autophagy initiation pathway likely to provide nutrients for bacterial growth.