During macroautophagy (herein referred to as autophagy), cytoplasmic contents can be sequestered in a unique double-membrane structure, the autophagosome. Through autophagy, cytoplasmic cargo in autophagosomes can be degraded by fusion of autophagosomes and lysosomes, and activation of the lysosomal degradation pathway (^{Levine and Deretic, 2007}). In addition to degradation of damaged organelles or materials, autophagy has received attention as a crucial component of innate defense against a variety of infectious agents. Autophagy of foreign micro-organisms, i.e., xenophagy, has emerged as a powerful method of eliminating intracellular bacteria (^{Gutierrez et al., 2004}; ^{Nakagawa et al., 2004}). However, there is a complex interplay between host autophagy mechanisms and pathogens. Numerous microorganisms have evolved strategies to evade or subvert host autophagy to survive and establish a persistent infection (^{Campoy and Colombo, 2009b}; ^{Orvedahl and Levine, 2009}). Thus, identification of mechanisms or virulence factors exploiting autophagy may provide a new strategy for therapeutic intervention in infectious diseases.

Host defense against pathogens requires coordination of multiple innate immune signaling pathways (^{Jo et al., 2007}). During infection, host cells recognize the pathogen-associated molecular patterns of a variety of microbes through the expression of various pattern recognition receptors. The recognition of foreign or danger molecules by innate receptors can trigger an intracellular signaling cascade, leading to activation of antimicrobial effector mechanisms to promote clearance of the infection (^{Jo et al., 2007}; ^{Brodsky and Medzhitov, 2009}). Autophagy is considered to be one of the effector mechanisms downstream of these receptors, and plays an integral role in both innate and adaptive immunity to various pathogens (^{Orvedahl and Levine, 2009}).

Autophagy also crosstalks with intracellular signaling molecules and effectors (^{Brodsky and Medzhitov, 2009}; ^{Shahnazari and Brumell, 2011}). Through these interactions, autophagy carries out not only direct microbial degradation but also other protective mechanisms, such as lysozyme secretion, the ubiquitin-mediated pathway, and antigen presentation (^{Brodsky and Medzhitov, 2009}; ^{Shahnazari and Brumell, 2011}). In particular, the autophagic or cargo receptors have received much attention, since they play an important role in transporting ubiquitinated microbial cargos to the autophagy machinery (^{Pankiv et al., 2007}; ^{Ichimura et al., 2008}; ^{Kirkin et al., 2009b}; ^{Thurston et al., 2009}; ^{von Muhlinen et al., 2010}).

Recent studies have provided evidence that autophagy acts as a 'tuning module' in the regulation of innate immunity to prevent excessive inflammatory responses and inflammasome signaling (^{Sumpter and Levine, 2010}). Generation of reactive oxygen species (ROS) of cellular and mitochondrial origin is thought to play an important role in autophagy regulation, thus influencing innate defense (^{Azad et al., 2009}; ^{Scherz-Shouval and Elazar, 2011}).

The specific role and regulatory mechanisms of autophagy in connection with innate immune pathways during infection will be discussed here. Additionally, the molecular mechanisms by which multiple bacteria and viruses evade and/or resist autophagy will be covered in this review.

Autophagy is a mechanism for adjusting cellular quality and quantity through capture and degradation of portions of the cytosol or organelles in response to diverse stress or stimuli, including infection (^{Deretic and Levine, 2009}). Autophagy is a means of bulk degradation of cytoplasmic components within lysosomes. During this process, cytoplasmic constituents are delivered to lysosomes for degradation (^{Mizushima and Levine, 2008}). Although autophagy is fundamentally a 'self-eating' process, it also facilitates degradation of pathogens as a 'xenophagy' process (^{Huang and Brumell, 2009}).

Accumulating evidence indicates that autophagy acts as a defense mechanism against multiple invading microbes (^{Deretic and Levine, 2009}). During infection, host autophagic responses can target several steps of bacterial invasion. For example, autophagy can target bacteria within vacuolar membranous compartments. Intracellular pathogens, such as Mycobacterium tuberculosis (M. tuberculosis), can actively survive within host cells and exploit host defense by inhibition of phagosomal maturation (^{Gutierrez et al., 2004}; ^{Yuk et al., 2009b}). Autophagy activation by nutrient starvation or rapamycin treatment leads to co-localization of mycobacterial phagosomes with autophagosomes to overcome the inhibition of phagosomal maturation by mycobacteria, thereby resulting in suppression of intracellular bacterial survival (^{Gutierrez et al., 2004}; ^{Yuk et al., 2009b}). Stimulation of host autophagy by treatment with vitamin D significantly enhanced the antimicrobial responses against M. tuberculosis in human macrophages. This effect was mediated by cathelicidin, a peptide that is induced by vitamin D and promotes co-localization of bacterial phagosomes and autophagosomes (^{Yuk et al., 2009b}). These data suggest that autophagy contributes to elimination of bacteria by overcoming bacterial resistance to lysosomal access.

Another example is group A Streptococcus (GAS). When GAS enters epithelial cells via endocytosis, they can escape from the endosomes to the cytoplasm by secretion of streptolysin O, a poreforming toxin (^{Nakagawa et al., 2004}). GAS is then trapped in autophagosome-like compartments, which mature into autolysosomes and so eliminate the bacteria (^{Nakagawa et al., 2004}). Mechanistically, the host small G proteins, Rab5 and Rab7, are associated with autophagosome formation and the progression of endosome maturation in cells infected with GAS (^{Sakurai et al., 2010}). Two endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors, VAMP8 from lysosomes and Vti1b from autophagic compartments, are required for the successful fusion of GAS-containing xenophagosomes with lysosomes, which facilitates xenophagy (^{Furuta et al., 2010}).

The third mode of anti-bacterial action of autophagy is receptor-mediated recruitment of bacteria to autophagosomes. Some populations of Salmonella enterica serovar Typhimurium (S. Typhimurium) are known to invade mammalian cells and replicate in Salmonella-containing vacuoles (SCVs). During this process, two kinds of Salmonella pathogenicity island (SPI) encoding type III secretion systems (T3SSs) are involved: the SPI-2 encoded T3SS facilitates bacterial growth, whereas the SPI-1 encoded T3SS causes damage to SCV (^{Birmingham et al., 2006}), which activates the autophagic process. The autophagy system targets the intracellular bacteria present within damaged SCVs, and contributes to restriction of bacterial growth during infection (^{Birmingham et al., 2006}). The autophagy receptor p62/SQSTM1, an adaptor protein for degradation of cargo tagged with ubiquitinated protein, is recruited to autophagy-targeted S. Typhimurium and required for the restriction of intracellular bacterial replication through induction of efficient antibacterial autophagy (^{Zheng et al., 2009}). The autophagy receptor NDP52 can also target distinct compartments in cells infected with Salmonella, and is required for autophagy activation (^{Cemma et al., 2011}). These data suggest that xenophagy is required for efficient targeting and killing of cytosolic pathogens. The post-translational modification (ubiquitination) of intracellular pathogens and the roles of cargo receptors will be discussed in detail in the latter part of this review.

Apart from its initial description, which is that autophagy is a response to amino acid deprivation, to recycle cytoplasmic constituents and synthesize energy for cell survival under stressful/starved conditions, autophagy is also being recognized as an innate immune process that is activated upon recognition of microbial infection (^{Brodsky and Medzhitov, 2009}; ^{Shahnazari and Brumell, 2011}). Bacteria and viruses possess molecules termed 'pathogen associated molecular patterns' (PAMPs) that bind to pattern-recognition receptors such as toll-like receptors (TLRs) on innate immune cells. Ligation of TLRs with PAMPs is known to activate innate immune cells and subsequently facilitate adaptive immune responses. So far, 10 TLRs in humans and 12 in mice have been identified. On the basis of subcellular localization, TLRs can be divided into extracellular and intracellular receptors: TLR1, 2, 4, 5, 6, and 10 are located on the cell surface. In contrast, TLR3, 7, 8, and 9 are expressed in intracellular endosomal/lysosomal compartments and the endoplasmic reticulum (^{Manavalan et al., 2011}). Bacterial PAMPs are recognized by five TLRs in humans: lipopolysaccharide (LPS) is the main ligand of Gram-negative bacteria for TLR4; lipotechoic acid and diacylated lipopeptides are sensed by TLR2/6; triacylated lipopeptides are sensed by TLR2/1; CpG motifs are sensed by TLR9; and flagellin is sensed by TLR5 (^{Takeuchi and Akira, 2010}). Interestingly, stimulation of TLR2, 4, and 7 with their ligands can stimulate autophagy, which functions to eliminate intracellular mycobacteria (^{Xu et al., 2007}; ^{Shi and Kehrl, 2008}; ^{Delgado and Deretic, 2009}; ^{Shin et al., 2010b}). Thus, TLR responses, which are well-known immune-activating signals, can also directly activate autophagy resulting in killing of invading bacteria.

In TLR signaling, several TIR domain-containing adaptor molecules, such as MyD88, TIRAP, and TRIF, lie downstream of TLR and deliver activation signals. (^{Takeda and Akira, 2004}). Upon stimulation of TLR4 with LPS, MyD88, TRIF, and tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6), associate with ubiquitinated autophagy-related protein (Atg)6/Beclin-1, which results in activation of autophagy (^{Shi and Kehrl, 2010}). The roles of further downstream signaling molecules of the TLR signaling pathway in the regulation of autophagy remain to be determined. Among them, the role of nuclear factor (NF)-κB, an important downstream mediator of TLR signaling (^{Takeda and Akira, 2004}), in autophagy is still being debated. Although the NF-κB signaling pathway inhibits the autophagy pathways in tumor cells (^{Djavaheri-Mergny et al., 2006}) and keratinocytes (^{Lee et al., 2011}), this has not been confirmed in primary innate cells, such as macrophages or dendritic cells. Mitogen-activated protein kinase signaling pathways may play a role in autophagy, since they are important in the activation of innate immune signaling during bacterial infection (^{Jo et al., 2007}). The stress-activated signaling molecule, c-Jun N-terminal protein kinase 1 (JNK1), pathway has been known to activate starvation- or ceramide-induced autophagy through Bcl-2 phosphorylation and subsequent dissociation of Bcl-2 from Beclin-1 (^{Wei et al., 2008}; ^{Pattingre et al., 2009}). More comprehensive data are necessary to gain a complete understanding of the roles and regulatory mechanisms of signaling modules in antibacterial autophagic activation.

Stimulation of other pattern recognition receptors, including the cytosolic receptor NOD2, regulates autophagy. NOD2 receptor recognition by muramyldipeptide induces autophagy in dendritic cells through receptor-interacting serine-threonine kinase-2, ATG5, ATG7 and ATG16L1 (^{Cooney et al., 2010}). Notably, NOD2-induced autophagy activation is required for both direct antimicrobial activities and antigen presentation to T cells via MHC class II antigen to induce adequate CD4+ T cell responses (^{Cooney et al., 2010}). These studies suggest that specific PAMP recognition through membrane and cytosolic innate receptors can regulate innate and adaptive immune responses through autophagy activation.

Numerous cytokines and chemokines, such as interleukin (IL)-12, IL-23, IL-17, interferon (IFN)-α, IFN-β, IFN-γ, IL-10, IL-27, IL-6, TNF, lymphotoxin, and IL-1 family cytokines, are produced by innate immune cells during bacterial infection (^{Cooper et al., 2011}; ^{Deretic, 2011}). Notably, several cytokines are key regulators of autophagy pathways (^{Deretic 2009}). For example, the Th1 cytokine IFN-γ is essential for host defense against intracellular pathogens. IFN-γ can overcome the blocking of phagosomal maturation induced by M. tuberculosis through autophagy activation (^{Gutierrez et al., 2004}; ^{Harris et al., 2009}). In contrast, the T helper (Th) 2 cytokines IL-4 and IL-13 contribute to inhibition of autophagy and autophagy-mediated suppression of mycobacteria in macrophages (^{Harris et al., 2007}).

The mechanisms by which IFN-γ activates autophagy are: IFN-γ activates several anti-bacterial proteins involved in autophagy, such as IRG (p47 GTPases) and the 65-kilodalton (kD) guanylate-binding protein (Gbp) gene family members (Gbp1, Gbp6, Gbp7, and Gbp10). The IRG proteins (p47 GTPases), transcription of which is induced by IFN-γ stimulation, play an essential role in the elimination of intracellular pathogens in mice (^{Deretic, 2009}). Murine IRG proteins are involved in innate defense against M. tuberculosis and Toxoplasma gondii (^{Gutierrez et al., 2004}; ^{Ling et al., 2006}). Although the single human IRG member, IRGM, is not induced by IFN-γ, it is essential for induction of autophagy in response to intracellular bacterial infection (^{Singh et al., 2006}). Moreover, recent studies have revealed that the Gbp gene family members (Gbp1, Gbp6, Gbp7, and Gbp10) are involved in oxidase-dependent killing and autophagy pathways in phagocytes to combat listerial or mycobacterial infection. Thus, 65-kD Gbps function to coordinate a potent oxidative and vesicular trafficking program to protect the host from infection (^{Kim et al., 2011}). Reciprocally, autophagy facilitates IFN-γ-activated Jak2-STAT1 pathways and inflammatory responses, suggesting a link between autophagy and cellular inflammation (^{Chang et al., 2010}).

Antimicrobial peptides are major components of the bactericidal machinery of mammalian phagocytes, and play an important role as innate effectors during bacterial and viral infections (^{Cederlund et al., 2011}). Of the antimicrobial peptides, cathelicidins exert antimicrobial activities against mycobacteria (^{Liu et al., 2006}; ^{Yang et al., 2009}; ^{Sonawane et al., 2011}). Interestingly, vitamin D activates anti-mycobacterial autophagy through induction of cathelicidin, which contributes to the promoter activation of the autophagy-related genes Atg5 and Beclin-1, as well as co-localization of bacterial phagosomes and autophagosomes (^{Yuk et al., 2009b}). Future studies are necessary to fully elucidate the roles of the numerous antimicrobial peptides in the context of autophagy activation leading to collaboration with innate immunity.

Intracellular bacterial pathogens have developed multiple strategies for exploiting or hijacking eukaryotic functions in order to survive and multiply inside host cells (^{Knodler et al., 2001}). Consistently, numerous pathogens antagonize the initiation and maturation of autophagic processes, evade autophagic recognition, or hijack the autophagy pathway to facilitate intracellular survival or replication (^{Levine et al., 2011}).

The various strategies used by bacterial pathogens are summarized in Figure 1. In this section, we will summarize two strategies by which bacterial pathogens can escape from or interact with autophagic pathways: 1) physical escape from degradative endocytic compartments and replication within the cytoplasm (Shigella, Listeria, Rickettsia); and 2) survival and replication within membrane-bound compartments (Mycobacteria, Salmonella, Francisella, Legionella, Brucella, Chlamydia, Porphyromonas gingivalis).

Recent reports have suggested that the autophagy pathway involves selective recognition and degradation of the autophagic cargo (^{Johansen and Lamark, 2011}). In selective autophagy, modification of target proteins or intracellular bacteria with ubiquitin is necessary for its autophagic clearance (^{Kirkin et al., 2009b}). Autophagy receptors including p62/SQSTM1 (sequestome 1), NBR1 (neighbour of Brca1 gene) and NDP52 (nuclear dot protein 52 kDa) can simultaneously bind intracellular ubiquitinated cargos and the autophagy modifiers, such as microtubule-associated protein LC3 and γ-aminobutyric acid receptor-associated proteins (GABARAP) (^{Kirkin et al., 2009b}; ^{Kraft et al., 2010}). Thus, autophagic receptors can mediate docking of ubiquitin-marked protein aggregates to the autophagosomes to induce selective autophagic degradation of ubiquitinated proteins, organelles, and intracellular bacteria (^{Kirkin et al., 2009a}, ^2009b; ^{Thurston et al., 2009}).

As reviewed above and elsewhere (^{Sumpter and Levine, 2010}), the cargo receptors p62 and NDP52 can target intracellular bacteria, such as S. typhimurium, S. flexneri, and L. monocytogenes, by addition of a molecular tag (such as polyubiquitin) to autophagic machinery (^{Thurston et al., 2009}; ^{Zheng et al., 2009}). Recently, it has been shown that optineurin promotes the selective autophagy of ubiquitin-coated cytosolic Salmonella enterica through phosphorylation of optineurin by protein kinase, TANK binding kinase 1 (^{Wild et al., 2011}). Additionally, an ubiquitin-independent pathway mediated by a lipid second messenger, diacylglycerol, generated from SCV membranes, is essential for the initiation of autophagy against S. typhimurium (^{Shahnazari et al., 2010}). Ogawa et al. (²⁰¹¹) revealed that the Atg5 binding partner Tecpr1 (tectonin domain-containing protein) is a cargo receptor for targeting bacterial pathogens to the selective autophagy system in Shigella-infected cells. Therefore, several autophagy receptors, which may interconnect with other pathways or mechanisms involved in autophagy, contribute to the xenophagic elimination of cytosolic bacteria in mammalian cells. The mechanisms by which autophagy receptors activate host defenses against bacterial pathogens other than S. typhimurium remain to be determined.

Intracellular ROS, produced by incomplete reduction of oxygen, can oxidize and damage various macromolecules such as proteins, lipids, and DNA, and eventually cause cell death (^{Azad et al., 2009}; ^{Scherz-Shouval and Elazar, 2011}). Accumulating evidence points to a key role for ROS as signaling messengers, including the initiation of autophagy and various intracellular processes (^{Azad et al., 2009}; ^{Scherz-Shouval and Elazar, 2011}). Several stimuli and stresses, including nutrient starvation, infection, and other pathological conditions, result in ROS generation (^{Azad et al., 2009}). Cellular ROS is involved in innate immune signaling through the TLR-dependent ASK activation (^{Yuk et al., 2009a}) and NADPH oxidase 2 pathways (^{Yang et al., 2009}). Recently, it was revealed that ROS of mitochondrial origin are a critical factor in the antibacterial response, mediated by interaction with TRAF6 and ECSIT (evolutionarily conserved signaling intermediate in Toll pathways), which are themselves involved in the mitochondrial respiratory chain assembly (^{West et al., 2011}).

Interestingly, these are generally accepted as activating signals for autophagy in a variety of physiological and pathological settings (^{Azad et al., 2009}; ^{Scherz-Shouval and Elazar, 2011}). Notably, TLR signaling from phagosomes can lead to initiation of antibacterial autophagy through generation of NAPDH oxidase NOX2-dependent ROS (^{Huang and Brumell, 2009}). Excessive ROS of cellular and mitochondrial origin is associated with activation of proinflammatory responses, autophagy, and cell death in macrophages infected with enhanced intracellular survival (eis)-deficient mycobacteria (^{Shin et al., 2010a}).

In contrast, recent evidence suggests that autophagy is also essential for prevention of excessive inflammation. Depletion of the autophagy proteins LC3B and beclin 1 significantly enhances NLRP3-dependent inflammasome activation and IL-1β and IL-18 levels, and increases susceptibility to LPS-induced septic shock in vivo (^{Nakahira et al., 2011}). Intriguingly, in this case, mitochondrial ROS are also thought to be important for mediating inflammatory responses, even in the autophagy-deficient host (^{Nakahira et al., 2011}). This effect was dependent on the release of mitochondrial DNA into the cytosol and increased mitochondrial ROS generation (^{Nakahira et al., 2011}). Thus, it seems that coordinated interplay between autophagy activation and ROS-dependent signaling is necessary for the homeostatic regulation of innate defense and inflammation.

Since the roles of autophagy have been revealed in host immune responses, a number of recent in vitro, in vivo, and molecular studies have provided evidence of selective autophagic degradation of bacteria and viruses (xenophagy). Moreover, rapid advances have been made in elucidating the mechanisms underlying the cell's defensive response to intracellular bacteria. In this review we have emphasized that numerous bacterial pathogens possess multiple strategies for avoiding or circumventing host defense pathways to ensure their survival and replication within the host. Thus, it is not surprising that autophagy pathways cross talk with innate immunity to contribute to the combat of invading pathogens. Various immune effectors have been identified as cooperating with and regulating the autophagic responses against invading bacterial infection, although additional host factors and detailed mechanisms remain to be delineated. Here, we propose that the host autophagy system has evolved strategies to manipulate certain autophagic adaptors or cargo receptors to promote autophagy targeting to bacteria, proteins, and damaged organelles. Several important questions related to the extended and distinct roles of immune effectors or cargo receptors, i.e., whether they are involved in defense against specific pathogens or general microbial invaders, should be answered in future studies. Current and future revelations regarding the interplay between host autophagy and its evasion by microbial pathogens will provide opportunities for the development of novel antibacterial agents.

We are indebted to both current and past members of our laboratory for the discussions and investigations that contributed to this article. This research was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) through the Infection Signaling Network Research Center (2011-0006228) at Chungnam National University.