Autophagy and Bacterial infections

Abstract

Autophagy is an evolutionarily conserved cellular process that is prominent during bacterial infections. In this review article, we discuss how direct pathogen clearance via xenophagy and regulation of inflammatory products represent dual functions of autophagy that coordinate an effective antimicrobial response. We detail the molecular mechanisms of xenophagy, including signals that indicate the presence of an intracellular pathogen and autophagy receptor-mediated cargo targeting, while highlighting pathogen counterstrategies, such as bacterial effector proteins that inhibit autophagy initiation or exploit autophagic membranes for replication. Pathways that are related to autophagy, including LC3-associated phagocytosis (LAP) and conjugation of ATG8 to single membranes (CASM), are expanding the role of autophagy in antimicrobial defense beyond traditional double-membrane autophagosomes. Examination of Crohn disease-associated genes links impaired autophagy to inflammation and defective bacterial handling. We propose emerging concepts, such as effector-triggered immunity, where autophagy inhibition by pathogens triggers inflammatory defenses and discusses the therapeutic potential of modulating autophagy in infectious and inflammatory diseases.

Keywords: Autophagy; CASM; Crohn disease; LC3 associated phagocytosis; bacteria; xenophagy.

Figure 1.

Molecular mechanisms of xenophagy. (A) Carbohydrates on the ruptured pathogen-containing vacuole act as eat-me signals to recruit members of the galectin-family of carbohydrate binding proteins. LGALS8 acts as a damage detecting receptor, interacting with CALCOCO2 to induce xenophagy. (B) Ubiquitin, deposited either directly on the lipopolysaccharide of bacteria like Salmonella, or on unknown substrates, initiates the recruitment of other E3 ligases, including linear ubiquitin chain assembly complex (LUBAC). This recruits autophagy receptors including CALCOCO2 (and TAX1BP1), SQSTM1 and OPTN (optineurin), as well as the proinflammatory signaling regulator, IKBKG/NEMO. (C) CALCOCO2, via its interactions with RB1CC1/FIP200-ULK and TBKBP1/SINTBAD-TBK1, initiates the in situ biogenesis of autophagosomes.

Figure 2.

Invasion, CASM, and xenophagy during Streptococcus pneumoniae infection. Following the invasion of cells, S. pneumoniae exit the endocytic pathway due to the lytic action of pneumolysin (Ply). Initially, CASM is initiated, which depends on LC3A and GABARAPL1 (GBRPL1). The action of GABARAPL2/GATE16 (GBRPL2)) ensures these early pneumococci-containing LAPosome-like vesicles (PcLvs) become pneumococci-containing autophagic vesicles (PcAvs) following the induction of xenophagy. This process is inhibited by the bacterial protein CbpC. NanA is a pneumococcal sialidase that trims the sialylated glycans of endosomal or endo-lysosomal membrane proteins, suppressing Ply binding and promoting survival of the bacteria.

Figure 3.

Autophagy mechanisms and functions in plant-bacteria interactions. (A) Autophagy is involved in pattern-triggered immunity and plant tolerance mechanisms. Selective autophagy mediates the turnover of the pattern recognition receptor FLS2 via the cargo receptors ORM1 and ORM2 and contributes to NBR1-mediated basal resistance responses that likely rely on the removal of as yet unknown host proteins. The cargo receptor NBR1/Joka2 is also able to directly target the bacterial effector protein XopL of Xanthomonas campestris pv. vesicatoria in a process referred to as “effectorphagy.” In addition, autophagy facilitates the removal of detrimental ER-associated HEM1 condensates via the interaction of ATG6 with the HEM1-binding partner BI-1, leading to improved tissue health during bacterial infection. (B) Autophagy contributes to effector-triggered immunity (ETI) and the regulation of the hypersensitive response (HR). Autophagy has been linked both to the promotion and restriction of the HR, yet the ATG8-interacting cargo receptors and degraded host proteins responsible for the associated cell death and survival activities remain largely unknown. However, non-catabolic functions of autophagy play important roles in ETI and HR processes. Autophagosomes mediate the vacuolar delivery of the death-promoting VPE protease, although the direct link to HR execution during avirulent bacterial infection awaits to be demonstrated. Autophagic vesicles are also involved in the secretion of monolignol precursors, which are required for the formation of a lignin barrier in the cell wall to restrict cell death and pathogen spread. Furthermore, the autophagy component ATG6 promotes the formation of NPR1-dependent condensates (SINCs) to limit immunogenic cell death to infected tissue. Interaction of ATG6 with NPR1 also increases NPR1 pools in the nucleus and subsequently, leads to enhanced defense gene expression and resistance against avirulent bacterial infection. (C) Autophagy has pro-bacterial functions both in pathogenic and symbiotic relationships. During Pseudomonas syringae pv. tomato (Pst) infection, the effector HopM1 inactivates proteasomes and triggers their autophagic degradation (proteaphagy), leading to the suppression of SA-dependent defense responses. HopM1 is also involved in the effector-mediated activation of autophagy that could help maintain cellular survival during the biotrophic phase of the bacterial infection. However, the exact mechanisms, cargo receptors and/or autophagic substrates mediating such pro-bacterial cytoprotective functions, are not known. During beneficial rhizobial infection of the legume Medicago truncatula, the selective degradation of damaged mitochondria seems to provide carbon skeletons and nitrogen for bacteroid development and formation of nitrogen-fixing cells. These processes are triggered by myotubularin phosphatase (MP)-mediated dephosphorylation of PtdIns3P on the developing autophagosome. Successful rhizobial colonization of Medicago truncatula involves also the MtNAD1-mediated removal of immunity-related proteins. (D) Bacterial pathogens use multiple effector-based strategies to subvert and hijack autophagy processes. Autophagy activation is mediated by the Ralstonia solanacearum (Rs) effector Avr5 through TOR inhibition or by the Pst HrpZ1 and Candidatus Liberibacter asiaticus (CLas) SDE4405 effectors via binding to ATG8 proteins. The Rs effector RipD activates autophagy by unknown mechanisms and influences the homeostasis of the ACD11-BPA1 complex by competitive binding to BPA1. While low levels of RipD promote ACD11 accumulation and cell death suppression, high levels lead to ACD11 and BPA1 degradation and the induction of autophagy-dependent cell death. Autophagy suppression is achieved by Pst AvrPtoB through ATG1 phosphorylation, by Xcv XopL through ubiquitination and subsequent proteasomal degradation of the autophagy regulator SH3P2, and by Pst HopF1 through ´inhibitory´ interaction with a subset of ATG8 proteins. Finally, CLas SDE4405 interacts with GAPCs, which prevents their degradation by ATG8 and likely promotes their inhibitory effect on the autophagy protein ATG3. See text for further details. Graphical elements are partly adapted from Kushwaha et al. [147].

Figure 4.

Crohn disease risk genes in anti-bacterial autophagy and innate immune homeostasis. NOD2, ATG16L1, and IRGM, key CD-associated proteins, interact with autophagy machinery to initiate antimicrobial autophagy (xenophagy). Simultaneously, bacterial infection induces NOD and RIPK2 oligomerization, forming NODo/RIPosomes that drive NFKB/NF-κB-mediated inflammation, a critical antibacterial response. The system is tightly regulated by selective autophagy, which degrades NODo/RIPosomes (inflammophagy) to prevent excessive inflammation and maintain innate immune homeostasis.

Figure 5.

Autophagy and LAP provide different ways to control infection. Canonical autophagy conjugates LC3 to the lipid phosphatidylethanolamine (PE) to generate double-membraned autophagosomes that engulf pathogens in the cytosol. Non-canonical CASM pathways that include LC3 associated phagocytosis (LAP) conjugate LC3 to PE and phosphatidylserine (PS) on single-membraned endosomes and phagosomes containing extracellular pathogens as they enter cells. In addition to differences in membrane structures (single versus double, indicated by arrows), these pathways are distinguished by the location of the pathogen at the time of initiation. In both cases, conjugation of LC3 to vacuoles containing pathogens facilitates fusion with lysosomes leading to pathogen degradation. Therefore, by mediating both autophagy and CASM, the autophagy machinery can target both cytosolic and non-cytosolic pathogens. Figure adapted from Wang et al. [343].

Figure 6.

Pathways for conjugation of LC3 to membranes during autophagy and the CASM pathways that use the V-ATPase:ATG16L1 axis. (A) autophagy. Autophagy is activated in response to a fall in amino acids which leads to inhibition of mTOR and activation of the ULK1:RB1CC1 (FIP200) initiation complex (i) and downstream activation of the PtnIns3kinase complex containing BECN1 (Beclin1), ATG14 and PIK3C3 (VPS34). The PtnIns3 kinase activity of PIK3C3 generates PtdIns3P in autophagosome membranes which provide a platform for binding of WIPI2 (ii). WIPI2 binds to the coiled coil domain (CCD) of ATG16L1 leading to recruitment of the LC3 conjugation complex (ATG5-ATG12, ATG3, ATG7). The conjugation reaction converts LC3I to LC3II leading to covalent binding of LC3 to phosphatidylethanolamine (PE) in the autophagosome membrane (iii-iv). (B) LC3 conjugation via CASM and the V-ATPase:ATG16L1 axis. LC3 conjugation by CASM in phagocytic cells (LAP) is activated by TLR signaling through a complex containing RUBCN, BECN1, UVRAG, PIK3R4, and PIK3C3 (i). TLR signaling activates PIK3C3 leading to generation of PtdIns3 in phagosome membranes (ii). This generates a binding site for NCF4/p40phox which stabilizes the NADPH complex (CYBB/NOX2, NCF1/p47phox, NCF4/p40phox, NCF2/p67phox). at the same time binding of RUBCN to CYBA/p22phox increases production of reactive oxygen species (ROS). Generation of ROS increases the pH in the lumen of the phagosome (iii) stimulating assembly of the V_o V₁ subunits of the V-ATPase (iv). the V-ATPase binds to the WD domain of ATG16L1 leading to recruitment of the LC3 conjugation complex (ATG12–ATG5, ATG3, ATG7) to the phagosome and conjugation of LC3 to phosphatidylserine (PS) and PE in the phagosome membrane (v). a similar ROS-dependent pathway involving assembly of the V-ATPase operates in non-phagocytic cells, but the precise components of the NADPH oxidase complex are unclear. Figure adapted from Wang et al. [343].

Figure 7.

Secretory epithelial cells that protect the intestinal barrier are dependent on autophagy. Left panel: fluorescent microscopy of a mouse small-intestinal crypt with LYZ (lysozyme) labeled in red, LC3 in green, and DNA in blue. LYZ is packaged in granules along with other antimicrobial proteins and can be seen secreted from Paneth cells at the crypt bottom into the lumen. Autophagosomes can be seen as green puncta. This process is disrupted upon inhibition of autophagy, such as through mutation of the Crohn disease gene ATG16L1. Image contributed by Shai Bel, Bar-Ilan University. Right panel: cartoon rendering of a small intestinal crypt showing the positioning of Paneth cells relative to epithelial stem cells, goblet cells, and enterocytes. Through their antimicrobial properties, Paneth cells protect the crypt and the epithelial stem cell niche. They work together with the mucus-secreting goblet cells and other immune mechanisms to establish a chemical barrier against invasive microbes.