Manipulation of Autophagy by Bacterial Pathogens Impacts Host Immunity

Abstract


42!
Maintaining cellular homeostasis requires a coordinated control of metabolic pathways.Thus 43! the cell needs anabolic processes to build new components, and catabolic processes to discard 44! long-lived or damaged components.Autophagy is one of these catabolic processes that leads 45! to lysosomal degradation of unnecessary or damaged proteins, lipids and organelles (Ohsumi,

46!
Autophagosome formation, elongation and maturation is a complex process involving 91!

97!
Autophagy induction can be triggered by different signals ranging from nutrient limitation 98! (non-specific autophagy) to the recognition of pathogens or damaged cellular components 99! (Deretic et al., 2013).Most signals for the initiation of autophagy converge through the 100! functions of the mammalian target of rapamycin complex 1 (mTORC1).Under nutrient-rich 101! conditions the mTORC1 complex phosphorylates Unc-51-like kinase 1 (ULK1) and ATG13, 102! which together with the FAK family kinase-interacting protein of 200kDa (FIP200) form the 103!ULK1 complex.When mTORC1 phosphorylates ULK1 and ATG13, the complex cannot be 104!recruited to the phagophore assembly site (PAS), thus inhibiting autophagosome formation

124!
ATG12-ATG5 forms a complex with ATG16L1, which associates with the expanding 125! phagophore membrane and gets released after the autophagosome is formed (Mizushima et al., 126! 2001; 2003).The ATG12-ATG5-ATG16L1 complex is essential for the formation of the 127! second ubiquitin-like conjugation system, where the protein light chain 3 (LC3; Atg8 in 128! yeast) is conjugated to phosphatidylethanolamine (PE) in the phagophore membrane.This 129! conjugation system relies on ATG7 (E1-like protein) and ATG3 (E2-like protein) that activate 130! LC3.In addition, ATG4B cleaves the carboxy-terminus of LC3 and exposes a glycine residue.131! Activated LC3 is directed by ATG12-ATG5-ATG16L1 to the expanding phagophore, where 132! the E3-like activity of ATG12-ATG5 conjugates PE to the previously exposed glycine residue 133! of LC3 in the phagophore membrane.Once the phagophore is closed and the autophagosome 134! is formed, the ATG12-ATG5-ATG16L1 complex is released from the autophagosome.and its interplay with innate immunity, it is expected that autophagy is also a crucial process 287! in adaptive immunity where it acts on the modulation of antigen processing and presentation 288! to elicit the correct development and homeostasis of lymphocytes.

289!
T cells are the main effectors of the adaptive immune system.They scan through the 290! output of the proteolytic machineries of the cells to detect pathogen-derived peptides.CD4 + 291! helper T cells and cytotoxic CD8 + T cells display a diverse receptor repertoire that allows 292! them to recognize these peptides but this recognition is not direct as antigens need to be 293!loaded to major histocompatibility class (MHC) molecules before being presented to T cells.

294!
MHC class I ligands are commonly generated by the proteasome and are presented to CD8 + T 295! cells while MHC class II loading peptides are produced by lysosomes and presented to CD4 + 296! T cells (Münz, 2010).

297!
The classical concept suggests that intracellular antigens get processed and loaded onto

((((
been shown to deconjugate a proportion of the LC3-PE complexes from 136! mature autophagosomes, facilitating LC3 recycling for the formation of new autophagosomes 137! Fujita et al., 2008; Tanida et al., 2004).It is thought that recycling of LC3 is an important 138! step in the maturation of fusion-capable autophagosomes.It has also been shown that LC3 139! !6! mediates the hemifusion of vesicles and controls the size of autophagosomes in yeast 140! Nakatogawa et al., 2007; Xie et al., 2008).141! Finally, a series of fusion events of autophagosomes with endosomes and lysosomes leads 142! to their maturation into degrading autolysosomes.In mammalian cells the fusion of 143! autophagosomes with lysosomes requires the small GTPase RAB7 (Ypt7 in yeast), the 144! autophagosomal SNARE protein syntaxin 17, the lysosomal SNARE protein VAMP8 145! (vesicle-associated membrane protein 8) and several lysosomal membrane proteins, such as 146! the lysosomal-associated membrane glycoprotein 2 (LAMP2).After autophagosome-147! lysosome fusion the cargo is degraded by the lysosomal hydrolases now present in the 148! autolysosome, (Eskelinen et al., 2002; Itakura et al., 2012; Jager et al., 2004; Tanaka et al., 149! 2000).150!For selective autophagy, such as xenophagy, an additional step of cargo selection is 151! required, a process that is mediated by cargo receptors and adaptor proteins.Mammalian 152! cargo specific receptors usually contain a LC3-interacting region (LIR) motif allowing to 153! recruit LC3-containing autophagosomes to the cargo (Liu et al., 2012; Novak et al., 2009; 154! Polson et al., 2014).In addition, many cargos are ubiquitinated and then they are recognized 155! by ubiquitin-binding protein adaptors that also contain LIRs.One of those proteins is p62 that, 156! among other functions, directs bacteria to autophagosomes.Together with the adaptor 157! proteins NDP52 and optineurin (OPTN), p62 is involved in S. Typhimurium recognition and 158! its direction to autophagosomes (Boyle and Randow, 2013).NDP52 and p62 also target 159!Shigella flexneri and Listeria monocytogenes to autophagosomes.Whereas p62 and NDP52 160! are recruited together during S. flexneri infection, p62 and NDP52 are recruited independently 161! during L. monocytogenes infection, suggesting that Shigella and Listeria induce different 162! pathways for selective autophagy (Mostowy et al., 2011).Another example is the neighbour 163! of BRCA1 gene 1 protein (NBR1) that is known to target Francisella tularensis to autophagy 164! Chong et al., 2014) and also participates in the targeting of S. flexneri (Mostowy et al., 2011), 165! but it is not required for targeting of S. Typhimurium to autophagy (Zheng et al., 2009).These 166! studies highlight that selective autophagy uses distinct protein adaptors to target different 167! types of bacteria.168! Crosstalk between autophagy and the innate immune response 169!The first step of innate immune responses is the recognition of a pathogen that is mediated by 170! a variety of pattern-recognition receptors (PRRs) like Toll-like Receptors (TLRs), RIG-I-like 171! receptors (RLRs) and NOD-like receptors (NLRs) that bind pathogen-associated molecular 172! !7! patterns (PAMPs) such as bacterial lipopolysaccharide (LPS), peptidoglycan, lipoproteins, 173! flagellin or nucleid acids.As autophagic degradation of pathogens aids PAMP recognition by 174! PRRs, autophagy is believed to have an important role in innate immunity.Additionally, 175! binding of PAMPs to PRRs stimulates autophagy (Delgado et al., 2009).176! Toll-like Receptors (TLRs): TLRs are membrane-bound receptors found at the surface of 177! the cell or in endosomal compartments.TLRs respond to LPS, lipotechoic acid, flagellin and 178! bacterial nucleic acids (Deretic, 2012).After binding their cognate ligand, TLRs activate 179! proinflammatory responses by triggering the production of cytokines such as tumor necrosis 180! factor-α (TNF-α), Interleukin-6 (IL-6) and IL-1β (Figure 2).181! Phagocytosed and cytosolic PAMPs are sequestered by autophagosomes and are delivered 182! to the endosomally located and luminally oriented TLRs for PAMP recognition.Therefore 183! autophagy takes part in the first steps of TLR activation (Desai et al., 2015).On the other side, 184! autophagy can also be induced upon TLR activation, through a MyD88/TRIF adaptor 185! dependent process and their interaction with BECN1 (Shi et al., 2008).TLR-autophagy 186! crosstalk is supported by several other findings: TLR4 stimulates PI3KC3-dependent 187! formation of cytosolic LC3 aggregates, enhancing the elimination of mycobacteria from 188! macrophages (Xu et al., 2007).Downstream of TLR4 activation, the TANK binding kinase 1 189! (TBK1) also links the TLR signalling pathway and autophagy as TBK1 phosphorylates the 190! autophagic receptor OPTN, enhancing LC3 binding affinity and consequently the autophagic 191! clearance of cytosolic Salmonella enterica (Wild et al., 2011).TLR7 activation has also been 192!shown to promote autophagic degradation of Mycobacterium bovis BCG (Bacillus Calmette-193!Guerin) (Delgado et al., 2008), and TLR2 induces autophagy in an ERK-dependent 194! mechanism during Listeria monocytogenes infection (Anand et al., 2011).Altogether these 195! studies highlight that TLR activation elicits autophagy, thus activating cell-autonomous 196! immunity after PAMP detection.197!However, autophagy may also down-regulate TLR-induced responses.Inhibition of 198! autophagy in macrophages and dendritic cells (DCs) through 3MA (3-methyladenine) or 199! through siRNA knock-down of autophagy genes, leads to an increase in cytokine release in 200! response to TLR3 or TLR4 agonists (Harris, 2011; Saitoh et al., 2008).ATG16L1-deficient 201! mice were shown to excessively activate caspase-1 in response to TLR4 stimulation by LPS, 202! which led to increased IL-1β and IL-18 production.This suggests that ATG16L1 might have 203! a regulatory function on the TLR4 signalling pathway and its depletion/malfunction may lead 204! to increased inflammation (Saitoh et al., 2008).Mature IL-1β protein can also be engulfed by 205! !8! autophagosomes in macrophages stimulated with TLR ligands, showing another way by 206! which autophagy can downregulate the production and secretion of cytokines (Harris et al., 207! 2011) (Figure 2).Thus, autophagy influences TLR signalling and consequently impacts TLR-208! mediated cytokine production and secretion.209! RIG-I-like receptors (RLRs): RLRs sense cytosolic dsRNA or DNA, and thus recognize 210! also nucleic acids from pathogens.PAMP recognition by RLRs as RIG-I and MDA5 triggers 211! the production of type I interferon (IFN) by infected cells, as has been shown for cells 212! infected with the intracellular pathogen Legionella pneumophila (Monroe et al., 2009).This 213! type I IFN activation pathway can be directly suppressed by several autophagy factors.For 214! example, autophagy-defective Atg5 −/− cells exhibit enhanced RLR signalling and increased 215! IFN secretion, mostly due to the accumulation of dysfunctional mitochondria and increased 216! generation of reactive oxygen species (ROS) which were largely responsible for the enhanced 217! RLR signalling in Atg5 −/− cells (Tal et al., 2009).218! cGAMP synthase (cGAS): Bacterial or aberrant cytosolic DNA are also recognized by 219! direct binding to cGAS, a cytosolic protein that generates cyclic dinucleotides (CDNs) such as 220! cGAMP within the host cytosol (Tao et al., 2016).Host cell-generated CDNs activate the 221! downstream stimulator of interferon genes (STING), a receptor that can be also activated by 222! CDNs of intracellular bacteria (Burdette et al., 2011).STING controls the activation of above-223!mentioned TBK1, which upon nucleic acid sensing and cGAMP synthesis triggers both IFN 224! regulatory factor 3 (IRF3) phosporylation and type I IFN production.STING induced type I 225! IFN production occurs during infection by intracellular bacteria such as Mycobacterium 226! tuberculosis (Watson et al., 2015).On the other hand, the autophagy activator protein ULK1 227! phosphorylates STING, inhibiting sustained type I IFN activation in response to dsDNA 228! Konno et al., 2013) (Figure 2).Furthermore, cGAS binding to BECN1 facilitates autophagic 229! removal of cytosolic dsDNA and reduces excessive type I IFN responses (Liang et al., 2014).230! NOD-like receptors (NLRs): NLRs are a class of receptors initiating a quick and potent 231! inflammatory cytokine response to PAMPs.Upon sensing PAMPs, cytosolic NLR-receptors 232! form a signalling complex called the inflammasome.The inflammasome consists of several 233! oligomerized NLRs that bind Caspase-1 directly or through the adaptor protein called 234! apoptosis-associated speck-like protein (ASC), which contains a caspase recruitment domain 235! (CARD).These complexes cleave the protein precursor pro-Caspase-1 into p10 and p20 236! subunits, activating Caspase-1.Active Caspase-1 then cleaves the presynthesized pro-IL-1β 237! into the active form of the cytokine IL-1β, which is secreted (Rodgers et al., 2014).IL-1β 238! Autophagy crosstalks with adaptive immune responses 285! Considering the role of autophagy in the restriction and destruction of intracellular pathogens 286!