Direct targeting of autophagy by intracellular C3
It is therefore established that C3 entering the cytosol on the surface of invading pathogens is capable of triggering cell intrinsic immune responses. It was also recently shown that this response can include direct induction of xenophagy, via a direct interaction of C3 with ATG16L1. A yeast 2-hybrid assay was used to show that C3 is a ligand for ATG16L1 (Sorbara et al., 2018). ATG16L1 is involved in several stages of autophagosome biogenesis, including determining the site of autophagosome initiation (Fujita et al., 2008), and the lipidation of LC3-I into the autophagosome-associated form LC3-II (Fletcher et al., 2018). Considering its role in defining the site of LC3 lipidation, ATG16L1 is a key component of the autophagic response to invading pathogens that reach the cytosol64. Sorbara et al. went on to show that C3 opsonisation of cyto-invasive bacteria impacts the host response to infection through increased autophagy (Sorbara et al., 2018). Indeed, intracellular C3-opsonisedListeria monocytogenes and adherent-invasive E. coli(AIEC) were targeted by autophagy significantly more than unopsonised bacteria, and autophagy induction restricted intracellular bacterial growth via the C3/ATG16L1 interaction; the C3-mediated reduction in recovered viable bacteria required expression of ATG16L1. C3 was also shown to play a role in vivo in restricting Listeriainvasion in a murine model of intra-gastric infection. In this model, C3 expression was increased in cecal and colonic tissue in response to infection, as well as to dextran sodium sulfate-induced colitis, showing that mucosal inflammation could increase C3 expression, deposition of which can then protect mucosal barriers against infection. After 24 hrs of infection, C3-/- mice had higher numbers ofListeria colony forming units per gram of cecal and colonic tissue, as well as lower levels of autophagic turnover as assessed by LC3-II conversion.
Listeria expresses several virulence factors that participate in autophagy escape: ActA and InlK recruit host cell proteins - cytoskeletal proteins or Major Vault Protein respectively - at the bacterial surface, to disguise themselves and escape autophagic recognition (Birmingham et al., 2007; Dortet et al., 2011), while phospholipases PlcA and PlcB reduce autophagic flux and phosphatidylinositol 3-phosphate (PI3P) levels (Tattoli et al., 2013). Despite these autophagy escape mechanisms, Listeria growth can be restricted by autophagy in the presence of C3, perhaps due to the C3-dependent recruitment of ATG16L1, which acts downstream of the PI3K complex. In contrast, Sorbara et al. showed that two other cyto-invasive bacteria, Shigella flexneri and Salmonellaenterica serovar Typhimurium, escaped C3-dependent autophagy-mediated growth restriction. They first noticed that intracellular Shigella rapidly shed C3 upon invasion of epithelial cells. The omptin proteases IscP in Shigella and PgtE in Salmonella are partly responsible for C3 cleavage, that enables these bacteria to escape from C3-dependent autophagic restriction. Unlike Listeria , these two bacteria have therefore evolved C3-specific defence mechanisms in addition to their more general autophagy escape mechanisms. Loss of IscP expression in Shigella , or PgtE in Salmonella , prevented C3 shedding and sensitised the bacteria to ATG16L1-dependent intracellular killing. This paper therefore not only established the existence of an intracellular C3 detection mechanism directly targeting intracellular pathogens for xenophagy, but revealed that some pathogenic bacteria have evolved mechanisms to resist it. Similarly, some viruses have also evolved anti-C3 defenses to avoid intracellular detection: both human rhinovirus and poliovirus express cytosolic 3C proteases that cleave C3 and reduce intracellular C3-mediated MAVS-dependent NF-kB induction (Tam et al., 2014). Pathogen mechanisms for preventing C3 opsonization therefore also protect against these ’novel’ intracellular detection mechanisms, including anti-microbial intracellular signaling and autophagy-mediated bacterial growth restriction.