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The role of ‘eat-me’ signals and autophagy

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Article in Current opinion in microbiology · April 2013

DOI: 10.1016/j.mib.2013.03.010 · Source: PubMed

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 Available online at www.sciencedirect.com
The role of ‘eat-me’ signals and autophagy cargo receptors in
innate immunity
Keith B Boyle and Felix Randow
Selective autophagy is an important effector mechanism of cell
autonomous immunity, in particular against invasive bacterial
species. Anti-bacterial autophagy is activated by rupture of
bacteria-containing vacuoles and exposure of bacteria to the
cytosol. The autophagy cargo receptors p62, NDP52 and
Optineurin detect incoming bacteria that have become
associated with specific ‘eat-me’ signals such as Galectin-8 and
poly-ubiquitin and feed them into the autophagy pathway via
interactions with phagophore-associated ATG8-like proteins.
Here we review recent progress in the field regarding the origin of
bacteria-associated ‘eat-me’ signals, the specific roles of
individual cargo receptors and how disrupting cargo receptor
function may be important for bacterial evasion of autophagy.
Addresses
MRC Laboratory of Molecular Biology, Division of Protein and Nucleic
Acid Chemistry, Francis Crick Avenue, Cambridge Biomedical Campus,
Cambridge CB2 0QH, UK
Corresponding author: Randow, Felix (randow@mrc-lmb.cam.ac.uk)
Current Opinion in Microbiology 2013, 16:339–348
This review comes from a themed issue on Innate immunity
Edited by Kate Schroder and Vojo Deretic
For a complete overview see the Issue and the Editorial
Available online 23rd April 2013
1369-5274/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.mib.2013.03.010
Introduction
In metazoans the host immune response to infection
requires the coordinated action of specialized cells that
repress or eliminate invading pathogens by providing
innate and, in vertebrates, adaptive immunity. Unicellular
organisms, where such cellular specialization cannot occur,
must rely on cell-autonomous defence pathways. Most
metazoan cells are also equipped with cell-autonomous
anti-microbial activity, but this remains less well studied
due to the focus on specialized immune cells. The evolu-
tionarily conserved cellular process of autophagy is an
important innate, cell-autonomous effector mechanism
against various invasive bacteria, parasites and viruses [1–3].
Overview of autophagy
Macroautophagy (herein referred to as autophagy) is a
cellular process during which constituents of the cytosol
become encapsulated in a de novo-generated double-
membrane vesicle, known as the autophagosome.
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Subsequent fusion with the lysosome leads to degra-
dation of the autophagosomal contents. The molecular
mechanism underpinning the process has been primarily
delineated in yeast and mammalian cells, with over 30
AuTophaGy (ATG) genes now identified [4]. The com-
plex series of events required for the initiation,
elongation, closure and maturation of autophagosomes
are carried out by the coordinated action of multiple
protein complexes (Figure 1a). These include the
ULK/FIP200/ATG101/ATG13 protein kinase complex
and the Vps34/Beclin-1/Vps15/ATG14 lipid kinase com-
plex, which cooperate to produce phagophores rich in
phosphatidylinositol 3-phosphate (PI(3)P), and the
ATG5/ATG12/ATG16 complex, which conjugates ubi-
quitin-like ATG8 proteins to phosphatidylethanolamine
on the phagophore membrane [4]. The presence of mem-
brane-conjugated ATG8 distinguishes autophagosomal
membranes from most other membranes and provides
a polyvalent binding surface for the tethering of cargo.
While in the yeast genome only a single ATG8 gene is
present, humans encode six ATG8-like (ATG8L) ortho-
logs, namely LC3A, LC3B, LC3C, GABARAP,
GABARAP-L1 and GABARAP-L2 (GATE-16) [5].
Autophagy operates either non-selectively, as occurs
during cellular starvation when the cytosol is recycled
for nutritional purposes, or selectively, when specific
substrates are tagged for targeting to the autophagy path-
way (Figure 1a). Endogenous substrates for selective
autophagy range from misfolded proteins to larger toxic
protein aggregates and whole organelles, such as mito-
chondria and peroxisomes, which are targeted once they
become defective or are surplus to requirements [6].
Selective autophagy is also harnessed to neutralize and
remove pathogens, in particular bacteria, from cells and
we will review this process.
Cargo receptor-mediated control of selective
autophagy
Substrates for selective autophagy are recognized either
directly or indirectly in the cell. The yeast ATG11
protein directs mitophagy by interacting with the mito-
chondrial protein ATG32 [7,8], whereas in mammalian
cells ATG8 family members on the phagophore bind
directly to the mitochondrial proteins Nix and FUNDC1
[9,10]. On the other hand cargo receptors can bridge
ATG8 family members to poly-ubiquitin and other
‘eat-me’ signals on prospective cargo. Four autophagy
cargo receptors have been identified; p62 (SQSTM1),
NBR1, NDP52 and Optineurin (Figure 1b), and they all
Current Opinion in Microbiology 2013, 16:339–348
340 Innate immunity

Figure 1

(a)
initiation elongation closure maturation

lysosome

LC3

non-selective

eat-me
signal
cargo
receptor

bacterium

selective

(b) LC3/GBR Ub
p62 PB1 ZnF LIR UBA 440

LC3/GBR Ub
NBR1 PB1 ZnF CC CC LIR UBA 966

LC3C Gal8 Ub
NDP52 SKICH CLIR CC Gal8IR ZnF 577

LC3/GBR Ub
OPTN CC LIR CC CC UBAN ZnF 460

Current Opinion in Microbiology

Overview of autophagy and cargo receptor domain structure. (a) Autophagosome biogenesis is initiated from crescent-shaped double membrane
structures termed phagophores, to which LC3 and related ATG8-like proteins are conjugated. Phagophore membranes subsequently elongate and
enclose cytosolic material, thereby partitioning autophagy cargo from the rest of the cell. Cytosolic material becomes encapsulated either non-
selectively or is captured selectively by cargo receptors that detect cargo-associated ‘eat-me’ signals. Autophagosome maturation results in fusion
with lysosomes causing the degradation of the autophagosome content and its inner membrane. (b) Domain structure of cargo receptors. Highlighted
are binding sites for ‘eat-me’ signals and ATG8 family members. All cargo receptors bind ubiquitin-labelled cargo; only NDP52 detects the ‘eat-me’
signal Galectin-8 (Gal8). P62, NBR1 and Optineurin (OPTN) bind non-selectively LC3 and Gabarap proteins (LC3/GBR) via their LC3-interactin regions
(LIRs), while NDP52 preferentially interacts with LC3C via a LC3C-specific binding site (CLIR). Abbreviations: CC, coiled-coil; CLIR, LC3C-specific LIR;
Gal8, Galectin-8; Gal8IR, Galectin-8 interacting region; LIR, LC3-interacting region; OPTN, Optineurin; PB1, Phox and Bem1P; SKICH, skeletal muscle
and kidney enriched inositol phosphatase carboxyl homology; Ub, ubiquitin; UBA, ubiquitin-associated domain; UBAN, ubiquitin binding in ABIN and
NEMO domain

detect ubiquitin-dependent ‘eat-me’ signals (via either a
ZnF, UBA or UBAN domain), while NDP52 is unique in
also directing Galectin-8 labelled cargo to autophagy
[11,12,13]. The cargo receptors also possess short
LC3-interacting regions (LIR), which bind ATG8L
proteins. With the exception of the p62 and NBR1
paralogous proteins, the cargo receptors possess
limited similarity, indicating their shared autophagy
functions are due to convergent evolution. Further
receptor-specific domains allow the proteins to engage
in diverse cellular functions. The physiological import-
ance of the cargo receptors is highlighted by disease-
causing mutations in p62 and Optineurin found in
patients with Paget’s Disease of the Bone, Amyotrophic
Lateral Sclerosis and hepatocellular carcinoma [14,15].
There is evidence for both shared and distinct autophagy
functions for the cargo receptors. For example, homo-
polymerisation and hetero-polymerisation via their PB1

Current Opinion in Microbiology 2013, 16:339–348 www.sciencedirect.com

 ‘Eat-me’ signals and autophagy cargo receptors Boyle and Randow 341
domains allow p62 and NBR1 to promote the aggregation
and subsequent targeting of ubiquitylated protein cargo to
ATG8-positive autophagosomes [6]. In contrast, NBR1 is
necessary and sufficient for the autophagy of peroxisomes
[16]. A fifth cargo receptor, Tax1BP1 (T6BP), is proposed
to regulate NF-kB signalling [17], though its function in
selective autophagy remains unexamined.
Autophagy is instrumental to various aspects of innate and
acquired immunity. Here we focus on the role of cargo
receptors in cell-autonomous immunity, with particular
attention to the defence of the cytosol against bacterial
invasion. We will not discuss LC3-assisted phagocytosis
(LAP), the role of autophagy in antigen presentation or in
the unconventional secretion of IL-1 and other cytokines,
which have all been recently reviewed [1,3,18].
Cytosol-invading bacteria trigger autophagy
Selective autophagy is essential for cell-autonomous
defence against bacteria invading the cytosol. Salmonella
enterica serovar Typhimurium (S. Typhimurium) is a
Gram-negative bacterium with a facultative intracellular
life style that causes severe enterocolitis in humans. Upon
invasion of cells, S. Typhimurium establishes its intra-
cellular niche in a specialized vesicular compartment, the
Salmonella-containing vacuole (SCV). However, the limit-
ing SCV membrane frequently becomes damaged and
access to the cytosol promotes S. Typhimurium prolifer-
ation if autophagy is impaired [19,20]. Owing to this
marked phenotype S. Typhimurium has become the
model pathogen of choice for autophagy research. Myco-
bacteria, including M. tuberculosis, and Streptococcus pyogenes
are also restricted by autophagy [21,22,23]. Professional
cytosol-dwelling bacteria, such as Shigella flexneri, Listeria
monocytogenes and Francisella tularensis, have evolved eva-
sion strategies to overcome restriction by autophagy and
ensure their survival within the host cell cytosol [24].
However, not all intracellular bacteria can be strictly
classified as either ‘vesicular’ or ‘cytosolic’. Indeed, cer-
tain species, for example M. tuberculosis, previously con-
sidered to be entirely vacuolar also access the cytosol due
to their membrane damaging activities [25]. The mem-
brane permeabilization event that brings vesicular bac-
teria into contact with the cytosol requires either a
membrane-spanning secretion apparatus such as in S.
Typhimurium and M. tuberculosis, a pore-forming toxin
such as in S. pyogenes and L. monocytogenes, or lipolytic
enzymes as in Rickettsia spp. and L. monocytogenes [26].
Anti-bacterial autophagy — anti-microbial or
anti-danger?
Innate immunity relies on synergism between pattern-
recognition receptors (PRRs) and danger receptors to
detect pathogens. PRRs, such as Toll-like receptors
(TLRs), bind to so-called pathogen-associated molecular
patterns (PAMPs), that is, microbial ligands that are evol-
utionary conserved and essential for microbial viability.
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PRRs can therefore directly detect the presence of
microbes [27]. Danger receptors, in contrast, sense disturb-
ances in host cell homeostasis typically caused by infec-
tions such as, for example, the release of molecules from
cells dying in an uncontrolled fashion [28]. Danger recep-
tors therefore detect the presence of microbes indirectly.
Selective autophagy of pathogens evidently relies on both
PRRs and danger receptors, although it is clear that the full
spectrum of receptors contributing to anti-microbial autop-
hagy is not yet known, nor is it well understood how these
receptors trigger autophagy.
Vesicular damage as an ‘eat-me’ signal
Bacterial entry into the cytosol of host cells requires
significant damage to the limiting membrane of the phago-
some (Figure 2). Such membrane damage is sensed by
cytosolic lectins of the Galectin family, which bind to b-
galactoside-containing glycans exposed on damaged
vesicles. Galectin-3 was originally shown to be recruited
to the vesicle membrane remnants following cytosolic
entry of S. flexneri, but other Galectins, in particular Galec-
tin-1, Galectin-8, and Galectin-9, are also recruited to
damaged vesicles [29,30]. Furthermore, damage caused
by diverse bacterial species, including L. monocytogenes, S.
pyogenes and S. Typhimurium, is detected by Galectins
[30]. Galectins are therefore bona fide danger receptors
that survey the integrity of the cell’s endosomal and
lysosomal compartments and that may both alert the cell
and protect it against invasion by vesicle-damaging patho-
gens. However, only Galectin-8 restricts the proliferation
of S. Tymphimurium [30]. This protective function of
Galectin-8 depends on its ability to induce autophagy by
binding the cargo receptor NDP52, thereby acting as a
novel ‘eat-me’ signal specific for damaged vesicles
(Figure 2). Structural analysis shows a unique binding
pocket in Galectin-8, which interacts with a short peptide
motif in NDP52 (Figure 3) [31,32]. Whether the other
Galectins are involved in different aspects of danger sig-
nalling, or sense damage to other vesicular compartments
remains to be resolved. Further autophagy-inducing sig-
nals caused by invading bacteria have been discovered, but
their relevance is not well understood. Tecpr1, an ATG5-
binding protein, is required for the autophagy of S. pyogenes
and an autophagy-sensitive mutant strain of S. flexneri [33].
Salmonella-induced and Shigella-induced membrane
damage is suggested to trigger amino acid starvation and
mTOR-dependent autophagy [34], while accumulation of
the lipid diacylglycerol (DAG) on damaged SCVs triggers a
cargo receptor-independent pathway [35]. Finally,
depletion of the NADPH oxidase subunit p22phox in
non-phagocytic cells impairs autophagy of S. Tymphimur-
ium [36], though this may be a LAP-related process.
Induction of autophagy by cytosolic pattern
recognition receptors
Several cytosolic pattern recognition receptors for pepti-
doglycan induce anti-bacterial autophagy. In Drosophila,
Current Opinion in Microbiology 2013, 16:339–348
342 Innate immunity

Figure 2

1 2
Salmonella Salmonella Salmonella

Salmonella LRSAM1

Gal9
Gal3
Gal8
NDP52

4 Ub Ub

Salmonella Ub Ub Salmonella Ub
p62
NDP52
Ub Ub
Ub
Ub Ub
Nap1 / Sintbad Optn
Gal8 Ub
NDP52

P
p62 TBK1
Ub
Optn ATP
Nap1 / Sintbad
P
TBK1

ATP

LC3C

Salmonella NDP52
Ub Gal8
Ub Ub
Ub Ub
5
LC3C
Gal8
Ub NDP52
NDP52

Ub p62 LC3
Nap1 / Sintbad Optn
P GBR
TBK1 GBR
LC3

ATP

Current Opinion in Microbiology

Targeting of cytosol-exposed S. Typhimurium for cargo receptor-dependent autophagy. S. Typhimurium frequently causes damage to the limiting
membrane of its vacuole (1). Bacteria may either escape fully from the damaged vacuole (2) or they may stay associated with membrane remnants
whilst glycans previously hidden inside the vacuole are recognized by members of the cytosolic Galectin family of proteins (3). Galectin-8 comprises
the eat-me signal that recruits the cargo receptor NDP52 directly to damaged membranes. Ubiquitylation is directed against proteins of the
damaged membrane and/or bacterial proteins (4). Ubiquitylation may depend on danger receptors and/or PRRs that recruit E3 ligases or that
directly encode E3 ligase activity such as LRSAM1. Ubiquitylation recruits p62 and Optineurin and is also required for the maintenance of NDP52

Current Opinion in Microbiology 2013, 16:339–348 www.sciencedirect.com

 ‘Eat-me’ signals and autophagy cargo receptors Boyle and Randow 343

Figure 3

90°

Current Opinion in Microbiology

Cargo receptor binds ‘eat-me’ signal: NDP52 in complex with Galectin-8. The C-terminal carbohydrate recognition domain (CRD) of Galectin-8 (pink),
highlighting its bent b-sandwich structure, in complex with NDP52368–381 (blue) and lactose (grey) bound to the convex and concave faces of NDP52,
respectively. Model derived from superimposed binary complexes (PDB 4FQZ and 3VKM). Note that Galectin-8 employs its N-terminal, not its C-
terminal, CRD to bind glycans on damaged vesicles.

peptidoglycan recognition protein LE (PGRP-LE)
restricts the growth of L. monocytogenes in an autophagy-
dependent manner, while in mammalian cells NOD1 and
NOD2 antagonize bacteria [37–39]. How these PRRs
induce autophagy is not known, although NODs enable
the recruitment of ATG16L1 to the site of bacterial entry.
Importantly, polymorphisms in NOD2 and ATG16L1 are
major risk alleles for Crohn’s disease, suggesting that the
aberrant detection of bacteria in the cytosol and their
removal by autophagy contribute to disease [40]. Other
members of the NOD-like receptor (NLR) family form
large multi-protein complexes termed inflammasomes
that are required for the generation of interleukin-1b
and related cytokines. While certain NLRs detect bac-
terial PAMPS, for example flagellin with the help of
NAIP5 in the case of NLRC4, others, such as the arche-
typal NLRP3 inflammasome, are activated by danger
signals [41]. NLRP4 and other NLR proteins interact
with the Vps34-binding protein Beclin-1, with the inhibi-
tory effect of NLRP4 on Beclin-1 alleviated upon sensing
of S. pyogenes, thereby activating autophagy [42]. Further
connections between the NLRs, the inflammasomes and
autophagy are likely to exist.
The E3 ubiquitin ligase LRSAM1 has been implicated as
a PRR for Gram-positive and Gram-negative bacteria as it
localizes to invading bacteria and features leucine-rich
repeats (LRR), a domain well-known to bind PAMPs in
other PRRs [43,44]. However, LRSAM1’s potential
bacterial ligand as well as the substrate for its ubiquitin
ligase activity remain unknown. LRSAM1 also binds
NDP52 and may stabilize NDP52 on ubiquitin-positive
bacteria [43]. Baterial-derived DNA found associated
with the bacterial cell surface is a PAMP that contributes
to the ubiquitin coating of M. tuberculosis in a STING-
dependent manner [23]; the E3 ubiquitin ligase respon-
sible remains unknown.
Ubiquitin as an ‘eat-me’ signal
The deposition of poly-ubiquitin in proximity to cytosolic
bacteria is critical for their autophagic removal [19,20].
We refer to these deposits as the ‘ubiquitin coat’ to
indicate that the identity of the proteins undergoing
ubiquitylation in vivo remains unknown. The substrates
for ubiquitylation are likely bacterial surface proteins,
host proteins embedded in vacuolar remnants or proteins
that bind and accumulate on either the bacteria or the
membrane remnants (Figure 2). Ubiquitin can form eight
different chain types and multiple chain types are found
in the vicinity of cytosolic bacteria. K48 and K63 chains
are present on Mycobacterium marinum while K63 and
linear chains are produced on S. Typhimurium
[45,46]. LRSAM1 is the first E3 ligase shown to con-
tribute to the ubiquitin coating of S. Typhimurium and to
restrict bacterial proliferation, though the chain type(s)
produced remain uncharacterized [43]. The complex
scenario of bacteria entering the cytosol and damaging
host structures suggests that in addition to LRSAM1

(Figure 2 Legend Continued) once the Galectin-8 signal ceases. NDP52 recruits TBK1 via Sintbad and Nap1, thereby promoting the
phosphorylation of Optineurin, which enhances its affinity for LC3B. NDP52 selectively binds LC3C, the only ATG8 family member that individually is
essential for anti-Salmonella autophagy (5). The relative contribution of autophagy targeting either membrane-associated or free cytosolic bacteria
remains unknown. Abbreviations: ATP, adenosine triphosphate; Gal, Galectin; GBR, Gabarap; TBK1, TANK-binding kinase 1.

www.sciencedirect.com Current Opinion in Microbiology 2013, 16:339–348

344 Innate immunity
further E3 ligases are involved and generate multiple chain
types on bacteria-derived and host-derived proteins.
In addition to detecting bacteria displaying ubiquitin and
other ‘eat-me’ signals, cargo receptors may also sculpt the
ubiquitin landscape around bacteria. For example, p62
interacts with the ubiquitin ligase TRAF6, potentially
establishing a feed-forward loop of further ubiquitination
and p62 recruitment [47]. Optineurin, on the other hand,
interacts with the deubiquitinating enzymes CYLD and
A20 [15,48]. The sequential recruitment of ubiquitin
ligases and deubiquitinating enzymes with different link-
age specificity may result in editing of the ubiquitin coat,
potentially informing host cells how long bacteria have
persisted in their cytosol and whether alternative defence
mechanisms are required.
Non-overlapping roles of cargo receptors
NDP52, p62 and Optineurin are each required to restrict
the proliferation of S. Typhimurium in human cells
[13,49,50], indicating distinct and unique contributions
by each receptor to cell-autonomous defence. The non-
overlapping roles of the cargo receptors are likely due to the
differences in their interaction with upstream ‘eat-me’
signals and/or downstream effectors. A unique contribution
of NDP52 is its detection of Galectin-8-positive bacteria
[30], but specificity in the recognition of the ubiquitin
signal, conferred by the affinities of the receptors for
different poly-ubiquitin chain types may also exist. The
limited availability of ubiquitin chains with defined linkage
types for in vitro studies has prevented a comprehensive
analysis of the cargo receptors’ binding specificities. Cur-
rently available data suggest that p62 preferentially binds
K63 over K48 chains, while also binding linear chains
[12,51]. Similarly, Optineurin binds both K63 and linear
chains whereas the ubiquitin-binding preference of
NDP52 remains to be established [13,52]. The affinity
for different chain-types in vivo may be affected by post-
translational modifications, as shown for the increased
affinity of p62 for both K48 and K63 chains upon phos-
phorylation by casein kinase 2 [53]. The availability of
distinct ligands on cytosolic S. Typhimurium for the three
cargo receptors is supported by the accumulation of p62 in
ubiquitin-positive micro-domains distinct from those occu-
pied by both NDP52 and Optineurin [13,30,54].
Whether p62 and NBR1 co-localize on bacteria as they
do on proteinaceous autophagy cargo is unknown, though it
was recently shown that on ubiquitylated peroxisomes p62
and NBR1 micro-domains only partially overlap [16].
The contribution of NBR1 to anti-bacterial autophagy is
unclear. NBR1 is recruited to S. flexneri and F. tularensis
but not, unlike p62, to Mycobacterium bovis [55–57]. In
contrast to p62, NDP52 and Optineurin, NBR1 is not
essential to target S. tymphimurium for autophagy [50]. A
more functional analysis of the role of NBR1 in anti-
bacterial autophagy is still required.
Current Opinion in Microbiology 2013, 16:339–348
Do NDP52, p62 and Optineurin engage distinct
signalling pathways?
Distinct interactors for the cargo receptors have been
identified and their recruitment could contribute to the
non-redundant function of the cargo receptors. NDP52
was initially purified as a ubiquitin-binding adaptor of
Sintbad/Nap1/TBK1 complexes, an important immuno-
modulatory kinase complex, which it recruits to cytosolic
S. Typhimurium [49,58,59]. Cells lacking TBK-1 exhibit
profound hyper-proliferation of S. Tymphimurium while
depletion of TBK1 in macrophages impairs killing of M.
bovis [49,60,61]. TBK-1 controls autophagosome matu-
ration, in part through autophagosomal delivery of the
hydrolase cathepsin D, and phosphorylates p62 on
Ser403, a site previously shown to increase the affinity
of p62 for ubiquitin [53,61]. TBK-1 also phosphorylates
Optineurin at Ser177, thereby increasing its affinity for
L3CB [13]. Ser177 in Optineurin is also phosphorylated
by Polo-like kinase 1, which promotes the nuclear local-
ization and pro-mitotic function of Optineurin [62].
Whether Polo-like kinase 1 impacts upon Optineurin’s
anti-bacterial functions or whether cells contain distinct
pools of Optineurin remains to be established.
Further evidence for cross talk between NDP52 and
Optineurin is their shared ability to bind the microtubule
motor protein Myosin VI at sites overlapping with their
ubiquitin binding domains — another example of con-
vergent evolution amongst autophagy cargo receptors
[63]. NDP52 and Optineurin may therefore initially
recruit ubiquitylated cargo to the concave side of the
phagophore membrane while subsequently tethering
Myosin VI-positive microtubules to the outer autopha-
gosomal membrane in a step required for the fusion of
autophagosomes with lysosomes. Although the import-
ance of Myosin VI for anti-bacterial autophagy remains to
be tested, a NDP52/Optineurin/TBK1 complex (or sub-
complexes containing TBK1) may act as a distinct signal-
ling hub on bacteria coated with ubiquitin and possibly
also with Galectin-8.
Specific modes of action for p62 include its ability to
interact with atypical protein kinase C zeta, with Raptor
and also with the large scaffold protein ALFY that recruits
the ATG5–ATG12–ATG16 complex to PI(3)P-positive
phagophores, during the autophagy of protein aggregates
[64–66]. P62 controls yet another, quite distinct, aspect of
anti-bacterial autophagy, namely the delivery of cytosolic
proteins including the ribosomal protein Fau to autopha-
gosomes for the generation of anti-microbial peptides
[57,67].
The CLIR — a LC3C-specific binding site in
NDP52.
The tethering of cargo to the phagophore membrane is
the major downstream function of cargo receptors. Their
interaction with ATG8 family members is mediated by
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 ‘Eat-me’ signals and autophagy cargo receptors Boyle and Randow 345

Figure 4

aromatic
LC3/GBR
pocket

W
N LIR / CLIR C p62 LIR
L

aliphatic
pocket

aromatic LC3C
N pocket

C I V
NDP52 CLIR
L V

aliphatic
pocket

Current Opinion in Microbiology

How cargo receptors bind ATG8 family members and how NDP52 and LC3C selectively interact. (Left) Topology diagram for the interaction of cargo
receptor LIR/CLIR motifs (blue) with ATG8 orthologs (yellow). Alpha-helices are shown as cylinders and beta-strands as arrows. (Right) Enlargement of
the interaction area showing binding pockets (grey circles) in LC3C (lower panel) and its paralogs (LC3, GBR) (upper panel). The canonical LIR of p62
(WxxV) engages the aromatic and the aliphatic pocket that are present in all ATG8 family proteins. The NDP52 CLIR, lacking the aromatic residue, fails
to bind through the aromatic pocket and relies on compensatory contacts through binding pockets found solely in LC3C.

LC3-interacting regions (LIRs) that form an intermole-
cular beta-sheet upon binding. The canonical LIR motif
W/YxxL/I occurring in p62, NBR1 and Optineurin pro-
vides little selectivity towards individual ATG8 orthologs
[11–13]. In contrast, NDP52 specifically binds LC3C via
an LVV peptide, called a CLIR (Figure 4) [68]. The lack
of an aromatic residue in CLIR prevents it from occupy-
ing the major binding pocket of canonical LIRs, the
aromatic pocket. Compensatory hydrophobic contacts
can be established with LC3C but not other ATG8 family
members, thereby explaining the selective binding of
NDP52 to LC3C. LC3C is necessary for the restriction
of Salmonella proliferation and is required for the recruit-
ment of the other ATG8 orthologs to bacteria, thereby
establishing a hierarchy amongst ATG8 family members
[68]. Thus, NDP52 is unique amongst the cargo recep-
tors in its ability to both bind the ‘eat-me’ signal Galectin-
8 and LC3C. By discriminating in favour of LC3C
NDP52 controls the recruitment of all ATG8 orthologs
to bacteria. Such selectivity of cargo receptors for ATG8
orthologs may be more prevalent than appreciated, since
in vivo p62 binds selectively to lipidated, but not soluble,
LC3B compared to GABARAP-L2 [69]. Further
examples of selective binding to ATG8L proteins in vivo
were observed in a proteomic study [70]. It will be
interesting to test whether phosphorylation of Optineurin
enhances its affinity for ATG8 orthologs globally or
selectively.
The NDP52–LC3C pair seems to lie at the heart of a
selective autophagy pathway that, in human cells, appears
dedicated to the removal of membrane remnants and
invading bacteria. In rodents, however, LC3C has been
lost and NDP52, whose expression seems restricted to
early developmental stages (http://biogps.org/#goto=gen-
ereport&id=76815; http://www.ncbi.nlm.nih.gov/Uni-
Gene/ESTProfileViewer.cgi?uglist=Mm.296049), has
lost its binding sites for Galectin-8 and ubiquitin. The
finding that murine NDP52 localizes to ubiquitylated M.
tuberculosis and restricts its proliferation is therefore
especially interesting and requires further investigation
[23].
Professional cytosol-dwelling bacteria escape
autophagic restriction
The anti-bacterial function of selective autophagy must
provide significant protection to host cells, since a number
of bacterial species have evolved counter strategies to
evade detection and capture by autophagy. Escape of L.
monocytogenes from autophagy requires the bacterial actA
and inlK genes, possibly to disguise bacteria in a coat of
actin and major vault protein respectively [71,72]. S.
flexneri avoids autophagy by shielding an ATG5 binding
site in icsA, another actin polymerization-inducing
protein, with the help of icsB [73]. However, icsA-de-
pendent actin polymerization is detected by septins,
which leads to the immobilization of bacteria and their

www.sciencedirect.com Current Opinion in Microbiology 2013, 16:339–348

346 Innate immunity
association with NDP52, p62 and LC3 [24,55]. F. tular-
ensis also actively antagonizes autophagy, by an unknown
mechanism, as auxotrophic and chloramphenicol-treated
bacteria are targeted by p62 into autophagosomes [56].
Intriguingly, wild type F. tularensis are still decorated with
both ubiquitin and p62, but evade autophagic capture,
suggesting a defect downstream of p62, such as an
inability to recruit ATG8L-positive phagophores.
Indeed, interference with cargo receptor function may
be a common strategy for different bacteria to avoid
autophagy. For example, Legionella pneumophilia employs
the protease RavZ to irreversibly inactivate ATG8L
proteins by cleaving them from the autophagosomal
membrane, thereby removing the docking site for cargo
receptors [74]. On the other hand, the Cif proteins of
Burkholderia pseudomallei and enteropathogenic Escheri-
chia coli impair ubiquitin chain synthesis by deamidating
ubiquitin and NEDD8, which potentially could interfere
with labelling of bacteria with poly-ubiquitin ‘eat-me’
signals [75].
Future work
Linking cargo to the phagophore membrane is an import-
ant task of autophagy receptors. Although cells deficient
in components of the ATG8L lipidation machinery can-
not restrict the proliferation of S. Typhimurium, they still
assemble an autophagosomal membrane around the
damaged SCV [76]. Therefore, even in situations when
cargo receptors cannot detect phagophores via their LIR
or CLIR motifs, bacterially induced autophagy remains
activated. Yoshimori and colleagues have defined the
most proximal events required for anti-bacterial autop-
hagy in their ‘three-axis’ model where the ATG5–
ATG12–ATG16L1 complex, the ULK complex, and
the transmembrane protein ATG9L are independently
recruited to Salmonella [77]. We speculate that the cargo
receptors may help recruit the proximal autophagy com-
plexes, via undefined mechanisms, and thereby coordi-
nate multiple stages of the autophagy pathway.
Acknowledgements
We would like to thank members of our laboratory for critical comments on
the manuscript. This work was supported by the Medical Research Council
(U105170648) and The National Association for Colitis and Crohn’s Disease
(M/11/3).
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