REVIEWS
Phagosome maturation:
going through the acid test
Jason M. Kinchen and Kodi S. Ravichandran
Abstract | Phagosome maturation is the process by which internalized particles (such as
bacteria and apoptotic cells) are trafficked into a series of increasingly acidified
membrane-bound structures, leading to particle degradation. The characterization of the
phagosomal proteome and studies in model organisms and mammals have led to the
identification of numerous candidate proteins that cooperate to control the maturation of
phagosomes containing different particles. A subset of these candidate proteins makes up
the first pathway to be identified for the maturation of apoptotic cell-containing
phagosomes. This suggests that a machinery that is distinct from receptor-mediated
endocytosis is used in phagosome maturation.
Receptor-mediated
endocytosis
The process by which a
ligand-bound receptor is
internalized into a
membrane-bound vesicle.
These vesicles sequentially
acquire different Rab GTPases.
Pinocytosis
The process by which liquids
and small particles are
internalized by the cell.
Complement
A protein complex component
of the innate immune system
that can bind to foreign
particles and initiate their
phagocytosis.
Beirne B. Carter Center for
Immunology Research and
the Department of
Microbiology,
University of Virginia,
Charlottesville, Virginia
22902, USA.
e-mails:
kinchen@virginia.edu;
ravi@virginia.edu
doi:10.1038/nrm2515
Eukaryotic cells internalize a variety of particles during
their lifetime. The uptake of particles >0.5 µm in size
is termed phagocytosis, whereas particles <0.5 µm are
taken up by receptor-mediated endocytosis or pinocytosis.
Distinct types of phagocytosis tend to be ligand specific:
bacteria (~0.5–3 µm) or yeast (~3–4 µm) are internalized
by macrophages through scavenger receptors; micro­
organisms can also be coated with serum components
(for example, complement) or antibodies and then taken
up through complement1,2 or Fc receptors3,4, respectively.
Cells that undergo apoptosis, which can range in size
from 5 to 50 µm, must also be removed (BOX 1). In fact,
in humans ~200 billion cells are turned over each day
per individual, making apoptotic cell removal one of
the most common types of phagocytosis5. Thus, under­
standing how apoptotic cells are phagocytosed and pro­
cessed is a fundamentally important biological problem.
Apoptotic cell turnover begins with the induction of
an apoptotic programme or other cellular changes that
mark the cell for removal6,7. The subsequent recognition
of altered features by phagocytes leads to their highly
efficient and immunologically silent removal8. Apoptotic
cells can be taken up either by neighbouring cells or by
professional phagocytes, such as macrophages9–11 and
dendritic cells12,13. The contribution of each type of
phagocyte to clearance in vivo has not been extensively
addressed.
Phagosome maturation can be viewed as the end of
the phagocytic process, during which internalized apop­
totic cells or bacteria are efficiently degraded. After a
particle is internalized, it ‘matures’ through a series of
increasingly acidified membrane­bound structures
nATurE rEvIEWs | molecular cell biology
called phagosomes14. These phagosomes sequentially
acquire different proteins (for example, the Rab GTPases15)
during maturation and eventually fuse with acidic lyso­
some structures. Defects in degradation have a number
of consequences that depend on the type of particle to
be removed. For example, defects in lysosome­associated
membrane protein­2 (LAMP2; see below) block phago­
some maturation and have been linked to periodontal
disease, owing to decreased neutrophil­mediated bacte­
rial killing16. Defects in apoptotic cell degradation can
result in autoimmune disease17. For example, apoptotic
cell removal by dendritic cells has been implicated
in the establishment of tolerance, the process by which
the immune system does not respond to self­derived
antigens9,12,13,18,19. Phagosome maturation is therefore of
great importance to normal homeostasis of the immune
system, to the response to bacterial pathogens and in
influencing the immune response to our normal bacte­
rial flora. Moreover, as certain pathogens seem to use the
phagocytic machinery to enter and/or live within
the host, understanding the various steps of cargo
handling in the context of phagocytosis could be key in
therapeutic design.
We focus on how apoptotic cell­containing phago­
somes mature within the phagocyte, and on the recent
developments in our understanding of the genetic,
molecular and functional aspects of phagosome matura­
tion in various contexts. Extensive studies on receptor­
mediated endocytosis over the past few decades have
allowed the development of a general paradigm for this
process: after the binding of a soluble ligand (such as a
growth factor) to a receptor, the membrane invaginates
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Box 1 | Removal of apoptotic cells
This review mainly deals with phagosome maturation.
However, the recognition and subsequent internalization
of the target precedes the processing of the cell corpse.
Signalling during internalization (see figure) seems to
Apoptotic cell
regulate phagosome maturation, the specific post‑
engulfment responses of the phagocyte and, in turn,
CED-10 (RAC1)
important biological consequences.
In the nematode and mammalian models, numerous
CED-5 (DOCK180)
CED-1
CED-2 (CrkII)
receptors (for example, CED‑1, LRP1, CD36, TIM4, BAI1
(MEGF10, LRP1) CED-6 CED-7
(GULP) (ABCA1, CED-12 (ELMO)
and MER; reviewed in ReFs 8,128) and adhesion molecules
ABCA7)
(for example, CD14 (ReF. 129)) have been linked to the
CED-10
removal of apoptotic cells. However, the specific ligands of
RAB-5–GDP
these receptors and the intracellular signalling pathways
that lead to cell corpse uptake have only been defined in
some cases (BOX 2). Studies on the simpler nematode
model and further studies in mammals have identified two
Phagosome
partially redundant pathways that have an evolutionarily
conserved role in apoptotic cell removal. In the first
VPS-34
pathway, the proteins CED‑2 (CrkII in mammals), CED‑5
DYN-1
(DOCK180) and CED‑12 (ELMO) function to activate
(dynamin)
130,131
CED‑10 (RAC1)
. In mammals, this pathway functions
downstream of the G‑protein‑coupled‑receptor
133
BAI1 (ReF. 132) during apoptotic cell removal and has been linked to signalling downstream
integrins
. In the Cell
second
NatureofReviews
| Molecular
Biology
pathway, the candidate receptor CED‑1 (MEGF10 and LRP1 in mammals) binds to an unknown ligand on the apoptotic
104
104,134–136
cell and signals through its cytoplasmic tail to the adaptor protein CED‑6 (CED6 or GULP)
, which then signals to
CED‑10. CED‑7 (ABCA1 and ABCA7) is thought to have a role in membrane dynamics137. This second pathway has also been
linked to Rac activation in both nematode and mammalian models98,101, and both pathways seem to coordinately regulate
the actin cytoskeleton41. Studies on phagocytic signalling in other models (such as Drosophila melanogaster and
mammalian cells) have underlined the importance of this pathway for the evolutionarily conserved removal of apoptotic
cell corpses138. VSP‑34, vacuolar sorting protein‑34.
Fc receptors
A group of proteins that bind
to immunoglobulin (Ig)Gopsonized particles, leading to
the activation of phagocytosis.
Rab GTPases
A family of proteins that cycle
between GDP-bound inactive
and GTP-bound active
forms and have a role in
targeting transport to
membrane-bound organelles.
and the vesicle that contains the receptor and the ligand
or cargo from the plasma membrane is excised20–22
(FIG. 1a). Time­lapse studies have shown that this vesicle
moves within the cell through a series of stages (dur­
ing which they acquire rAb5 and rAb7 (ReF. 23)) that
lead to progressive acidification and eventually to fusion
with lysosomes24,25. During these acidification steps, the
cargo dissociates from the receptor, which might be
recycled back to the membrane and the cargo might
be trafficked for degradation or use within the cell26,27.
Although this paradigm has been useful for our current
knowledge of vesicular transport within cells, membrane
biogenesis and fusion events, the phagocytic process
poses several unique challenges.
The first challenge stems from the need of a plasma
membrane to temporarily extend over the entire surface
area of a neighbouring apoptotic cell during engulf­
ment; in many cases, the neighbouring cell is as large or
larger than the phagocyte14. This engulfment is accom­
plished by the mobilization of intracellular vesicles to
the plasma membrane; these vesicles also potentially
transport signalling proteins to the site of internaliza­
tion14,28,29. second, the contents of the phagocytic cargo
brought into the cell (for example, bacteria or apoptotic
cells) need to be ‘unpacked’ and processed in a way
that allows for either an immunological response (in
the case of microorganisms) or immunological toler­
ance (to self­antigens derived from apoptotic cells;
BOX 2)30,31. Third, the engulfment of larger particles,
such as apoptotic cells, often involves the simultaneous
782 | o CTobEr 2008 | voLuME 9
activation of multiple types of receptors that are distinct
from those involved in receptor­mediated endocytosis
or phagocytosis of bacteria8,32 (BOX 1). Therefore, the
paradigm that phagocytosed particles mature through
progressively acidic structures seems to be true 33
(FIG. 1b). However, how to define which molecules regu­
late phagosome maturation, and our knowledge of how
signalling events overlap or differ from endocytosis and
other phagocytic events, have become active areas of
investigation.
Insights from proteomics approaches
using targets such as whole bacteria or latex beads,
early biochemical and proteomics studies attempted
to identify proteins that are involved in phagosome
maturation33. Although this resulted in the identifica­
tion of a vast number of candidate proteins that are
localized to phagosomes, in general these studies did
not address the functional requirements for these pro­
teins in phagosome maturation. However, an excellent
descriptive analysis of the different stages of phagosome
maturation was obtained from these studies, which also
led to the development of a basic descriptive model
for protein recruitment and release from the nascent
phagosome33–35. various proteins must be delivered to
the phagosome, including vacuolar (v)­type ATPases
(which acidify the phagosome), acidic proteases (such
as cathepsins) and major histocompatibility complex
(MHC) class II molecules (for the presentation of
phagosome­derived antigens on the cell surface).
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a
b
Apoptotic cell
Dynamin
Dynamin
Phagocytic
receptor
Clathrin
Receptor-bound
cargo
Adaptins
Recycling
endosome
Clathrin
removal
RAB5 recruitment
RAB5
recruitment
RAB7 recruitment
Concentration
stage
RAB7
recruitment
LAMP1 recruitment
Loss of
RAB5
Recycling
endosome
Lysosome
Lysosome
Fusion to
lysosomes
Golgi
Golgi
Figure 1 | endocytosis as a paradigm for phagocytosis. Phagocytosis and receptor-mediated endocytosis are similar
processes that also have distinct features. a | During receptor-mediated endocytosis, the activated receptor sinks into
the cell, in this case into a clathrin-coated pit21. Clathrin is removed from the vesicle after dynamin-mediated scission
Reviews
| Molecular
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from the membrane20; receptors are then trafficked to the recycling endosome, whereNature
they are
eventually
sortedCell
back
to
the cell surface22. Activated endocytic receptors are also removed from phagosomes and presumably are also targeted
to recycling endosomes (see part b). Endocytosed vesicles acquire the GTPase RAB5, which is activated by guanine
nucleotide-exchange factors (GEFs; such as RABEX5 and GAPEX5)150. Active RAB5 allows homotypic fusion with other
RAB5-positive structures23,57, as well as heterotypic fusion with RAB4-positive recycling endosomes70. RAB5 is then
exchanged for RAB7 through the HOPS (homotypic fusion and vacuole protein sorting) complex23, which contains the
RAB7 GEF vacuolar sorting protein-39 (VPS39), resulting in the recruitment of the RAB7 effector RAB7-interacting
lysosomal protein (RILP) and, ultimately, fusion with acidic lysosomes151 and acquisition of the lysosomal markers
lysosome-associated membrane protein-1 (LAMP1) and LAMP2. b | By contrast, during phagocytosis the plasma
membrane is extended around the apoptotic cell, forming the phagosome8,14. Phagosomes also mature through
RAB5-positive and RAB7-positive stages, which result in fusion with lysosome structures28,33,41,52,57,58; however, phagosomes
seem to use a distinct mechanism for GTPase regulation. In the nematode, known RAB-5 GEFs are not required for
phagosome maturation, and the HOPS complex is not required for the recruitment of RAB-7 to the phagosome. This might
reflect the observations that phagosomes rarely fuse together and tend to mature individually. Other events have been
described43, such as the trafficking of proteins from the Golgi to the phagosome, although whether these occur during
endocytosis remains to be determined. A number of GTPases are associated with the phagosome during phagosome
maturation (BOX 3), but the roles of many of these on the endosome have not been described.
Recycling endosome
A membrane-bound organelle
for the recycling of receptors
following ligand dissociation.
The acid test and its consequences. Acidification of the
phagosome is perhaps the most common read­out used
for addressing protein function in phagosome maturation.
Acidification is essential during phagosome maturation: it
is only when the phagosome hits a sufficiently low pH that
the cathepsin family of acidic proteases become active and
nATurE rEvIEWs | molecular cell biology
the phagocytosed particle can begin to be degraded36. In
addition to degrading proteins into peptides for loading
onto class II and class I MHC molecules, cathepsin D also
has a role in the activation of MHC class II proteins, a role
that is essential for peptide loading37,38. Thus, acidification
is a marker of a ‘functional’ phagosome.
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Box 2 | The consequences of apoptotic cell removal
There are several consequences associated with the internalization of apoptotic cell
corpses. These consequences fall into three general classes that might also be affected
by signalling from the phagosome.
First, the removal of apoptotic cells has been linked to the enhanced secretion of
‘pro‑healing’ cytokines (such as transforming growth factor‑β10 and interleukin‑10
(ReF. 139)) that serve to reduce inflammation in the extracellular surroundings and to
promote wound healing. The signalling pathways that alter cytokine secretion are not
well characterized, but many phagocytic receptors (for example, stabilin‑2 (ReF. 140)
and CD36 (ReF. 139)) have been implicated. Intriguingly, endothelial cells have been
reported to induce pro‑inflammatory signalling (increased secretion of tumour‑
necrosis factor‑α) following recognition of apoptotic cells141; this signalling has
implications for atherosclerotic disease.
Second, phagocytes must also dispose of internalized components of the apoptotic
cell. Few studies have tracked the fate of apoptotic cell components, but two recent
studies have addressed signalling pathways that lead to cholesterol efflux from the cell
and suggest that internalization of apoptotic cells (or targets) activates the nuclear
receptor LXR. LXR activation results in enhanced transcription of the transporter
protein ABCA1 (ReFs 103,142).
Third, protein components of the cell are degraded and cross‑presented by major
histocompatibility complex (MHC) class I molecules11,143 and tend to be excluded from
class II presentation97. Processing of apoptotic cell‑associated antigens seems to be
especially important, and has been linked to the maintenance of self‑tolerance12,19,144.
Potential pathways for DNA degradation have been described in the nematode145–147
and mouse models17; defects in this process, as with defects in cell corpse removal,
have been linked to autoimmune disease, emphasizing that efficient phagosome
maturation is required for health of the organism.
Acidification of the phagosome occurs in two stages.
First, a poorly understood ‘early’ acidification step results
in a relatively small drop in pH39. second, v­type ATPases
—ATP­hydrolysis­driven proton pumps40 — are sub­
sequently trafficked to the phagosome at relatively late
stages. In the nematode Caenorhabditis elegans, phago­
some acidification begins quite early: rAb­5­positive
phagosomes stain weakly with acridine orange, a marker
for acidic organelles41. However, these phagosomes do
not stain as strongly as late­stage apoptotic cells, a pattern
that is consistent with multiple modes of acidification that
depend on the stage of maturation.
Programmed cell death
The process by which a healthy
cell is induced to die,
undergoes apoptosis and is
then cleared and degraded by
a phagocyte.
Vacuolar ATPases. The v­type ATPase is a multisubunit
complex composed of 14 subunits in two separate
structures: the v1 complex mediates ATP hydrolysis,
whereas the v0 complex transports H+ across the endo­
somal membrane40. v­type ATPases are trafficked to the
phagosome and acidify its contents, but where do these
ATPases (and other proteins trafficked to the phagosome)
come from?
In Dictyostelium discoideum, yeast­containing phago­
somes acquire v­type ATPases by fusion with v­type
ATPase­containing endosomes42. In multicellular organ­
isms, the trafficking of v­type ATPases is more complex,
because rather than delivery by endosomal fusion, these
proteins seem to be trafficked from the trans­Golgi to the
phagosome along with cathepsins43. Proteomics studies
have identified a large number of components that local­
ize to phagosomes33–35,44–48. However, the mechanism by
which many of these components arrive at the phago­
somes has yet to be addressed. Intriguingly, coatomer
protein complex­I (CoPI), which is required for vesicle
784 | o CTobEr 2008 | voLuME 9
budding from the Golgi, is also required for phagosome
maturation49, although a role for CoPI in vesicle budding
from the phagosome (or other organelles) is possible.
studies in zebrafish have suggested that v­type ATPases
might have roles in phagosome maturation in addition to
phagosome acidification: the v­type ATPase A1 subunit
is required for apoptotic cell­containing phagosome
fusion with lysosomes, although the exact stage at which
phagosomes are arrested is unknown50. A screen for
genes that are required in the early stages of phagosome
maturation identified C. elegans vha‑16, which encodes
the D subunit of the v0 ATPase41. A direct role for these
ATPases in vesicle fusion seems unlikely, but it is possible
that a low pH might induce conformational changes in
proteins that are required for phagosome maturation.
Alternatively, acidification might result in cleavage of a
target protein by activated cathepsins, which would then
induce maturation. These are mere speculations, however,
and the mechanism by which v­type ATPases regulate
maturation remains uncertain.
Proteomics and contamination. one must sound a note of
caution when analysing proteomics data. A recent study
has suggested that proteomics purification schemes suf­
fer from relatively high contamination from other cellular
membranes, which could be misleading51. Although trans­
ient protein localization to the phagosome might provide
an argument for a role in this process, further studies are
required to validate such candidates, either by rnA inter­
ference (rnAi) or dominant­negative approaches. In this
respect, recent studies in model organisms, such as the
nematode C. elegans, have for the first time identified a
functional pathway for phagosome maturation41,52,53.
Defining signalling pathways
Whereas studies in cultured cells have been instrumental
in identifying the protein components of the phagosome,
an understanding of a comprehensive pathway for how
phagosome maturation is regulated has been lacking.
recent genetic studies, combined with cell biological
approaches in model organisms, have begun to define
the basic pathway for the acidification of apoptotic
cell­containing phagosomes.
C. elegans has been a powerful genetic tool for the
identification of genes that are involved in programmed
cell death; and genes identified in this model have also
been shown to have a role in mammalian phagocytic
uptake (BOX 1). However, the events leading to cell corpse
degradation following phagocytosis have been addressed
only recently. one of the major impediments to these
studies has been the requirement of many endocytic
proteins for cell viability; these challenges necessitate
the identification of rare temperature­sensitive muta­
tions41 or time­consuming clonal screens28,54. rnAi has
bypassed this issue as it is remarkably efficient in the
nematode41, although complementary studies using
mutant alleles are preferable when such mutants are
available. rnAi­based approaches, combined with
genetic and fluorescent markers that define specific
steps in phagosome maturation, allow unbiased screens
to identify regulators at various steps in phagosome
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Table 1 | Genes and proteins required for phagosome maturation during apoptotic cell removal
categories
gene or
protein
name
mammalian
or nematode
orthologue
Function
loss- or reduction-of-function
phenotypes
refs
ced-1
LRP, MEGF10
ced-6
GULP, human
CED6
Recognition of the apoptotic cell and
intracellular signalling
Delayed phagocytosis and accumulation of
RAB-7 and FYVE marker
53,104,
134,136,
137,153
ced-7
ABCA1, ABCA7
ced-5
DOCK180
GEF for CED-10 (also known as RAC1)
Delayed phagocytosis and accumulation of
FYVE marker
53,131,
154,155
rab-2
RAB2
ER-to-Golgi transport and potentially
ER- or Golgi-to-phagosome transport
Delayed (or possibly abnormal) maturation
52,54
rab-5
RAB5
Early endosome maturation
Arrested phagosomes, block in acidification
41
rab-7
RAB7
Late endosome maturation
Arrested phagosomes at the RAB-5-positive
stage
41,53
dyn-1
Dynamin
Recruitment of RAB-5
Enlarged phagosomes, pre-RAB-5 arrest
41
RAB-5
effectors
vps-34
VPS34
PI3K
Blocked in RAB-5 recruitment, no
acidification
41
RAB-7
effectors
vps-11
VPS11
RING-motif-containing protein
41
vps-16
VPS16
Unknown
Arrested phagosomes at RAB-7-positive
stage
vps-18
VPS18
SNARE binding?
vps-33
VPS33
Sec1 homologue, SNARE binding?
vps-39
VPS39
GEF for RAB-7
vps-41
VPS41
Adaptor protein, vacuolar biogenesis
VPS45
Sec1 homologue, vacuolar biogenesis
No gross effect on recruitment of RAB-5 or
RAB-7 to phagosomes
41
vps-45
RAB5
recruitment
Dynamin
DYN-1
Required for RAB5 recruitment
Arrested nascent phagosome, block in
acidification
28,41
GTPases
RAB5
RAB-5
Required for maturation from nascent
phagosome to RAB7-positive stage
Block in acidification
41,61
RAB7
RAB-7
Maturation from the RAB5-positive to
LAMP1-positive phagosome
Unknown
89
RAB5 GEF
GAPEX5
RME-6
RAB5 activation
Blocked RAB5 activation during phagosome
maturation
61
Unknown
role
RhoA
RHO-1
ERM-1
Blocked RAB7 recruitment, phagosome
acidification and protease delivery
89
ERM proteins
Regulate early steps of phagosome
maturation
Caenorhabditis elegans
Phagocytic
genes
GTPases
Unassigned
genes
Mammals
Recent studies in the nematode Caenorhabditis elegans and mammalian systems have helped identify proteins that are involved in various steps of phagosome
maturation during apoptotic cell removal. The genes in the nematode were identified in genetic screens, whereas those in the mammalian systems were identified
using a candidate approach that targets individual molecules. ER, endoplasmic reticulum; ERM, ezrin–radixin–moesin; GEF, guanine nucleotide-exchange factor;
LAMP1, lysosome-associated membrane protein-1; PI3K, phosphoinositide 3-kinase; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein
receptor; VPS, vacuolar sorting protein.
maturation (TABLe 1). several groups have made great
strides towards developing the nematode as an in vivo
model for phagosome maturation41,52,53.
Prenylated
The process by which a
hydrocarbon moiety is
attached to a conserved CAAX
motif in the C terminus of
GTPases.
Phagosome maturation: Rab GTPases and protein
trafficking. rab GTPases have been implicated in con­
trolling transport between membrane­bound organelles.
These proteins function as ‘molecular switches’: in the
inactive conformation, the rab protein is GDP bound
and resides in the cytoplasm in complex with a rab GDP­
dissociation inhibitor (rabGDI), which keeps the protein
inactive55,56. The rabGDI releases the rab GTPase follow­
ing a signalling event, thereby exposing the prenylated tail
nATurE rEvIEWs | molecular cell biology
and allowing it to bind membranous organelles. rab
proteins can then bind to a guanine nucleotide­exchange
factor (GEF), resulting in the exchange of GDP for GTP55.
The now active rabGTP can bind to effector proteins57,
resulting in the recruitment of machinery for further steps
of maturation.
recent genetic studies have shown that rAb­5
(ReF. 41) and rAb­7 (ReFs 41,53) are required for the
removal of apoptotic cells in the nematode, with both
GTPases marking distinct (but partially overlapping)
stages of maturation41. similar to our current knowledge
of maturation of internalized particles in mammalian
systems15,58, rAb­5 functions upstream of rAb­7, as
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phagosomes are arrested at the rAb­5­positive stage
in rab‑7­deficient worms. based on the role of rAb­5
and rAb­7 on endosomes, rab GTPases seem to func­
tion as part of a fusion pore that determines the types
of interactions that vesicles have with other membrane­
bound organelles59. In this respect, rAb­5 and rAb­7
potentially serve as nodes for recycling proteins back to
the plasma membrane and as entry points for vesicles
to deposit lysosomal proteases, with ‘effector’ proteins
binding to the activated rab protein to mediate this
function (see below).
Dynamin
A large GTPase that is involved
in membrane scission, actin
cytoskeletal dynamics and
phagosome maturation.
Focal exocytosis
small vesicles are targeted to
the plasma membrane during
phagocytosis. In turn, this is
thought to produce a local
increase in the volume of
plasma membrane, thereby
allowing the cell to extend its
membrane around large
particles.
FYVE domain
A zinc-finger-containing protein
motif that binds to the
membrane lipid phosphatidylinositol-3-phosphate. This
results in the targeting of the
FYVe-containing protein to
the membrane.
A novel recruitment strategy for RAB5. one of the
earliest known maturation events in a number of sys­
tems is the recruitment of rAb5 to phagosomes41,58,60,61,
although a detailed mechanism for how this is accom­
plished during phagosome maturation is lacking.
recent studies in the nematode41 have implied an
unexpected role for dynamin and the rAb­5 effector
vacuolar sorting protein­34 (vPs­34) in rAb­5 recruit­
ment to nascent phagosomes (FIG. 2), which suggests
that phagosomal recruitment of GTPases is a complex,
regulated process41,62.
The GTPase DYn­1, the nematode orthologue of
dynamin, was identified in forward and reverse genetic
screens for genes that are involved in programmed cell
death28,41. Although initial reports linked DYn­1 to the
internalization of apoptotic cells (by having a role in
focal exocytosis28), this view has now been revised41,53.
The current view, based on studies in mammalian
cells and nematodes 41,62, is that DYn­1 (and mam­
malian dynamin) has a role not in exocytosis or the
internalization of cell corpses, but is instead essential
for phagosome maturation, namely the recruitment of
rAb­5 to the nascent phagosome. Phagosomes in dyn‑1
loss­of­function mutant worms appear arrested, without
recycling of the phagocytic receptor CED­1 back to the
plasma membrane41. Further genetic and cell biological
analysis identified the phosphoinositide 3­kinase (PI3K)
vPs­34 (an orthologue of human PIK3C3 or vPs34)
as another protein that regulates rAb­5 recruitment41.
biochemical analysis revealed a novel protein complex in
which DYn­1 mediates the recruitment of GDP­bound
rAb­5 through interaction with vPs­34, which serves
as a bridging protein. To complicate matters, rAb­5 also
seems to regulate vPs­34 activity63 (this is also shown
during removal of immunoglobulin (Ig)G­opsonized red
blood cells in mammals58,63), and knockdown of rAb­5
blocks the accumulation of PtdIns3P on the phagosome
(see below).
Although this novel complex explains how rAb­5
is recruited to apoptotic cell­containing phagosomes, it
does not suggest a mode of regulation, as rAb­5 must
still be converted to the GTP­bound form for phago­
somes to mature41,64. There are at least three canonical
rAb­5 GEFs in the nematode genome, all of which are
vPs9­domain­containing proteins41: rME­6, rAbX­5
and TAG­333 (orthologues of mammalian GAPEX5,
rAbEX5 and rIn1, respectively). However, not one of
these proteins is required (either singly or redundantly)
for the removal of apoptotic cells, suggesting that a novel
786 | o CTobEr 2008 | voLuME 9
GEF might have this role. regulation of rAb­5 is thus
distinct from other endocytic events (such as the inter­
nalization of proteins from the extracellular space) that
jointly require rME­6 and rAbX­5, suggesting that the
regulation of phagosome maturation differs from that of
receptor­mediated endocytosis.
Intriguingly, a recent study in which a fluorescence
resonance energy transfer (FrET) probe was used to
monitor rAb5 activation in swiss 3T3 cells suggested
that the GEF GAPEX5 might have a role in regulating
GTP loading of rAb5 during removal of apoptotic
cells61. one caveat to this is that the experimental system
is somewhat artificial, requiring overexpression of
integrin proteins (and opsonization with the integrin
ligand MFG­E8) rather than the monitoring of basal
engulfment. The importance of GAPEX5 in the context
of endogenous cell corpse removal (which does not
require the opsonin MFG­E8 in most cases65) therefore
remains to be determined.
Phosphoinositides and RAB5 effectors. The enrichment
of PtdIns3P on the surface of the phagosome can be
regarded as a sign of phagosome maturation from the
rAb5­positive to the rAb7­positive stage in both mam­
malian and nematode models (see below). The recruit­
ment of rAb5 effectors (such as rabenosyn­5 (ReF. 66))
by interaction with GTP­bound rAb5 (in conjunction
with PtdIns3P57) results in the subsequent recruitment
of rAb7 and continued maturation of the phagosome
(see below). There are many known rAb5 effectors in
cultured cells that serve diverse functions57. Effectors
typically mediate homotypic fusion events during endo­
cytosis — for example, among rAb5­positive vesicles,
as in the case of early endosome antigen­1 (EEA1)59,67,68
— or they mediate heterotypic fusion between structures
coated with different rab proteins (potentially between
rAb5­ and rAb7­staining structures, as in the case of
rabenosyn­5 (ReFs 66,69), or between rAb5­ and rAb4­
coated structures, as in the case of rabaptin­5 (ReF. 70)).
Typically, rAb5 effectors share a similar architecture;
they contain a FYVe domain for binding PtdIns3P that is
generated on the phagosome by vPs34 (ReF. 57).
In the nematode, vPs­34 is required for the accu­
mulation of PtdIns3P on the phagosome41, and green
fluorescent protein (GFP)­tagged constructs contain­
ing the FYvE domain from EEA­1 (a component of the
rAb­5 fusion pore) or HGrs­1 (which is required for
formation of multivesicular endosomes71) have been
successfully used to monitor this process41,53,54. vPs­34 is
currently the only rAb­5 effector to be linked to phago­
some maturation in the nematode, and worms that are
deficient in eea‑1 or hgrs‑1 show no defect in cell corpse
removal41, although EEA1 is required for maturation
of Mycobacterium species­containing phagosomes in
mammalian macrophages72. Knockdown of another
known rAb­5 effector, F22G12.4 (the nematode ortho­
logue of rabankyrin), which is required for maturation
of pinocytosed particles73, suggested that this protein
is dispensable for maturation41. Moreover, a targeted
screen of all FYvE­domain­containing proteins in the
genome failed to identify other candidate effectors.
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REVIEWS
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Figure 2 | activation of rab5 during phagosome maturation. During internalization of apoptotic cells, dynamin and
vacuolar sorting protein-34 (VPS34) are recruited to the phagosome by an unknown mechanism. VPS34 can interact with
inactive (GDP bound) or active (GTP bound) RAB5; the recruitment of dynamin to theCVijgZGZk^ZlhqBdaZXjaVg8Zaa7^dad\n
forming phagosome results in the
recruitment of VPS34 and RAB5–GDP to the phagosome41. Dynamin (and DYN-1 in nematodes) has a role in the
maintenance of the actin cytoskeleton; this role also has implications for phagocytosis41,152. Once on the phagosome, an
unidentified guanine nucleotide-exchange factor (GEF) probably activates RAB5 (converting it to the GTP-bound state),
thereby activating VPS34 (ReF. 61), a kinase that generates phosphatidylinositol-3-phosphate (PtdIns3P) on the
phagosome. It is noteworthy that GAPEX5 has been identified as a possible GEF for RAB5 in one mammalian cell context61.
Effector proteins bind to active RAB5 in the context of PtdIns3P on the phagosome57; the assembly of unidentified
effectors on the phagosome results in the exchange of RAB5 for RAB7 and leads to the continued maturation of the
phagosome. RabGDI, Rab GDP-dissociation inhibitor.
Phox homology (PX) domain
A lipid- and protein-interaction
domain that consists of
100–130 amino acids and is
defined by sequences that are
found in two components of
the phagocyte NADPH oxidase
(phox) complex.
other phospholipid­binding proteins, such as those
containing the phox homology (PX) domain, have been
described to function in endocytic events and can bind
to PtdIns3P. Targeted screening of these factors identi­
fied two previously uncharacterized genes with weak
defects in cell corpse removal, but could not place these
genes within the pathway for phagosome maturation41.
nATurE rEvIEWs | molecular cell biology
In summary, it seems that the function and regulation
of rAb­5 during phagosome maturation is distinct from
other endocytic events. Intriguingly, studies in mammalian
macrophages have suggested that Mycobacterium species
target EEA1 as a means of arresting phagosome matura­
tion72, which suggests that the requirements for some
proteins might vary depending on the phagosome cargo.
voLuME 9 | o CTobEr 2008 | 787
REVIEWS
a
CED-1 CED-2
CED-6 CED-5
CED-7 CED-12
CED-10
DYN-1
VPS-34
endocytosis
RABEX5
GAPEX5
RIN1
RAB5
VPS34
RAB-5
Concentrating
endosomes
EEA1
?
RAB-7
?
HOPS
complex
?
LMP-1
LMP-2
PtdIns3P­binding proteins and rAb­5 effectors, and
their roles in phagosome maturation, remain to be
determined.
b Receptor-mediated
Rabenosyn-5
HOPS
complex
RAB7
RILP
LAMP1/2
Figure 3 | Pathways for phagosome maturation and
Nature
Reviews | Molecular
Celland
Biology
endocytosis. Pathways
for phagosome
maturation
endosome maturation are derived from publications that
describe the requirements of each protein in the different
endocytic processes. a | In the nematode, the process of
acidification begins with the recruitment of RAB-5 to the
phagosome (through interaction with DYN-1 and vacuolar
sorting protein-34 (VPS-34)41); this leads to VPS-34
activation and the recruitment of unidentified effectors.
RAB-7 is then recruited to the phagosome, resulting in the
recruitment of the HOPS (homotypic fusion and vacuole
protein sorting) complex. The HOPS complex activates
RAB-7, which promotes further maturation events and the
eventual fusion of the phagosome with lysosome
structures. b | In mammals, a concentration step is begun
by the activation of RAB5 on endocytic vesicles (by the
guanine nucleotide-exchange factors (GEFs) RABEX5,
GAPEX5 or RIN1). During this step, vesicles merge and the
ligand is concentrated23, potentially through the formation
of a fusion pore by the RAB5 effector early endosome
antigen-1 (EEA1), which binds to VPS34-generated
phagosomal phosphatidylinositol-3-phosphate (PtdIns3P).
Another RAB5 effector, rabenosyn-5, binds the HOPS
complex component VPS45, which acts as a GEF and an
effector for RAB7. Rabenosyn-5 binding to VPS45 might
mediate RAB5 recruitment to the maturing vesicle23,66.
The HOPS complex member VPS39 activates RAB7,
resulting in the recruitment of the RAB7 effector
RAB7-interacting lysosomal protein (RILP), potentially
resulting in lysosome fusion151. Although some of these
proteins have been shown to have a role in mammalian
engulfment, a step-wise pathway for apoptotic
cell-containing phagosome maturation in mammalian
systems is only beginning to be defined.
Generation of PtdIns3P on phagosome surfaces must
have a function in maturation given that knockdown of
vPs­34 in nematodes generates a potent inhibition
of phagosome maturation41, but the identity of other
788 | o CTobEr 2008 | voLuME 9
Control of RAB7 activation and function. We do not
know how rAb7 is recruited to the phagosome, or how
recruitment is related to the release of rAb5. recent
studies in cultured cells suggest that rAb5 is simul­
taneously exchanged for rAb7 during endocytosis23
(a process called rab conversion), rather than by a series
of fusion events between rAb5­ and rAb7­positive
structures, as was previously proposed74. recruitment of
rAb­7 independent of vesicle fusion events has also been
observed during time­lapse studies during apoptotic cell
removal in the nematode53. one potential candidate to
regulate this process, the HoPs (homotypic fusion and
vacuole protein sorting) complex, has been implicated
in rAb7 recruitment during endocytosis in mammals23;
however, this complex seems to serve a different function
during phagosome maturation (FIG. 3).
The HoPs complex was first identified in the yeast
Saccharomyces cerevisiae75, and in the nematode it is com­
posed of the proteins vPs­11, vPs­16, vPs­18, vPs­33,
vPs­39, vPs­41 and vPs­45 (TABLe 1). A subset of these
proteins are also involved in transport from the Golgi to
endosomal structures as part of the CorvET (class C
core vacuole/endosome tethering complex)76. vps39 has
been shown to be a GEF for Ypt7 (the S. cerevisiae ortho­
logue of rAb­7)65, and vPs­33 and vPs­45 are sec1
orthologues; these orthologues have been proposed to
function in vesicular tethering during fusion events77,78.
Although the function of the HoPs complex has been
tied to rAb­5 function during endocytosis23, genetic
studies in the nematode suggest instead that this com­
plex functions downstream of rAb­7 recruitment during
phagosome maturation41. Worms with mutated vps‑11,
vps‑16, vps‑18, vps‑33 or vps‑39 contain phagosomes that
are arrested at the rAb­7­positive stage, which suggests
that these proteins might be required for maturation
of phagosomes to phagolysosomes (and/or fusion with
existing lysosome structures)41.
not all HoPs complex members function downstream
of rAb­7 recruitment. one member of the complex,
vPs­45, seems to function after the loss of rAb­5
from the phagosome and before rAb­7 recruitment41.
vPs45 interacts with the rAb5 effector rabenosyn­5
(known as rAbs­5 in C. elegans) during endocytosis
in worms23 and mammals69, providing a mechanism
for HoPs complex recruitment to rAb5­positive vesi­
cles. However, rAbs­5 is not required for phagosome
maturation in C. elegans41, suggesting that vPs­45 might
interact with another rAb­5 effector (or might have an
alternative, unidentified role) during phagosome matura­
tion. vPs­41, however, seems to be required downstream
of rAb­7 function, as phagosomes can recruit rAb­5 and
rAb­7 in vps‑41­depleted worms41. The molecular func­
tion of vPs­41 has not been described, although as with
most HoPs complex members it is required for vesicle
docking41, and further studies are needed to address how
this protein functions both during phagosome maturation
and endocytosis.
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REVIEWS
Box 3 | Rab-family GTPases implicated in phagosome maturation
Proteomics approaches have identified a number of different Rab GTPases (see table) that
are localized to the phagosome at different times. However, most of these GTPases seem
to be localized to the endoplasmic reticulum (ER) or Golgi bodies; these observations
might have biased previous reports of phagosome–ER fusion51,148.
What are the functions of these GTPases on the phagosome? The Rab family of GTPases
typically mediate transport between membrane‑bound organelles, so the most likely role
for them would be the delivery of cargo (for example, from the Golgi and ER to the
phagosome), potentially delivering lysosomal proteases and other degradatory
components. Recruitment of RAB5, potentially the most well‑characterized Rab GTPase,
seems to be related to the carefully timed acidification of the phagosome and might have
consequences for the generation of peptide antigens. Another tantalizing possibility is
that one (or multiple) of these GTPases mediates transport from the phagosome to the ER,
carrying processed antigens that will be presented on the cell surface (by major
histocompatibility complex class I proteins). Regardless of function, these GTPases are an
important target of bacterial pathogens; several bacterial pathogens require these
GTPases for phagosome arrest (for example, RAB14 and RAB22 by Mycobacterium
tuberculosis60,149 and RAB1 by Legionella pneumophila119).
Protein
Function
references
RAB1
ER-to-Golgi transport
15,35,46–48
RAB2
ER-to-Golgi transport
34,48,52,54,
81–84
RAB3
Exocytosis
RAB5
Intermediate between, for example, the plasma
membrane and early endosome and/or recycling
endosomes
RAB6
Retrograde transport, Golgi-to-ER transport
RAB7
Trafficking from early to late endosome or lysosome
RAB8
Trafficking between Golgi, endosomes and plasma
membrane
RAB9
Lysosomal enzyme trafficking, late endosome-to-transGolgi transport
RAB10
Transport from Golgi to polarized membrane (basolateral
face; might cooperate with RAB8)
15,34,35,48
RAB11
Transport from Golgi to polarized membrane (apical face),
vacuolar-ATPase transport
15,34,35,
44, 47
RAB14
Phagosome and early endosome fusion, trafficking
between early endosomes and Golgi
15,34,48
RAB20
Localized to apical membranes and Golgi complex,
potentially involved in vacuolar-ATPase trafficking
15
RAB22
Trans-Golgi-to-endosome transport
15
RAB23
Localized to endosomes and plasma membrane
RAB32
Localized to the mitochondrion
RAB33
Retrograde transport from Golgi to ER
15
RAB35
Cytokinesis
15
Retrograde transport
Transport from the Golgi to the
endoplasmic reticulum (eR).
This process is the reverse of
the normal mode of
eR-to-Golgi transport.
34
15,34,35,41,
44,47,48
35
15,34,35,41,
44,46–48
35,47
15,44,48
15
15,48
signalling pathways that are required for the matu­
ration (or fusion) of rAb7­positive phagosomes into
acidic lysosomes have not been studied in depth; to
date, the HoPs complex has not been implicated in the
maturation of bacteria­ or bead­containing phagosomes.
one rAb7 effector, rAb7­interacting lysosomal protein
(rILP), is required following Fcr­mediated uptake of
opsonized red blood cells; the Gram­negative bacteria
Brucella abortus has been shown to require rILP for
conversion of the phagosome into an endoplasmic
nATurE rEvIEWs | molecular cell biology
reticulum (Er)­derived replicative organelle, suggesting
that this protein is relevant for phagosome maturation79.
similarly, Mycobacterium bovis secretes a protein that
interferes with rAb7 activation, blocking recruitment
of rILP to the phagosome80. The nematode possesses
an orthologue of rILP (J.M.K. and K.s.r., unpublished
observations), but the function of this protein has yet to
be linked to phagosome maturation or to endocytosis
in the nematode.
Other Rab GTPases and the phagosome. Whereas the
roles of rAb5 and rAb7 in phagosome maturation
can be inferred from mammalian studies, the role of
rAb2 is something of an enigma. A number of differ­
ent rab GTPases (including rAb2) have been identified
in proteomics studies34,48,81–84 (BOX 3); genetic studies in
C. elegans have shown that the loss of rAb­2 delays
lysosome fusion54, but how rAb­2 mediates this effect
has not been addressed. In these studies, matura­
tion markers, such as PtdIns3P­binding proteins, are
recruited to the phagosome with normal kinetics54,
which suggests that the defect that leads to lysosome
fusion delay occurs after rAb­5 recruitment. Consistent
with this, knockdown of rab‑5 blocks the recruitment of
rAb­2 to phagosomes (J.M.K. and K.s.r., unpublished
observations).
rAb­2 colocalizes with rAb­5, rAb­7, LMP­1 (the
nematode orthologue of LAMP1; see below) (ReF. 52)
and PtdIns3P markers54 on the phagosome, suggesting
that rAb­2 localizes to phagosomes throughout their
lifespan, rather than at a distinct stage. Indeed, deple­
tion of other phagosome maturation markers invariably
results in rAb­2­positive arrested phagosomes (J.M.K.
and K.s.r., unpublished observations). rAb­2 is also
not required for phagosome acidification per se: recent
studies have shown that apoptotic cell­containing
phagosomes in rab‑2­deficient worms are acidified
and stain with acridine orange 52. However, another
group has suggested that in rab‑2‑deficient worms,
phagosomes might be less acidified compared with
wild­type worms54.
What is the function of rAb2 on phagosomes, if it
does not mark a particular stage of maturation? studies
in mammalian cells have shown that rAb2 is specifically
localized to the Er and Golgi, and might have a role
in retrograde transport or in the movement of proteins
from the Er to the Golgi81,82. It is thus conceivable that
the function of rAb2 might be to direct vesicles from
the Er or Golgi to the nascent phagosome, delivering
cargo­containing degradatory proteases (and, poten­
tially, MHC molecules in mammals). In this context, it
should be mentioned that many rab GTPases that are
associated with the Er and Golgi have been identified
on phagosomes using proteomics approaches15,34,35,45–48,60
(BOX 3). rAb­10, another GTPase that is associated with
Golgi transport85 and recycling endocytosis86, has also
been identified in screens for factors that are involved
in cell corpse removal in C. elegans41. However, at this
time we have no clear understanding of which mole­
cular process rAb­2 (and its mammalian orthologue)
regulates during phagosome maturation.
voLuME 9 | o CTobEr 2008 | 789
REVIEWS
Efficient targeting of vesicles to the phagosome (and
appropriate acidification) also has important implica­
tions for antigen presentation. Dendritic cells isolated
from rAb27A­deficient mice show defects in antigen
presentation related to overacidification of the phago­
some87. Another factor, noX2, is an nADPH oxidase
that generates reactive oxygen species, contributing to
the alkalinization of the phagosome88. Fusion of latex
bead­containing phagosomes with noX2­positive
structures slows acidification, allowing the controlled
generation of antigenic peptides and loading onto MHC
molecules. Loss of rAb27A, which is required for this
fusion event, leads to overacidified latex bead­containing
phagosomes, thereby greatly reducing efficiency of
antigen presentation 87. This work emphasizes the
complex sorting requirements of phagosomes and the
importance of rab proteins to phagosome maturation
(BOX 3). other studies have suggested that this method of
delayed acidification might also be relevant in apoptotic
cell removal, as apoptotic cell­containing phagosomes
are acidified more slowly in dendritic cells compared
with macrophages89.
LAMP proteins and lysosome fusion. LAMP1 and LAMP2
(and the nematode orthologues LMP­1 and LMP­2)
are glycoproteins that are specifically localized to acidic
lysosome structures90, although the precise molecular
functions of these proteins are unknown. LAMP1 and
LAMP2 (and nematode LMP­1) are recruited to the
phagosome; intriguingly, in Lmp1–/– Lmp2–/–‑mutant
mice, phagosomes that contain IgG­opsonized beads
appear arrested at the rAb5­positive, PtdIns3P­positive
stage91, which suggests that these two proteins have roles
in the recruitment of rAb7 to the phagosome. rAb7
and its effector rILP can be found on LAMP­positive
structures91, although a molecular mechanism for LAMP
function during phagosome maturation remains to be
determined.
Phagocytic receptors and maturation
Conceptually, recognition of the target during phago­
cytosis is a desirable signalling mechanism for distin­
guishing cargo. Therefore, the possibility that phagocytic
receptors could have a key role in determining how a
given target is processed for degradation is enticing.
In endocytosis, ligand­bound receptors (for example,
epidermal growth factor receptor) have been shown to
control rAb5 recruitment, although the mechanisms
by which this occurs are sketchy92–94, and the nerve
growth factor receptor actively signals from the endo­
some to activate the ras–MAPK signalling pathway95.
signalling during internalization might be particularly
important for generating either a tolerogenic (apoptotic
cells) or an immunogenic (bacteria) immune response.
recent studies in both nematode53 and mouse models96,97
have led to the proposal of a ‘phagosome autonomous’
model, in which signalling proteins that are enriched
on the phagosome during internalization determine how
the phagosome will mature. This becomes especially
relevant in the case of internalized pathogens, some
of which can inject factors outside of the phagosome,
790 | o CTobEr 2008 | voLuME 9
thereby potentially altering signalling and disrupting
maturation. However, our knowledge of how early
steps in phagocytosis dictate later events of phagosome
maturation is minimal.
Do phagocytic receptors control phagosome maturation?
The contribution of proteins that are involved in apoptotic
cell phagocytosis to phagosome maturation was recently
addressed. In engulfment­deficient worms (for example,
a ced‑1 or ced‑10 mutant), the efficiency of phago­
cytosis is greatly decreased, although many cells are
eventually engulfed53,98. using time­lapse microscopy of
embryogenesis as a model, it was shown that CED­1,
a transmembrane protein that is thought to function as a
phagocytic receptor (BOX 1), was required for the recruit­
ment of a FYvE­domain marker and of rAb­7 to the
phagosome53. This work also suggested that another key
engulfment protein, CED­5 (DoCK180 in mammals; a
racGEF that functions in an alternate engulfment path­
way downstream of an unknown receptor), also showed
reduced phagosomal PtdIns3P and a mild delay in
rAb­7 recruitment28. studies of apoptotic cell removal
in mammalian macrophages have shown that signalling
from rhoA (which negatively regulates apoptotic cell
removal99) and ErM (ezrin–radixin–moesin) proteins
also have a role in the timely recruitment of rAb­7 to
the phagosome89, suggesting that there might be feed­
back between phagosome maturation and apoptotic cell
uptake. Indeed, studies using non­digestible latex beads
have shown that the inability to degrade a target can
result in decreased uptake100.
Many of the studies of this process focus on localiza­
tion of rAb7 or other lysosomal markers (rather than
on earlier markers such as rAb5 or vPs34). studies on
endocytosis have revealed that internalized receptors
can modulate rAb5 activity on vesicles93,94, and one
tantalizing possibility is that a receptor (or co­receptor)
would recruit rAb5 or a rAb5­regulatory machinery
(such as a GEF or GTPase­activating protein (GAP)) to
the phagosome. It is intriguing to note that the punc­
tate localization of dynamin on the phagosome appears
similar to the localization pattern of phosphatidylserine
(Ps) or LrP1 on the phagocytic cup41 (Ps is exposed
by the apoptotic cell during apoptosis and LrP1 is a
phagocytic receptor that is required for apoptotic cell
removal). These localization patterns suggest that the
recruitment of proteins involved in phagosome matur­
ation might indeed be mediated by receptors. studies
in mammalian systems have also suggested that GuLP,
a mammalian orthologue of the adaptor protein CED­6
(which binds to the phagocytic receptor CED­1 (LrP1
in mammals; see BOX 1)), can interact with the endocytic
machinery102,103. Whether this is a mechanism for recy­
cling CED­1 to the cell surface or a mechanism to recruit
maturation proteins remains to be addressed.
one caveat with these studies is that they do not
address how delays in phagocytosis might affect phago­
some maturation. For example, one recent study has
shown that microtubules are required for delivery of
GAPEX5 (a GEF for rAb5) to the phagosome61. In
this respect, a delay in microtubule attachment to the
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REVIEWS
phagosome could affect the timing of maturation with­
out a ‘direct’ role in signalling. Addressing this issue is
likely to be difficult and might require the identification
of mutant proteins that can affect phagosome maturation
but leave phagocytosis intact. In the case of CED­1 (LrP1
in mammals), one study has analysed functional domains
of the protein that are required for cell corpse removal104.
Further clarification of whether higher numbers of
apoptotic cells in this case represent defects in uptake
versus maturation would strengthen the hypothesis
that these proteins direct phagosome maturation.
Toll-like receptor
A transmembrane protein that
recognizes bacterial pathogens
and induces an inflammatory
response.
Mammalian TLRs and phagosome maturation. Toll-like
receptors (TLrs) have many functions and can recog­
nize multiple bacterial antigens to mediate their effects.
During phagocytosis, TLrs serve as co­receptors for
bacteria (in conjunction with a primary receptor such
as CD14 or CD36)105; typically, TLrs do not function as
phagocytic receptors, but instead induce macrophage
activation in response to bacterial antigens, such as
lipopolysaccharide (LPs), resulting in the induction of
an inflammatory response106. Most importantly in this
context, TLr2 and TLr4 seem to control phagosome
maturation and steer the bacterial cargo into the ‘fast
lane’ for both antigen presentation and the induction
of an inflammatory immune response96. When bacteria
and apoptotic cells (which do not engage TLrs) are co­
incubated with phagocytes, the different targets are not
present within the same phagosome, and each target
matures at a different rate. It is important to note that
fast­tracking a phagosome might require multifactorial
signalling events, as studies have shown that the addi­
tion of TLr agonists alone is insufficient to enhance the
maturation of IgG­opsonized beads and other partic­
les107, although this needs to be addressed in greater
detail in vivo.
one of the consequences at the end of phagosome
maturation is the generation of peptide antigens that are
subsequently loaded onto MHC molecules and targeted
to the cell surface38,108. The differential maturation of
phagosomes seems to be an important regulatory mecha­
nism that excludes apoptotic cell antigens from class II
presentation (and avoids the subsequent stimulation of a
self­reactive response). MHC class II molecules are typi­
cally activated by a proteolyic event on the phagosome
(as a result of cathepsin activation37); engagement of TLr
signalling and fast­track maturation results in steps that
favour MHC class II loading, leading to phagocyte activa­
tion and induction of an immune response. Apoptotic
cell­containing phagosomes do not possess peptide­
loaded MHC class II, and apoptotic cell antigens are not
presented on the cell surface on class II molecules97.
normally, peptides derived from antigens that enter
an antigen­presenting cell (for example, macrophages
or dendritic cells) are presented on class II MHC mole­
cules. However, it is well documented that antigens
derived from apoptotic cells are presented by a pro­
cess referred to as antigen cross­presentation on class I
MHC molecules, which generally present Er­derived or
endogenously produced proteins11. This again suggests
that early steps in the recognition of targets entering
nATurE rEvIEWs | molecular cell biology
the phagocyte and/or phagosome maturation dictate
how the antigens derived from the cargo would be pro­
cessed. This influence on phagosome maturation could
have important implications for immune responses to
antigens that are derived from the target.
similar to mammals, the fly has developed primi­
tive innate and cellular immune systems to target and
remove both foreign and self particles109. A number of
genes that encode phagocytic receptors have been iden­
tified in the fly, including croquemort (for the removal
of apoptotic cells and Staphylococcus aureus)110,111, draper
(apoptotic cells and injured axons)112,113, eater (S. aureus,
Escherichia coli and Serratia marcescans)114, nimrod C1
(S. aureus)115 and peste (Mycobacterium fortuitum)116.
The corresponding receptors have yet to be linked to
phagosome maturation in the fly, but orthologues of the
Croquemort protein in mammals (CD36) have been
linked to TLr2 and TLr6 signalling, which suggests
that these receptors might also modulate phagosome
maturation in the fly. In addition, TLrs have been impli­
cated in the innate immune response to bacteria in the
nematode: tol‑1‑deficient worms show significant inva­
sion of Salmonella species into the pharynx117. Whether
this is true ‘invasion’ or due to deficiencies in phagosome
maturation and bacterial degradation remains to be
determined.
Overlap between signalling pathways. The outcomes of
recognizing bacteria (immunogenic response) and apop­
totic cells (tolerogenic response) are different; the differ­
ential activation of signalling during both phagocytosis
and phagosome maturation might be the molecular basis
of this difference. Little is known about how apoptotic
cells generate a tolerogenic state, but much work has gone
into the identification of signalling pathways that regu­
late the inflammatory response to bacteria (reviewed in
ReFs 105,118). The activation of MHC class II molecules
on bacterial phagosomes (but not phagosomes that con­
tain apoptotic cells) supports distinct signalling during
each process97. The molecular basis for this difference is
difficult to understand because MHC class II molecules
are present on phagosomes that contain apoptotic cells97,
but apoptotic cell­derived antigens are not loaded; this
suggests a negative regulatory event. other signalling
events on the phagosome seem to be conserved: the
GTPases rAb5 and rAb7 are present on both bacteria­
and apoptotic cell­containing phagosomes; further, both
types of phagosomes accumulate and lose PtdIns3P dur­
ing maturation and require vPs34 (ReFs 8,9,15,41,58).
How these rab proteins are regulated during bacterial
phagocytosis has yet to be addressed.
Escaping a timely death
The immune system is incredibly efficient at seeking
out and destroying potential pathogens. However, cer­
tain pathogens have developed mechanisms to escape
degradation, persisting (and often replicating) within
phagosomes by co­opting the phagosome­maturation
machinery (TABLe 2) . Legionella pneumophila, for
example, changes the composition of the phagosomal
membrane so that it contains many features of the Er,
voLuME 9 | o CTobEr 2008 | 791
REVIEWS
Table 2 | Bacteria have evolved mechanisms to modify the phagosome
bacteria
mode of action
refs
Brucella abortus
Converts the phagosome into an
autophagosome or ER-like vesicle
156,157
Chlamydia spp.
Form inclusions that are enriched in
sphingomyelin
158,159
Coxiella burnetii
Converts phagosome into a hybrid
autophagosome–RAB7-positive
phagosome
160,161
Cryptococcus neoformans
Phagosome is extruded from living cells by
an unknown mechanism
162,163
Helicobacter pylori
Sequesters RAB7 to block delivery of
proteases
164,165
Legionella pneumophila
Converts phagosome into rough ER
120–122
Leishmania spp.
Inhibits phagosome maturation
166,167
Mycobacterium spp.
Block at the RAB5-positive stage
Salmonella spp.
Blocks maturation at the RAB5-positive
phagosome, potentially by influencing
RAB7 function
166,169
Toxoplasma spp.
Phagosome has characteristics of host
membrane (non-receptor-mediated
uptake)
164,165
168
ER, endoplasmic reticulum.
ARF GTPases
(ADP-ribosylation factor).
A family of small GTPases that
regulate aspects of membrane
trafficking that are related to
the budding of vesicles from
membranes.
allowing it to persist and replicate within the cell119. To
achieve this, the bacteria have developed proteins that
act specifically on rAb1, although how this leads to
phagosome transformation is not well understood120–122.
Many other bacteria similarly modify signalling
to block their degradation within the cell (TABLe 2).
Furthermore, the yeast Cryptococcus neoformans,
rather than blocking phagosome maturation, can induce
expulsion of the phagosome from the cell. one can
speculate that this yeast might convert the phagosome
into an exocytic vesicle, although the mechanism of how
this would be accomplished is unknown.
Intriguingly, recent work has shown that one patho­
gen, Shigella species, seems to use some of the proteins
required for cell corpse removal, for example ELMo1
and DoCK180, for entry into the mammalian cells123
(BOX 1). As phagocytic receptors and co­receptors have
been linked to maturation events in other contexts, and
ced‑5 is required for aspects of phagosome maturation
in the nematode53, it is possible that some bacteria might
use the apoptotic cell­uptake machinery to disguise itself
and avoid detection. A recent study showed that vaccinia
virus might disguise itself in a Ps coat to mimic an apop­
totic cell124, but the consequence of such Ps­dependent
entry to phagosome maturation and presentation of
bacterial or viral epitopes is not yet defined.
one of the major problems in designing drugs to treat
diseases that are associated with these pathogens is our
poor understanding of how they adapt the phagosome to
their life cycle. A more thorough evaluation of the func­
tional role of different proteins on the phagosome, and
an assembly of these proteins into a coherent pathway,
is of key importance to the development of therapeutics
that can restore ‘normal’ phagosome maturation, rather
than inhibiting bacterial growth or replication.
792 | o CTobEr 2008 | voLuME 9
Where do we go from here?
The recent identification of genes and proteins involved
in phagosome maturation is of key importance to basic
science and for the understanding of bacterial patho­
genesis and drug design. unbiased genetic screening
approaches have identified >60 proteins that are poten­
tially involved in phagosome maturation in C. elegans.
Further characterization of proteins identified by genetic
and proteomics approaches and testing them in mam­
malian systems is likely to shed light on the control of
phagosome maturation.
Most studies have focused on the acidification of
the phagosomes as the primary read­out. However,
there are a number of other steps in phagosome matu­
ration, such as the accumulation of specific proteins
on the phagosome (for example, ATPases involved in
phagosome acidification, myosin motors and acidic
proteases89,125,126) or other steps in processing (BOX 2),
that could provide new insights. Indeed, a recent study
in zebrafish in which v0 ATPase function was moni­
tored revealed a novel role for the protein in phagosome
fusion50, although the molecular details (or the stage at
which the phagosome is arrested) remain to be defined.
Moreover, whereas significant attention has been paid
to the rab family of GTPases in phagosome matura­
tion (and justifiably so, given the importance of rab
GTPases in vesicular traffic in other systems; BOX 3), the
role of the ARF GTPases, another family that is linked to
vesicular traffic in cells, is less well defined. As one of the
engulfment proteins, GuLP, has been linked to ArF6­
mediated signalling103, this might be worthy of future
investigation. similarly, the role of microtubules, which
have recently been suggested to have a role in delivery
of a rAb5 GEF to the phagosome61 during phagosome
maturation, needs more detailed investigation.
Another important topic is whether phagosome
maturation and autophagy might share a common
mechanism. both processes share a similar aetiology:
the degradation of a target (internalized particles or
organelles, respectively) within a double­membraned
vesicle. Markers for autophagy (such as LC3 and beclin)
are recruited to mammalian phagosomes127, and loss of
autophagy protein­5 (ATG5) or ATG7 impairs phago­
some acidification. How these proteins are recruited to
the phagosome (and whether they overlap with rAb5,
rAb7 or LAMP1) has not been addressed. However,
these studies suggest that the view of phagosome
maturation as the movement from a phagosome into a
lysosomal structure is simplistic, and signalling might
be more complex than envisioned.
Current knowledge of how bacteria can adapt phago­
cytic and phagosomal signalling to their own designs
is sketchy (BOX 3), and further research on this topic is
important for the generation of novel therapeutic tar­
gets. removal of apoptotic cells is important for innate
immunity, and defects in apoptotic cell phagocytosis and
degradation have been shown to result in autoimmune
disease. Comparative studies on how apoptotic cells
and bacteria are degraded is therefore of great clinical
importance, and further studies into the basic biology of
phagosome maturation are crucial to the identification
www.nature.com/reviews/molcellbio
REVIEWS
of relevant therapeutic targets for persistent bacterial
diseases. How signalling proteins that are required for
phagosome maturation (TABLe 1) can be co­opted by
bacterial pathogens (TABLe 2) is a topic of great interest.
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Acknowledgements
The authors wish to thank colleagues in the engulfment field
and members of our laboratory for insightful discussions. This
work was supported by grants from the National Institute of
General Medical Sciences/National Institutes of Health and
the Strategic Program for Asthma Research (K.S.R), and an
Arthritis Foundation Postdoctoral Fellowship (J.M.K).
DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
croquemort | draper | eater | nimrod C1 | peste | vha-16
UniProtKB: http://www.uniprot.org
DYN-1 | EEA-1 | GAPEX5| LAMP1 | LAMP2 | LRP1| RAB2 |
RAB5 | RAB7 | RAB27A | RABEX5 | RILP | RIN1 | TLR2 | TLR4 |
VPS-34 | VPS-45
FURTHER INFORMATION
Kodi S. Ravichandran’s homepage: http://www.
healthsystem.virginia.edu/internet/cmb/immunology.
cfm?uva_id=kr4h&hideintro=1
all links are active in the online PdF
voLuME 9 | o CTobEr 2008 | 795