Biologia Celular de La Fagocitosis - Flannagan2012

PM07CH03-Grinstein ARI 16 December 2011 7:56
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• Other articles in this volume
• Top cited articles Ronald S. Flannagan,∗ Valentin Jaumouillé,∗
Annu. Rev. Pathol. Mech. Dis. 2012.7:61-98. Downloaded from www.annualreviews.org
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• Our comprehensive search and Sergio Grinstein
Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada;
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email: sergio.grinstein@sickkids.ca
Annu. Rev. Pathol. Mech. Dis. 2012. 7:61–98 Keywords

First published online as a Review in Advance on antigen presentation, antimicrobial, endocytosis, innate immunity,
September 9, 2011
phagosome, receptor
The Annual Review of Pathology: Mechanisms of
Disease is online at pathol.annualreviews.org Abstract
This article’s doi:
Engulfment and destruction of invading microorganisms by phagocyto-
10.1146/annurev-pathol-011811-132445
sis are critical components of the innate immune response. In addition,
Copyright c 2012 by Annual Reviews.
phagocytosis is also required for the clearance of apoptotic bodies, an
All rights reserved
essential aspect of tissue homeostasis and remodeling. Here, we sum-
1553-4006/12/0228-0061$20.00
marize the current knowledge of the cellular and molecular basis of
∗
These authors contributed equally to this work. phagosome formation and maturation. We discuss the manner in which
phagocytosis is subverted by certain pathogens and consider congenital
disorders that affect phagocyte function.
61
INTRODUCTION description of phagocytosis, most of these

mechanisms are poorly differentiated and
Phagocytosis was first observed by Élie Metch-
only superficially understood. Yet, the de-
nikoff (1) more than 100 years ago. Since then,
tailed analysis of a few specific models has
it has been recognized as a critical component
allowed the establishment of key principles of
of the innate and adaptive immune responses
phagocytosis, such as the role of spatiotem-
to pathogens. In addition, more recent stud-
porally coordinated signaling, the need for
ies have revealed that phagocytosis is crucial
cytoskeletal rearrangement, and the occur-
for tissue homeostasis and remodeling. Clearly,
rence of membrane remodeling. Here, we
the phagocytic process is of great biological
describe in detail our current understanding
importance.
of the molecular mechanisms implicated in
Phagocytosis is defined as the ingestion
the recognition, uptake, and degradation of
by cells of large (≥0.5-μm) particles. Unlike
particles by professional phagocytes.
macropinocytosis, phagocytosis involves the

recognition and binding of prey by receptors
on the cell surface. Foreign bodies such as bac- PARTICLE RECOGNITION: A
teria or fungi can be cleared from infection sites RECEPTOR-MEDIATED PROCESS

by professional phagocytes such as neutrophils, Phagocytosis is a receptor-mediated event. Be-
macrophages, and dendritic cells. Phagocytosis cause particles of widely different natures can
thereby contributes to the first line of defense be taken up by phagocytosis, it is not surprising
against infection. It also plays a key role in the that numerous receptor types can mediate this
initiation of the adaptive immune response; by process. Because of space constraints, and be-
promoting the release of proinflammatory cy- cause the existing evidence is woefully incom-
tokines, engagement of phagocytic targets can plete, we describe only a handful of prototypical
attract lymphoid cells. Moreover, professional receptors. Receptors involved in the phagocy-
phagocytes can present to lymphoid cells anti- tosis of foreign bodies and those mediating the
gens derived from the degradation of engulfed uptake of apoptotic (endogenous) corpses are
particles. discussed separately, as these processes lead to
Although they are unable to internalize very different outcomes.
microbes, fibroblasts, epithelial cells, and en-
dothelial cells can also perform phagocyto-
sis. These cells can ingest apoptotic bodies, Receptors That Engage
thereby contributing to the clearance of bil- Foreign Bodies
lions of cells that are turned over every day. Detection and clearance of foreign bodies, such
Apoptotic bodies are also targets of professional as infectious microbes, were the first acknowl-
phagocytes. Remarkably, whereas professional edged functions of phagocytosis. These pro-
phagocytes unleash an inflammatory reaction cesses involve a variety of receptors that detect
when engulfing foreign bodies, they release microbial molecules either directly or through
anti-inflammatory mediators when confronted the intermediate of opsonins.
with apoptotic bodies, preventing further tissue
damage. Pattern-recognition receptors. Foreign par-
Owing to the number of different phagocyte ticles such as bacteria, fungi, and parasites
types, the variety of their targets, and the com- display many molecules that are never found in
plexity of their interactions, these engulfment higher organisms. These pathogen-associated
processes are not always identical. Indeed, the molecular patterns (PAMPs) can be detected
term phagocytosis encompasses a diversity of by several receptors, in particular the Toll-
related yet distinct molecular mechanisms. like receptors (TLRs), but also by some
It is striking that, a century after the first phagocytic receptors (Table 1). For instance,
62 Flannagan · Jaumouillé · Grinstein

Table 1 Human receptors mediating phagocytosis, and their ligands

Receptors Ligands Reference(s)
Pattern-recognition receptors
Mannose receptor (CD206) Mannan 2
Dectin-1 (CLEC7A) β1,3-glucan 3
CD14 Lipopolysaccharide-binding protein 215
Scavenger receptor A (CD204) Lipopolysaccharide, lipoteichoic acid 4, 216
CD36 Plasmodium falciparum–infected erythrocytes 217
MARCO Bacteria 218
Opsonic receptors
FcγRI (CD64) IgG1 = IgG3 > IgG4 6
FcγRIIa (CD32a) IgG3 ≥ IgG1 = IgG2 6
FcγRIIc (CD32c) IgG —

FcγRIIIa (CD16a) IgG 6
FcαRI (CD89) IgA1, IgA2 219
FcεRI IgE 220

CR1 (CD45) Mannan-binding lectin, C1q, C4b, C3b 221
CR3 (αM β2 , CD11b/CD18, Mac-1) iC3b 7
CR4 (αV β2 , CD11c/CD18, gp150/95) iC3b 7
α5 β1 Fibronectin, vitronectin 222
Apoptotic corpse receptors
TIM-1 Phosphatidylserine 20
TIM-4 Phosphatidylserine 20
BAI1 Phosphatidylserine 22
Stabilin-2 Phosphatidylserine 21
Mer Gas6, protein S 24
αV β3 MFG-E8 23
αV β5 Apoptotic cells 223
CD36 Oxidized lipids 25
Abbreviations: BAI, brain-specific angiogenesis inhibitor; CR, complement receptor; Ig, immunoglobulin; MFG, milk fat
globule; TIM, T cell immunoglobulin mucin.
polysaccharides present on the surface of some response that is mediated by other receptors
yeast cells attach to the mannose receptor or (5).
to Dectin-1 (2, 3), and the lipopolysaccharide At present, the repertoire of known phago-
(LPS) displayed by gram-negative bacteria is cytic receptors for PAMPs remains limited and
detected by the scavenger receptor A (4). Some does not cover the full spectrum of infectious
of these receptors, such as Dectin-1, suffice to agents that professional phagocytes confront.
trigger phagocytosis. Indeed, their heterolo- The list of PAMPs and their cognate receptors
gous expression confers phagocytic properties will probably expand in the future.
to cells that are otherwise unable to recognize
and engulf PAMP-bearing particles (2, 3). In Opsonic receptors. Foreign bodies can also
other cases, the contribution of PAMP recep- be recognized by soluble molecules circulating
tors to phagocytosis is controversial and may in the blood and in interstitial fluids. Im-
occur indirectly, either by tethering the prey munoglobulins recognize extraneous antigens,
to the cell surface or by priming the phagocytic and components of the complement cascade
www.annualreviews.org • Cell Biology of Phagocytosis 63

deposit on foreign surfaces. Following their Even before lateral clustering occurs, multi-
deposition on particles, these molecules— ple receptors need to attach to the prey to secure
termed opsonins—are in turn engaged by it onto the phagocyte surface. The low affin-
receptors on the membrane of phagocytes, ity of most phagocytic receptors accounts for
which thereby indirectly latch onto the this need; several receptors have to be engaged
surface of their prey. Receptors to the Fc simultaneously to counteract the tendency of
portion of immunoglobulin G (IgG) and the particle to detach either by Brownian mo-
receptors that bind complement com- tion or by pathogen motility. Engagement of
ponent iC3b are particularly effective multiple receptors is facilitated by the dynamic
and have been analyzed in greatest detail nature of phagocytes, which continuously ex-
(Table 1) (6, 7). For simplicity, these receptors tend actin-dependent membranous projections
were studied in isolation by use of model sys- to probe their surroundings (13–15). Particle
tems in which engagement of other receptors binding, which was long thought to be merely
can be avoided or at least minimized. In nature, passive, is therefore aided by such flailing.
however, multiple opsonic and nonopsonic re-
Pathogen strategies to avoid recognition
ceptors are engaged simultaneously, producing

by phagocytes. Considering the importance
a complex and probably synergistic response.
of phagocytosis in the prevention and clear-
ance of infection, it is not surprising that
Modulation of particle binding. The effec- multiple pathogenic organisms have developed
tiveness of the interaction between receptors means of avoiding recognition by phagocytes
and their ligands is dictated by their mutual (Table 2). The most common strategy is to
affinity and by their density on the surface of the interfere with opsonin binding. Several
phagocyte and prey. However, other factors can bacterial and fungal species synthesize
contribute to the efficiency of recognition. For polysaccharide-based capsules that act as a
instance, integrin receptors such as the receptor shield, preventing the deposition of opsonins.
for the complement component iC3b (CR3) Other bacteria express specific surface pro-
exhibit significant affinity for their ligand only teins that inhibit receptor binding. Group
after they undergo so-called inside-out activa- A streptococci escape complement-mediated
tion, which can be elicited by external stimuli phagocytosis by means of M proteins (16).
that activate TLRs (8), IgG receptors, or CD44 Some M proteins recruit C4b-binding protein,
(9, 10). Signaling downstream of these recep- an inhibitor of complement activation. Others
tors activates the small GTPase Rap1, which bind extracellular matrix proteins, such as
in turn induces conformational changes in the fibronectin, albumin, and plasminogen, that
extracellular domain of the integrin, increasing prevent complement deposition. Similarly,
its affinity for the appropriate ligand (8). the Yersinia enterocolitica adhesin YadA binds
Although this mechanism is not fully under- fibrinogen and collagen, preventing deposition
stood, it is clear that association of talin with of the complement component iC3b (17).
the cytosolic region of the integrin is a crucial A few pathogens interfere with Fcγ recep-
step (11). tor (FcγR)-mediated phagocytosis. The best-
Although affinity for the ligand is essential, known mechanism is that of Staphylococcus
other properties of the receptors, such as their aureus, which deploys protein A on its sur-
dynamics on the surface of the phagocyte, also face. Protein A binds the Fc portion of IgG
influence the binding response (12). Phagocytic with very high affinity, preventing its interac-
receptors generally activate when forming mul- tion with Fcγ receptors (18).
timers, the result of coalescence in the plane of
the membrane. Thus, receptors undergoing fast Receptors of Apoptotic Corpses
lateral diffusion are more likely to be activated In healthy human individuals, more than
than are poorly mobile or immobile receptors. 10 billion cells die every day. The resulting

Table 2 Examples of virulence factors employed by pathogenic microbes to avoid uptake by phagocytosis
Effectors Functions Species Reference(s)
Inhibition of attachment
Protein A Binds Fc region, preventing normal interaction Staphylococcus aureus 18
with FcγR
Capsule Prevents complement deposition Cryptococcus neoformans, Streptococcus —
pneumoniae, Escherichia coli K1,
Klebsiella pneumoniae, Neisseria
meningitidis, S. aureus, Haemophilus
influenzae, Treponema pallidum
M proteins Prevents binding to CRs Streptococcus pyogenes 16
YadA Prevents deposition of C3b Yersinia enterocolitica 17
Inhibition of signaling
YopH Tyrosine phosphatase for Cas, Fyb, SKAP-HOM, Yersinia sp. 224
paxillin, and FAK
YopE GAP for RhoA, Rac, and Cdc42 Yersinia sp. 225
YopT Cysteine protease of Rho, Rac, and Cdc42 Yersinia sp. 226
YpkA (YopO) Serine/threonine kinase of actin, RhoA, and Rac Yersinia sp. 227, 228
ExoT GAP for RhoA, Rac, and Cdc42 Pseudomonas aeruginosa 229
ExoS GAP for RhoA, Rac, and Cdc42 Pseudomonas aeruginosa 230
EspJ Inhibits FcγR- and CR3-mediated phagocytosis Escherichia coli 231
EspB Inhibits myosin–actin interactions Escherichia coli 232
EspH Inactivates Rho GEFs Escherichia coli 233
T4SS Delays phagocytosis Helicobacter pylori 115
LspA1 and -2 Inhibit Src-family kinases Haemophilus ducreyi 234
Nef Inhibits membrane delivery to the phagosome HIV-1 102
Abbreviations: CR, complement receptor; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; HIV, human immunodeficiency
virus; T4SS, type 4 secretion system.
apoptotic bodies must be cleared by phagocytes. several molecules—termed “eat me” signals—
Despite its obvious importance, phagocytosis that distinguish them from healthy cells. The
of apoptotic corpses has been extensively stud- most characteristic marker of apoptotic cells is
ied only recently. Nevertheless, this process ap- phosphatidylserine (PS). This lipid is virtually
parently involves a wide complexity of ligand- restricted to the inner leaflet of the plasma
receptor interactions. membrane in healthy cells but becomes scram-
bled during apoptosis, appearing on the outer
Receptors and coreceptors mediating leaflet. Multiple phagocytic receptors bind to
“eat me” signals. Recognition of apoptotic PS. Direct binding to PS can be mediated by
corpses involves a complex combination of sig- receptors of the TIM (T cell immunoglobulin
nals. First, cells undergoing apoptosis release mucin) family, BAI1, and Stabilin-2 (20–22). In
various molecules such as ATP, lysophos- other instances, soluble extracellular molecules
phatidylcholine, fractalkine, and sphingosine bind to PS and to surface receptors, serving
1-phosphate (19). These soluble “find me” as bridging elements. One such molecule,
signals act as chemoattractants that recruit MFG-E8 (also known as lactadherin), connects
phagocytes to the vicinity of dying cells. In PS to αV β3 integrins, which are effective
addition, apoptotic cells display on their surface phagocytic receptors (23). Similarly, molecules

such as Gas6 and protein S can bridge PS to ligand. Lessons learned from such model sys-
phagocytic receptors of the TAM (Tyro3, Axl, tems are summarized in the following sections.
Mer) family (24). Derivatives of PS metabolism
also contribute to the recognition of apoptotic Signal Transduction During Fcγ
bodies. PS exposed on the surface of apoptotic Receptor–Mediated Phagocytosis
corpses seemingly undergoes oxidation, and
Internalization of IgG-opsonized particles by
some phagocytic receptors such as CD36 and
FcγRs is by far the best-understood model of
CD68 bind modified lipids, including oxidized
phagocytosis. This process can be conceptually
PS (25). In conclusion, numerous phagocytic
separated into three steps: (a) binding of ligand-
receptors bind apoptotic bodies directly or
coated particles to receptors; (b) clustering of
indirectly, and professional phagocytes such as
the FcγR, inducing a signaling cascade; and
macrophages coexpress most of these receptors.
(c) engulfment of the particle by an actin-driven
Notably, PS is not completely absent from
process. Importantly, activation of FcγR is an

the surface of healthy cells. However, apoptosis
iterative process that is replicated numerous
increases the amount of PS exposed to the sur-
times as the membrane “zippers” around the
face by ≈300-fold. This observation suggests

target particle; extension of pseudopods leads
the existence of a threshold that precludes PS-
to additional encounters between unoccupied
mediated phagocytosis of healthy cells (26).
receptors and available ligands on the surface
of the particle. Receptor engagement around
“Don’t eat me” signals. In a few instances, the entire particle appears to be required for
healthy cells expose considerable amounts of the completion of internalization (29).
PS on their surface. This is the case for acti-
vated T or B cells. To avoid phagocytosis, these Receptor clustering and tyrosine phospho-
cells display “don’t eat me” signals. CD31 is one rylation. Like other immunoreceptors, FcγRs
such receptor; it prevents phagocytosis when it are not activated by monovalent ligands (30);
undergoes homotypic (self-) interactions (27). bivalent or multivalent ligands are required to
Cells that display the membrane protein CD47 elicit downstream signaling (31). An important
also escape phagocytosis. CD47 binds the re- body of work, which used FcεRI as a model,
ceptor SIRPα (signal regulatory protein α), led to the conclusion that Fc receptor cluster-
which is expressed on the phagocyte surface ing is required to elicit cellular responses (32).
(28). This interaction activates tyrosine phos- Lateral clustering brings the cytosolic domain
phatases, leading to a dephosphorylation cas- of multiple Fc receptors into close proximity.
cade and inhibition of myosin II, a contractile This cytosolic domain encompasses a unique
protein associated with actin. region known as the immunoreceptor tyrosine-
based activation motif (ITAM), which is char-
acterized by a tandem YxxI/L motif that is a
substrate for phosphorylation by tyrosine ki-
PARTICLE INTERNALIZATION nases of the Src family. The ITAM can be
Phagocytic receptors differ not only in the na- part of the same polypeptidic chain that en-
ture of the ligands they recognize but also in gages the ligand, as in the case of FcγRIIA and
the signals they generate. Under physiological FcγRIIC, or of a separate γ subunit that as-
conditions, several receptor types are activated sociates noncovalently with the ligand-binding
in parallel, and multiple signaling cascades are subunit of the receptor, as in the case of FcγRI
triggered concomitantly. Analysis of this com- and FcγRIIIA. Upon receptor clustering, the
plex profile is an ambitious project. Fortunately, ITAM associates with and becomes phospho-
this task has been made easier by the imple- rylated by Hck, Lyn (33, 34), and/or Fgr (35).
mentation of simplified systems in which single However, other kinases of the Src family are
receptors engage particles coated with a select probably able to phosphorylate the ITAM,

given that bone marrow–derived macrophages cups form but become arrested at an early
from triple-knockout (hck−/− , fgr−/− , lyn−/− ) stage (45). On the one hand, this observation
mice show only partial impairment of FcγR- suggests that, whereas Syk is essential for
mediated phagocytosis (36). Importantly, both phagocytosis, some early signaling is induced
tyrosines of the ITAM are required for optimal independently of this kinase. On the other
signaling and phagocytosis (37, 38). hand, phosphorylation of the FcγR γ chain
The mechanism whereby receptor clus- is greatly reduced in syk−/− macrophages, and
tering leads to phosphorylation of the ITAM Syk can phosphorylate the receptor ITAM in
tyrosines remains elusive. Clustering may vitro (46). It is therefore possible that once
induce association between the receptors some ITAMs are phosphorylated by Src-
and cholesterol-enriched lipid rafts, where family kinases, Syk is recruited and can itself
Src-family kinases are concentrated. Two types phosphorylate additional neighboring ITAMs,
of evidence support this model. First, FcγRII thereby amplifying the signaling cascade.
becomes associated with detergent-resistant

Adaptor proteins. Phosphorylation of Syk
membranes (DRMs) upon activation by cross-
leads to a striking recruitment of additional
linking (39, 40). Second, cholesterol depletion

signaling proteins to the activated FcγR
with methyl-β-cyclodextrin inhibits FcγRII
complex. Several adaptor proteins bind directly
phosphorylation in response to clustering (39).
to the tyrosine kinases and act as platforms
Association of FcγRIIA with DRMs depends
for the recruitment of downstream signaling
on its palmitoylation on a cysteine residue
components (Figure 1). The transmembrane
(41). Despite this suggestive evidence, several
protein linker of activated T cells (LAT) binds
caveats must be considered: (a) Among the
to and is directly phosphorylated by Syk (47,
FcγRs, only FcγRIIA is palmitoylated; there-
48). Phosphorylation of LAT induces docking
fore, other FcγRs may not associate with lipid
of additional adaptors: Grb2 binds to LAT
rafts or would do so by a different mechanism.
via its SH2 domain and in turn recruits the
(b) Whether DRMs indeed reflect the segre-
Grb2-associated binder Gab2. The binding
gation of lipids in intact membranes or are
of Gab2 to the signaling complex is further
artifactually induced by the detergents used
buttressed by its interaction with plasmalem-
in their preparation is unclear. (c) Because of
mal phosphatidylinositol-3,4,5-trisphosphate
the harshness of the treatment, conclusions
[PI(3,4,5)P3 ], which, as detailed below, is also
based on cholesterol extraction with methyl-β-
generated in the vicinity of activated receptors.
cyclodextrin treatment are increasingly being
Gab2 associates with PI(3,4,5)P3 via a pleckstrin
questioned. In view of these limitations, the
homology (PH) domain. Once attracted to the
leading specialists in the field regard the raft
receptor complex, Gab2 is phosphorylated in a
model with caution (42).
Syk-dependent manner, which in turn leads to
The event that follows FcγR phospho-
recruitment of other proteins (49). The adaptor
rylation by Src-family kinases involves the
CrkII is also attracted to the phagocytic cup
spleen tyrosine kinase (Syk) (Figure 1). Syk
(Figure 1), probably via its SH2 domain (50).
displays two Src homology 2 (SH2) domains,
Adaptors appear to be critical for signal-
which bind doubly phosphorylated ITAMs
ing downstream of FcγR, as phagocytosis is
(43). This cytosolic kinase is expressed mostly
significantly impaired in bone marrow–derived
in hematopoietic cells and becomes activated
macrophages from gab2−/− mice (51). More-
by phosphorylation upon FcγR cross-linking
over, dominant-negative mutants of CrkII or
(43, 44). Studies in macrophages from syk−/−
its silencing by small interfering RNA also in-
mice demonstrated that Syk is required for
hibits phagocytosis (50).
FcγR-mediated phagocytosis (45, 46). Inter-
estingly, however, actin polymerization is not Lipid signaling. Lipids play a critical role in
totally eliminated, and incipient phagocytic orchestrating the signaling events that trigger

PI(3,4)P2
PC
SHIP PI(3,4,5)P3
PA
PI(4,5)P2 PI(4,5)P2
DAG
PLD
ITAM
ITAM
P P
LAT
PI3K PI4P-5Kα SFK P IP3 PI4P-5Kα
P PLCγ
Syk Syk PKCε
Grb2
Gab2 ARF6
Myosin X
CrkII
Dock180 Cdc42
WASP
Rac Vav
N-WASP
Scar
WAVE Arp2/3
Actin polymerization
Figure 1
Signaling leading to actin polymerization during Fcγ receptor–mediated phagocytosis. Aggregated receptors trigger activation of
tyrosine kinases (orange), including Src-family kinases (SFKs), followed by recruitment of adaptor proteins ( green). The resulting
signaling platforms activate multiple lipid-modification enzymes (blue) and guanine nucleotide exchange factors (lavender) for small
GTPases (brown). Nucleation-promoting factors ( purple) activate the actin nucleation complex Arp2/3 (red ), which in turn elicits actin
polymerization that drives pseudopod extension. Abbreviations: DAG, diacylglycerol; ITAM, immunoreceptor tyrosine-based
activation motif; LAT, linker of activated T cells; PA, phosphatidic acid; PC, phosphatidylcholine; PI3K, phosphatidylinositol 3-kinase;
PI(3,4)P2 , phosphatidylinositol-3,4-bisphosphate; PI(3,4,5)P3 , phosphatidylinositol-3,4,5-trisphosphate; PI(4,5)P2 ,
phosphatidylinositol-4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; SHIP, Src homology 2
domain–containing inositol 5 -phosphatase; Syk, spleen tyrosine kinase; WASP, Wiskott-Aldrich syndrome protein.
phagocytosis (Figures 1 and 2). Phos- the most abundant in macrophages, followed
phatidylinositol-4,5-bisphosphate [PI(4,5)P2 ] is by PI4P-5Kα (54). Macrophages from PI4P-
present in substantial amounts in the inner 5Kα-deficient animals have defective particle
leaflet of the plasma membrane of resting ingestion, whereas PI4P-5Kγ−/− cells have im-
phagocytes. During phagocytosis, the concen- paired particle binding (54).
tration of PI(4,5)P2 transiently increases in the Before and during the initial steps of
pseudopods that form the phagocytic cup but phagocytosis, the inner leaflet of the plasma
then decreases abruptly (52). This local synthe- membrane is negatively charged, owing
sis probably arises from stimulation of phos- to the presence of anionic phospholipids
phatidylinositol 4-phosphate 5-kinases (PI4P- such as PS and phosphoinositides, mostly
5K) because their inhibition prevents PI(4,5)P2 PI(4,5)P2 (55). The uniquely negative charge
accumulation and impairs phagocytosis (53). of the plasmalemma fosters the electrostatic
Four isoforms of PI4P-5K exist; PI4P-5Kγ is association of PI4P-5K, which displays

a b RAW 264.7
PI(4,5)P2 Plasmalemma PH-PLCδ-GFP Akt-PH-RFP

High Low
PI(3,4,5)P3
High Low
PI(3)P
High Low
0s
330 s
480 s
Formed
phagosome
Figure 2
Dynamic changes in phospholipid composition define stages of phagosome maturation. (a) Schematic
illustration of the changes in phosphoinositide abundance at the forming phagosome. Early in phagosome
formation, a transient increase in phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2 ] occurs, followed by its
depletion from the base of the phagosome. Concomitantly, phosphatidylinositol-3,4,5-trisphosphate
[PI(3,4,5)P3 ] increases and persists until after the phagosome seals. At later stages in phagosome formation
(between 1 and 10 min after sealing), phosphatidylinositol 3-phosphate [PI(3)P] accumulates, becoming
markedly enriched in early phagosomes. (b) Confocal fluorescence images of a RAW 264.7 macrophage
ingesting immunoglobulin G–opsonized sheep red blood cells. The image series on the left shows the
distribution of PH-PLCδ-GFP, a probe that specifically detects PI(4,5)P2 . The series on the right shows the
accumulation of Akt-PH-RFP, which detects PI(3,4,5)P3 , in the same cell. The arrow points to the base of
the forming phagosome, where PI(4,5)P2 becomes depleted while PI(3,4,5)P3 levels remain high. Scale bar is
3.4 μm. Abbreviations: GFP, green fluorescent protein; PH, pleckstrin homology; PLC, phospholipase C;
RFP, red fluorescent protein.
polycationic patches on its surface (56). Less are active in phagocytosis, but whether they
is known about the mechanism whereby PI4P- function upstream of PI4P-5K remains unclear
5K is activated during phagocytosis. In other (57–59).
systems, PI4P-5K isoforms can be activated The drastic disappearance of PI(4,5)P2 fol-
by the small GTPases ARF6 and Rac1 and by lowing its modest initial accumulation is essen-
phosphatidic acid (PA); all of these stimulants tial to allow particle internalization, probably

by facilitating actin disassembly (60). Several ing demonstrated that lipid-anchored proteins
pathways contribute to the disappearance of diffuse more slowly within the cup than else-
PI(4,5)P2 . The phosphoinositide-specific phos- where in the plasma membrane (69). However,
pholipase Cγ (PLCγ) is phosphorylated and this reduced diffusion rate cannot fully explain
recruited to the phagocytic cup in a Syk- the observed lipid confinement, and other bar-
dependent manner, probably by interaction of riers probably exist. In this regard, the phago-
its SH2 domain with LAT (52, 61), while its cytic cup appears to be a separate compartment
PH domain binds PI(4,5)P2 , stabilizing it at the from the bulk plasma membrane; the former
membrane. PLCγ hydrolyzes PI(4,5)P2 , yield- experiences local signaling events and restricts
ing diacylglycerol (DAG) and inositol-3,4,5- their spread.
trisphosphate. PLCγ activity is critical because
its inhibition prevents DAG production and Small GTPases. Small GTPases of the Rho
ablates phagocytosis (52). Notably, DAG pro- family are key factors in the regulation of actin
motes the recruitment of protein kinase Cε dynamics. They alternate between an active
(PKCε), which enhances phagocytosis inde- (GTP-bound) state and an inactive (GDP-
pendently of actin polymerization (62). bound) state. Nucleotide exchange is essential

PI(4,5)P2 is also consumed when it becomes for activation of Rho-family proteins, and
phosphorylated by class I phosphatidylinositol guanine nucleotide exchange factors (GEFs)
3-kinase (PI3K), producing PI(3,4,5)P3 at the catalyze the release of GDP and its replace-
phagocytic cup (63, 64). Interestingly, inhibi- ment by GTP. In their active state, Rho-family
tion of PI3K abolishes phagocytosis of large GTPases are targeted to membranes in part by
(≥3-μm) particles but has only a limited ef- prenylation, a hydrophobic posttranslational
fect on smaller ones (65, 66). p85, the regu- modification that anchors the GTPases to the
latory subunit of PI3K, is recruited to form core of the bilayer. In the GDP-bound state,
phagosomes via interaction of its SH2 domain however, they associate with a soluble factor,
with Syk and Gab2. Recruitment is followed the guanine dissociation inhibitor (GDI),
by phosphorylation and consequent activation which stabilizes the inactive species in the
of p85 (51, 67). The PI(3,4,5)P3 produced by cytosol and precludes the access of GEFs,
PI3K stabilizes Gab2, creating a signaling am- thereby limiting the ability of Rho-family pro-
plification loop. teins to become activated. Rho-family proteins
In addition to the phosphoinositides, glyc- are inherently poor GTPases, but their activity
erophospholipids play a role in FcγR-mediated is greatly enhanced through interactions
phagocytosis. Phospholipase D (PLD) is re- with GTPase-activating proteins (GAPs); the
cruited at the phagocytic cup, where it hy- resulting rapid hydrolysis of GTP to GDP
drolyzes phosphatidylcholine (PC) to generate terminates the activity of the Rho GTPases
PA, a metabolically active lipid (57). Inhibi- until another activation cycle is initiated.
tion of PLD1 or PLD2 reduces phagocytosis Several GTPases play a role in FcγR-
(68). PA, a cone-shaped lipid, may induce neg- mediated phagocytosis. Cdc42, which stimu-
ative curvature of the membrane and thus favor lates formation of filopodia in various cell types,
membrane fission. In addition, as mentioned is activated early in phagocytosis, mostly at the
above, PA can stimulate PI4P-5K activity (58). rims of the cup (70). Inactivation of Cdc42, ei-
Dynamic fluorescence microscopy revealed ther by expression of a dominant-negative mu-
that the changes in lipid composition are re- tant or by knockdown, greatly inhibits phagocy-
stricted to the phagocytic cup area (Figure 2). tosis (71, 72). Rac1 and Rac2, which induce the
This finding suggests the existence of a diffu- formation of lamellipodia, are also stimulated
sion barrier, either within the cup or at the rim during FcγR-mediated phagocytosis, although
of the nascent phagosome. Measurements of with different kinetics (70). Shortly after Cdc42
fluorescence recovery following photobleach- is stimulated, Rac1 is activated throughout the

entire nascent phagosome, whereas Rac2 is ac- actin polymerization, but rather by promoting
tivated later, mostly at the base of the cup the delivery of endomembranes to the nascent
(70). Transfection of dominant-negative Rac1 phagosome (see the section entitled Membrane
inhibits phagocytosis (71). However ingestion Sources, below) (81).
is not impaired in neutrophils from rac1−/−
mice, which may reflect a compensatory ef- The actin polymerization machinery. Actin
fect of Rac2 (73). Accordingly neutrophils and filaments are dynamic polymers formed by
macrophages from double-knockout (rac1−/− , a constant association of actin monomers at
rac2−/− ) or even single-knockout (rac2−/− ) mice one end and dissociation from the other end,
display significantly impaired phagocytosis a process known as treadmilling. Filament
(73–75). formation requires the activity of nucleators.
During FcγR-mediated phagocytosis, the During FcγR-mediated phagocytosis, the
adaptor CrkII recruits the Dock180-ELMO1 Arp2/3 nucleator complex (78) supports actin
complex, a bipartite GEF for Rac (50). In ad- polymerization. This seven-protein complex
dition, Vav proteins, which can also function promotes the formation of branched actin
as Rac GEFs, bind to and are activated di- filaments by nucleating polymerization from
rectly by Syk (76). The role of Vav in FcγR- the side of an existing filament. Arp2/3 can
mediated phagocytosis, however, is the subject be activated by nucleation-promoting fac-
of controversy. Experiments using dominant- tors: the Wiskott-Aldrich syndrome protein
negative mutants suggested a role for Vav in Rac (WASP)/N-WASP and the Scar/WAVE-
activation (77). However, macrophages from family proteins, which are downstream
double-knockout (vav1−/− , vav3−/− ) or triple- effectors of Cdc42 and Rac, respectively.
knockout (vav1−/− , vav2−/− , vav3−/− ) mice in- In macrophages from patients affected by
gest IgG-opsonized erythrocytes normally (75). the Wiskott-Aldrich syndrome, which have de-
Because neutrophils from vav1−/− , vav3−/− fective WASP, FcγR-mediated phagocytosis is
mice are deficient in FcγR-mediated phago- significantly impaired (82). WASP is stimulated
cytosis, the apparent discrepancies may reflect by the combined action of GTP-bound Cdc42
cell-specific differences (74). and PI(4,5)P2 . In the presence of these ligands,
Conflicting results also exist regarding the WASP undergoes a conformational change, ex-
role of RhoA. The canonical model, based posing its VCA (verprolin homology, cofilin
on experiments using chemical inhibitors and homology, and acidic) domain, which binds and
dominant-negative mutants, states that RhoA activates Arp2/3 (83, 84). In addition, WASP
is dispensable in FcγR-mediated phagocyto- can become phosphorylated; this event pro-
sis (71, 78, 79), yet inhibition of Rho with motes its association with WASP-interacting
TAT-C3 significantly affected phagocytosis of protein. Formation of this complex is impor-
IgG-opsonized erythrocytes (75). In addition, tant for the development of the phagocytic cup
p115-RhoGEF, which specifically activates (85).
RhoA, is recruited to the phagocytic cup and Although the need for Rac in actin remod-
its knockdown inhibits phagocytosis (80). eling during phagocytosis has been firmly es-
The same study also identified a role for yet tablished, its downstream effector has not been
another GTPase, RhoG, in FcγR-mediated identified. Impairment of WAVE function
phagocytosis (80). has no discernible effect on FcγR-mediated
Importantly, Rho-family GTPases are not phagocytosis (86).
the only small GTPases involved in phago- That actin polymerization is required for
some formation. ARF6 is activated early in phagocytosis is beyond dispute. It therefore
FcγR-mediated phagocytosis (59), and its in- seems paradoxical that sustained polymeriza-
hibition strongly affects engulfment. However, tion prevents internalization, particularly of
ARF6 does not exert its effects by modulating large particles (62). Video microscopy revealed

that actin is cleared from the base of the cup estingly, both SHIP and SHP-1 also bind
prior to sealing (62). Removal of actin from the directly to monophosphorylated ITAM (94,
base may eliminate a possible barrier for mem- 95), suggesting a built-in regulatory feedback
brane deformation, enabling the membrane- of the stimulatory receptors.
bound particle to sink into the cytoplasm. Actin Overexpression or overstimulation of
clearance from the base of the cup requires the FcγRIIB has an inhibitory effect on FcγR-
disappearance of PI(4,5)P2 , which is mediated mediated phagocytosis. Conversely, impair-
by PLCγ and stimulated by PI3K (60). ment of its function causes disproportionate
stimulation, which may lead to autoimmune
Role of myosins. Myosins are actin- disease. Indeed, a polymorphism of FcγRIIB
associated contractile proteins. Myosin II, (I232T) is associated with lupus erythematosus
IXb, and IC have been detected on forming (96).
phagosomes, suggesting that they play a role
in engulfment (87–88). Myosin X has been

Membrane sources. Phagosome formation
convincingly demonstrated to participate in
around large particles entails internalization of
phagosome formation (91); it has been detected

a considerable membrane area. The phagoso-
around nascent phagosomes and seems to be
mal membrane derives mostly from the plasma
required for completion of ingestion. Indeed,
membrane, at least initially (97). However,
phagocytosis is depressed by introduction of
additional membrane from several intracellu-
antibodies or truncated mutants that inhibit
lar compartments is delivered to the form-
myosin X (91). Interestingly, myosin X is an
ing phagosome by focal exocytosis, an event
effector of PI3K and, like the phosphoinositide
that is more noticeable in the case of larger
kinase, seems to be essential for the internal-
particles. In particular, recycling endosomes
ization of large but not small particles. There
and late endosomes fuse with the phago-
is also evidence for the involvement of myosin
cytic cup, as indicated by the delivery of the
II and its regulator, myosin light-chain kinase,
specific markers vesicle-associated membrane
in FcγR-mediated phagocytosis (89, 90).
protein 3 (VAMP3) and/or Rab11 and TI-
VAMP/VAMP7, respectively (98–100). Mem-
Inhibitory signaling. Not all FcγRs stim-
brane delivery is important because inhibition
ulate phagocytosis. Unlike other FcγRs that
of exocytosis reduces phagocytosis in a size-
contain an ITAM with tandem tyrosines,
dependent manner (98, 100, 101). The role of
FcγRIIB displays a single-tyrosine signal-
this mechanism in the context of infectious dis-
ing region known as an immunoreceptor
eases has been highlighted by the observation
tyrosine-based inhibition motif (ITIM). When
that human immunodeficiency virus (HIV)-1
phosphorylated, ITIM recruits the phos-
inhibits phagocytosis by abrogating the deliv-
phatases Src homology 2 domain–containing
ery of VAMP3-positive vesicles (102).
inositol 5 -phosphatase (SHIP) and the Src
homology 2 domain–containing protein
tyrosine phosphatases SHP-1 and -2 (Fig-
ure 3). SHIP is an inositol 5-phosphatase Dectin-1-Mediated Signaling
that inhibits phagocytosis by hydrolyzing During Phagocytosis
PI(3,4,5)P3 into phosphatidylinositol-3,4- Dectin-1 has been identified as an important
bisphosphate [PI(3,4)P2 ] (92). SHP-1 and -2 phagocytic receptor for fungi. Its cytosolic do-
are phosphotyrosine-specific protein phos- main bears an ITAM-like motif, suggesting that
phatases. Overexpression of SHP-1 inhibits it shares signaling features with FcγRs. How-
phagocytosis (93). The full range of its targets ever, the membrane-distal tyrosine of Dectin-1
during phagocytosis is not known, but the is atypical, and mutagenesis studies demon-
ubiquitin ligase Cbl is one of them. Inter- strate that it is dispensable for phagocytosis (3).

PI(3,4)P2
PI(3,4,5)P3 PI(4,5)P2
ITIM
ITIM
ITAM
ITAM
P P P P
LAT
SHIP P SFK P
PI3K
Syk Syk
Gab2
Grb2
Vav
SHP
Figure 3
Inhibitory signaling mediated by the immunoreceptor tyrosine-based inhibition motif (ITIM)-bearing receptor FcγRIIB. Upon
receptor aggregation, the ITIM of FcγRIIB is monotyrosine-phosphorylated and recruits the phosphatases SHP and Src homology 2
domain–containing inositol 5 -phosphatase (SHIP). The inositol 5-phosphatase SHIP inhibits phosphatidylinositol 3-kinase
(PI3K)-dependent signaling by hydrolyzing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3 ] into phosphatidylinositol-3,
4-bisphosphate [PI(3,4)P2 ]. The tyrosine phosphatase SHP inhibits upstream FcγR-mediated signaling at multiple levels by
dephosphorylating the ITAM of stimulatory receptors, as well as activation sites on Src-family kinases (SFKs), spleen tyrosine kinase
(Syk), Vav, and/or PI3K ( faded regions). Abbreviations: ITAM, immunoreceptor tyrosine-based activation motif; LAT, linker of
activated T cells.
Dectin-1 also signals via Syk. Because both that Syk is required for zymosan phagocytosis
SH2 domains of this kinase are required for by dendritic cells, but not by macrophages (3,
its recruitment by Dectin-1, it is assumed that 104, 105). Further studies will be necessary
two receptors are bridged by one Syk molecule to clarify these apparent discrepancies and
(103). As is the case for FcγR, phagocytosis to understand how phagocytosis proceeds in
mediated by Dectin-1 involves Src-family ki- macrophages in a Syk-independent manner.
nases, Syk, PI3K, PKC, Cdc42, and Rac1 (but
not RhoA), and ultimately, actin remodeling
(3). Yet, in macrophages, Dectin-1-mediated Complement-Mediated Phagocytosis:
phagocytosis of zymosan appeared to be Outside-In Signaling
less dependent on Src, Syk, PI3K, and Rho CR3, also known as αM β2 integrin or Mac-1,
GTPases than was the uptake of IgG-opsonized is the best-studied phagocytic receptor after
erythrocytes via FcγR (3). These results must the FcγR. The signaling events elicited by
be interpreted cautiously because zymosan CR3 and FcγR are markedly different. Early
particles are smaller than red cells, and a size studies of CR3-mediated phagocytosis by
threshold for signaling may exist, as described electron microscopy suggested that particles
for PI3K in FcγR-mediated phagocytosis. sink into the phagocyte without extending
Cell-type differences may also influence the the pseudopods that are characteristic of
results: Studies using knockout mice indicated FcγR phagocytosis (106, 107). However, this

canonical difference has been questioned by may account for the reported contribution of
more recent observations from both elec- microtubules to CR3-mediated phagocytosis
tron (75) and light microscopy studies (14, (113, 114). Thus, microtubules, together with
108), which showed membrane protrusions two types of actin nucleators—one forming a
encircling the targets during CR3-mediated branched network and the other forming a non-
phagocytosis. branched network—function cooperatively in
Remarkably, when integrins are activated CR3-mediated phagocytosis.
by phorbol esters, CR3-mediated phagocytosis The mechanism leading to RhoA activation
is abrogated by PKC inhibitors but not by is not fully understood. Two different cytosolic
general tyrosine kinase inhibitors (107, 109). domains of the β2 subunit of the integrin recep-
In addition, CR3-mediated phagocytosis tor are required for RhoA recruitment and acti-
is not affected in syk−/− macrophages (46). vation (112). In addition, the Rac/Rho GEF Vav
However, a more recent study found that is seemingly required for Arp2/3 recruitment,
Syk is phosphorylated during CR3-mediated F-actin accumulation, and iC3b-opsonized par-

phagocytosis and that Syk antagonism by ticle uptake (75). Because RhoA is important
knockdown or by expression of a dominant- for Arp2/3 recruitment and actin polymeriza-

negative allele inhibits particle uptake (110). tion by CR3 (78), and because Vav is a substrate
Syk can be indirectly activated by integrins of Syk (76), these observations suggest that Syk
via the ITAM-bearing FcR γ chain and/or may attract and activate Vav proteins, leading
DAP12 (111). Thus, syk−/− macrophages may to RhoA and perhaps Rac activation (Figure 4).
compensate for the absence of the kinase by
overexpressing other molecules, such as the
closely related kinase Zap70. Strategies Applied by Pathogens to
During CR3-mediated phagocytosis, F- Inhibit Signaling During Phagocytosis
actin accumulation and particle uptake depend Several pathogenic microbes can evade phago-
on RhoA and are ostensibly independent of Rac cytosis by inhibiting the signaling pathways
or Cdc42 (71, 78, 79). Accordingly, binding of that lead to their uptake. These microbes in-
iC3b-opsonized erythrocytes increased levels clude Yersinia spp., Pseudomonas aeruginosa, and
of Rho-GTP but not of Rac-GTP (112). Yet, enteropathogenic Escherichia coli, which use a
the uptake of iC3b-opsonized erythrocytes is type III secretion system, a syringe-like molec-
severely depressed in rac1−/− , rac2−/− double- ular nanomachine, to inject a cocktail of effec-
knockout cells, challenging the classical model tors into the phagocyte. Some of these effec-
that CR3-mediated phagocytosis is solely de- tors inhibit Rho-family GTPases or antagonize
pendent on RhoA (75). tyrosine phosphorylation and PI3K-dependent
RhoA activates actin polymerization by two signaling (Table 2), often by mimicking the
distinct mechanisms (Figure 4). First, it ac- function of inhibitory host proteins. Helicobac-
tivates Rho kinase, which stimulates myosin ter pylori uses another injection machinery, the
II by phosphorylation of its light chain (90). type IV secretion system, to prevent phagocy-
Intriguingly, inhibition of Rho kinase or of tosis (115), although the effector(s) remain to
myosin light-chain kinase abrogates Arp2/3 be identified.
recruitment and actin cup assembly (90). Not every microbe has an injection sys-
Second, the formin mDia1 is recruited in a tem. Some pathogens, such as HIV and
RhoA-dependent manner (79). Silencing the Haemophilus ducreyi, use a Trojan horse–like
expression of mDia1 reduces F-actin accumu- strategy. Such pathogens are initially engulfed
lation and particle uptake. The microtubule- normally, but once internalized their effectors
associated protein CLIP-170 binds directly abolish the phagocytic capacity of the host cell,
to mDia1 and controls its recruitment at thereby preventing further uptake of additional
the phagocytic cup (113). This connection microbes (102, 116).

α
β
ITAM
P
SFK P
Talin
Syk
Microtubules
RhoA Vav
ROCK
Myosin II CLIP-170
Arp2/3 mDia
Actin polymerization
Figure 4
Signaling leading to actin polymerization during phagocytosis mediated by the receptor for the complement component iC3b (CR3).
Binding of iC3b-opsonized particles to αM β2 integrins activates RhoA in a tyrosine kinase-independent manner or, possibly, via the
spleen tyrosine kinase (Syk) kinase pathway, involving recruitment of the immunoreceptor tyrosine-based activation motif (ITAM)-
bearing DAP12 and/or Fc receptor γ chain. RhoA stimulates the activity of the serine/threonine kinase Rho kinase (ROCK), which
activates myosin II by phosphorylation of its light chain. Myosin II seemingly participates in the recruitment of Arp2/3, which nucleates
actin polymerization. RhoA also activates the actin nucleator mDia, which is recruited to the phagocytic cup in a CLIP-170-
dependent manner.
PHAGOSOME MATURATION: is followed by a rapid succession of biochemical

BIOGENESIS OF A MICROBIAL modifications that convert the nascent phago-
KILLING ENTITY some into a potent microbicidal organelle that
is central to both innate and adaptive immunity.
Although the receptors that elicit phagocytic This remodeling process, termed phagosome
responses can vary, the internalization process maturation, proceeds through an ordered
in all instances culminates in the formation series of strictly choreographed membrane
of a membrane-bound vacuole termed the fusion and fission events that radically alter the
phagosome. Immediately after scission from composition of the phagosome, while keeping
the surface membrane, the newly formed its size nearly constant (117). Remarkably,
phagosome is innocuous, as its limiting mem- newly formed phagosomes are refractory to
brane resembles the plasmalemma from which fusion with late endosomes and lysosomes and,
it was derived, and its fluid-phase contents are instead, have a propensity to interact with early
a reflection of the extracellular milieu. As such, endosomes, giving rise to early phagosomes.
phagocytosis per se is insufficient to mediate Only later in phagosome maturation do late
the destruction of microbes; however, scission endosomes and lysosomes begin to fuse with

early phagosomes, converting them into late seminal studies employing model organisms—
phagosomes and phagolysosomes, respectively such as Caenorhabditis elegans—have shaped
(Figure 5). In many regards, the sequential our understanding of this complex process and
events that yield mature phagolysosomes are also considered here.
parallel those that occur during endosomal
progression (reviewed in Reference 120), and
much has been learned about phagosomes by The Early Phagosome
comparing them with the endocytic pathway. Metamorphosis of the phagosome commences
At present, it is unclear whether maturation immediately after membrane scission, and
is driven by the complete fusion of endocytic shortly thereafter its biochemical properties are
organelles with phagosomes or whether a markedly altered (119). Changes in both the
limited exchange of constituents occurs during membrane and the contents are brought about
the course of transient, reversible interactions, by vesicular traffic to and from the vacuole;
as predicated by the so-called kiss-and-run these processes are coordinated by a family

model (118). Regardless of the precise mech- of molecular switches, the Rab GTPases (re-
anism, it is generally accepted that, as a result viewed in Reference 120). Like the Rho-family
of the sequential interaction with discrete GTPases described above, Rab GTPases al-
endosomal compartments, the phagosome ternate between an active (GTP-bound) state
becomes markedly acidic (luminal pH ≈4.5), and an inactive (GDP-bound) state and are
highly oxidative, and enriched with hydrolytic controlled by GDIs, GEFs, and GAPs. Once
enzymes that ultimately degrade its contents. activated, Rab proteins associate with various
Although phagocytosis normally results in effector molecules that serve many functions,
the eradication of microbes, some remarkable including motor-driven vesicular traffic, vesic-
pathogens have evolved strategies to alter ular fusion, and fission, and that can even pro-
phagosome maturation and usurp host cell mote the activation of other Rab GTPases
functions for their own benefit (Table 3). through a process termed Rab conversion.
Furthermore, a number of heritable human During maturation, early phagosomes gen-
diseases cause phagocytic defects that com- erated through fusion of nascent phagosomes
promise the immune response (Table 4). with early endosomes acquire Rab5. Rab5
In this section, we review the key molecular coordinates endocytic traffic and early phago-
events that define and underlie the maturation some biogenesis. It is activated by the GEF
of phagosomes; we also describe instances GAPVD1 (GAP and VPS9 domain–containing
in which infectious agents or genetic lesions protein 1), at least in the context of phago-
perturb this process. Although the majority of somes that contain apoptotic bodies (121). The
advances in our understanding of phagosome precise mechanism that triggers early recruit-
maturation have stemmed from studies of ment of Rab5 to phagosomes has remained
professional mammalian phagocytes, some elusive, but some active Rab5 may exist on the
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 5
Stages of phagosome maturation. Upon internalization of particulates, the phagosome undergoes an acute transformation, creating a
potent microbicidal and degradative organelle. The maturing phagosome interacts in succession with various subcompartments of the
endocytic pathway to generate vacuoles with biochemically distinct properties. The recognized stages of phagosome maturation are
early, intermediate, late, and phagolysosomal. As the phagosome matures, it becomes increasingly acidic due to delivery of H+ into its
lumen via the vacuolar ATPase (V-ATPase). Furthermore, the phagosome becomes increasingly degradative due to enrichment with
various hydrolases and other antimicrobial proteins. Abbreviations: EEA1, early endosomal antigen 1; ESCRT, endosomal sorting
complex for transport; M6PR, mannose 6-phosphate receptor; LAMP, lysosome-associated membrane protein; MVB, multivesicular
body; ORPL1, oxysterol-binding protein–related protein 1; PI(3)P, phosphatidylinositol 3-phosphate; RILP, Rab7-interacting
lysosomal protein; TfR, transferrin receptor.

Pathogen
(e.g. Staphylococcus aureus)
~ pH 7.4
V-ATPase
TfR EEA1
Rab11
PI(3)P
Early
EE
~ pH 5.4 RE
Vps34
Rab5
Syntaxin 13 GAPEX
ESCRT MVB
Mon1a/b
CD63
Intermediate
LBPA
M6PR
To TGN
Syntaxin 7
LE
ORPL1
LAMP-1/2
Dynein/
dynactin
Late
Rab7
~ pH 5.0
RILP
Microtubules
LY
Cathepsins/
hydrolases
Phagolysosome
Antimicrobial
~ pH 4.5 peptide

Table 3 Representative human pathogens and the biochemical properties of pathogen-containing phagosomes
Strategy Organism Mechanisms of survival and phagosome properties Reference(s)
Altered phagosome Mycobacterium Phagosome is mildly acidic and continuously interacts with 205
maturation tuberculosis recycling endosomes bearing the TfRs that provide the bacteria
with iron
M. tuberculosis–containing phagosomes are Rab5 positive but
devoid of PI(3)P and EEA1
Maturation arrest is mediated by the expression of numerous
effector proteins (e.g., SapM and PknG) and lipids (e.g.,
lipoarabinomannan)
Burkholderia Delay maturation of their vacuole 235, 236
cenocepacia
B. cenocepacia–containing phagosomes acquire early markers [i.e.,
Rab5, PI(3)P, and EEA1], but Rab7 activation and phagosome

acidification are perturbed
LAMP proteins are eventually acquired, indicating lysosomal
fusion
Require the expression of unknown bacterial effectors secreted
through type IV and/or type VI secretion systems
Coxiella burnetii Internalized C. burnetii transit through Rab5- and Rab7-positive 205
compartments
Ultimately reside within an acidic lysosome-like compartment
Bacteria-containing vacuole acquires markers of autophagy (e.g.,
LC3), indicating intersection with autophagosomes, ostensibly
for nutrient acquisition
Histoplasma capsulatum Evade formation of late phagosomes and phagolysosome fusion 237
Perturb V-ATPase activity, blocking acidification, which
prevents vesicular fusion and the acquisition of lysosomal
markers (i.e., LAMP-2)
Leishmania donovani Leishmania spp. impair phagosome maturation by blocking the 238
and L. major fusion of late endosomes and lysosomes
The blockade in maturation requires the expression of a unique
surface glycolipid, LPG
F-actin accumulation around the parasite-containing phagosome
also occurs in an LPG-dependent manner, ostensibly to form a
cage that blocks fusion of late endosomes and lysosomes
Phagosome lysis Listeria monocytogenes Express a pore-forming toxin, LLO, that in conjunction with 205
various lipases solubilizes the limiting membrane of the
phagosome
Maturation of the phagosome is rapidly prevented by
LLO-dependent pore formation and perturbation of luminal
H+ and Ca2+ concentrations
Shigella flexneri Lyse phagosomes in which they reside, entering the cytosol and 239
disseminating via actin-based motility
Phagosome escape requires the function of the Mxi-Spa type III
secretion system and the secreted effector proteins IpaB, IpaC,
and IpaD
(Continued )

Table 3 (Continued )
Strategy Organism Mechanisms of survival and phagosome properties Reference(s)
Theileria parva Rapidly escape phagosomes through an unknown mechanism 240
and replicate in the cytosol
Induce uncontrolled proliferation of infected cells, and through
the expression of the protein TaSE, attach to host cell
microtubules to disseminate between daughter cells
Genesis of a unique Chlamydia trachomatis Upon phagocytosis, C. trachomatis construct a unique vacuole, 241
vacuole termed the inclusion, in which the bacteria replicate
Inclusion is devoid of early and late phagosome markers (Rab5
and Rab7, respectively) and does not resemble a proper
phagosome
Many effector proteins (e.g., IncA) are required for C. trachomatis
to usurp host cell functions and to construct its inclusion

Toxoplasma gondii Invade phagocytes and reside in a unique vacuole that, although 240
derived from the host cell plasma membrane, is largely devoid
of host transmembrane proteins

The vacuole does not acidify, and T. gondii induces the formation
of a pore in its vacuole to gain access to diffusible nutrients from
the host
Host cell microfilaments and vimentin associate with the
T. gondii–containing vacuole, ostensibly to dock it in proximity
to the nuclear membrane
Abbreviations: EEA, early endosomal antigen; LAMP, lysosome-associated membrane protein; LLO, listeriolysin O; LPG, lipophosphoglycan;
PI(3)P; phosphatidylinositol 3-phosphate; TfR, transferrin receptor.
plasmalemma prior to particle engulfment that although their role in phagosome maturation
is carried over to the phagosomal vacuole upon awaits clarification (124). The function of
scission (122). In any case, the density of Rab5 other Rab5 effectors, such as early endosomal
on the phagosomal membrane is much greater antigen 1 (EEA1), the p150–Vps34 complex,
than that detectable at the plasmalemma, so and Mon1a/b, is better understood.
additional recruitment must occur. Irrespective mVps34, a class III PI3K, is transiently re-
of the mechanism underlying its recruitment, cruited to Rab5-positive early phagosomes, in
Rab5 is vital to phagosome development, as which it catalyzes the formation of the signaling
perturbation of its function can arrest matura- lipid phosphatidylinositol 3-phosphate [PI(3)P]
tion, precluding formation of late phagosomes from phosphatidylinositol (125). mVps34 and
and phagolysosomes (123). Rab5 stimulates the its product, PI(3)P, are essential to phago-
fusion of nascent phagosomes with early endosome progression, as ablation of mVps34 ki-
somes (also known as sorting endosomes). The nase activity dramatically impairs maturation
fusogenic ability of Rab5 is best illustrated by (125, 126). The catalytic activity and targeting
the observation that heterologous expression of mVps34 to the limiting membrane of early
of a constitutively active (GTP-locked) mutant phagosomes are enhanced by the Vps15-like
induces the formation of giant endosomes serine/threonine kinase p150, which can form a
and can also cause the formation of enlarged complex with mVps34 and has been suggested
phagosomes (118). Rab5 exerts its effects by to bind directly to active Rab5 (125). Indeed,
recruiting and activating multiple effector pro- accumulation of PI(3)P on early phagosomes is
teins. Accordingly, Rabaptin and Rabankyrin strictly dependent on active Rab5, whereas re-
have been detected on isolated phagosomes, cruitment of Rab5 is independent of mVps34

Table 4 Heritable human diseases and their associated innate immune defects
Disease Etiology Innate immune defect(s) Reference
Chediak-Higashi Mutation of the LSYT gene; it encodes Poor granule fusion with phagosomes, 242
syndrome a protein involved in vesicular traffic causing impaired bacterial clearance
Glycogen storage disease Genetic lesions affecting Recurrent infection due to impaired 243
1b/c glucose-6-phosphate production microbicidal function of neutrophils and
macrophages
Impaired Ca2+ mobilization
Wiskott-Aldrich Mutations affecting WASP expression Phagocytes demonstrate severe defects in 244
syndrome WASP functions as a scaffolding many cellular processes, including cell
protein required for actin migration and phagocytosis
rearrangement
Hermansky-Pudlak Mutation of the gene ADTB3A coding Susceptibility to infection due to altered 245
syndrome type 2 for the β subunit of the AP3 complex vesicular/protein transport
AP3 is required for vesicle formation Characterized by increased CD63
and protein trafficking from the expression on the plasma membrane and
trans-Golgi network reduced protein (e.g., elastase) delivery to

neutrophil granules and phagosomes
Chronic granulomatous Mutations affecting genes encoding Susceptibility to infection by numerous 246
disease subunits of the NOX2 NADPH bacterial and fungal pathogens
oxidase ROS production by phagocytes is impaired
Abbreviations: AP, activator protein; ROS, reactive oxygen species; WASP, Wiskott-Aldrich syndrome protein.
(123); these findings imply that association engages PI(3)P and is thus efficiently recruited
of the GTPase with the phagosome precedes to early phagosomes, where it facilitates dock-
that of the phosphatidylinositide kinase. In this ing and fusion of early endosomes (130). The
manner, Rab5 coordinates the membrane local- fusion-promoting function of EEA1 stems in
ization and activation of mVps34, harnessing its part from its direct interaction with syntaxin
catalytic activity to synthesize PI(3)P locally; ac- 13, a soluble N-ethylmaleimide-sensitive factor
cumulation of this signaling phosphoinositide, attachment protein receptor (SNARE) (133).
in turn, facilitates subsequent steps in phago- SNARE proteins are universal mediators of
some maturation. membrane fusion; they function by forming
There is a striking accumulation of PI(3)P hairpin-like complexes composed of v-SNARE
on the cytosolic leaflet of the limiting mem- proteins expressed on the donor membranes
brane of the early phagosome, which has been (e.g., early endosomes) and t-SNARE proteins
verified through the use of fluorescent pro- expressed on acceptor membranes (e.g., nascent
teins such as green fluorescent protein fused phagosomes). By forming the hairpin complex,
to specific PI(3)P-binding domains, including the SNAREs bring donor and acceptor mem-
some PX and FYVE domains. Proteins that branes into direct apposition, reducing the free-
bear such domains, such as the p40 subunit of energy barrier for membrane fusion. Syntaxin
the NADPH oxidase, EEA1, and hepatocyte 13 probably plays an equivalent role in phago-
growth factor–regulated tyrosine kinase sub- some maturation (134).
strate (Hrs), also accumulate on early phago- As a consequence of these initial fusion
somes (127–129). EEA1 is a well-characterized events, early phagosomes become biochem-
Rab5 effector that binds the GTPase di- ically distinct from their predecessors, the
rectly via an N-terminal CH2 zinc-finger motif nascent phagosomes. But despite repeated
(130–132). Furthermore, EEA1 simultaneously rounds of vesicular fusion, the surface area of

the maturing phagosome remains noticeably to form a recycling-competent retromer (148).

constant, implying that some of the phagosomal The operation of this system in mammalian
membrane must be removed concomitantly. phagosomes has not been directly documented,
Indeed, as described for endosomes, certain but retromer-dependent recycling of the Ced-
components of the early phagosome, such as 1 receptor from apoptotic body-containing
transferrin receptors, are retrieved back to the phagosomes back to the plasma membrane has
plasma membrane via a recycling pathway. been demonstrated in C. elegans (149).
This pathway consists of specialized tubular In summary, an elaborate network of sig-
and vesicular endosomes that tend to con- naling, budding, and tubulating components,
centrate in a juxtanuclear location by means including Rab4, Rab5, Rab11, EHDs, COPI,
of microtubule-associated motors such as the and the retromer complex, underlies the out-
dynein/dynactin system (135, 136). Recycling ward fission of material from phagosomes. The
is regulated in part by Rab4 and Rab11, which importance of this process is not to be underes-
are also found on the limiting membrane of timated, as impairment of recycling profoundly
early phagosomes (98, 137). Protein retrieval inhibits the ability of phagocytes to ingest IgG-
from early phagosomes is enhanced by the opsonized phagocytic targets or to process in-
Rab-coupling protein, which can bind directly gested apoptotic cells (98, 146, 149).
to Rab4 and Rab11 (137, 138). Membrane budding and the emergence of
Other cellular factors, such as the Eps15 ho- recycling compartments from early phago-
mology domain proteins (EHDs), also function somes are important contributors to the
in the retrieval of components from endosomes maintenance of phagosome size, but they
and, although not yet directly demonstrated, are not the sole mechanism. Phagosomes
presumably also from phagosomes. EHDs also direct membrane-associated cargo that
are lipid-binding proteins that stimulate is destined for degradation to intraluminal
membrane tubulation and influence recycling vesicles (ILVs). ILVs emerge via invagination
from Rab4- and Rab11-positive endosomes and pinching of the limiting phagosomal
(139–141). Interestingly, EHD1 can also membrane, and the formation of these luminal
bind the promiscuous Rabenosyn-5, a Rab4 bodies ostensibly occurs through a process
and Rab5 effector, potentially mediating the akin to multivesicular body (MVB) formation.
generation of recycling endosomes from early MVB generation requires the endosomal
phagosomes (142, 143). Other factors, such sorting complex for transport (ESCRT) ma-
as the hetero-oligomeric complex COPI, the chinery (reviewed in Reference 150). Factors
GTPase adenosine ribosylation factor, and the that initiate ILV formation are recruited to
retromer (a multisubunit protein sorting ma- phagosomes at an intermediate stage, when
chine), are also required for efficient retrieval of the phagosome no longer resembles an early
endosomal and ostensibly phagosomal proteins compartment but is not yet endowed with the
to either the plasmalemma or the trans-Golgi properties of a late phagosome (Figure 5).
network (TGN) via recycling pathways (144– Proteins targeted for degradation through
147). The retromer complex, composed of the MVB formation are mono- or polyubiqui-
vacuolar protein sorting–associated protein 26 tylated; these posttranslational modifications
(Vps26), Vps29, Vps35, and the sorting nexin facilitate the sorting of cargo destined for ILVs.
(SNX) subcomplexes SNX1/2 and SNX5/6, Accordingly, phagosome-associated FcγRIIA,
delivers specific endosomal proteins, such as when ubiquitylated, is sorted into a multi-
the cation-independent M6PR, to the TGN vesicular compartment and is subsequently
(147, 148). Recruitment of the SNX complexes degraded (151). Ubiquitin-dependent sorting
depends on Rab5-mediated activation of of substrates targeted for lysosomal degrada-
mVps34 and PI(3)P production (148) and is tion requires the protein Hrs, which contains a
necessary for the association of the Vps proteins FYVE (Fab1, YOTB, Vac1, EEA1) zinc-finger

domain that targets it to PI(3)P-bearing this transition. The protein Mon1a/b (known
phagosomes (152). Additionally, SNX3, an- as SAND-1 in C. elegans) is a novel Rab5 effec-
other PI(3)P-binding protein, is required for tor that interacts solely with active Rab5. Upon
ILV formation and may act in concert with recruitment to early phagosomes, Mon1 exerts
Hrs to initiate ILV genesis (153). Experimen- a two-pronged effort to facilitate the acquisi-
tal depletion of Hrs produced phagosomes tion and activation of Rab7 (157, 158). First,
that retained early markers (e.g., EEA1) for Mon1 induces the recruitment of the Rab7-
inordinately long periods and had an impaired binding protein Ccz-1 to the early phagosome.
ability to acidify and to acquire markers of late When associated with the Rab5/Mon1 com-
stages of maturation [e.g., lyso-bisphosphatidic plex, Ccz-1 functions as a tether to capture
acid (LBPA); see below] (152). GDP-bound Rab7 (157). Second, Mon1 is in-
Membrane deformation leading to invagi- trinsically able to displace from the membrane
nation of the endosomal (and, presumably, the Rab5 GEF, Rabex-5, thereby inactivat-
also the phagosomal) membrane is catalyzed ing phagosome-bound Rab5 and suppressing
by the ESCRT-I and -II complexes, whereas Rab5-regulated early events (158). Whereas the
scission of ILV appears to be mediated by Mon1/Ccz-1 complex does not mediate Rab7
ESCRT-III proteins that are then recycled by nucleotide exchange, Ccz-1 appears to promote
the ATPase Vps4 (154; reviewed in Reference dissociation of the GTPase from its GDI, which
155). ESCRT-III and ILV formation may is an important initiating step toward Rab7 ac-
be subject to phosphoinositide regulation, tivation (157). Nucleotide exchange and activa-
given that Vps24 (CHMP3 in mammals)— tion of Rab7 may be effected by another protein
an ESCRT-III member—binds directly complex, termed homotypic fusion and pro-
to phosphatidylinositol-(3,5)-bisphosphate tein sorting (HOPS), that might operate simul-
[PI(3,5)P2 ], the product of the FYVE finger– taneously to mediate Rab conversion. Indeed,
containing phosphoinositide kinase (156). the HOPS complex is required for the acquisi-
tion of Rab7 by endosomes (159) and, presum-
ably, is similarly required for Rab conversion
The Late Phagosome on phagosomes. HOPS, composed of the core
Eventually, the early phagosome transitions to complex of proteins Vps21, Vps16, Vps18, and
a more mature late phagosome that is defined Vps33, is predicted to be tethered to phago-
by the acquisition of distinct biochemical mark- somes, in which it associates with the accessory
ers, including the small GTPase Rab7, with the proteins Vps41 and Vps39; the latter possesses
concomitant loss of early markers such as Rab5. Rab7 GEF activity (160). Importantly, recent
Compared with early phagosomes, late phago- evidence suggests that Mon1 interacts directly
somes are more acidic (pH 5.5–6.0), which is with core proteins of the HOPS complex (158).
a consequence of the acquisition of additional In this regard, Mon1 appears to function as an
proton pumps. Proton pumping is catalyzed by effector that links Rab7 recruitment to early
the vacuolar ATPase (V-ATPase), a multimeric phagosomes via Ccz-1 and, through its ability
protein complex that translocates H+ (inward) to directly associate with the Rab7 GEF com-
across endosomal and phagosomal membranes plex HOPS, mediates nucleotide exchange and
at the expense of ATP. Rab7 activation (157, 158).
The mechanism whereby an early Rab5- Rab7 and its effectors are vital for the
positive/Rab7-negative phagosome transitions completion of phagosome maturation. Indeed,
to become a late Rab7-positive/Rab5-negative impairment of Rab7 activation blocks phago-
phagosome has been the subject of much de- some/lysosome fusion and proper acidification
bate. However, recent experimental evidence (161). Even though Rab7 is of critical impor-
employing C. elegans coelomocytes revealed tance to maturation, the characterization of its
some critical molecular players that mediate effectors is lagging, and to date few have been

identified. Rab7-interacting lysosomal protein above, Rab5 and its early effectors are essen-
(RILP) and oxysterol-binding protein–related tial for the recruitment of the SNX subcomplex
protein 1 (ORPL1) are two effectors that to early phagosomes. Recently, however, it has
accumulate in a Rab7-dependent manner on been revealed that association of retromer pro-
late degradative endosomes, phagosomes, and teins Vps26, Vps29, and Vps35 with assembled
lysosomes (162, 163). Together, RILP and SNXs requires active Rab7 (148).
ORPL1 coordinate microtubule-dependent Within the lumen of late phagosomes are
vesicular traffic of Rab7-positive compartments ILVs, which are characterized by the presence
through direct association with the molecular of the unique lipid LBPA and the tetraspanin
motor dynein/dynactin (163). Centripetal protein CD63. The precise function of LBPA is
movement and the perinuclear localization of unclear, but its perturbation drastically impairs
phagosomes are needed for efficient lysosomal traffic within the endosomal network and alters
fusion and require active RILP, dynein, and intracellular cholesterol homeostasis (166). In-
intact microtubules (161). deed, endocytosis of antibodies to LBPA results

Although the importance of Rab7 for phago- in aberrant intracellular cholesterol accumula-
some maturation is unquestionable, its acquisition in LBPA-containing compartments (166),

tion of and activation on phagosomes alone are a feature that is strikingly reminiscent of the cel-
insufficient to mediate phagosome/lysosome lular phenotype of Niemann-Pick type C dis-
fusion (161). Antagonists of PI3K that block ease, a lipid-storage disorder (166, 167). Impor-
phagosome maturation do not eliminate Rab7 tantly, abnormal cholesterol transport blocks
recruitment and activation, which indicates phagosome maturation at the level of Rab7 acti-
that there are additional phosphoinositide- vation, preventing phagosome/lysosome fusion
dependent events that must operate in con- (168). This response conceivably contributes to
cert to mediate phagosome maturation. These the development of infections observed in some
events remain to be identified (123). Niemann-Pick type C disease patients. Once
Additional factors, which were thought the appropriate material has been dispersed
to be mostly structural components of late from the phagosome either to the TGN, via
phagosomes, are also required for maturation. recycling, or to the phagosome lumen by
Lysosome-associated membrane proteins 1 and ILV formation, the late phagosome proceeds
2 (LAMP-1 and -2) are heavily glycosylated further along the maturation pathway by
integral membrane proteins that are abun- fusing with lysosomes to form the microbicidal
dant in late endosomes, lysosomes, and mature organelle, termed the phagolysosome.
phagosomes. Although it had been tacitly as-
sumed that LAMP proteins serve mainly to
preserve lysosomal membrane integrity, recent The Phagolysosome and Its Arsenal
experimental evidence demonstrates that they of Microbicidal Effectors
are in fact essential for Rab7 recruitment and The mature phagosome is the ultimate mi-
for the acquisition of microbicidal functions crobicidal and degradative organelle; it can
by phagosomes (164, 165). Indeed, LAMP- digest proteins, lipids, and carbohydrates.
deficient cells are incapable of eradicating Neis- Acquisition of a complete armamentarium of
seria gonorrhoeae, a bacterial pathogen that is antimicrobial and hydrolytic effectors by the
otherwise killed by wild-type cells (165). phagosome requires fusion with preformed
Continuous remodeling of the phagosome lysosomal compartments. Fusion of lysosomes
via recycling and MVB biogenesis also occurs at with endosomes (and ostensibly phagosomes)
the late phagosomal stage. Although retromer- is coordinated in part by the formation of a
mediated retrieval of M6PRs to the TGN is SNARE complex composed of syntaxin 7 and
initiated on early phagosomes, completion of VAMP7 and requires Ca2+ , which is released
this process requires active Rab7. As described from the lumen of the fusing organelles.

Both syntaxin 7 and VAMP7 are present in Proton pumping and the resulting acidifi-
phagosomes, and interference with syntaxin 7 cation serve multiple purposes in the context
function blocks phagolysosome fusion (28, 29). of phagosome physiology. The acidity di-
Biochemically, the phagolysosome can be rectly restricts microbial growth and indirectly
defined by its marked acidity (pH 4.5–5.0); its enhances the degradative capacity of the phago-
enrichment with active cathepsins; and the ab- some by activating proteolytic enzymes such as
sence of PI(3)P, M6PR, and LBPA (169–171). cathepsins D and L (176, 177). The proton gra-
Phagolysosome acidification, which itself con- dient across the phagosomal membrane further
tributes to target degradation, is also required constrains microbial growth by stimulating
for the activation of lysosomal hydrolases such natural resistance–associated macrophage pro-
as cathepsins that function optimally at low tein 1 (NRAMP), a divalent metal transporter.
pH. The impressive destructive capacity of the NRAMP, which is described in more detail
phagolysosome is attributed to the concerted below in the section entitled Antimicrobial Pro-
activity of numerous effectors, including hy- teins and Peptides, utilizes the electrochemical
drolytic enzymes, oxidants, and cationic pep- proton gradient as a source of energy to eject
tides. Note, however, that the relative contri- metals from the phagosomal lumen, thereby
bution of the individual lytic mechanisms varies depriving bacteria of the essential cofactors
among the different types of phagocytes, de- they require for survival and proliferation
pending on their primary function: for instance, (178). Intraphagosomal protons also partici-
pathogen eradication in the case of neutrophils pate in the generation of lethal reactive oxygen
versus antigen presentation for dendritic cells. species (ROS), another important component
The arsenal of antimicrobial effectors at the dis- of the antimicrobial arsenal of phagocytes (179,
posal of phagocytes is discussed below. 180). Finally, acidification is not merely a con-
sequence of phagolysosome fusion, but appears
Phagosome acidification. The acidification also to be required for the maturation process.
of the phagosomal lumen is generated by V- Indeed, exposure of cells to weak bases or
ATPases. Mature phagosomes have a reduced chemical inhibitors of the V-ATPase not only
passive proton permeability, often referred to as blocks acidification but also perturbs endoso-
proton leak, which enables the protons pumped mal traffic and phagosome maturation (181).
by the V-ATPases to greatly accumulate (172). Given the importance of phagosome acidifica-
The V-ATPase is a massive (≈103 -kDa) multi- tion, it is not surprising that many notorious
meric protein complex consisting of two major intracellular pathogens—including Salmonella
functional subcomplexes, the V1 and the V0 . and Mycobacterium—exhibit a variety of
The cytosolic V1 subcomplex comprises eight strategies to sense, respond to, and circumvent
different protein subunits and mediates ATP the effects of phagosome acidification (182–
hydrolysis. The V0 subcomplex is integral to 184). H. pylori, noted for its ability to survive
the membrane and constitutes the pore through exposure to stomach acid, also manipulates
which protons are pumped, at the expense of the acidic environment of the phagolysosome
ATP, into the lumen of the phagosome (173). through the expression of urease. Through
In addition to the V-ATPase, the generation the production of ammonium from urea,
and maintenance of the extremely acidic lumi- H. pylori can buffer the phagosomal pH to pre-
nal pH require other ion-transport processes vent maturation and allow for intraphagosomal
to prevent the buildup of a prohibitive electri- survival (185).
cal potential across the phagosomal membrane. The magnitude of the H+ gradient
The inward flux of anions (mainly Cl− ), as well across the phagosomal membrane can vary
as the efflux of cations (probably K+ and Na+ ) greatly, depending on the type of phagocyte.
through conductive pathways, neutralizes the Macrophages, which are effective antimicro-
electrogenic effect of the V-ATPase (174, 175). bial cells, fully acidify their phagosomes. In

contrast, the phagosomes of dendritic cells, Whereas p67phox and p47phox are required
whose primary function is to present antigens, for activation of flavocytochrome b558 at the
are considerably more alkaline (pH ≈ 6.0). plasma membrane (e.g., at the phagocytic cup),
The more modest acidification of dendritic cell p40phox is necessary for NOX2 activity in
phagosomes limits the extent of proteolysis, formed (sealed) phagosomes. The function of
leaving antigens of appropriate size for load- p40phox at the phagosomal membrane is de-
ing onto major histocompatibility complex for pendent on mVps34 and its product, PI(3)P
cross presentation. The more alkaline pH of (188). In addition, other immunomodulatory
dendritic cell phagosomes has been attributed proteins, such as the signaling lymphocyte-
to NOX2, an NADPH oxidase that consumes activation molecule family (SLAMF), can alter
luminal protons by generating superoxide oxidase activity in response to proinflammatory
anions (186, 187). stimuli (189). SLAMF1 is expressed by most
hematopoietic cells, in which it functions as
a sensor of highly conserved outer-membrane

Reactive oxygen and nitrogen species. proteins of gram-negative bacteria. SLAMF1
The importance of ROS production in innate stimulation modulates NOX2 activity at the
immunity became firmly established once the phagosome and affects phagosome maturation
molecular basis of chronic granulomatous through direct recruitment of mVps34, thereby
disease was revealed. Patients suffering from promoting local PI(3)P production (189).
this disease, a recessive genetic disorder, are Despite the complexity and sophistication
exquisitely susceptible to life-threatening of the phagocyte oxidative arsenal, many suc-
bacterial and fungal infections due to an im- cessful intracellular pathogens have developed
paired ability to assemble a functional NOX2 mechanisms to evade ROS toxicity. To this end,
oxidase. As mentioned above, this isoform of pathogens have implemented means of either
the NADPH oxidase is responsible for the chemically altering the various ROS (e.g., by
production of ROS in the phagosome. NOX2 expressing superoxide dismutase and catalase)
is a multimeric protein complex that assembles or preventing ROS production altogether by
in response to proinflammatory stimuli to me- perturbing oxidase function directly (190–192).
diate the production of superoxide anion (O− 2) Like ROS, reactive nitrogen intermediates
by transferring electrons from nicotinamide (RNI) contribute to pathogen eradication by
adenine dinucleotide phosphate to molecu- nonselectively damaging cellular proteins,
lar oxygen. NOX2 consists of the integral lipids, and nucleic acids and are an important
membrane proteins gp91phox and gp22phox , element of the phagocyte antimicrobial arma-
which together form flavocytochrome b558 , mentarium (193). The formation of nitrous
and the cytosolic components p40phox , p47phox , oxide (NO· ) in phagocytes is catalyzed by
and p67phox . Upon activation, the cytosolic nitrous oxide synthase 2, also termed inducible
components assemble together with the small NOS (iNOS). In contrast to ROS, which are
GTPases Rac1 and Rac2 and associate with the produced through rapid assembly of preexist-
flavocytochrome b558 to shuttle electrons across ing components of the NADPH oxidase, RNI
the limiting membrane of the phagosome to production is delayed because cells under basal
form O− −
2 . O2 itself is a cytotoxic species, but it conditions do not express iNOS. Indeed, the
can also be dismutated to form highly reactive transcription and translation of the iNOS gene
H2 O2 . H2 O2 can react directly with phagoso- are stimulated only through the activation of
mal contents and can lead to the generation of p38 mitogen-activated protein kinase, nuclear
toxic hydroxyl radicals via Fenton chemistry. factor κB, or the JAK-STAT-IRF1 ( Janus-
Additionally, the granular enzyme myeloper- activated kinase–signal transducer and activator
oxidase utilizes H2 O2 to convert chloride ions of transcription–interferon regulatory factor
into deadly hypochlorous acid (179). 1) signaling cascades (194–196). Induction of

iNOS expression requires phagocyte detection for RNI in immunity to various bacterial, fun-
of proinflammatory cytokines (e.g., tumor gal, and protozoan infections (203, 204). These
necrosis factor α) and/or conserved pathogen- and other studies demonstrated that RNI-
associated molecules, such as LPS and dependent killing of intracellular pathogens
lipoteichoic acid, by their cognate receptors. features prominently in the macrophage re-
The iNOS protein comprises an N-terminal sponse to infection and functions in neutrophil
oxygenase domain that binds the substrate and dendritic cell immunobiology.
L-arginine, haem, and tetrahydrobiopterin.
The C-terminal region consists of a reductase Antimicrobial proteins and peptides.
domain that possesses binding sites for calmod- Phagocytes have at their disposal an impressive
ulin, flavin mononucleotide, flavin adenine array of proteins and peptides that antagonize
dinucleotide, and the electron donor NADPH. microbial fitness from within the phago-
Functionally, iNOS operates as a homodimer some (205). Neutrophils are particularly well
and, as a consequence of electron transfer from equipped, as they possess various specialized
NADPH, oxidizes L-arginine to yield NO· and secretory organelles (granules) that contain a
L-citrulline (197, 198). The delivery of NO· to broad spectrum of bactericidal and degradative
its intended site of action is unique in that it is proteins, some of which are also expressed
synthesized on the cytosolic face of the phago- by macrophages. In some instances, these
somal membrane and subsequently diffuses into antimicrobial factors act as effectors that kill
the phagosome lumen. Although NO· is itself ingested pathogens, whereas others may hinder
a toxic radical gas, it readily reacts further with growth by depriving internalized microbes of
ROS to form various toxic RNI, such as peroxy- essential nutrients. Iron is an essential metal
nitrite (ONOO− ). As microbicidal mediators, that functions as cofactor in several bacterial
RNI and ROS operate in concert, reacting processes including DNA replication, and
with a plethora of proteins and with DNA to bacteria have specialized systems that facilitate
curtail pathogen replication and viability. For iron acquisition (206). Neutrophils strive to
instance, NO· can react with integral compo- restrict luminal iron availability through the
nents of the respiratory chain of many bacteria delivery of lactoferrin, an iron (Fe3+ )-binding
and of the eukaryotic parasites Leishmania protein found within granules that fuse with
major and Trypanosoma spp., thereby blocking phagosomes (207). Lactoferrin is a multital-
electron transport and ATP biosynthesis ented antimicrobial protein that also curtails
(199, 200). Additionally, ONOO− can oxidize pathogen survival through direct interaction
nucleic acids and induce double-stranded with the microbial cell surface (208). Additional
DNA breaks that lead to bacterial genome mechanisms that restrict nutrient availability
instability. to microbes trapped within the phagosome
Although iNOS and its reactive products include NRAMP1 (also known as SLC11A1),
restrict microbial survival within the phago- an integral membrane protein that is highly
some, various pathogens have evolved strate- enriched in endosomes and lysosomes of
gies to protect themselves from RNI-mediated phagocytes (209, 210). NRAMP1 exhibits
toxicity. One striking example is provided by bacteriostatic and antiparasitic effects, which
mycobacteria: M. tuberculosis impairs the gen- are attributed to its ability to extrude from the
eration of RNI by interfering with the ability phagosomal lumen important divalent cations
of the scaffolding protein EBP-50 to recruit such as Zn2+ and Mn2+ (178).
iNOS to the phagosome (201, 202). Although Phagocytes also express a plethora of
increased susceptibility to infectious diseases in antimicrobial proteins that react directly with
humans as a consequence of iNOS deficiency proteins, membranes, or carbohydrates of the
has not been described, studies employing ingested microorganisms, directly jeopardizing
iNOS-deficient mice clearly demonstrate a role their structural integrity. Lysozyme, which

hydrolyzes β(1-4) glycosidic linkages between than 50 different lysosomal acid hydrolases.
the main structural components of the bacterial The concerted action of proteases, lipases,
peptidoglycan layer, compromises the integrity nucleases, glycosidases, and phosphatases can
of gram-positive bacteria. In the case of mediate the complete disintegration of large
gram-negative organisms, the outer membrane complex structures such as dead or dying
confers protection against lysozyme-mediated microbes. The very important and widely
attack. However, phagocytes also express studied cathepsin family of lysosomal enzymes
cationic antimicrobial peptides (CAPs), such as consists of serine proteases (cathepsins A and
the defensins, that can permeabilize the outer G), aspartate proteases (cathepsins D and E),
membrane. In general, CAPs are highly toxic and cysteine proteases (cathepsins B, C, F, H,
effectors that kill bacteria through a broad K, L, O, S, V, X, and W). Although they are
spectrum of mechanisms (211). Because they stimulated by some of the conditions prevailing
are highly cationic, CAPs such as defensins inside phagosomes (e.g., acidity), cathepsins
bind electrostatically to negatively charged are adversely affected by others. Specifically,

bacterial cell-surface components and exert the redox state of the phagolysosome imposed
their microbicidal effect by forming pores that by the NADPH oxidase diminishes their pro-
allow the diffusion of ions across the bacterial teolytic activity (214). This complex regulation
membrane, dissipating essential gradients and may be needed to ensure that proteases such as
creating an osmotic imbalance (212). cathepsin H and S that are delivered to early
The immense degradative capacity of the and late phagosomes, respectively, are fully
phagolysosome reflects the joint efforts of more active only when the time is right (215).
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
Original work in the authors’ laboratory is supported by the Canadian Cystic Fibrosis Foundation
and the Canadian Institutes of Health Research (CIHR). The authors thank M. Bohdanowicz
for providing fluorescence images of RAW cell transfectants. R.S.F. is the recipient of a CIHR
fellowship. V.J. is the recipient of a Heart and Stroke Foundation of Canada fellowship. S.G. is
the current holder of the Pitblado Chair in Cell Biology.
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15:578–84

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Molecular Mechanisms of Fragile X Syndrome:
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Pathogenesis of NUT Midline Carcinoma
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ATM and the Molecular Pathogenesis of Ataxia Telangiectasia
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Tuberculosis Pathogenesis and Immunity
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Psoriasis
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Caveolin-1 and Cancer Metabolism in the Tumor Microenvironment:

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Indexes
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Cumulative Index of Chapter Titles, Volumes 1–7 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 500
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• Top downloaded articles

Annu. Rev. Pathol. Mech. Dis. 2012. 7:61–98 Keywords

INTRODUCTION description of phagocytosis, most of these

macropinocytosis, phagocytosis involves the

teria or fungi can be cleared from infection sites RECEPTOR-MEDIATED PROCESS

62 Flannagan · Jaumouillé · Grinstein

Table 1 Human receptors mediating phagocytosis, and their ligands

FcγRIIc (CD32c) IgG —

FcεRI IgE 220

www.annualreviews.org • Cell Biology of Phagocytosis 63

ceptors are engaged simultaneously, producing

64 Flannagan · Jaumouillé · Grinstein

www.annualreviews.org • Cell Biology of Phagocytosis 65

process. Importantly, activation of FcγR is an

face by ≈300-fold. This observation suggests

66 Flannagan · Jaumouillé · Grinstein

becomes associated with detergent-resistant

linking (39, 40). Second, cholesterol depletion

www.annualreviews.org • Cell Biology of Phagocytosis 67

68 Flannagan · Jaumouillé · Grinstein

PI(4,5)P2 Plasmalemma PH-PLCδ-GFP Akt-PH-RFP

www.annualreviews.org • Cell Biology of Phagocytosis 69

pendently of actin polymerization (62). bound) state. Nucleotide exchange is essential

70 Flannagan · Jaumouillé · Grinstein

www.annualreviews.org • Cell Biology of Phagocytosis 71

in engulfment (87–88). Myosin X has been

phagosome formation (91); it has been detected

72 Flannagan · Jaumouillé · Grinstein

www.annualreviews.org • Cell Biology of Phagocytosis 73

Syk is phosphorylated during CR3-mediated F-actin accumulation, and iC3b-opsonized par-

knockdown or by expression of a dominant- for Arp2/3 recruitment and actin polymeriza-

74 Flannagan · Jaumouillé · Grinstein

PHAGOSOME MATURATION: is followed by a rapid succession of biochemical

www.annualreviews.org • Cell Biology of Phagocytosis 75

as predicated by the so-called kiss-and-run these processes are coordinated by a family

76 Flannagan · Jaumouillé · Grinstein

www.annualreviews.org • Cell Biology of Phagocytosis 77

Rab5, PI(3)P, and EEA1], but Rab7 activation and phagosome

78 Flannagan · Jaumouillé · Grinstein

to usurp host cell functions and to construct its inclusion

of host transmembrane proteins

www.annualreviews.org • Cell Biology of Phagocytosis 79

trans-Golgi network reduced protein (e.g., elastase) delivery to

80 Flannagan · Jaumouillé · Grinstein

the maturing phagosome remains noticeably to form a recycling-competent retromer (148).

www.annualreviews.org • Cell Biology of Phagocytosis 81

82 Flannagan · Jaumouillé · Grinstein

intact microtubules (161). deed, endocytosis of antibodies to LBPA results

some maturation is unquestionable, its acquisi- tion in LBPA-containing compartments (166),

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