Come On In Get Killed Death in the Shredder

Phagocytosis is defined as the ingestion of a particle by a cell (see Chapter 3). Phagocytosis in vertebrates is typically carried out by professional phagocytic cells, that is, polymorphonuclear cells (particularly neutrophils), dendritic cells, monocytes and macrophages, but is not unique to them. Most ingested particles are apoptotic cells, microorganisms and denatured protein complexes larger than some 0.4 mm in diameter. Their surface molecules are recognized by a variety of phagocyte surface receptors. Spatial accumulation of such receptors ("clustering") by the local crowding of their microbial partner molecules ("ligands") can then trigger signaling into the cell followed by membrane deformation, cytoskeleton rearrangement and a wrapping of the particle in host cell membrane. Eventually, the particle is ingested by

Phagocytic Vacuoles Dendritic Cell
Figure 10.1 Phagosomes in macrophages. Murine J774E macrophage-like cells have been infected with Afipiafelis for 24 h. A communal vacuole harboring several bacteria is found next to vacuoles containing single bacteria (arrowheads). Reprinted with permission from [2].

fusion at the tip of the nascent membrane bag and a phagosome is formed (Figure 10.1; see also Chapter 3).

In terms of cell biology, the newly created phagosome is an endosome specialized in microbial killing and, particularly in dendritic cells, in supporting antigen presentation. Ligation of only certain phagocyte receptors triggers the flushing of the phagosome lumen with antimicrobial compounds. As a central event in phagosome biogenesis, the phagosome lumen is strongly acidified (to pH ~4.5) by a membrane-embedded proton-pumping ATPase (Figure 10.2). An NADPH oxidase complex produces large quantities of superoxide radicals from molecular oxygen, and, in activated macrophages of some species, a nitric oxide synthase produces NO radicals from arginine. Through the cooperation of reactive oxygen and nitric oxide species, highly reactive peroxynitrite can be formed within the phagosome. Neu-trophils are especially aggressive. They possess myeloperoxidase which, together with the above enzyme activities, leads to the formation of hyperchlorous acid and chloramines, hydroxyl radicals, and ozone, all ofwhich are biocidal. As non-oxidative tools, some 50 different lysosomal hydrolases (proteases, DNases, lipases, etc.) compromise microbial viability and integrity, and defensin peptides and cationic proteins add to the killing potential [1, 2].

The development of the phagosome into a fully degradative phagolysosome is a temporally and spatially ordered process and parallels endosome maturation [3] (see Chapter 3). The newly formed "early" phagosome develops vectorially into a "late" phagosome after fusion with late endosomes and will finally become a phagolysosome by fusion with lysosomes (the normally terminal organelles of the endocytic pathway). Recent data suggests that more complex fusion patterns may exist [4, 5], but most features seen can be well explained by operational definition of

Host Cell Definition

Figure 10.2 Outline ofthe phagocytic pathway in mammalian cells. When a harmless (non-interfering) microorganism (MO) is ingested by a phagocytic cell, it is wrapped in part ofthe host cell plasma membrane and a phagosome is formed. This will fuse successively with early (sorting) endosomes (SE), late endosomes (LE), and lysosomes (Lys). Some recycling of host cell surface components may occur via the recycling endosome (RE). The phagosome maturation steps are operationally defined as early and late phagosome and phagolysosome stages. Each of these phagocytic compartments is characterized by a set of marker proteins and lipids (as indicated) with a typical drop in pH along the continuum. NADPH oxidase, acidification, and acidic hydrolases plus defensins act at various steps of phagosome development. Killing of ingested microorganisms is likely most pronounced once the phagolysosome stage has been reached. Comprehensive recyclingfrom the phagosomes to the previous stage of maturation is indicated by backwards arrows; this kind of recycling has not been experimentally addressed but likely happens as it is required to ensure compartment identity. ECV, endosomal carrier vesicle, a vesicle population which has been described to transport membrane proteins to be degraded between sorting and late endosomes. The roles of ECVs in phagosome maturation require further analysis. Blue lettering: killing mechanisms. Marker molecule abbreviations: Early endosome antigen-1 (EEA1), lysosome-associated membrane proteins (LAMP) 1 and 2, phosphatidylinositol 3-phosphate (PI3P), mannose 6-phosphate receptor (M6PR), Ras-like protein from rat brain (Rab) 5, 7, and 11, vesicle-associated membrane protein (VAMP) 3 and 7, proton-pumping vacuolar adenosine triphosphatase (vATPase), Scheme modified from [2].

these three phagocytic maturation stages. These stages can be experimentally discriminated by the presence or absence of stage-specific lipids and proteins or by chasing endocytic tracers into the endocytic continuum for different periods of time (Figure 10.2). Phagosomes containing the same cargo in the same cell type acquire and loose their maturation markers with almost identical kinetics, though exceptions to this rule may apply [5-7]. While a limited fusion competence between endocytic and phagocytic organelles of different maturation stages may actually be a prerequisite for phagosome maturation to occur, early phagosomes fuse mostly "homotypically" with early endosomes and, with much lower frequency, "hetero-typically" with late endosomes [8, 9]. Late phagosomes are likely to fuse relatively avidly with late endosomes and less frequently with lysosomes, which again fuse predominately homotypically. Overall, fusion competence decreases as a phagocytic (or endocytic) compartment matures [9-11].

Rab proteins (mainly Rab 5, 7, and 11) are key regulators of phagosome fusion as they are in other fusion events of the endocytic and exocytic pathways (Figure 10.2). Rab proteins identify and, together with tethering factors, specifically bridge membranes to be fused [12]. SNARE proteins (e.g., syntaxins 7,8 and 13, VAMPs 4, 7 and 8) on both partner membranes, as Rab downstream effectors, form extremely stable four-helix bundle complexes between the membranes and pull them together to a distance of a few nanometers [13]. This event may, in itself, be sufficient to catalyze membrane fusion and is likely supported by additional factors such as calcium-dependent proteins (e.g., calmodulin), ion channels (e.g., proton-pumping ATPase), and phospholipases. In addition to these key players, phagosome acidification can also contribute to maturation to some degree [14], as has been observed with early endosomes [15,16]. While the contributions of cytoskeletal elements to phagocytosis, and phagosome maturation in particular, are still poorly defined, recent studies shed some light on the crucial role in phagosome maturation of the polymerization of tubulin [17] and actin [18,19] (see also Chapter 8). Finally, protein phosphorylation [10] and lipid composition also change with and probably regulate phagosome maturation [10]. Some 1000 different lipid species have been identified on latex bead-containing phagosomes (LBP) from macrophages, adding to the amazing complexity of this organelle (cited after [3]). While the above describes the normal progression of phagosome maturation, some pathogenic, "intracellular" pathogens have specialized in diverting phagosome development. These will be presented below and in many other chapters in this book.

As models for host factors required for phagosome biogenesis both in physiological and pathological states, non-mammalian organisms have started to be used (for details, see Chapter 4). Dictyostelium (a slime-mold), in particular, has developed into an important model, as it is strongly phagocytic and a readily manipula-ble unicellular amoeba. Trafficking is a bit different from that in macrophages: Microorganisms, in particular bacteria, are taken up as food and this process seems to be more a particle-indiscriminating macropinocytosis than a (receptor-mediated) phagocytosis, although phagocytosis may be important as well [20]. Soon after ingestion, the macropinosome or phagosome acidifies and phagolysosome formation is initiated. After digestion of food or if the ingested materials have been unsuccessfully dealt with for some time, lysosomal hydrolases are recaptured and recycled by binding to specific receptors followed by alkalinization of the phago-some lumen. Nondigestible remainders are expelled. This is not normally so for macrophages, yet it should be mentioned that macrophages and likely neutrophils can exocytose a major proportion of their lysosomes when cytosolic calcium levels increase. Cells use this capability to release the contents of lysosome-like melano-somes or lytic granules from toxic Tcells [21]. While release of indigestible material to "get rid of it" would work for Dictyostelium, as debris is expelled into the bulk extracellular world, it does not work well for macrophages, where another phagocyte would likely ingest the particle and then have the same problems.


Professionals and Laypersons

Professional phagocytes are not the only cells that intracellular pathogens have evolved to colonize. In fact, some pathogens, such as Salmonella, are better known for their abuse of epithelial cells than that of macrophages, although both cell types are targets and the pathogen may thrive in either environment. Even more impressive, some pathogens have developed mechanisms to invade strictly nonphagocytic cells, the paradigm of which is red blood cell (RBC) colonization by Plasmodium, the malaria parasite. The question is: How can eukaryotic cells be either professionally phagocytic, occasionally phagocytic or not phagocytic at all?

Professional Phagocytes

The professional phagocytes of vertebrates have been designed by nature to sniff out, bind to and ingest microbial foreign or nonphysiological particles, to decompose them and, where appropriate, present their antigenic structures to cells of the acquired immune system. Therefore, an arsenal ofphagocytic receptors has evolved that takes care of the ingestion of a wide variety of particulate matter (see Chapter 3). These receptors either recognize particle structures directly or they identify "opsonizing" host proteins that have attached to the particles and branded them as material destined to be destroyed. While some receptors are more specific for certain ligands (e.g., complement receptor, immunoglobulin Fc receptor), others are not as discriminating (e.g., scavenger receptors). The various receptors are parts of different signaling pathways which regulate the reactions to the ingested particles. For example, a particle that has been covered with immunoglobulin G (IgG) can be interpreted as an enemy whose identity is known to the infected organism and that has previously activated an immune response and, therefore, is likely dangerous. Not surprisingly therefore, the macrophage's response to an IgG-covered particle is quite strong and mounts a strong production of oxygen radicals. On the other hand, an apoptotic cell which does not pose an infection threat to the organism is removed in an "immunologically silent" way after recognition by apoptosis-dedicated receptors such as phosphatidylserine receptors [22].

Nonprofessional Phagocytes

Nonprofessional phagocytes were discovered when formaldehyde-fixed RBCs were fed to macrophages and fibroblasts [23]. These particles were surprisingly taken up by either cell type. Coating RBCs with specific antibodies made them a particularly good meal for macrophages, while they were less avidly ingested by fibroblasts which do not possess immunoglobulin receptors. A recent paper by Gratton et al. [24] demonstrated that synthetic particles of up to 5 mm diameter can be ingested by epithelial (HeLa) cells, albeit with varying efficiency. Of 1 mm particles, a surprising 70% were taken up by these "nonprofessional^" phagocytic cells. However, the above studies used cell lines for analysis and it is possible that immortalization affects phagocytosis, although some cases of phagocytosis with primary "nonphagocytic" cells are clearly documented [25]. In general, it is probably fair to say that professional phagocytes have a much wider array of surface receptors at higher density connected to internalization signaling pathways and therefore ingest more types ofparticles more avidly than nonprofessionals do.

Nonprofessionally phagocytic cells, on the other hand, can be turned into professional phagocytes, at least with respect to some particles, when they are transfected with suitable receptor genes such as the immunoglobulin receptor [26, 27] or complement receptor 3 [27]. Such experimental model cells are termed "engineered phagocytes."

As particulate matter can, in principle, be ingested by nonprofessional phagocytes, experimental uptake of microorganisms by nonprofessional phagocytes, in particular low-level uptake, does not necessarily constitute evidence for a microbial strategy to force its uptake into these cells. For example, fibronectin is a very "sticky" protein of serum and can opsonize particles such as bacteria [28] and mediate phagocytosis [29], likely via host cell integrins. Most ofthe fibronectin is a constituent of the extracellular matrix, where it promotes the adhesion of cells such as fibroblasts. Therefore, phagocytosis of fibronectin-coated latex beads [30] by nonprofessional phagocytes maybe a mimic of colonization of substratum which, more by chance, results in phagocytosis when two different portions of the plasma membrane that have surround a fibronectin substratum fuse with each other. As with other virulence pathways, some pathogens, such as Staphylococcus aureus (a more extracellular pathogen), have "learned" to abuse this normality by expressing on their surfaces fibronectin-binding proteins [31].

Another important pathway of forced entry into nonprofessionals is macropino-cytosis which is triggered by Salmonella. Salmonella uses a "type III protein secretion system" (T3SS) to inject effector proteins into the host cell that induce the formation of large lamellipodial structures that will eventually fuse with each other and form a large vesicle, the macropinosome [32]. Macropinocytosis can be normally induced in many cells [33], usually by exposure to growth hormones, such as epithelial growth factor. Therefore, Salmonella opens a host front door by using a fake key. A microbe's fate can be vastly different depending on which port of entry has been used into which type of host cell.

What happens when a microbe is finally inside, say, an epithelial cell? Some microorganisms will be killed [34] and others might thrive [35]. This means that the capability to enter a nonprofessional phagocyte does not necessarily ensure pathogen multiplication. In fact, the resulting host cell vacuoles are not automatically hospitable places, just because the cell around them is not specialized in killing microbes [36, 37].

"Not Phagocytic Cells at All"

Colonization of RBCs is the supreme discipline when it comes to colonizing vertebrate cells and only few pathogens, such as the malaria parasite Plasmodium falciparum (see Chapter 33), the apicomplexan Babesia spp. (see Chapter 33), or the pathogenic bacterium Bartonella spp. [38], have mastered it. The reason for this is that RBCs do not endocytose and, hence, this pathway cannot be hijacked by pathogens. They have to enter via mechanisms that involve major contributions from themselves (like the actin-propelled and gliding motilities of apicomplexans). The entry process of Plasmodium merozoites into RBCs has been studied in detail and involves secretion of parasite granules and shedding of a protein surface coat [39] (see Chapter 33). Important features of the RBC as a home are the lack of lysosomes and antimicrobicidal processes such as production of reactive oxygen metabolites. RBCs further provide a relatively long-lived (60-120 days life span) membrane-surrounded and protected place to live with no antigen-presentation machinery in the immediate neighborhood. RBCs can also be taken up in great numbers by bloodsucking insects and, hence, provide an appealing mode of distribution.

Although the above shows that, in principle, pathogens can thrive in any of these host cell types, macrophages are still the preferred host cells for many intracellular pathogens because they

• are professionally phagocytic and will, in contrast to nonprofessional phagocytes or to "phagocytosis-inert" cells such as RBC, actively ingest most particles so that entry ofthe phagocyte does not necessarily require evolution of invasion or "forced entry" mechanisms;

• can have a considerably longer lifetime (months to years) than neutrophils (12-72 h), a cell type which would be another obvious choice. This feature is particularly important for slow-growing pathogens such as Mycobacterium tuberculosis which would possibly not even start multiplying when the host cell was already dying by causes unrelated to the infection;

• are less microbicidal than neutrophils;

• are likely better food suppliers than RBCs, which contain plenty of hemoglobin but little else and which cannot supply a microbe-containing phagosome with nutrients via endocytosis.


Come On In, Have Fun! Life in a Golden Cage

In the following, intracellular pathogenic microbes will be categorized according to the prevailing characteristics oftheir vacuoles (Figure 10.3; Table 10.1). Some vacuoles share features of different compartments (Table 10.2), but for the sake ofsimplicity, this review



Phagosome Definition

Figure 10.3 The subcellular compartments of a mammalian cell and the "phagosome zoo" within it. Shown is a scheme of a mammalian cell. Note that red blood cells do not have elaborate subcellular compartments but that, nevertheless, RBC-infecting eukaryotes have been included. The secretory pathway for the production and location of proteins to the endoplasmic reticulum (ER), the Golgi apparatus (c/s-Golgi, CGN and trans-Golgi network, TGN), secretory vesicles (SV), and to the plasma membrane (PM) are shown on the left. Secretory proteins are synthesized on the (rough) ER, encased in transport vesicles that are shuttled to the c/s-Golgi apparatus, further modified in the Golgi apparatus and finally secretory vesicles are released from the trans-Golgi network for transport to the plasma membrane. Some specific vesicles from the TGN fuse with sorting or late endosomes (as defined in Figure 10.2) and bring new proteins for endosomes and lysosomes. The second major pathway, the continuum of endocytic vesicles which includes the formation of small vesicles on the plasma membrane followed by fusion with early sorting lysosomes (SE) is shown on the right. These early endosomes mature into late endosomes (LE) and finally into lysosomes (Lys). Phagocytic compartments of different maturation stages possess a similar protein and lipid composition to their corresponding endosomal maturation stages. Macropinosomes (MPs) bring large quantities of liquid into the cell and can be hijacked as well. Some pathogens (Toxoplasma) enter the cell by true invasion and create a special parasitophorous vacuole which, in some cases, can associate with mitochondria (M). Autophagosomes (AP) are formed by mammalian cells when nutrients are scarce or the phagocyte is suffering from other forms of distress. These autophagosomes enclose cytoplasm (CYTO), possibly containing organelles, in a process which is only little understood. These autophagosomes then fuse with lysosomes to form autophagolysosomes (APL) in which the contents are degraded and from which the degradation products are made available to the cell for biosynthesis. Staphylococcus aureus is normally an extracellular pathogen but, in small quantities, can also be incorporated into autophagosomes, concomitant with inhibition ofautophagolysosome formation. Subsequent escape into the cytoplasm allows for limited multiplication [125]. Pathogens are placed in this scheme according to which compartment resembles most their protein composition once they have established a stable compartment. Further compartments: nucleus (N), peroxisomes (P). For details, see text and corresponding chapters in this book.

Table 10.1 Intracellular vacuolar pathogens.

Pathogen Host cells Vacuole Marker molecules Vacuole pH

(only genus) identity of mature and illness vacuoles caused by it

Afipia (cat scratch disease)

Anaplasma (3) (anaplasmosis)

Brucella (brucellosis)

Chlamydia (3) (urogenital infections, conjunctivitis)

Macrophages, likely freshwater amoebae

Not canonically endocytic compartment

Granulocytes, endothelial cells

Macrophages, dendritic cells, epithelial cells Epithelial cells


Endoplasmic reticulum

Post-Golgi compartment

No marker enriched, compartment negative for, e.g., LAMP1/2, proton-pumping ATPase, TfR, Rab5, Rab7, ca-thepsin D, EEA1. Ganglioside GM1 is present at least early in infection. LC3, beclin-1

Calnexin, calreti-culin, Rabl, Rab2, BIP, Sec6ip Multiple chlamydial proteins (Incs), host lipids sphingomyelin and cholesterol

Not acidic

Average Average Vacuole Time How does vacuole size number localization spent in the microbe of microor- in cell host cell exit?

ganisms / vacuole

10 min after 1-5 Perinuclear ND ND

0.3-8 urn 1-50 ND 4-7 ds Host cell rupture, exocytosis

2 1 Perinuclear 3 ds in vitro, Unknown several dd in vivo

1-40 1-1000 Perinuclear 2-3 ds Lysis or exocytosis


Table 10.1 (Continued)

Pathogen Host cells Vacuole Marker molecules Vacuole pH

(only genus) identity of mature and illness vacuoles caused by it

Ehrlichia (3) (ehrlichiosis)


Macrophages monocytes

Lysosomal, autophago-lysosomal

Early endosome

thepsin D, LC3,

Rab7, Rab24, proton-pumping

ATPase, cholesterol

Rab5, EEA1, TfR Slightly acidic

Francisdla (tularemia)

Histoplasma (histoplasmosis)

(legionnaire's disease)

Leishmania (3) (leishmaniosis)


Human macrophage

Freshwater protozoa and macrophages

Unusual late endosome, then cytosolic ND

Endoplasmic reticulum

Macrophages Lysosomal


Proton-pumping ATPase is excluded ARF1, Rabl, Calnexin, KDEL motif proteins, BIP, PDI, PI(4)P Promastigote stage: poorly fusogenic with endosomes and lysosomes; rare

Promastigote stage: ?





How does

vacuole size



spent in

the microbe

of microor

in cell

host cell





200-1000 (2)


>1 wk



0.3-8 jim



4-7 ds

Host cell






2-3 ds







2-3 d

Cell rupture


by dividing





24 h



vesicles and

cell necrosis







M. marinum (swimming pool granuloma)

M. tuberculosis (tuberculosis)

Nocardia (nocardiosis)

Dictyostelium, macrophage


Macrophages, endothelial cells

Amoebal post-lysosome, later cytoplasmic

Recycling endosome, later possibly cytoplasmic ND

acquisition of TfR, EEA1 or Rab7, slow acquisition of LAMP1

Amastigote stage: Rab7, LAMP1/2, proton-pumping ATPase, cathe-psins B, D, H, L, annexins I and VI, lysosomal preloaded tracer; membrane made permeable for small substances by parasite pore proteins

Predicted copper transporter p80, flotillin-like va-cuolin, NRAMP1

Rab5, TfR, pro-cathepsin D, exclusion of proton-pumping ATPase ND

Amastigote stage: 4.7-5.2

1-20 mm 1-30 Throughout ~30h cytoplasm

1 mm 1 Perinuclear 2-6 d

ND Mostly Throughout ND

singular cytoplasm

Nonlytic release

Apoptosis, u host cell lysis !

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