The first step in phagocytosis is the detection of a microorganism by phagocytes. Microbial pathogens are recognized directly by receptors that bind PAMPs or indirectly by receptors that bind opsonins. Receptors that directly bind PAMPs are known as pattern-recognition receptors and among these receptors, we find lectin-like recognition molecules, such as CD169 and CD33; C-type lectins, such as Dectin-2, Monocyte-INducible C-type LEctin (Mincle), or DNGR-1; scavenger receptors (26), such as scavenger receptor A, which detects lipopolysaccharide (LPS) on some Gram-negative bacteria (27), and on Neisseria meningitidis (27); and Dectin-1, which is a receptor for fungal beta-glucan (28, 29). Mannose receptors bind to mannan (30), and CD14 binds to LPS-binding protein (31). Toll-like receptors (TLRs), although recognize microorganisms, do not function as phagocytic receptors (32), however, they can cooperate with other non-opsonic receptors to stimulate phagocytosis (33).

Opsonins are soluble molecules that bind to microorganisms, marking them for phagocytosis. Antibody [immunoglobulin (Ig)] molecules and complement components are important opsonins that induce efficient phagocytosis (2). The most studied opsonic phagocytic receptors are the Fc receptors (FcRs), and the complement receptors (CRs), respectively (34). FcγRs bind to the constant (Fc) portion of IgG (35–38), while FcαRs bind IgA antibodies (39). CRs, such as CR3, bind to iC3b deposited on the microorganism after complement activation (40). Crosslinking of FcγR on the surface of cells activates efficient phagocytosis and other effector functions. These effector functions, such as activation of the oxidative burst, cell degranulation, antibody-dependent cell-mediated cytotoxicity, and activation of genes for production of cytokines and chemokines, are aimed toward the destruction of pathogens and the induction of an inflammatory response that is beneficial during infections (37, 41, 42). CRs, such as the integrin α_Mβ2 (also known as CD11b/CD18, CR3, or Mac-1), bind the complement component iC3b deposited on pathogens to promote phagocytosis (34, 43).

After phagocyte receptors engage a microorganism, signaling events are triggered to initiate phagocytosis. Important changes in membrane remodeling and the actin cytoskeleton take place leading to the formation of pseudopods that cover the microorganism. Lipids associate and dissociate from the membrane of phagosomes in an orderly fashion (44), and the small GTPases Rho, Rac, and cell division cycle 42 (Cdc42), which are important regulators of the actin cytoskeleton, get activated and recruited to the forming phagosome (9, 10). At the point of contact, a depression of the membrane (the phagocytic cup) is formed. Then, F-actin polymerization allows formation of pseudopods that surround the target microorganism and within few minutes, the membrane protrusions fuse at the distal end (11, 12, 45) to seal the new phagosome (Figure 1).

The new phagosome quickly develops the characteristics of early endosomes, through a series of fusion and fission events with endosomes (5, 6). The early phagosome is marked by the presence of the small GTPase Rab5 (13, 14). This membrane GTPase regulates the fusion events between the phagosome and early endosomes by recruiting early endosome antigen 1 (EEA1) (15). Rab5 also recruits the class III PI-3K human vacuolar protein-sorting 34, which in turn, generates phosphatidylinositol 3-phosphate (16). This lipid then promotes recruitment of other proteins involved in phagosome maturation, including Rab7, a marker of late endosomes (19, 20). The early phagosome also becomes a little acidic (pH 6.1–6.5) by the action of V-ATPase accumulating on its membrane and also by transient fusions with more acidic vesicles (56). This V-ATPase translocates protons (H⁺) into the lumen of the phagosome using cytosolic ATP as an energy source (17, 18) (Figure 2).

As maturation continues, Rab5 is lost, and Rab7 appears on the membrane. Rab7 mediates the fusion of the phagosome with late endosomes (21). At the same time, proteins that will be recycled are separated in sorting (recycling) vesicles, while proteins intended for degradation are eliminated in intraluminal vesicles, directed into the lumen of the phagosome (24). Furthermore, the lumen of the late phagosome gets more acidic (pH 5.5–6.0), due to the action of more V-ATPase molecules on the membrane (17) (Figure 2). Rab7 also recruits other proteins to the membrane. One such protein is Rab-interacting lysosomal protein (RILP), which brings the phagosome in contact with microtubules (57, 58), and lysosomes (57, 58). In addition, lysosomal-associated membrane proteins (LAMPs) and luminal proteases (cathepsins and hydrolases) are incorporated from fusion with late endosomes (7, 59) (Figure 2). LAMPs are a family of heavily glycosylated proteins that accumulate on the lysosomal membrane. They all contain a conserved intracytoplasmic tyrosine-based lysosome-targeting motif YXXφ (where φ represents a bulky hydrophobic residue) that directs the trafficking of the molecule through an endosome/lysosome pathway (60). LAMPs are fundamental in regulating membrane fusion events (61) and are required for fusion of lysosomes with phagosomes (22, 23).

The late phagosomes gradually fuse with lysosomes, to become phagolysosomes, the definitive microbicidal organelles (Figure 2) (47, 53, 62). Phagolysosomes possess many sophisticated mechanisms directed to eliminate and degrade microorganisms. They are acidic (pH 5–5.5) thanks to the large number of V-ATPase molecules on their membrane (18) and contain many degradative enzymes, including various cathepsins, proteases, lysozymes, and lipases (17). Other microbicidal components of the phagosome are scavenger molecules, such as lactoferrin that sequesters the iron required by some bacteria (63), and the NADPH oxidase that generates superoxide (O2−) (25), and other reactive oxygen species (ROS) (64, 65) (Figure 2). Although the low pH is clearly microbicidal, it is important to note that phagosome acidification is highly regulated. The lysosomal pH may cycle between acidic and neutral conditions, allowing for the optimal activity of the different hydrolases (66). Within the hybrid degradative vesicle (phagolysosome) (46), most of these enzymes are active at pH 5–5.5; while in primary lysosomes that function as storage vesicles, the lower pH of 4.5 induces enzyme aggregation and inactivation (66).

The most distinctive characteristic of phagolysosomes is their low pH. During the maturation process, the membrane of the phagosome accumulates more and more active molecules of V-ATPase. The molecular complex of this ATPase translocates protons (H⁺) into the lumen of the phagosome using cytosolic ATP as an energy source (17, 18) (Figure 3). The acidic content of the phagosome creates an adverse environment for most microorganisms. The low pH disrupts the normal metabolism of many bacteria and fungi and prevents the use of several essential microbial nutrients (67). In addition, the low pH is required for the activation of many hydrolytic enzymes, which will degrade the ingested pathogen. The V-ATPase also favors the generation superoxide (O2−) (25) by neutralizing the negative charges generated by the NADPH oxidase and by combining O2− with H⁺ to generate other ROS (Figure 3) (as discussed later).

In addition to an acid environment, the phagolysosome can concentrate ROS to destroy microorganisms. Production of ROS is achieved by the NADPH oxidase (NOX2) on the membrane of phagosomes (68, 69, 84). The relevance of this antimicrobial mechanism is evident in patients with chronic granulomatous disease, who have mutations that result in malfunction of the NADPH oxidase. These patients suffer from severe recurrent infections that can cause death in many cases (85, 86). The NADPH oxidase is an enzymatic complex formed by two transmembrane proteins, CYBB (cytochrome b-245 heavy chain/gp91-phox) and CYBA (cytochrome b-245 light chain/p22-phox), that upon activation bring together three cytosolic components: NCF-4 (neutrophil cytosol factor 4/p40-phox), NCF-1 (neutrophil cytosol factor 1/p47-phox), and NCF-2 (neutrophil cytosol factor 2/p67-phox) (69, 87) (Figure 3). In addition, Rac1 and Rac2 are also required for efficient activation of the enzyme complex (70, 71). The oxidase transfers electrons from cytosolic NADPH to molecular oxygen to generate O2− inside the phagosome (25). In the acid conditions of the phagosome, O2− quickly dismutate into H₂O₂, which can then produce hydroxyl radicals (OH⁻) by a Fenton reaction (88) catalyzed by iron released from proteins in the phagosome (89). Also, myeloperoxidase can transform H₂O₂ into hypochlorous acid and chloramines (65) (Figure 3). In addition, O2− can also react with nitric oxide (NO) to form peroxynitrite (ONOO⁻), both of which are highly reactive agents. The various forms of ROS are together efficient antimicrobial agents because they can damage proteins, lipids, and DNA of microorganisms in the phagosome (89).

In addition to ROS, phagocytes, such as macrophages, can also generate nitrogen-based radicals or reactive nitrogen species (RNS) that contribute to microbial destruction. Nitric oxide radicals (NO^⋅) are produced by the inducible nitric oxide synthase 2 (iNOS or NOS2) (72). This enzyme is not present in the resting phagocyte and is only produced in response to proinflammatory agonists (90). NOS2 catalyzes the conversion of l-arginine and oxygen into l-citrulline and NO^⋅ (Figure 3). Contrary to O2−, NO^⋅ is produced on the cytoplasmic side of phagosomes, but it can diffuse across membranes and accumulate inside the phagosome (91). As mentioned earlier, once inside the phagosome, NO^⋅ can react with O2− to generate the highly toxic peroxynitrite (ONOO⁻) that can alter proteins and DNA of ingested microorganisms (73, 74). Also, NO^⋅ can directly impair bacterial enzymes and affect microbial growth (92).

Not only the low pH and the oxidative conditions are used to harm the ingested pathogen but also a series of enzymes and peptides are delivered into the phagosome to limit its growth. Microbial growth can be limited by reducing the amount of essential nutrients inside the phagosome. Nutrients are eliminated from the phagosome by special capture molecules delivered into the phagosome or by transporters present on the phagosome membrane. Perhaps, lactoferrin is the best characterized capture molecule that prevents growth of some bacteria (75). Lactoferrin is a non-heme Fe²⁺-binding glycoprotein present in the specific granules of neutrophils (93), and it can be delivered into the phagolysosome. In there, lactofferin captures Fe²⁺ that is essential for bacterial growth (75, 94) (Figure 3). Other metals, such as Mn²⁺, are also important for microbial growth. Thus, during maturation, phagosomes become gradually depleted of Fe²⁺ and Mn²⁺ by the action the transporter natural resistance-associated macrophage protein 1 (NRAMP-1; also known as SlC11A1) (76) (Figure 3). NRAMP-1 is a membrane protein present on phagolysosomes that transports divalent cations, such as Fe²⁺, Zn²⁺, and Mn²⁺ out of the phagolysosome. This transporter requires H⁺ ions to function, thus NRAMP-1 is more efficient in the acid environment of the phagolysosome (76). Removal of these metal ions prevents microbial growth. However, some microorganisms present a mechanism to counteract the phagocyte function and retain these nutrients (see next section). Microorganisms secrete siderophores, specialized molecules that capture Fe²⁺ and target it for pathogen use (95). The phagocyte in turn presents yet another strategy to control microbial growth. The phagocyte protein lipocalin binds selectively catechol type siderophores expressed by some bacteria, such as Escherichia coli and Mycobacterium tuberculosis. Consequently, the Fe²⁺-loaded siderophore is still sequestered from the bacteria (96, 97).

As described earlier, the phagolysosome interior is an inhospitable environment for most microorganisms. Enzymes and peptides delivered to the phagolysosome have potent antimicrobial effects by destroying the different components of the microbial cell.

Antimicrobial peptides are small (<10 kDa), cationic, and amphipathic polypeptides, often broadly classified based on structure (82, 98). In phagocytes, the main types include defensins (disulfide-stabilized peptides) (77, 78) and cathelicidins (α-helical or extended peptides) (99). Defensins are subdivided into α and β groups and are stored in primary granules of neutrophils. These peptides disrupt the integrity of pathogens by attaching to negatively charged molecules on their membrane. Defensins form multimeric ion-permeable channels that cause membrane permeabilization on both Gram-positive and Gram-negative bacteria (100) (Figure 3). Cathelicidins are stored in secondary granules of neutrophils as inactive precursors. They are then converted into active antimicrobial peptides in the lumen of the phagosome by elastase (99), a primary granule enzyme (93). Active cathelicidins permeabilize the cell wall and inner membrane of Gram-positive bacteria (100). In particular, the cathelicidin LL-37 (hCAP) has drawn particular attention because of its multiple functions. Not only LL-37 works as a broad-spectrum antibiotic but also it has potent chemotactic and immunomodulatory properties (101). Neutrophil-produced LL-37 can be internalized by macrophages (102) and can induce phagocytosis of IgG-opsonized Gram-negative and Gram-positive bacteria by these phagocytes (103). LL-37 also promotes leukotriene B4 and thromboxane A2 generation by human monocyte-derived macrophages (104), thus regulating the inflammation response during infections. Recently, it was also found that macrophages could also produce LL-37. Mice deficient in the Cramp (cathelicidin-related antimicrobial peptide) gene, the murine functional homolog of human LL-37, had increased susceptibility to M. tuberculosis; and macrophages from these mice were unable to control M. tuberculosis growth in an in vitro infection model (105).

Among the many degradative enzymes, the cathepsins are perhaps the most extensively characterized group of lysosomal proteases. These are cysteine proteases that play a direct role in microbial killing by inducing the disruption of bacterial membranes (Figure 3). For example, cathepsins L and K were found to be involved in phagocytosis and non-oxidative killing of Staphylococcus aureus (80), while cathepsin D controlled Listeria monocytogenes intracellular growth (79), probably by degrading the pore-forming toxin listeriolysin O of L. monocytogenes and thus preventing bacterial escape from the phagosome (see next section).

The phagolysosome also contains many lysosomal hydrolases, which help destroy ingested pathogens (106). An important enzyme of this group is lysozyme, an antibacterial protein that can be expressed and secreted by several cell types (81, 82). Lysozyme degrades peptidoglycan, a primary building block of the cell wall of bacteria (Figure 3). By breaking the bonds between N-acetylmuramic acid and N-acetyl-d-glucosamine, lysozyme disrupts the peptidoglycan integrity (107), and then other enzymes can complete the lysis of bacterial cells. One such enzyme is the type IIA secreted phospholipase A2 (sPLA2-IIA) (Figure 3), which exhibits potent antimicrobial activity against Gram-positive and Gram-negative bacteria (83). This remarkable property is due to the unique preference of sPLA2-IIA for anionic phospholipids such as phosphatidylglycerol, the main phospholipid component of bacterial membranes. The importance of this mechanism is highlighted by the fact that transgenic mice overexpressing human sPLA2-IIA are resistant to infection by S. aureus, E. coli, and Bacillus anthracis, the etiological agent of anthrax (83). Thus, antimicrobial peptides and degradative enzymes work together in the lumen of the phagolysosome to completely degrade most phagocytized microorganisms (Figure 3).

The best way to escape from the destructive power of phagocytosis would be just to prevent ingestion by phagocytes from happening. Some pathogens try just to do that by producing substances that extracellularly intoxicate phagocytes. S. aureus can secrete various membrane damaging toxins that will cause cell lysis and death. These toxins include the leukocidins (120) (named this way because they can kill leukocytes) and α-hemolysin (121) (Figure 4). Although there are different leukocidins, they all are dimer proteins (e.g., LukAB, LukED, HlgAB, HlgCB, and LukSF-PV) that induce membrane permeability by the formation of octameric β-barrel pores on the cell membrane (120, 122, 123). Interestingly, leukocidins do not attack any membrane indiscriminately. They must bind first to specific membrane receptors, and therefore, only cells with these receptors are targeted for intoxication (124, 125). For example, LukE binds to the chemokine receptor CCR5 on macrophages, marking these cells for lysis by the active leukocidin LukED (124, 126). Similarly, LukA binds only to the CD11b subunit of the CR Mac-1, which is expressed on both macrophages and neutrophils (125) (Figure 4). The α-hemolysin is another toxin from S. aureus that also forms pores on the membrane of macrophages. It uses phagocyte protein ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) as a receptor (127, 128), and then it assembles into a β-barrel pore of seven identical monomers across the cell membrane (129, 130) (Figure 4).

Phagocytosis is most efficient when microorganisms have been opsonized by antibodies or complement. Microorganisms have also evolved mechanisms to prevent opsonization. The first strategy displayed by S. aureus to block opsonization is simply to degrade opsonins. Some staphylococcal proteases seem capable of directly attacking opsonins. However, a more efficient instrument for this function is the protein staphylokinase, a bacterial plasminogen activator that converts host plasminogen to the active serine protease plasmin. Activated plasmin can then degrade IgG or C3b on the bacterial surface (139) (Figure 4). Another mechanism is to capture the opsonin, so that it does not bind to the bacteria. S. aureus Protein A is a well-known protein expressed on the bacterial cell wall. Protein A specifically binds to the Fc region of IgG, preventing the antibody from engaging FcγRs. In consequence, phagocytosis is effectively blocked (131) (Figure 4). In addition, Protein A can obstruct complement activation by the classical pathway, since the Fc portion of IgG is no longer accessible to the complement component C1q. This will result in less deposition of C3b on the bacteria (140). In addition to Protein A, most S. aureus strains express the Staphylococcal binder of IgG (Sbi) protein, which also binds to the Fc portion of IgG (132–134) (Figure 4). Inhibition of complement activation is an important strategy also used by Staphylococcus. The secreted metalloprotease aureolysin functions as an effective C3 convertase. Aureolysin cuts C3 in the α-chain at a different cleavage site from the C3 convertase, leaving C3a′ and C3b′ fragments (135). Unfortunately, the aureolysin-generated C3b′ fragment is rapidly degraded and not deposited on the bacteria (Figure 4). In addition, the staphylococcal complement inhibitor, a 10-kDa protein, can inhibit complement activation and efficiently prevent phagocytosis and killing of staphylococci (136) (Figure 4). As if all this were not enough, S. aureus can also hide the C3b deposited on its surface. The bacteria secrete the extracellular fibrinogen binding protein (Efb), which binds the serum protein fibrinogen (137). In this way, the bacterium creates a proteinaceous shield that covers the surface bound opsonin and prevents phagocytosis (137, 138) (Figure 4). This impressive array of anti-phagocytic effectors has been described for individual molecules. However, there is not enough information on when and how bacteria decide to use each one of them. The external elements that regulate the expression of each factor are not known. Novel techniques, such as expression profiling, should bring new light into these topics, as discussed later.

Another way to prevent ingestion by phagocytes from happening is to inactivate the cell machinery that creates the phagosome around the microorganism. Some pathogens have developed strategies to prevent actin polymerization and thus avoiding phagocytosis (141). The role of the actin cytoskeleton is fundamental for constructing a phagocytic cup and then extending membrane protrusions around the target particle. The small GTPase Rho family (10) controls formation of F-actin fibers required for phagocytosis. The GTPases Rho, Rac1, and Cdc42 act as molecular switches alternating between an active (GTP-bound) state and an inactive (GDP-bound) state (142, 143). For activation, they need to release GDP and replace it with GTP. This action is catalyzed by guanine nucleotide exchange factors (GEFs). Later, GTP is hydrolyzed to GDP returning the GTPase to its inactive state. This last step is enhanced through interactions with GTPase-activating proteins (GAPs). During phagocytosis, these GTPases are activated and recruited to the forming phagosome, where they activate nucleation-promoting factors such as Wiskott–Aldrich Syndrome protein (WASp) (144). WASp, in turn activates the actin-related protein 2/3 (Arp2/3) complex for actin polymerization (145, 146). As the new actin fibers grow, the plasma membrane is forced out, extending the membrane as pseudopodia around the particle to be ingested. Due to their central role in controlling actin dynamics, these small GTPases are the chosen target of some bacterial toxins. These toxins can alter the activity of the GTPases through covalent modifications or regulation of the nucleotide state. For example, the bacterium Clostridium difficile, which causes pseudomembranous colitis and is responsible for many cases of nosocomial antibiotic-associated diarrhea, produces two glycosylating exotoxins. Toxin A and toxin B modify Rho by glycosylation and inactivate its function. Rho inactivation causes disorganization of actin reducing phagocyte cell migration and phagocytosis (147). Similarly, the bacterium Photorhabdus asymbiotica, an emerging pathogen in humans, produces a toxin (PaTox) that tyrosine glycosylates Rho causing its inactivation. PaTox actions result in actin disassembly and inhibition of phagocytosis (148).

Another group of bacterial toxins regulate the nucleotide state and thus the function of the GTPases by functioning as GAPs or GEFs. For example, the enteropathogenic bacteria Yersinia spp. have type III secretion systems that inject Rho GAP toxins into cells. One such toxin (virulence factor) is YopO, which prevents Rac activation and in consequence prevents phagocytosis (149). Similarly, the Gram-negative bacteria Pseudomonas aeruginosa, an opportunistic pathogen that causes life-threatening infections in cystic fibrosis patients, burn victims, and immunosuppressed individuals, produces the type III virulence factor ExoS that is injected into cells. ExoS is a Rho GAP for Rho, Rac, and Cdc42 that causes the reorganization of the actin cytoskeleton by inhibition of Rac and Cdc42, and actin stress fiber formation by inhibition of Rho (150). An additional example recently described of pathogens disrupting Rho GTPase function comes from the opportunistic bacteria Burkholderia cenocepacia that has a propensity to infect cystic fibrosis patients. B. cenocepacia was shown to disrupt Rac and Cdc42 activation through perturbation of GEF function. Inactive Rac and Cdc42 led to inhibition of phagocyte function (151).

Besides bacteria, several fungal pathogens also display mechanisms for evading phagocytosis. Candida albicans, a commensal ascomycete, is part of the normal microbiota associated with mucosal tissues. It causes opportunistic infections, known as thrush, on superficial mucosas, and systemic infections, named candidiasis. C. albicans is normally phagocytized by macrophages, but it can decrease being recognized by phagocytes with a thick cell wall. The cell wall antigen, β-glucan is hidden among manno-proteins, thus reducing phagocytosis (152). In addition, C. albicans can limit phagocyte chemotaxis during transition from the yeast to the hyphal forms (153). Another fungus, Aspergillus fumigatus, also can mask antigenic proteins and carbohydrates to avoid recognition by phagocytes. RodA hydrophobin is a hydrophobic protein expressed on the surface of A. fumigatus conidia. This hydrophobin efficiently prevents recognition and phagocytosis (154). Similarly, the yeast basidiomycete Cryptococcus neoformans can also avoid recognition by macrophages. The basidiospores of C. neoformans produce a polysaccharide coat (capsule) that forms a thick barrier from phagocytes (155). This capsule can also be shed to prevent macrophage detection and phagocytosis (155). In addition, C. neoformans secretes antiphagocytic protein 1, a protein that binds to CR Mac-1 and inhibits phagocytosis (156).

Once a microorganism is ingested, it will be exposed to the very harsh environment of the phagolysosome. Thus, many pathogens present strategies directed to avoid the formation of this final antimicrobial organelle. Phagosome maturation can be blocked at different points and there are examples of pathogens blocking acidification, reducing activation of the NADPH oxidase, and preventing phagosome to lysosome fusion. Perhaps the most studied example of inhibition of phagosome maturation occurs in M. tuberculosis. The first report was published more than 40 years ago (157), and since then several mycobacterial factors interfering with the process have been found, such as mannose-capped lipoarabinomannan (ManLAM), phosphatidyl-myo-inositol-mannosides (PIMs) (115, 158, 159), and trehalose-6,6′-dimycolate (TDM) (160).

As mentioned earlier, one of the earlier features of phagosome maturation is the rapid and gradual acidification of the phagosome. The number of V-ATPase molecules increases on the phagosome membrane as the maturation process takes place. The low pH directly affects many pathogens (67), and it is also required for the activation of many hydrolytic enzymes. In the case of M. tuberculosis, acidification is inhibited by preventing the accumulation of V-ATPase on the phagosome membrane (161) (Figure 5). Although the complete mechanism is unknown, the M. tuberculosis secreted protein tyrosine phosphatase (PtpA) plays an important role. PtpA binds to subunit H of the macrophage vacuolar V-ATPase (162), and then it dephosphorylates human vacuolar protein sorting 33B (VPS33B) (163), leading to subsequent exclusion of the V-ATPase from the phagosome (Figure 5).

The Gram-positive bacteria Streptococcus pyogenes blocks the V-ATPase activity through expression of surface proteins regulated by the virulence factor Mga (a transcription factor) (181). Similarly, Rhodococcus equi, Gram-positive bacteria that cause severe pneumonia in horses, and the dimorphic fungus Histoplasma capsulatum are also able to maintain a non-acidic phagosome by excluding the V-ATPase (182, 183) (Figure 6). Other pathogens can avoid acidification of phagosomes, including Yersenia pestis, the Gram-negative bacteria causing bubonic plague (184), and C. albicans (185), by mechanisms not completely described.

Phagosome maturation is also inhibited by interfering with the proper accumulation of molecules responsible for vesicle fusion, thus keeping the new phagosome with characteristics of an early phagosome. M. tuberculosis blocks phagosome maturation at a stage between the expression of Rab5 and Rab7, by preventing the delivery of the molecule EEA1 to the membrane (165) (Figure 5). This effect is mediated in part by the action of nucleoside diphosphate kinase (Ndk), which exhibits GAP activity toward Rab5 and Rab7. Ndk inactivates both Rab5 and Rab7 thus preventing recruitment of their respective effectors EEA1 and RILP and in consequence inhibits phagosome maturation and fusion with lysosomes (164) (Figure 5). This blockage also involves ManLAM (171), and it seems to require binding of ManLAM to the mannose receptor (169). Recently, the adhesin PstS-1, a 38-kDa mannosylated glycolipoprotein, was also found to bind the mannose receptor (170) (Figure 5). The connection between the mannose receptor and the lysosome fusion machinery is obscure. Because, capping of the ManLAM with mannose receptor was necessary during phagocytosis to maintain the blockade (169), it seems that the initial engagement of the mannose receptor directs, in an unclear manner, M. tuberculosis to a selective initial phagosomal niche, where other molecules can be excluded. Also, Mincle was recently identified as a receptor for the mycobacterial glycolipid TDM (172). Recruitment of Mincle by TDM coupled to IgG-opsonized beads during FcγR-mediated phagocytosis interfered with phagosome maturation (160). This inhibition involved the SH2-domain-containing inositol polyphosphate 5′ phosphatase (SHP-1) and the FcγRIIb (160), strongly suggesting an inhibitory downstream signaling of Mincle during phagosome formation (Figure 5). Without EEA1, delivery of the V-ATPase or enzymes such as cathepsin D does not take place (196). Therefore, the M. tuberculosis-containing phagosome is kept with a pathogen-friendly environment (Figure 5). Other microorganisms can also arrest phagosome maturation at early stages. For example, the Gram-negative bacteria Neisseria gonorrhoeae express a porin that induces phagosomes to keep larger amounts of Rab5 and low levels of Rab7 (186) (Figure 6). In addition, this bacterium also secretes proteases that digest LAMPs (187) (Figure 6). As mentioned earlier, LAMPs are fundamental for fusion of lysosomes to phagosomes (22), thus its degradation prevents formation of a mature phagolysosome (187). Similarly, the Gram-negative bacteria Legionella pneumophila intercepts vesicular traffic from endoplasmic reticulum (ER) (189) to create an organelle that allows the bacteria to have access to cysteine for survival (190) (Figure 6). This bacterium is the cause of Legionnaires’ disease, a severe form of pneumonia. When the bacteria are phagocytized, the phagosome is rapidly associated with mitochondria and the rough ER, thus getting decorated with ribosomes (197). This effect seems to be mediated by DotA, a bacterial product that is part of the type IV secretion system (T4SS) transporter. T4SS exports various bacterial effector proteins, including RalF, a GEF for the phagocyte ADP-ribosylation factor (ARF1) (198). Active ARF1 promotes vesicle traffic between the ER and the Golgi (199). Therefore, the ER-like phagosome does not get acidic and it does not fuse with lysosomes. Another example of phagosomes fusing with the ER is found in the Gram-negative bacteria Brucella melitensis (Figure 6). This bacterium is the etiological agent of brucellosis, a zoonotic infection that can cause muscle pain, fever, weight loss, and fatigue in people, but can also induce abortion and infertility in animals. In the macrophage cell line J774, B. melitensis alters vesicle trafficking (200) to create a modified phagosome known as a Brucella-containing vacuole (BCV) that fuses with the ER (191) (Figure 6). The mechanism for creating a BCV is not completely known, but it involves several virulence factors such as VirB, an element of the bacterial type III secretion system (191), and cyclic β-1,2-glucan, a cell wall component (201).

Since the phagolysosome is the most harmful organelle for microorganisms, many pathogens have mechanisms to prevent fusion of lysosomes with the phagosome. The best-known example is again M. tuberculosis that avoids lysosome fusion by maintaining an early phagosome (115) (Figure 5). The mechanism for this effect is multifactorial and complex. We only have a partial understanding of it with the identification of some key virulent factors involved. One such virulent factor is the lipoprotein LprG, which binds to lipoglycans, such as lipoarabinomannan (LAM), increasing the surface expression of LAM (166). A M. tuberculosis null mutant for LprG (Mtb ΔlprG) had lower levels of surface-exposed LAM and impaired phagosome–lysosome fusion (167). How LprG prevents phagosome–lysosome fusion is only partially known. It is possible that its effect is indirect via Ndk, which inactivates both Rab5 and Rab7 (164), or is direct by binding to LAMP-3 and modulating the traffic machinery in the host cell (168) (Figure 5). One more virulent factor is PtpA which, as mentioned earlier, dephosphorylates VPS33B, a regulator of membrane fusion events and leads to inhibition of phagosome–lysosome fusion (163) (Figure 5). Another way M. tuberculosis prevents phagosome–lysosome fusion involves inhibition of Rab7 recruitment to prevent autophagy-mediated degradation. The maturation of mycobacteria-containing autophagosomes into autolysosomes requires recruitment of Rab7, but this is blocked by the virulence factor early secretory antigenic target-6 (ESAT-6) (173) (Figure 5). Again, the molecular events for this blockage are not known. However, for another virulence factor of M. tuberculosis, the secretory acid phosphatase (SapM) the inhibition of autophagosome-lysosome fusion (202) is achieved via direct binding to Rab7 (174). Molecularly, Rab7 is blocked by SapM through its cytoplasmic domain preventing its involvement in autophagosome–lysosome fusion (174) (Figure 5). In addition, SapM is known to dephosphorylate phosphotidylinositol 3-phosphate present on phagosomes (158). This phospholipid is also required for membrane fusion events, thus SapM also prevents lysosome fusion in this manner (Figure 5).

Mycobacterium tuberculosis has also evolved other ways to prevent autophagy from happening. One recently described way is the activation or inhibition of cell host microRNAs (miRNAs). Upon infection, macrophages increased several miRNAs and inhibited pathways involved in autophagy. These miRNAs include miR-30A (175), miR-33 (177), and miR-125a (176) (Figure 5). At the same time, another miRNA, miR-17, is downregulated with the same result, blockage of autophagy (178). The signaling pathways affected by these miRNAs are only beginning to be described. For example, miR-125a targets UV radiation resistance-associated gene (UVRAG) (176) to block autophagy, while miR-17 activates a PKCδ/STAT3 pathway to regulate autophagy (178). Thus, inhibition of miR-17 leads also to reduce autophagy (Figure 5). How M. tuberculosis usurps cell host signaling pathways to alter expression of these miRNAs is not known. It seems, however, that the initial signal for this comes from TLRs (176) (Figure 5).

Similarly, S. pyogenes can also prevent lysosome fusion by expressing the virulence factor M1, which regulates vesicle trafficking (188) (Figure 6). M1 can also inhibit activation of the nuclear factor κB and in consequence reduce the macrophage inflammatory response (188). The Gram-negative bacteria Coxiella burnetti, the causative agent of Q fever, resides inside a large phagolysosome-like vesicle known as parasitophorous vacuole (192). This modified phagosome concentrates Rab5 on the membrane and avoids lysosome fusion (193) (Figure 6). The fungi A. fumigatus (203) and the parasitic protozoa Leishmania (204) seem also able to avoid being killed by macrophages by preventing fusion between phagosomes and lysosomes. In the case of A. fumigatus, the molecule dihydroxynaphthalene–melanin on the surface of the pathogen has been reported as responsible for altering vesicle fusion events (205). For Leishmania, the promastigote is efficiently internalized by receptor-mediated phagocytosis (204). Complement and mannose receptors participate in macrophage ingestion (195). Once internalized, promastigotes insert lipophosphoglycan (LPG) into the phagosome membrane. LPG inhibits depolymetization of F-actin (194), and in consequence prevents lysosome fusion (195) (Figure 6). This allows enough time for the promastigote to transform into the other life-cycle form, the amastigote, which can then replicate inside the phagosome.

In addition to preventing phagolysosome formation, pathogens also possess various mechanisms to resist the microbial components found in the phagolysosome lumen. A prominent example is S. aureus that can resist the lytic effect of lysozyme on the cell wall peptidoglycan. These bacteria express the enzyme O-acetyltransferase A (OatA), which causes O-acetylation of the peptidoglycan. This modification makes the peptidoglycan resistant to the muramidase activity of lysozyme (206, 207) (Figure 7). S. aureus also can block the action of antimicrobial peptides. First, the enzyme staphylokinase directly binds α-defensins, blocking almost completely their bactericidal effect (208) (Figure 7). Second, bacteria alter the composition of its membrane. Phosphatidylglycerol is modified with l-lysine, causing a reduction in the negative charge of the membrane (209). In addition, the cell wall is also modified by incorporation of teichoic acids and lipoteichoic acids (210), making it more positively charged. These modifications reduce interaction of α-defensins with the bacterial surface. Third, the metalloprotease aureolysin can degrade LL-37, an antimicrobial peptide with potent activity against staphylococci (211) (Figure 7).

Also, several pathogens express urease, an enzyme that catalyzes the hydrolysis of urea to form ammonia, resulting in the pH neutralization of the phagosome (Figure 7). Important examples of microorganisms using this strategy to survive in the phagosome are S. aureus (218), Helicobacter pylori, bacteria known for causing gastric and duodenal ulcers (221), C. neoformans (222), and Coccidioides posadasii (223).

The oxidative environment of the phagolysosome is also very damaging to most microorganisms. Yet, some pathogens have evolved ways to fight back the effects of ROS and RNS. For example, S. aureus has the golden pigment staphyloxanthin, which works as an antioxidant and prevents damage from peroxide (212) (Figure 7). Also, the protein SOK (surface factor promoting resistance to oxidative killing), that is expressed on the bacteria surface, was recently described as a virulence factor that blocks the effects of ROS (224). In addition, S. aureus express the enzymes super oxide dismutases, sodA and sodM, which convert O2− into H₂O₂ (213), and the enzyme catalase (KatA), which breaks down H₂O₂ into oxygen and water (214, 215) (Figure 7). A phagocytized bacterium has also to prevent the effects of iNOS-derived RNS. S. aureus can detect NO^⋅ by the two component system SsrAB (225), which regulates the expression of the gene hmp coding for a flavohemoglobin that functions as an NO^⋅ scavenger (216, 217) (Figure 7).

Similarly, M. tuberculosis can resist in various ways the microbicidal components within the phagolysosome. A novel glycosylated and surface-localized lipoprotein, Lprl can inhibit the lytic activity of lysozyme (226) (Figure 8). Also, at least two proteins have been found to prevent the formation of ROS by inhibiting the NADPH oxidase. The type I NADH dehydrogenase (NDH-1) blocks ROS production to inhibit tumor necrosis factor alpha (TNF-α)-mediated host cell apoptosis (227) (Figure 8), while the enhanced intracellular survival (eis) gene product (Eis) abrogates production of both ROS and proinflammatory cytokines leading to arrest in apoptosis. These effects seem to depend on the N-acetyltransferase domain of the Eis protein (228) (Figure 8). In both cases, apoptosis is inhibited, but the mechanisms are different. In the case of NDH-1, apoptosis is dependent on caspase-3 and caspase-8 (227), while for Eis, apoptosis seems to be caspase independent (228). M. tuberculosis can also block RNS by interfering with EBP50, a scaffolding protein that controls the recruitment of iNOS at the membrane of phagosomes in macrophages. Interestingly, overexpression of EBP50 by a recombinant lentivirus had no effect on the iNOS recruitment to M. tuberculosis-containing phagosomes, but significantly increased the generation of NO^⋅ and the level of apoptosis in macrophages (229). The EBP50-induced apoptosis was NO^⋅-dependent and mediated by Bax and caspase-3 (229) (Figure 8). The mechanism for iNOS inhibition is not completely elucidated, but it seems to involve both having less iNOS on the membrane and blocking its enzymatic activity. The way M. tuberculosis prevents EBP50 functions remains a mystery.

Other pathogens are also known to display similar mechanisms against ROS and RNS. Streptococcus agalactiae (Group B Streptococcus) is an important cause of pneumonia and meningitis in neonatal humans (239). S. agalactiae expresses a superoxide dismutase (SodA), an orange carotenoid pigment, and glutathione. The latter two compounds functions as ROS scavengers (240, 241). H. pylori can also express a superoxide dismutase (SodB) (242), a catalase (KatA) (243), and the arginase RocF, which transforms the iNOS substrate arginine into urea (244, 245). Similarly, the yeast C. albicans expresses a copper and zinc containing superoxide dismutase (Sod1) (246), and a catalase (Cta1p) (247, 248), while H. capsulatum also secretes two catalases, CatB and CatP (249). The fungus C. neoformans produces a superoxide dismutase (250) and covers itself in a thick polysaccharide and melanin capsule that absorbs ROS (251). Also, the dimorphic fungus Blastomyces dermatitidis seems to be able to inhibit the enzyme iNOS to prevent the production of RNS (252). In all these pathogens, the expression of these enzymes and virulent factors effectively reduces the levels of ROS and RNS within the phagosome. Yet, very little is known about the mechanisms that induce expression of these virulent factors in each pathogen and the molecular details by which they inhibit NADPH oxidase and iNOS enzymes.

The phagolysosome is a place where microbial nutrients are eliminated to arrest pathogen growth. As mentioned earlier, divalent cations, such as Fe²⁺, Zn²⁺, and Mn²⁺, are actively transported out of the phagolysosome (76). In response to this, several microorganisms have evolved mechanisms to retain these important nutrients. One strategy to acquire Fe²⁺ relies on the production of siderophores, which are low-molecular weight Fe²⁺-binding molecules of extremely high affinity, that remove Fe²⁺ from host proteins, such as hemoglobin, and transferrin (233). S. aureus produces two citrate-based siderophores, staphyloferrin A (SA) and staphyloferrin B (SB) (219, 220) (Figure 7). Together, SA and SB can trap enough Fe²⁺ to allow bacterial survival. These siderophores are very efficient because they avoid detection by the phagocyte siderophore-binding protein lipocalin (96, 97). In addition, S. aureus is also able to acquire Mn²⁺ through the action of Mn²⁺ transporters encoded by the bacterial gene loci mntABC and mntH (253). In M. tuberculosis, two groups of siderophores, mycobactins and carboxymycobactins, exist to overcome Fe²⁺ deficiency. The type VII secretion system Esx-3 contributes to siderophore production and release from these bacteria (230, 231) (Figure 8). Recently, another siderophore export system was identified in M. tuberculosis. The MmpS4 and MmpS5 transporters are required for biosynthesis and secretion of siderophores (Figure 8). Because a M. tuberculosis mutant lacking the mmpS4 and mmpS5 genes did not grow under low Fe²⁺ conditions and experienced Fe²⁺ starvation even under high-Fe²⁺ conditions, it seems that these transporters are the primary source of siderophores in mycobacteria (232). The importance of siderophore synthesis for Fe²⁺ acquisition is clear, but Fe²⁺ must find a way back into the bacteria. In M. tuberculosis an ABC transporter system, irtAB (product of the genes irtA and irtB), has been described for efficient utilization of Fe²⁺ from Fe²⁺ carboxymycobactin (Figure 8). Inactivation of the irtAB system decreases the ability of M. tuberculosis to survive Fe²⁺-deficient conditions (234, 235). Similarly, other microorganisms such as A. fumigatus (254) and H. capsulatum (255) can produce siderophores for Fe²⁺ capture.

Intracellular bacteria have also evolved various means to take nutrients from the host cell. Lipids are important building blocks for bacterial membrane formation and an energy source (256). Upon infection, M. tuberculosis alters the phagosome to divert host lipids for its own benefit. A virulent factor was identified within the gene cluster, mce4, because it was specifically required for bacterial survival during prolonged infection. It was found that mce4 encodes a cholesterol import system that enables these bacteria to derive both carbon and energy from this lipid in host membranes (236) (Figure 8). Also, mycobacteria-infected macrophages acquire a “foamy” phenotype characterized by the accumulation of lipid bodies, which serve as source of nutrients. This foamy phenotype is caused by bacterial manipulation of host cellular metabolism to divert the glycolytic pathway toward ketone body synthesis (237). This deregulation results in feedback activation of the anti-lipolytic G protein-coupled receptor GPR109A, causing changes in lipid homeostasis and accumulation of lipid bodies in the cell. ESAT-6, a secreted M. tuberculosis virulence factor, mediates the enforcement of this feedback loop via an unknown mechanism (237) (Figure 8). Another strategy used by M. tuberculosis to exploit host lipids involves the bacterial enzymes isocitrate lyases (ICLs). These ICLs are catalytically bifunctional isocitrate and methylisocitrate lyases that allow bacteria survival on even (acetate) and odd (propionate) chain fatty acids (238) (Figure 8). Moreover, the miR-33 induced by M. tuberculosis also inhibited fatty acid oxidation to support bacterial growth by a mechanism not yet described (177) (Figure 5). In addition, M. tuberculosis has yet another strategy to acquire lipids even from outside the cell in the lung environment. Alveolar macrophages are not only responsible for phagocytosis of these bacteria but also for catabolizing lung surfactant, a lipid–protein complex that lines the alveolar spaces. Recently, it was found that the scavenger receptor CD36 is redistributed to the macrophage cell membrane following exposure to surfactant lipids and participated in surfactant lipid uptake by these cells (180) (Figure 5). These macrophages also supported better bacterial growth in a CD36-dependent manner (180). Thus, it seems that CD36 mediates surfactant lipid uptake by human macrophages and that M. tuberculosis exploits this function for growth.

In addition to resisting all the microbial effectors within a phagolysosome, several pathogens such as C. neoformans, L. monocytogenes, or M. tuberculosis can also completely escape from it. By getting out of the phagosome, these microorganisms can in the cytoplasm travel to other cell sites and finally leave the host cell.

As mentioned earlier, the fungus C. neoformans is well equipped to replicate inside the phagosome. In addition, it can subsequently escape the cell by a non-lytic tactic known as vomocytosis (257, 258). Vomocytosis allows for the pathogen escape leaving the phagocytic cell alive (259). Although the molecular details of vomocytosis are not completely described, the process involves an exocytic fusion of the phagosome with the plasma membrane, thus releasing the fungus (259) (Figure 9). Vomocytosis also involves microtubules, but apparently not actin polymerization. Nevertheless, the formation of dynamic actin cages (“actin flashes”) around the phagosome is observed in many cases. These actin structures actually prevent vomocytosis. Yet, fungus strains with high rates of vomocytosis induce more actin flashes, suggesting that these flashes are a reaction from the cell to contain the phagosome. Still, at the end, the fungal phagosome is fused with the cell membrane and the pathogen is liberated (259). Also, the secreted phospholipase B1 (PLB₁) is required for vomocytosis (260). It is thought that PLB₁ helps permeabilizing the fungal phagosome to neutralize its lumen and to allow nutrients to come in (111, 261). Although vomocytosis is a unique escape function known only for cryptococci, a similar process has recently been described for C. albicans (262) and Candida krusei (263).

Another intracellular pathogen capable of escaping from the phagosome and then from the infected cell is L. monocytogenes (268). This bacterium uses its virulent factor listeriolysin O (LLO) to escape the phagosome (264) (Figure 9). LLO is a pore-forming toxin that permeabilizes the phagosome membrane. It is a potent toxin capable of also degrading the cell membrane, thus its expression and activity are strictly regulated. LLO expression is limited to the intraphagosomal phase of the bacteria, where it is induced by the low pH and high Ca²⁺ conditions of the phagosome (264). Also, LLO activation requires cooperation of host factor such as GILT (γ-inducible lysosomal thiol reductase) (271). In addition, several phospholipases are activated to completely degrade the phagosomal membrane and allow the bacterial escape (265). Once in the cytosol, the bacterium is propelled by the formation of actin tails that push it across the cell. This process is known as “actin rocketing” and it is initiated by the Listeria surface protein ActA (266, 267) (Figure 9). The actin fibers pushing the bacteria are called “comet tails” and propel the bacteria with enough force, allowing it to transfer between cells (268). In the same way, the Gram-negative bacteria Shigella flexneri can disrupt the phagosome membrane and escape into the cytosol (272), where it induces “comet tails” similar to Listeria. The bacterial protein IscA induces activation of N-WASp to initiate actin polymerization by the complex Arp2/3 (273). The actin “comet tails” then propel the bacteria across the cytosol and into neighboring cells. Bacteria from the genus Rickettsia are obligate intracellular pathogens that can also escape phagosomes. Rickettsia uses a secreted phospholipase A2 to disturb the phagosome membrane (274). Once in the cytosol, Rickettsia produce actin tails that allow them direct cell to cell transfer. The bacterial protein RickA is able to activate the Arp2/3 complex to initiate actin polymerization (275). Another microorganism that seems capable of phagosome escaping from neutrophils but not macrophages is S. aureus (269). These bacteria produce phenol soluble modulins (PSMs), which are peptides with lytic activity toward many mammalian cells (270). In particular, the α-PSM was found to induce a strong destruction of neutrophils after phagocytosis, allowing the escape of the phagocytized bacteria (276) (Figure 9).

Other bacteria, such as M. tuberculosis (277) and Mycobacterium marinum (278), can also escape phagosomes. After escaping the phagosome into the cytosol, M. marinum is able to move around by actin-mediated propulsion (279). The M. marinum actin tail formation involves activation of WASp proteins (280) and requires a functional region of difference 1 (RD1) loci (281). This RD1 locus encodes for a secretion system called the ESAT-6 system-1 (ESX-1) or type VII secretion system, which can induce pore formation on host-cell membranes (282). Thus, it was thought that all mycobacteria could escape from phagosomes using the pore-forming activity of ESX-1. However, this has to be formally proven experimentally. M. tuberculosis could be found in increasing numbers in the cytosol of dendritic cells and macrophages when infection was allowed to proceed beyond 2 days in culture (283), and the presence of cytosolic bacteria was also shown to occur in vivo (284). Therefore, there is no doubt about the capacity of mycobacteria to escape into the cytosol but the significance of this phenomenon is still a matter of debate. A simple idea is that bacteria need to leave the phagosome to replicate and then leave the cell. However, bacilli escape the phagosome at later times of infection and this is followed by cell lysis and release of bacilli (278). In consequence, escaping from the vacuole is not a requirement for either survival or growth of M. tuberculosis (285). Instead, it was proposed that the escape from the vacuole represents a transient state that could be critical to the rapid expansion of the bacterial population (285). If this is the case, then escaping from the phagosome is just an important step in the pathology that accompanies progression of tuberculosis infection to active disease. How, mycobacteria kill the cell to allow its release is not clear. Yet, recently, it was reported that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) is required for survival and cytotoxicity of M. tuberculosis in macrophages (286). CpnT consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. This secreted portion is also named tuberculosis-necrotizing toxin (287). It can, in the cytosol of mycobacteria-infected macrophages, hydrolyze the essential coenzyme NAD(+) and induce cell necrosis. However, the mechanism for this cell lysis remains to be elucidated. Clearly, CpnT has a dual function in M. tuberculosis. It is used for uptake of nutrients within the phagosome and for induction of host cell lysis in the cytosol. The regulation of CpnT functions becomes then a topic of important research for controlling M. tuberculosis infections. Another M. tuberculosis virulence factor has also been found to participate in phagosome escape. The unique cell wall lipid phthiocerol dimycocerosates greatly augmented the bacteria escape from its intracellular vacuole (288), by a process not well understood. The mechanism for phagosome lysis is clearly complex as indicated by the fact that host molecules are also recruited by the bacteria to aid in its escape. Activation of host cytosolic phospholipase A2 rapidly led to phagosome lysis for bacteria moving into the cytoplasm of the host cell (116).