Control of Phagocytosis by Microbial Pathogens

Abstract

Phagocytosis is a fundamental process of cells to capture and ingest foreign particles. Small unicellular organisms such as free-living amoeba use this process to acquire food. In pluricellular organisms, phagocytosis is a universal phenomenon that all cells are able to perform (including epithelial, endothelial, fibroblasts, etc.), but some specialized cells (such as neutrophils and macrophages) perform this very efficiently and were therefore named professional phagocytes by Rabinovitch. Cells use phagocytosis to capture and clear all particles larger than 0.5 µm, including pathogenic microorganisms and cellular debris. Phagocytosis involves a series of steps from recognition of the target particle, ingestion of it in a phagosome (phagocytic vacuole), maturation of this phagosome into a phagolysosome, to the final destruction of the ingested particle in the robust antimicrobial environment of the phagolysosome. For the most part, phagocytosis is an efficient process that eliminates invading pathogens and helps maintaining homeostasis. However, several pathogens have also evolved different strategies to prevent phagocytosis from proceeding in a normal way. These pathogens have a clear advantage to perpetuate the infection and continue their replication. Here, we present an overview of the phagocytic process with emphasis on the antimicrobial elements professional phagocytes use. We also summarize the current knowledge on the microbial strategies different pathogens use to prevent phagocytosis either at the level of ingestion, phagosome formation, and maturation, and even complete escape from phagosomes.

Keywords: bacteria; infection; inflammation; macrophage; neutrophil; phagolysosome; phagosome maturation.

Figure 1

Initiation of phagocytosis. After receptor engagement (A) (2), the plasma membrane covers the microorganism to be ingested (B) (9, 10) and closes at the distal end (C) (11, 12), forming a vacuole where the microorganism is internalized (D).

Figure 2

Phagosome maturation. The new phagosome quickly develops the characteristics of early endosomes, through a series of fusion and fission events with sorting and recycling endosomes (5, 6). The early phagosome is marked by the presence of the small GTPase Rab5 (13, 14), early endosome antigen 1 (EEA1) (15), and the class III PI-3K human vacuolar protein-sorting 34 (hvPS34) (16). The early phagosome also becomes a little acidic (pH 6.1–6.5) by the action of V-ATPase accumulating on its membrane (17, 18). The late phagosome is marked by the presence of Rab7 (–21) and lysosomal-associated membrane proteins (LAMPs) (22, 23). Proteins that will be recycled are separated in sorting (recycling) vesicles, while proteins intended for degradation are eliminated in intraluminal vesicles (ILVs), directed into the lumen of the phagosome (24). 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. Phagolysosomes are formed when late phagosomes fuse with lysosomes. Phagolysosomes are acidic (pH 5–5.5) and contain many degradative enzymes, including various cathepsins, proteases, lysozymes, and lipases. Other microbicidal component of the phagosome is the NADPH oxidase that generates reactive oxygen species (25).

Figure 3

Antimicrobial effectors inside the phagolysosome. The most distinctive characteristic of phagolysosomes is their low pH. The V-ATPase translocates protons (H⁺) into the lumen of the phagosome (17, 18). The NADPH oxidase is an enzymatic complex formed by two transmembrane proteins, such as CYBB and CYBA, and three cytosolic components: NCF-4, NCF-1, and NCF-2 (68, 69). Rac is also required for efficient activation of the enzyme complex (70, 71). Myeloperoxidase (MPO) can transform H₂O₂ into hypochlorous acid (65). Nitric oxide radicals (NO^⋅) are produced by the inducible nitric oxide synthase 2 (iNOS) (72), and NO^⋅ reacts with ${\text{O}}_2^{-}$ to form peroxynitrite (ONOO⁻) (73, 74). Lactoferrin captures Fe²⁺ that is essential for bacterial growth (75), and the transporter natural resistance-associated macrophage protein 1 (NRAMP-1) takes Fe²⁺ out of the phagosome (76). Defensins are antimicrobial peptides that form multimeric ion-permeable channels on bacteria (77, 78). Cathepsins are lysosomal proteases (79, 80). Lysozyme (81, 82) degrades peptidoglycan, a primary building block of the cell wall of bacteria, and the type IIA secreted phospholipase A2 (sPLA2-IIA) (83) degrades anionic phospholipids such as phosphatidylglycerol, the main phospholipid component of bacterial membranes.

Figure 4

Staphylococcus aureus blocks opsonic phagocytosis. S. aureus secrete toxins, leukocidins (120, 125) and α-hemolysin (121), which induce membrane permeability by forming pores on the cell membrane. To be fully active, leukocidin A binds to the complement receptor Mac-1 (125), while α-hemolysin binds to protein ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) (127, 128). Staphylokinase converts host plasminogen to the active serine protease plasmin, which in turn degrades IgG or iC3b on the bacteria (127, 129). Protein A (SpA) (131) and staphylococcal binder of IgG (Sbi) protein specifically bind to the Fc region of IgG (–134), blocking Fc receptor (FcR) engagement and activation. Aureolysin functions as a C3 convertase, leaving non-functional C3b′ fragments (135). Also, the staphylococcal complement inhibitor (SCIN) prevents complement activation on the bacteria (136). Finally, the extracellular fibrinogen binding protein (Efb) binds the serum protein fibrinogen (Fg), creating a proteinaceous shield that covers surface-bound opsonins (137, 138).

Figure 5

Mycobacterium tuberculosis interferes with phagosome maturation. M. tuberculosis inhibits acidification by preventing the accumulation of V-ATPase on the phagosome membrane (161), in part through the action of protein tyrosine phosphatase (PtpA) (162). PtpA also dephosphorylates human vacuolar protein sorting 33B (VPS33B) leading to the inhibition of phagosome-lysosome fusion (163). The nucleoside diphosphate kinase (Ndk) is a GAP for Rab5, and by inactivating this GTPase (164), it prevents recruitment of early endosome antigen 1 (EEA1) to the membrane (165). The lipoprotein LprG increases the surface-expression of mannose-capped lipoarabinomannan (ManLAM) (166) and can directly bind to lysosomal-associated membrane proteins (LAMPs) to modulate the traffic machinery of the cell (167, 168). Also, ManLAM (169) and the adhesin PstS-1 (170) bind the mannose receptor, which is involved in the lysosome fusion machinery by an unknown mechanism (171). The mycobacterial glycolipid TDM binds the receptor Monocyte-INducible C-type LEctin (Mincle) (172), activating the SH2-domain-containing inositol polyphosphate 5′ phosphatase (SHP-1) to interfere with phagosome maturation (160). The virulence factor early secretory antigenic target-6 (ESAT-6) inhibits recruitment of Rab7 to the phagosome membrane, preventing autophagy-mediated degradation (173). Also, the secretory acid phosphatase (SapM) direct binds to Rab7 (174) and prevents autophagosome-lysosome fusion (174). In addition, SapM can block the effects of phosphotidylinositol 3-kinase (PI3K) present on phagosomes (158). Upon infection, mycobacteria induce upregulation of several microRNAs (miRNAs) (–177) and downregulation of others (178) to block autophagy. miR-125a targets UV radiation resistance-associated gene (UVRAG) (176) to block autophagy, while miR-17 activates a protein kinase Cδ (PKCδ)/signal transducer and activator of transcription 3 (STAT3) pathway to regulate autophagy (178). The miR-33 also inhibits fatty acid oxidation to support bacterial replication by a mechanism not yet described (177). How M. tuberculosis alters cell signaling to control miRNAs is not known, but the initial signal might come from TLR2 (176, 179). Finally, the scavenger receptor CD36 participates in surfactant lipid uptake by alveolar macrophages, and M. tuberculosis exploits this function for growth (180).

Figure 6

Inhibition of phagosome maturation. (a) Several pathogens, such as Mycobacterium tuberculosis (161), Histoplasma capsulatum (182), and Rhodococcus equi (183) inhibit acidification by preventing the accumulation of V-ATPase on the phagosome membrane. M. tuberculosis also blocks early endosome antigen 1 (EEA1) on the membrane (165), while Neisseria gonorrhoeae express a porin that induces large amounts of Rab5 (186) and also proteases that digest lysosomal-associated membrane proteins (LAMPs) (187). Another bacteria, Streptococcus pyogenes, express the virulence factor M1, which regulates vesicle trafficking (188). Each of these actions effectively will block lysosome fusion to the phagosome. (b) Other pathogens, such as Legionella pneumophila (189, 190) and Brucella melitensis (191), induce the rapid association of the phagosome with the endoplasmic reticulum (ER). (c) The bacteria Coxiella burnetti (192, 193), and the parasite Leishmania reside inside a phagolysosome-like vesicle known as parasitophorous vacuole (PV) that concentrates Rab5 on the membrane. Leishmania promastigotes also insert lipophosphoglycan (LPG) into the phagosome membrane (194). These actions, in consequence, prevent lysosome fusion (195).

Figure 7

Resistance of Staphylococcus aureus to phagolysosome contents. The bacteria S. aureus modifies the composition of its cell wall to resist the action of lysozyme (206, 207) and alters the composition of its membrane, with l-lysine and lipoteichoic acids, to reduce the negative charge of the membrane (209, 210); thus resisting antimicrobial peptides, such as the cathelicidin LL-37. Also, it secretes staphylokinase and aureolysin to block α-defensins and LL-37, respectively (208, 211). In addition, S. aureus has the golden pigment staphyloxanthin (Sx), which works as an antioxidant (212), two super oxide dismutases (Sod) (213), and a catalase (214, 215) that together protect against reactive oxygen species. In addition, flavohemoglobin functions as an NO^⋅ scavenger (216, 217). The bacterial urease catalyzes the hydrolysis of urea to form ammonia, resulting in pH neutralization (218). Finally, S. aureus produces siderophores (SA) (219, 220) that trap enough Fe²⁺ to allow bacterial survival.

Figure 8

Resistance of Mycobacterium tuberculosis to phagolysosome contents. M. tuberculosis inhibits acidification by preventing the accumulation of V-ATPase on the phagosome membrane (161), in part through the action of protein tyrosine phosphatase (PtpA) (162). The bacterial lipoprotein, Lprl, can inhibit the lytic activity of lysozyme (226). The secretion system Esx-3 (230, 231) and the MmpS4/S5 transporters (232) are required for biosynthesis and secretion of the siderophores mycobactins (Mbac) and carboxymycobactins (Cabac), which seize Fe²⁺ from host proteins, such as lactoferrin (233). Then, the transporter system irtAB takes Fe²⁺ from Fe²⁺-carboxymycobactin into the bacterium (234, 235). The type I NADH dehydrogenase (NDH-1) (227) and the Eis protein (228) inhibit the NADPH oxidase, preventing formation of ROS. Also, M. tuberculosis prevents the generation of NO^⋅ and apoptosis by interfering with EBP50, a scaffolding protein that controls the recruitment of iNOS at the membrane of phagosomes (229). In addition, M. tuberculosis alters the phagosome to divert host lipids for its own benefit through mce4, a cholesterol import system (236), and through accumulation of lipid bodies via the early secretory antigenic target-6 (ESAT-6) (237). The enzymes isocitrate lyases (ICLs) allow bacteria survival on even (acetate) and odd (propionate) chain fatty acids in lipid bodies (238).

Figure 9

Escape from the phagosome. Several pathogens escape from the phagosome to persist in the less harsh environment of the cytoplasm. (a) The intracellular pathogen Listeria monocytogenes uses its virulent factor listeriolysin O (LLO) (264) and phospholipases (PLC and PLD) (265) to escape the phagosome. Once in the cytosol, the bacterium is propelled by the formation of actin “comet tails” that push it across the cell, allowing it to transfer between cells (–268). (b) Staphylococcus aureus can escape from neutrophil phagosomes (269) by producing phenol soluble modulins (PSM), which are peptides with lytic activity toward many mammalian cells (270). (c) The fungus Cryptococcus neoformans escapes the cell by vomocytosis (257, 258). Here, the phagosome fuses with the cell membrane with assistance of the secreted phospholipase B1 (PLB₁) (260), leaving the pathogen free and the phagocytic cell alive.