Formation and Maturation of the Phagosome: A Key Mechanism in Innate Immunity against Intracellular Bacterial Infection

Abstract

Phagocytosis is an essential mechanism in innate immune defense, and in maintaining homeostasis to eliminate apoptotic cells or microbes, such as Mycobacterium tuberculosis, Salmonella enterica, Streptococcus pyogenes and Legionella pneumophila. After internalizing microbial pathogens via phagocytosis, phagosomes undergo a series of 'maturation' steps, to form an increasingly acidified compartment and subsequently fuse with the lysosome to develop into phagolysosomes and effectively eliminate the invading pathogens. Through this mechanism, phagocytes, including macrophages, neutrophils and dendritic cells, are involved in the processing of microbial pathogens and antigen presentation to T cells to initiate adaptive immune responses. Therefore, phagocytosis plays a role in the bridge between innate and adaptive immunity. However, intracellular bacteria have evolved diverse strategies to survive and replicate within hosts. In this review, we describe the sequential stages in the phagocytosis process. We also discuss the immune evasion strategies used by pathogens to regulate phagosome maturation during intracellular bacterial infection, and indicate that these might be used for the development of potential therapeutic strategies for infectious diseases.

Keywords: bacterial infection; innate defense mechanism; innate immunity; lysosome; pathogen; phagocytosis; phagosome maturation.

Figure 1

Fc receptor (FcR)-mediated signaling for phagocytosis. Engagement between FcR and the IgG immune complex induces FcR crosslinking and phosphorylation of ITAM on the cytoplasmic tail of FcR by SFKs, such as Lyn, Lck and Hck. The phosphorylation of ITAM is responsible for the recruitment and phosphorylation of Syk. Phosphorylated Syk triggers the conversion of PI(4,5)P₂ to PI(3,4,5)P₃ through PI3K. PI(3,4,5)P₃ regulates the activation of Rac and myosin. Vav is activated by Syk to further activate Rac. Rac activates the NF-κB- and JNK-mediated signaling pathways. Rac and Cdc42 contribute to actin polymerization. Phosphorylated Syk also generates DAG and IP3 from PI(4,5)P₂. DAG induces PKC-mediated activation of p38 and ERK signaling. IP₃ triggers the release of Ca²⁺ from the ER to the cytosol.

Figure 2

Complement receptor (CR)-mediated signaling for phagocytosis. CR binds to various complement components and initiates intracellular signaling cascades via Rho. Rho activates ROCK, and activated ROCK induces myosin II phosphorylation, and recruits the Arp2/3 complex, which leads to branched actin polymerization. Activated Rho also recruits mDia1, which mediates actin polymerization into a linear network. mDia also interacts directly with CLIP-170, which interacts with microtubules, to promote actin polymerization.

Figure 3

Schematic representation of phagosome maturation. Following the internalization of foreign particles, the phagosome sequentially forms early phagosomes, late phagosomes and phagolysosomes. The newly formed phagosome acquires the proteins needed for its maturation via a series of fusions and fissions with endocytic organelles (early endosome, late endosome, lysosome and recycling vesicles), and the late phagosome fuses with the lysosome for the removal of foreign particles, using various hydrolytic enzymes, antimicrobial peptides and ROS.

Figure 4

Maturation of the early phagosome. (A) (a) During the process of ‘probing’, the membrane expands via cytoskeletal rearrangement to recognize foreign particles using phagocytic receptors. (b) The interaction between phagocytic receptors and ligands derived from foreign particles induces receptor clusters and activates intracellular signaling pathways responsible for the formation of phagocytic cups. (c) The expansion of pseudopods is induced by the continuous assembly of actin filaments, which leads to the elongation of membranes. (d) Finally, the pseudopod surrounds the target particles and seals the distal tip of the phagocytic cup. (B) Following the internalization of foreign particles, the nascent phagosome begins to acquire the proteins needed for its maturation into the early phagosome. GTP-bound Rab22a recruits Rab5 GEF Rabex-5 into the phagosomal membrane. Rabex-5 converts GDP-bound Rab5 to GTP-bound Rab5, an activated form of Rab5. GTP-bound Rab5 induces the recruitment of Rabaptin-5 to further promote the activation of Rab5. (C) PI3K Vps34 accumulates in phagosomal membranes via Rabaptin-5, and generates phosphatidylinositol 3-phosphate (PI(3)P) from phosphatidylinositol (PI). (D) PI(3)P and GTP-bound Rab5 promote the recruitment of EEA1, which enhances the interaction between early endosomes and early phagosomes, by docking at the membrane of early endosomes. GTP-bound Rab5 also recruits the CORVET complex, to strengthen the fusion of early endosomes.

Figure 5

Transition from the early to the late phagosomes. (A) The transition of GTP-bound Rab5 to GTP-bound Rab7 is mediated by Mon1 and Ccz1. Mon1 and Ccz1 are recruited into early phagosomes to interact with Rab5. Mon1 binds to Rabex-5 to reduce Rab5 activity, and is then exchanged with Rabex-5 on early phagosomes. Mon1-Ccz1 acts as a Rab7 GEF, which induces Rab7 association and Rab5 dissociation. (B) The conversion of Rab5 to Rab7 is also mediated by the replacement of the CORVET complex with the HOPS complex. Vps3 and Vps8 of CORVET are exchanged by Vps39 and Vps41 of HOPS. (C) The HOPX complex enhances the interaction with Rab7-expressing late endosomes. Late phagosomes acquire LAMP-1 and LAMP-2 through fusion with late endosomes and lysosomes. Late phagosomes also exhibit strong interactions with late endosomes and lysosomes using SNAREs, including VAMP7, VAMP8, syntaxin7 and syntaxin8. SNARE proteins support the formation of phagolysosomes by tethering between late phagosomes and lysosomes.

Figure 6

Model of the formation of phagolysosomes. For the fusion of late phagosomes and lysosomes, GTP-bound Rab7 interacts with new proteins, such as RILP and ORP1L. RILP recruits the dynein-dynactin motor complex, which moves Rab7-expressing late phagosomes toward the (−) end of the microtubule. The late phagosome then interacts and fuses with the lysosome.