LC3-associated phagocytosis: host defense and microbial response

Abstract

The innate immune system has evolved to recognize diverse microbes and destroy them. At the same time, microbial pathogens undermine immunity to cause disease. Here, we highlight recent advances in understanding an antimicrobial pathway called LC3-associated phagocytosis (LAP), which combines features of autophagy with phagocytosis. Upon phagocytosis, many microbes, including bacteria, fungi, and parasites, are sequestered in an LC3-positive, single-membrane bound compartment, a hallmark of LAP. LAP depends upon NADPH oxidase activity at the incipient phagosome and culminates in lysosomal trafficking and microbial degradation. Most often LAP is an effective host defense, but some pathogens evade LAP or replicate successfully in this microenvironment. Here, we review how LAP targets microbial pathogens and strategies pathogens employ to circumvent LAP.

Figure 1.. Xenophagy and LC3-associated phagocytosis

A. Xenophagy is a form of canonical autophagy, in which microbes are selectively captured into a double membrane autophagosome. When microbes disrupt the phagosomal membrane and gain access to the cytosol, damaged membrane remnants and bacterial surface proteins are ubiquitinated by host E3 ubiquitin ligases. Autophagy adaptors such as p62 and NDP52 bind ubiquitinated cargo and also LC3, thereby linking cargo to the emerging autophagosomal membrane. Damaged phagosomes also expose luminal glycans to the cytosol, which are recognized by cytosolic galectins, which also bind NDP52. Thus, autophagy adaptors target microbes and damaged phagosomes to the LC3-decorated double membrane compartment. Formation of this compartment requires the ULK1 pre-initiation complex (ULK1/2, FIP200, ATG13 and ATG101), which translocates from the cytoplasm to the endoplasmic reticulum. The ULK1 complex and the PI3KC (ATG14L, BECLN1, VPS15 and VPS34) promote autophagy initiation during xenophagy. The autophagosome fuses with lysosomes to degrade sequestered microbes. B. LAP is initiated on host phagocytes by engagement of surface receptors such as TLR2, DECTIN-1, FcγR and TIM4 by bacteria, fungi, immune complexes, and dead cells, respectively. The signaling cascade initiated upon receptor engagement results in recruitment and assembly of NADPH oxidase complex on incipient phagosomes, which is stabilized by RUBICON. RUBICON is also a component of the PI3KC (RUBICON, BECLN1, VPS15 and VPS34), which generates PI3P. PI3P binds the p40^phox subunit of the NADPH oxidase and is required for LAP. NADPH oxidase generates ROS, which is required for recruitment of the LC3 conjugation machinery to the phagosome. LC3 becomes lipidated and decorates the single-membrane structure referred as a LAPosome, which undergoes fusion with lysosomes to degrade phagocytosed cargo.

Figure 2.. Role of calcium signaling and lipid second messengers in LAP

Studies of LAP in the context of Aspergillus, Salmonella, and Listeria infection have revealed the importance of calcium signaling and lipid second messengers. Melanin in the cell wall of A. fumigatus dormant conidia sequesters intra-phagosomal calcium. Upon germination, melanin is shed from the conidial cell surface, leading to calcium flux from the lumen to the cytosol, which activates CaM/CAMKII signaling that regulates recruitment of downstream PI3KC and NADPH oxidase assembly. Listeria engagement of the Mac-1 receptor activates acid sphingomyelinase (ASMase), which converts sphingomyelin into ceramide and phosphorylcholine. Ceramide-enriched membrane platforms promote NADPH oxidase assembly and activation, resulting in ROS generation. Phosphorylcholine can be converted to DAG by the sequential action of Phospholipase D and Phosphatidic acid phosphatase. DAG activates PKCδ, which promotes activation of NADPH oxidase via phosphorylation of p47 ^phox subunit.