Identification of ATG16L1 interacting proteins involved in the control of microbial infection

Ramos, Maria João Ferreira Gonçalves (2024) Identification of ATG16L1 interacting proteins involved in the control of microbial infection. Doctoral thesis, University of East Anglia.

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Abstract

The delivery of pathogens to lysosomes for degradation is enhanced by non-canonical autophagy pathways that conjugate LC3/ATG8 to the limiting membrane of endosomes/phagosomes. Depending on cell type, this process has been called LC3-associated phagocytosis (LAP), LC3-associated endocytosis (LANDO) or conjugation of ATG8 to endo-lysosome single membranes (CASM). These pathways can be activated through TLR signalling and/or a raise in pH in the lumen of the vacuole which triggers the assembly of the vacuolar ATPase (V-ATPase) on the endosomal membranes. The V-ATPase provides a binding site for the WD domain of ATG16L1, leading to the recruitment of the ATG16L1:ATG5-ATG12 LC3 conjugation machinery. Previous work in our lab showed that mice lacking the WD domain of ATG16L1 (ΔWD mice) have systemic loss of LAP/LANDO/CASM and show high sensitivity to low-pathogenicity Influenza virus, demonstrating the importance of this domain in infection control in vivo. This project has used proximity-dependent biotinylation to investigate how protein interactions with the WD domain of ATG16L1 may protect from infection.

Proximity-dependent biotinylation identified the TBC1D15 as a protein interacting with ATG16L1. TBC1D15 inhibits Rab7 activity by converting the active form Rab7-GTP to the inactive form Rab7-GDP, preventing interaction between Rab7 and downstream effector proteins. The Rab-interacting lysosomal protein (RILP) is a Rab7 effector protein and regulates the recruitment of the V-ATPase subunit V1G to late endosomal and lysosomal membranes. Experiments showed that the cellular distribution of TBC1D15 and V1G change after activation of LAP/LANDO/CASM by raising vacuolar pH with chloroquine. Chloroquine induced recruitment of V1G subunit from the Golgi to the limiting membrane of lysosomes and caused cytoplasmic TBC1D15 to aggregate into puncta. These changes in distribution required the WD domain of ATG16L1 and V1G transport could be restored by expression of constitutively active Rab7-GTP in ATG16L1 ΔWD cells. A role for the WD domain in assembly of the V-ATPase was also evident from slowed acidification of phagosomes in bone-marrow derived macrophages isolated from ΔWD mice.

The role played by the WD domain of ATG16L1 in recruiting proteins to the Salmonella containing vacuoles (SCV) was investigated using a ΔSopF Salmonella strain that lacked the Sop-F effector, which prevents interaction of the WD domain of ATG16L1 with the V-ATPase and prevents LC3 lipidation. SopF reduced recruitment of LC3 to the SCV but had no effect on V-ATPase recruitment. In addition, canonical autophagy independent of LAP/CASM/LANDO provided only a small percentage of the early LC3 recruitment to SCVs.

Overall, this project identified new roles for the ATG16L1-WD domain in the activation of Rab7, assembly of the V-ATPase and in the acidification of late endosomes/phagosomes, highlighting the importance of the ATG16L1 WD domain in the early responses to invading pathogens.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User:	Chris White
Date Deposited:	02 Apr 2025 10:18
Last Modified:	02 Apr 2025 10:18
URI:	https://ueaeprints.uea.ac.uk/id/eprint/98922
DOI:

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