A non-canonical autophagy-dependent role of the ATG16L1^T300A variant in urothelial vesicular trafficking and uropathogenic Escherichia coli persistence

Abstract

50% of Caucasians carry a Thr300Ala variant (T300A) in the protein encoded by the macroautophagy/ autophagy gene ATG16L1. Here, we show that the T300A variant confers protection against urinary tract infections (UTIs), the most common infectious disease in women. Using knockin mice carrying the human T300A variant, we show that the variant limits the UTI-causing bacteria, uropathogenic Escherichia coli (UPEC), from establishing persistent intracellular reservoirs, which can seed UTI recurrence. This phenotype is recapitulated in mice lacking Atg16l1 or Atg7 exclusively in the urothelium. We further show that mice with the T300A variant exhibit urothelial cellular abnormalities, including vesicular congestion and aberrant accumulation of UPK (uroplakin) proteins. Importantly, presence of the T300A variant in humans is associated with similar urothelial architectural abnormalities, indicating an evolutionarily conserved impact. Mechanistically, we show that the reduced bacterial persistence is independent of basal autophagic flux or proinflammatory cytokine responses and does not involve Atg14 or Epg5. However, the T300A variant is associated with increased expression of the small GTPase Rab33b; RAB33B interacts with ATG16L1, as well as other secretory RABs, RAB27B and RAB11A, important for UPEC exocytosis from the urothelium. Finally, inhibition of secretory RABs in bladder epithelial cells increases intracellular UPEC load. Together, our results reveal that UPEC selectively utilize genes important for autophagosome formation to persist in the urothelium, and that the presence of the T300A variant in ATG16L1 is associated with changes in urothelial vesicle trafficking, which disrupts the ability of UPEC to persist, thereby limiting the risk of recurrent UTIs.Abbreviations: 3-PEHPC: 3-pyridinyl ethylidene hydroxyl phosphonocarboxylate; ATG: autophagy; ATG16L1: autophagy related 16 like 1; BECs: bladder epithelial cells; dpi: days post infection; hpi: hours post infection; IF: immunofluorescence; IL1B: interleukin 1 beta; IL6: interleukin 6; MAP1LC3B/ LC3B: microtubule-associated protein 1 light chain 3 beta; MVB: multivesicular bodies; T300A: Thr300Ala; TNF: tumor necrosis factor; QIR(s): quiescent intracellular reservoir(s); siRNA: short interfering RNA; UPEC: uropathogenic Escherichia coli; UTI(s): urinary tract infection(s); TEM: transmission electron microscopy; WT: wild type ARTICLE HISTORY

Matrix stiffness regulates endosomal escape of uropathogenicE. coli

“…For UPEC, initial internalization is dependent on the Rho GTPases Rac and Cdc42 (Martinez & Hultgren, ), which in turn can be regulated by substrate stiffness (Kim et al, ; Klein et al, ). During later stages of infection, the establishment of persistent quiescent intracellular reservoirs (QIRs) within transitional epithelial cells utilizes the autophagy gene atg16L1 (Wang et al, , ) and the autophagy pathway is similarly affected by matrix stiffness (Pavel et al, ; Ulbricht et al, ). Our microindentation‐based estimates of the overall bladder stiffness cannot determine whether cellular stiffness varies across the uroepithelial layers; however, one possible mechanism for the establishment of QIRs may be that the immature transitional epithelial cells experience higher stiffness which could support UPEC residence within endosomes.…”

Proteomics of autophagy deficient macrophages reveals enhanced antimicrobial immunity via the oxidative stress response

“…Genetic loss-of-function of core autophagy genes including Atg16l1 increases secretion of pro-inflammatory cytokines by macrophages in response to toll-like receptor (TLR) activation (Lim et al, 2019;Saitoh et al, 2008). This contributes to increased mucosal inflammation, driving resistance to extracellular pathogens such as Citrobacter rodentium (Marchiando et al, 2013;Martin et al, 2018) and pathogenic Escherichia coli (Wang et al, 2019). reconciled with observations made in cell-based systems to fully describe the roles of autophagy in antimicrobial immunity.…”

Autophagy—A Story of Bacteria Interfering with the Host Cell Degradation Machinery

“…The ATG16L1 T300A variant found in humans was shown to mimic ATG16L1 deficiency in mice. Expression of this variant in knockout mice abolished UPEC persistence and hence, UTI recurrence [ 175 ]. Furthermore, loss of ATG7 but not ATG14 or ectopic P granules protein 5 (EPG5) led to a decrease in intracellular UPEC in bladder epithelial cells [ 175 ].…”