RESUMEN
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.
Asunto(s)
Autofagia/genética , Infecciones por Escherichia coli/metabolismo , Infecciones Urinarias/metabolismo , Escherichia coli Uropatógena , Urotelio/microbiología , Animales , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Línea Celular , Células Epiteliales/metabolismo , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Femenino , Variación Genética , Humanos , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Cuerpos Multivesiculares/genética , Cuerpos Multivesiculares/microbiología , Cuerpos Multivesiculares/patología , Vejiga Urinaria/microbiología , Infecciones Urinarias/genética , Infecciones Urinarias/microbiología , Infecciones Urinarias/patología , Uroplaquinas/metabolismo , Urotelio/citología , Urotelio/metabolismo , Urotelio/ultraestructura , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Given their obligate intracellular lifestyle, Chlamydia trachomatis ensure that they have access to multiple host sources of essential lipids by interfering with vesicular transport. These bacteria hijack Rab6-, Rab11- and Rab14-controlled trafficking pathways to acquire sphingomyelin from the Golgi complex. Another important source of sphingolipids, phospholipids and cholesterol are multivesicular bodies (MVBs). Despite their participation in chlamydial inclusion development and bacterial replication, the molecular mechanisms mediating the interaction between MVBs and chlamydial inclusions remain unknown. In the present study, we demonstrate that Rab39a labels a subset of late endocytic vesicles - mainly MVBs - that move along microtubules. Moreover, Rab39a is actively recruited to chlamydial inclusions throughout the pathogen life cycle by a bacterial-driven process that depends on the Rab39a GTP- or GDP-binding state. Interestingly, Rab39a participates in the delivery of MVBs and host sphingolipids to maturing chlamydial inclusions, thereby promoting inclusion growth and bacterial development. Taken together, our findings indicate that Rab39a favours chlamydial replication and infectivity. This is the first report showing that a late endocytic Rab GTPase is involved in chlamydial infection development.