Salmonellasubverts autophagy balancing bacterial fate and cellular inflammation

Abstract

SummarySalmonella Typhimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of host cell types and adopting different intracellular lifestyles for survival. Host endocytic trafficking and autophagy have been implied to regulate the S. Typhimurium subcellular localization and survival. To reveal alternative host regulators on S. Typhimurium lifestyle, we combined a novel fluorescent reporter, Salmonella Intracellular Analyzer (SINA) with haploid forward genetic screening. This identified transcription factor c-MYC as a negative regulator of S. Typhimurium cytosolic lifestyle via stabilizing the Salmonella-containing vacuole (SCV). We further confirmed that c-MYC downstream regulated LC3 acts to maintain SCV stability and limits S. Typhimurium cytosolic lifestyle. We demonstrated that LC3 is recruited to the SCV prior to the endomembrane damage marker Galectin 3, and it regulates SCV stability independent of the autophagosome adaptor NDP52. The LC3 processing enzymes ATG3 and ATG4 reciprocally act on SCV stability, where the loss of LC3-PE conjugation in the absence of ATG3 limits SCV damages. We further identified the dosage-dependent function of the S. Typhimurium effector SopF in mediating SCV stability by actively avoiding LC3 recruitment to the proximity of the SCV to reduce its catastrophic rupture and host cell death. Altogether, we offer insights on the significance of cellular transcription profile in the determination of S. Typhimurium pathophysiology as well as the underlying host-evasion strategy of S. Typhimurium.

Perturbation of ATG16L1 function impairs the biogenesis of Salmonella and Coxiella replication vacuoles

“…In the absence of ATG16L1, the proportion of Δ sopF and WT bacteria accessible to the cytosol/in damaged vacuoles (CHQ resistance assay and Gal8 recruitment) were comparable, indicating that ATG16L1 contributes to the enhanced cytosolic access of ∆ sopF bacteria. Furthermore, it has been shown that interfering with LC3 conjugation by ATG3‐silencing (ATG3 functions in the step upstream of ATG5‐ATG12‐ATG16L1 in the autophagy cascade) reduced the levels of cytosolic Δ sopF bacteria in a similar manner to that seen in our ATG16L1 KO cell lines (Luk et al., 2020). In addition, we utilized SopF point mutants that fail to inhibit ATG16L1 recruitment to the V‐ATPase (Xu et al., 2019).…”

“…2020 have also recently described residual LC3 conjugation to S . Typhimurium upon ATG3 knockdown in epithelial cells (Luk et al., 2020). Altogether, the data demonstrate that LC3‐conjugation on the SCV in epithelial cells requires, but is not absolutely dependent on, ATG3 and ATG16L1.…”

“…Luk et al. 2020 have also recently described residual LC3 conjugation to S . Typhimurium upon ATG3 knockdown in epithelial cells (Luk et al., 2020).…”

“…This may be explained by the transient nature of galectin association with damaged vacuoles (Thurston et al., 2012). Additionally, it was recently reported that LC3 is recruited to the SCV prior to the galectin‐3 marker (Gal3), which acts similarly to Gal8 as an indicator of membrane damage (Luk et al., 2020; Paz et al., 2010). Furthermore, LC3 accumulation on the SCV is the sum of xenophagic detection and autophagy‐mediated repair of the vacuole (Kreibich et al., 2015).…”

“…However, the direct action of RavZ on LC3 makes it unable to discriminate between different mechanisms of LC3 lipidation. Since its first description (Cheng et al., 2017), SopF has been used as a molecular tool to study the interactions between autophagy, xenophagy, and viral and bacterial pathogenesis (Fischer et al., 2020; Lau et al., 2019; Luk et al., 2020; Ulferts et al., 2020; Xu et al., 2019). In this study, we continued this application to confirm the role of SopF in the maintenance of the SCV and avoidance of ATG16L1‐mediated LC3‐lipidation.…”