The Crohn's disease: associated ATG16L1 variant and Salmonella invasion

Abstract

Objective: A common genetic coding variant in the core autophagy gene ATG16L1 is associated with increased susceptibility to Crohn's disease (CD). The variant encodes an amino acid change in ATG16L1 such that the threonine at position 300 is substituted with an alanine (ATG16L1 T300A). How this variant contributes to increased risk of CD is not known, but studies with transfected cell lines and gene-targeted mice have demonstrated that ATG16L1 is required for autophagy, control of interleukin-1-β and autophagic clearance of intracellular microbes. In addition, studies with human cells expressing ATG16L1 T300A indicate that this variant reduces the autophagic clearance of intracellular microbes.

Design/results: We demonstrate, using somatically gene-targeted human cells that the ATG16L1 T300A variant confers protection from cellular invasion by Salmonella. In addition, we show that ATG16L1-deficient cells are resistant to bacterial invasion.

Conclusions: These results suggest that cellular expression of ATG16L1 facilitates bacterial invasion and that the CD-associated ATG16L1 T300A variant may confer protection from bacterial infection.

Figure 1

Generation and characterisation of ATG16L1^Δ/Δ and ATG16L1^300A/300A HCT116 cells. (A) Targeting strategy to generate ATG16L1^Δ/Δ cells. A floxed-promoterless-neomycin resistance cassette was cloned into 2 kB of homology around exon 9. The construct deleted a splice acceptor site and generated unstable mRNA with a stop codon in the middle of exon 9. The neo cassette was removed by Cre recombinase and targeting was repeated for the second allele. For the generation of the ATG16L1^300A/300A cells, a different construct was generated that targeted the 3′ side of exon 9, did not delete any intronic sequence and changed the nucleotide sequence from ACT (threonine) to GCT (alanine), as shown in the dendrogram of DNA sequence from these cells. (B) Northern blot showing lower expression of ATG16L1 mRNA in ATG16L1^Δ/Δ cells compared to both ATG16L1^300T/300T and ATG16L1^300A/300A HCT116s. (C) Immunoblot for ATG16L1 protein (full-length isoform-001 and splice isoform-003 lacking exon 8) in (left panel; whole cell lysates collected in Laemmli buffer) lysates from ATG16L1^300T/300T, ATG16L1^Δ/Δ or ATG16L1^300A/300A cells; or (right panel) ATG16L1^300T/300T, ATG 16L1^Δ/Δ, ATG16L1^Δ/Δ cells complemented with control PWPI lentivirus (pWPI) or lentivirus encoding ATG16L1 300T (pWPI 300T). (D) Immunoblot for LC3B showing conversion of LC3I (upper band) to LC3II (lower band) indicative of LC3 lipidation in; (upper panel) ATG16L1^300T/300T, ATG16L1^Δ/Δ, pWPI complemented and pWPI 300T -complemented cellular lysates or; (lower panel) ATG16L1^300T/300T, ATG16L1^Δ/Δ or ATG16L1^300A/300A HCT116 cellular lysates from standard cell culture media or in media containing the lysosome inhibitor NH₄Cl (50 mM) to allow the accumulation of LC3II. (E) Immunoblots showing accumulation of p62 in these cell types under the same conditions.

Figure 2

CD-associated ATG16L1^300A/300A variant limits Salmonella typhimurium infection in human IECs. (A) ATG16L1^300T/300T (WT), or gene-targeted ATG16L1^Δ/Δ and ATG16L1^300A/300A HCT116 cells were exposed in culture to increasing MOI of S typhimurium for 10 min, followed by gentamycin treatment to kill extracellular microbes and infecting cytosolic bacteria collected and counted as colony forming units. (B) CD-associated ATG16L1^300A/300A variant limits S typhimurium infection in human IECs grown in transwell culture. ATG16L1^300T/300T, ATG16L1^Δ/Δ or ATG16L1^300A/300A HCT116 cells were grown on Transwell filters and exposed in culture, at 100 MOI, to S typhimurium for 10 min, followed by gentamycin treatment to kill extracellular microbes. Infecting cytosolic bacteria were collected and counted as colony forming units. (C) Representative images, using epifluorescence microscopy in flow (ImageStream), to identify cell surfaces (TmR-WGA) and internalised Salmonella (GFP) in 10 000 cells per sample. (D) Quantification of the number of ATG16L1^300T/300T, ATG16L1^Δ/Δ and ATG16L1^300A/300A cells with internalised S typhimurium. (E) Count of the number of S typhimurium per cell per sample.

Figure 3

Complementation with CD-associated ATG16L1^300A/300A variant limits Salmonella typhimurium infection in ATG16L1^Δ/Δ cells. ATG16L1^Δ/Δ cells complemented with lentiviral expression vectors encoding ATG16L1 300T (PWPI 300T), the CD-associated ATG16L1 (PWPI 300A) variant, or empty vector (PWPI) were examined for rates of S typhimurium infection as described.

Figure 4

CD-associated ATG16L1^300A/300A variant limits clearance of intracellular Salmonella typhimurium. Cells were exposed in culture to S typhimurium for 10 min, washed in gentamycin to kill extracellular microbes and then left in culture for indicated times. Intracellular S typhimurium were collected by cell lysis and further washing and quantified by colony formation assays, *p<0.05.