Invasive bacteria, including Salmonella enterica
serovar Typhimurium (S. Typhimurium), trigger
autophagy in intestinal enterocytes. This is indicated by abundant epithelial cell autophagosomes,
marked by LC3 (microtubule-associated protein
1 light chain 3), that capture and eliminate invad-
ing bacteria (7). S. Typhimurium also invaded
Paneth cells (fig. S1), and invasion was associated
with elevated numbers of LC3+ puncta in Paneth
cells (Fig. 1, A and B). The puncta numbers were
comparable to those in mice subjected to fasting,
a trigger of canonical autophagy (8) (Fig. 1, A and
B). However, many of the LC3+ structures in
infected Paneth cells were markedly larger (0.2
to 7 mm in diameter) than the LC3+ puncta in
LC3 LYZ Merge
+S. Tm -S. Tm
LC3 LYZ DAPI
-S. Tm +S. Tm
LYZ IgG Input LC3 LYZ IgG
+S. Tm -S. Tm
Fig. 1. Large LC3+ vesicles in S. Typhimurium–infected mice contain
lysozyme. (A) Immunofluorescence of LC3 in intestinal crypts. Nuclei are
stained with DAPI (4′,6-diamidino-2-phenylindole). Scale bars, 10 mm.
(B) Quantification of LC3+ puncta. Each data point represents one mouse.
(C) Immunofluorescence of LC3 in intestinal crypts. Scale bars, 5 mm.
(D) LC3+ vesicle diameter measurements. (E) Immunofluorescence of LC3
and lysozyme in S. Typhimurium–infected intestinal crypts. A Paneth cell is
outlined. Arrows indicate a lysozyme-filled LC3+ vesicle; arrowheads
indicate an autophagosome that does not contain lysozyme. Scale bars,
5 mm. (F) Colocalization of LC3 and lysozyme in intestinal crypts from
S. Typhimurium–infected mice. Each point represents one lysozyme
granule. (G) Coimmunoprecipitation (IP) of intestinal lysates using the
indicated antibodies. Immunoblot (IB) was performed with anti-lysozyme
antibody. IgG, immunoglobulin G. (H) Transmission electron microscopy of
Paneth cells from uninfected (-S. Tm) and infected (+S. Tm) mice.
Asterisks indicate secretory granules; arrowheads indicate surrounding
membranes. (I) Immunofluorescence of lysosomes (cathepsin D+), LC3,
and lysozyme in S. Typhimurium–infected intestinal crypts. Arrows
indicate a lysozyme-filled LC3+ vesicle with no lysosome (cathepsin D)
signal; arrowheads indicate lysosomes that are not coincident with
lysozyme-filled LC3+ vesicles. Scale bars, 5 mm. (J) Quantification of
lysosome (cathepsin D), LC3, and lysozyme colocalization in (I). Each data
point represents one lysozyme-containing granule. Two points connected
by a line represent the same granule. The dashed line denotes the limit of
strong colocalization. (K) Immunofluorescence of LC3 and lysozyme in
intestinal crypts. Scale bars, 10 mm. *P < 0.05; **P < 0.01; ***P < 0.001;
****P < 0.0001; one-way analysis of variance (ANOVA) [(B) and (D)].
S. Tm, Salmonella Typhimurium; LYZ, lysozyme.