(A) BMDMs were infected with L. pneumophila JR32 or the legA9 mutant with MOI of 0.5. CFUs were scored at 1, 24, 48, and 72 h. (B) BMDMs were infected with JR32 or the legA9 mutant harboring empty PL, or legA9+ legA9 pBC‐KS+ PL. (A, B) Data are presented as means ± SD of at least three independent experiments. Asterisks indicate significant differences, (*p < 0.05, **p < 0.01, ***p < 0.001; two‐tailed t‐test).

Four female C57BL/6 mice/group received 1 × 106 of JR32 or legA9 mutant bacteria intratracheally. Lungs were homogenized and plated for CFUs counting at (C) 4 or (D) 48 h postinfection. (C, D) Data are shown as mean + SD of n = 4 and are representative as one of two independent experiments. Asterisks indicate significant differences (**p < 0.01 two‐tailed t‐test)

(A) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria. Nuclei were stained blue with DAPI and L. pneumophila stained green with L. pneumophila antibody. Lyso−tracker red was used to stain acidified lysosomes. White arrows show L. pneumophila colocalization with lysotracker.(B) Percent colocalization of L. pneumophila with lyso‐tracker.(C) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria. LC3 is stained red with LC3 antibody. White arrows show L. pneumophila colocalization with LC3.(D) Percent colocalization of L. pneumophila with LC3. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm. (A-D) Data are presented as mean + SD of n = 3 and are representative of three independent experiments. Asterisks indicate significant differences (**p < 0.01, ***p < 0.001; two‐tailed t‐test).

Induction of autophagy in macrophages restricts legA9 mutant replication, and rescues colocalization with LC3 and fusion with lysosomes. BMDMs were untreated or pretreated with rapamycin (Rap) for 1 h and kept throughout the infection. BMDMs were infected with L. pneumophila JR32 or the leg A9 mutant for (A) 1 or (B) 24 h with an MOI of 0.5. CFUs were scored at these time points.

(C, D) Percent colocalization of L. pneumophila with (C) lyso‐tracker and (D) LC3. Hundred bacteria were scored from each coverslips. (A-D) Data are presented as mean + SD of n = 3 and are representative of three independent experiments. Asterisks indicate significant differences (**p < 0.01; two‐tailed t‐test).(A) BMDMs derived from WT and Atg5−/− mice were infected with L. pneumophila JR32 or legA9 mutant at an MOI of 0.5. Data are shown as mean ± SD of n = 3 and are representative of two independent experiments. Asterisks indicate significant differences (*p < 0.05; two−tailed t−test.(B) Western blot showing the absence of Atg 5 in the knockout. Data are representative of three independent experiments.(A) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria. Polyubiquitin is stained red. White arrows show L. pneumophila colocalization with polyubiquitin.(B) Percent colocalization of L. pneumophila with polyubiquitin. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm. Data are presented as means + SD of n = 3 and are representative of two independent experiments. Asterisks indicate significant differences (*p < 0.05, **p < 0.01, ***p < 0.01; two‐tailed t‐test).(A) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria. P62 is stained red with p62 antibody. White arrows show L. pneumophila colocalization with p62.(B) Percent colocalization of L. pneumophila with p62. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm.

(C)BMDMs were infected with JR32 or the legA9 mutant for 6 h and the level of p62 protein was quantified in cell lysates by ELISA (performed in triplicate and the SD are too small to detect).

BMDMs were nucleofected with scrambled control (sictrl) or siRNA against p62 (sip 62). Twenty‐four hours after nucleofection, murine macrophages were infected with JR32 or legA9 mutant with an MOI of 0.5 for 1 h (D and F) or 24 h (E and G).

(H) Western blot analysis showing the downregulation of p62 at 24 and 48 h postnucleofection. (A-B and D-H) Data are shown as mean + SD of n = 3 and are representative of three independent experiments. (C) Data are representative of one experiment n = 3. Asterisks indicate significant differences (*p < 0.05, **p < 0.01; two−tailed t−test).(A) Caspase−1 KO (casp−1−/−) macrophages were infected with L. pneumophila JR32 or legA9 with an MOI of 0.5 for 1, 24, 48, and 72 h.

(B) CFUs were measured at the indicated time points. Levels of IL‐1β were detected in supernatants of WT or casp‐1−/− BMDMs infected with JR32 or the legA9 mutant after 24 h. WT BMDMs were either not treated (NT) or infected with L. pneumophila JR32 or legA9 mutant bacteria for 2 h.

Activation of casp‐1 or casp‐7 was detected in cell extracts using (C) casp‐1 or (D) casp‐7 antibodies, respectively. (A-D) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments.

(A) BMDMs were not infected (NT) or infected with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5, 1 or 5 for 24 h. Apoptosis was quantified by Photometric enzyme immunoassay analysis of cytoplasmic (apoptosis) histone−associated−DNA fragments (ELISA).(B) BMDMs were either not treated (NT) or treated with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5 or 5 for 24 h. The fold change in LDH release was measured from the overall population of macrophages. (A-B) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments.