Loss of MTOR function triggers a host cell death response that requires bacterial replication.

Abstract

<p><b>(a)</b> Experimental scheme for the results shown in <b>(b-e and g-j)</b>. <b>(b-j)</b> BMMs were serum-starved and infected under serum-free conditions either at MOI = 20 <b>(b-e and g-j)</b> or MOI = 10 <b>(f)</b>. <b>(b)</b> Micrographs showing representative of live and dead macrophages infected with <i>Legionella</i> for 12 hrs and stained with anti-<i>L</i>. <i>pneumophila</i> (L.p), anti-ubiquitinated proteins (FK2) antibodies and Hoechst 33342. Arrowheads indicate LCVs, Bar = 5μm. (*) marks the condensed nucleus of the dead cells. The mean nuclear volumes ± s.d of at least 100 live and 100 dead cells are graphed. <b>(c-d)</b> Quantitation of infected and neighboring uninfected <i>Myd88</i>^-/- <b>(c-d)</b>, C57BL/6 <b>(d)</b> and <i>Mtor</i>^-/- <b>(d)</b> macrophages with condensed nuclei after infections with <i>ΔflaA</i> <b>(c-d)</b> or Δ<i>dotA</i> <b>(d)</b>. Means ± s.d of technical replicates of dead cell as percentage of total cells in each condition are shown. <b>(e)</b> Quantitation of infected <i>Myd88</i>^-/- BMMs with condensed nuclei after infection with <i>L</i>. <i>dumoffii</i> and treatment with inhibitors or vehicle as indicated. <b>(f-g)</b> Kinetics of the cell death response in C57BL/6 BMMs under serum starvation conditions infected as indicated. Quantitation of infected cells with condensed nuclei <b>(f)</b> and LDH released in the culture supernatants <b>(g)</b> are shown. <b>(h-i)</b> Analyses of ubiquitin recruitment <b>(h)</b> and LCV size <b>(i)</b> in live and dead <i>Myd88</i>^-/- BMMs infected with <i>ΔflaA</i> and treated with PP242. <b>(j)</b> Cell death in infected <i>Myd88</i>^-/- BMMs with <i>thyA ΔflaA</i> strain treated with vehicle (DMSO) or PP242 in the presence or absence of thymidine. <b>(c, e, h-j)</b> Rapamycin (250nM), PP242 (2.5μM), LY294002 (10μM), Torin2 (300nM). <b>(c-j)</b> Means ± s.d of technical triplicates for each condition are shown. At least 50 cells <b>(e, h-j)</b> or 200 cells <b>(c-d)</b> were analyzed for each condition. A representative of two <b>(e-j)</b> or three <b>(c-d)</b> biological replicates is shown for each experiment. <b>(b-j)</b> n.s—not significant, ** p<0.005 (unpaired T-test).</p