YlfA and YlfB are required for efficient formation of the <i>L</i>. <i>pneumophila</i>-containing vacuole.

<p>A) Bone marrow-derived macrophages derived from A/J mice were infected with wild type <i>L</i>. <i>pneumophila</i>, a Δ<i>ylfA</i> Δ<i>ylfB</i> mutant, and the Δ<i>ylfA</i> Δ<i>ylfB</i> mutant complemented by knock-in of both <i>ylf</i> ORFs onto the <i>L</i>. <i>pneumophila</i> chromosome. Duplicate samples were fixed at 2 and 10 h post-infection. The index of replicative vacuole formation was calculated by dividing the number of observed replicative vacuoles at 10 h by the total number of infected cells at 2 h. Due to variability in the overall efficiency of infection between individual experiments, the index for each strain is presented as a percent of wild type. Data represent the average of three independent experiments ± SD, n = 500 per time point, per experiment. Δ<i>ylfA</i> Δ<i>ylfB</i> mutant R.V. formation is significantly less efficient compared to wild type in an unpaired Student’s <i>t</i> test.*<i>p</i><0.0001. B) Whole cell lysates were prepared from stationary phase broth cultures of wild type <i>L</i>. <i>pneumophila</i>, Δ<i>ylfA</i>, Δ<i>ylfB</i>, Δ<i>ylfA</i> Δ<i>ylfB</i>, and Δ<i>ylfA</i> Δ<i>ylfB</i> in which one or both <i>ylf</i> open reading frames have been reintroduced into the genome. YlfA and YlfB protein levels in these strains were detected by anti-YlfA and anti-YlfB immunoblot. Relative loading efficiency was monitored by anti-DotA immunoblot.