Single mutations selected during growth in macrophages impact growth in amoebae.

<p>Intracellular growth of macrophage-passaged <i>L. pneumophila</i> strains in (<b>A</b>) primary A/J bone marrow-derived macrophages and (<b>B</b>) <i>Acanthamoeba castellanii</i>. Host cells were inoculated with <i>L. pneumophila</i> at MOI = 0.05 in 96 well plates: d0 = progenitor; d133A = A lineage clone from 133 days; d133B = B lineage clone from 133 days; d180C = C lineage, clone from 180 days containing <i>fleN(V107A)</i>; d180C′ = C lineage, clone from 180 days containing <i>fleN⁺</i>; d180D = D lineage clone from 180 days. (The genotypes of each of these clones is indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002731#ppat.1002731.s001" target="_blank">Table S1</a>.) Solid arrowhead (d0) and open arrowheads (d133B and d180D). In multiple independent experiments, cultures were incubated at 37°C, 5% CO₂ in a Tecan M200 Pro plate-reader and luminescence of each well was measured every 20 minutes. The data are plotted as the average of 3 or more replicates of each strain at each time-point from a representative experiment. Error bars represent the standard error of the mean. (<b>C–E</b>) Individual <i>argD</i> and <i>lysC/A</i> mutations recapitulate the amoebal growth defects displayed by the B and D lineages from which they were initially isolated. (<b>F</b>) Competitions were performed by co-inoculating <i>A. castellanii</i> with the progenitor strain and strains harboring either a point mutation in <i>fleN</i> or the <i>lysC</i> region of <i>lysC/A</i> gene. Total bacteria from the inoculum and from 3 days post inoculation were plated onto non-selective media. Ratios of each strain were determined by imaging each plate with or without illumination and counting colony forming units under each condition. Competitive index = ratio output/ratio input, normalized to competitions between non-luminescent (d0⁻) and luminescent (d0) progenitor strains. Each bar represents the geometric mean of data from 3 independent infections (points). Difference from d0/d0⁻ as tested by an unpaired Student's t-test of log-transformed values: *: p<0.05, ***: p<0.001.