Emergency of morphological diversity in the bacterial populations adapting to MΦ.

<p>(A) Examples of the variability for colony morphology that emerged in <i>E. coli</i> populations adapting to MΦ, from left to right – ANC stands for morphology of ancestral, SCV for the small colony variants morphology and MUC for the mucoid colony morphology. (B) Dynamics of frequency change of the evolved phenotypes in each replicate evolving populations (M1 to M6): white squares indicate ANC, black triangles SCV, black circles MUC phenotypes.</p

Mutations acquired by evolved clones identified through whole genome re-sequencing (WGS).

<p>Mutations in intergenic regions have the two flanking genes listed (e.g., <i>clpX</i>/<i>lon</i>). SNPs are represented by an arrow between the ancestral and the evolved nucleotide. Whenever a SNP gives rise to a non-synonymous mutation the amino acid replacement is also indicated. The symbol Δ means a deletion. For intergenic mutations, the numbers in the Mutation row represent nucleotides relative to each of the neighboring genes, here + indicates the distance downstream of the stop codon of a gene and − indicates the distance upstream of the gene, that is relative to the start codon. Insertions of IS elements are denoted by the specific IS element followed by the number of repeated bases caused by its insertion.</p

Predictions of model of clonal interference for changes in mucoid frequencies with time.

<p>Simulations of the adaptive dynamics over the period of the experiment (30 days). The frequencies of mucoid phenotypes are plotted and can be compared to those observed in the experiments (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003802#ppat-1003802-g001" target="_blank">Fig. 1B</a>). The values of parameters used and the dynamics of haplotypes that compete for fixation are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003802#ppat.1003802.s009" target="_blank">Figure S9</a>.</p

<i>In vitro</i> evolved <i>E. coli</i> show increased virulence <i>in vivo</i>.

<p><b>A</b>) Survival of mice infected with different doses of ancestral (ANC, in blue), mucoid bacteria evolved in the presence of MΦ (MUC, in red) or bacteria evolved in the absence of MΦ (CON, in green). The number of mice are shown inside the bars, <b>B</b>) Survival probability of mice infected with ANC, MUC and CON, represented as lines from the fit of a binomial General Linear Model used to infer LD₅₀, <b>C</b>) Kaplan-Meier curves and <b>D</b>) % maximum reduction in temperature or weight at the LD₅₀ dose for the MUC (n = 10), ANC (n = 11) and CON (n = 5) (Error bars correspond to 2SE, * indicates p<0.05).</p

Genetic characterization of adaptive mutations and the dynamics of their appearance.

<p>(A) Mutations identified in MUC1 to MUC6 clones isolated from M1 to M6 populations (evolved for 450 generations), represented along the <i>E. coli</i> chromosome. For simplicity, the genomes are represented linearly and are horizontally drawn. The types of mutations are represented in the following way: SNPs are shown as crosses, IS insertions as inverted triangles and deletions as triangles. Filled symbols represent mutation in the coding region of the gene and empty symbols in the regulatory region. (B) Emergence and spread of adaptive mutations in M1 to M6 populations. Dynamics of haplotype frequencies in evolving populations at different days of evolution experiment are represented by circles. The color and symbol (IS insertions are represented as circles and other mutations as crosses) of each sector represents different haplotypes and the area of the circle their frequency in the population. Grey area represents the frequency of clones in the population that were typed for existing mutations in the population and did not differ from ancestral haplotype.</p

Phenotypic characterization of evolved populations.

<p>(A) Fitness increase of M1 to M6 populations relative to the ancestral clone at 285 (white bars) generations and 450 (black bars) generations. Error bars correspond to 2SE. (B) Competitive fitness of SCV clones in presence of MΦ. The change in frequency (ΔX) of the evolved bacteria against the ancestral in the intracellular (black bars) and extracellular (white bars) niche of the MΦ at MOI (1∶1). Clones are ranked in the following order: SCV_M1_D8 and SCV_M3_D5. Because SCV clones revert to ancestral looking colonies, frequencies of those phenotypic revertant SCV_REV colonies are shown in grey. (C) MUC clones overproduce colanic acid. After purification from the growth medium of each clone (SCV_M1_D8, MUC_M3_D19 and ANC), the amount of colanic acid was determined by measuring non-dialyzable methylpentose (fucose) absorbance at 396 and 427 nm after reaction with sulfuric acid and cysteine hydrochloride. Measurements were repeated three times for each clone. Obtained values (ΔA396–ΔA427) were directly correlated with fucose calibration curve (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003802#ppat.1003802.s002" target="_blank">Fig. S2</a>) and normalized for CFUs. (D) Evidence that MUC clones adapted to better escape MΦ phagocytosis. Rr represents the relative abundance (R_r) of evolved clones to that of the ancestral at 3 h of infection. Clones MUC1 to MUC6 were sampled from each independent evolution. In black bars the relative abundance inside MΦ and in white bars outside MΦ. All evolved clones show a smaller abundance inside MΦ, suggesting that these are better adapted to escape MΦ phagocytosis. Error bars correspond to 2SE.</p

A – Closure of cutaneous wounds in the HO-1^+/+ wild type and HO-1^Tg mice.

<p>Each bar represents mean+SD. N = 10 animals per group. * P<0.05, ** P<0.01, *** P<0.001 in comparison to HO-1^+/+ mice. B – Representative pictures demonstrating CD31 staining of endothelial cells in the wounded skin (3 days after wounding) in the 3-month old mice of different genotypes. Scale bar = 100 µm. C – Number of vessels in wounded skin (3 days after wounding, CD31 staining) in the 3-month old mice of different genotypes. Each bar represents analysis of samples from 5–8 animals. Data are presented as mean+SD. * P<0.05 in comparison to HO-1^+/+ animals.</p

Closure of cuteneous wounds in the HO-1^+/+ (WT), HO-1^+/− (HT), or HO-1^−/− (KO) C57BLxFVB mice.

<p>A – 3-month old animals. B – 6-month old animals. Each point represents individual animal (N = 4–5), lines connect the median values. Crossed points represent animals subjected to euthanasia. * P<0.05, ** P<0.01 in comparison to WT. C – representative pictures showing the wounds in 6-month old animals immediately after wounding and on day 10^th. Scale bar = 5 mm.</p

Activity of primary murine keratinocytes isolated from HO-1^+/+ and HO-1^Tg newborns and cultured <i>in vitro.</i>

<p>A – Migration of cells measured by time of gap closure in the presence of hydroxyurea (10 mmol/L). Scratch assay. B – Spontaneous proliferation of cells cultured for 48 h. BrdU incorporation assay. C – Viability of cells cultured in hypoxia (1% O₂) for 24 h. MTT reduction assay. D – Concentration of VEGF in media harvested from cell cultures after a 24 h incubation. Each bar represents mean+SD of 3–5 experiments. * P<0.05, ** P<0.01 in comparison to HO-1^+/+.</p

Effect of HO-1 transgene delivery on wounds.

<p>A – Effect of HO-1 transgene delivery on wound closure in the db/db diabetic mice. Adenoviral vectors (2.3×10⁷ IU in 100 µL of PBS) were injected subcutaneously near the wound immediately after injury. Control animal were injected with the same amount of AdGFP carriers. Each bar represents mean+SD; N = 5–8 animals per group. * P<0.05 in comparison to control, AdGFP treated mice. B – representative pictures showing blood vessels in the wounded skin of db/db mice injected with AdHO-1 or AdGFP vectors. CD31 staining of the skin cross-section. Scale bar = 100 µm. C – Number of vessels in wounded skin in the db/db mice injected with AdHO-1 or AdGFP, on the 3^rd and 14^th days after wounding. Analysis of specimens stained for CD31 to visualize endothelial cells. Each bar represents mean+SD values for 5–8 animals. * P<0.05 in comparison to control, AdGFP injected animals.</p