The Genetic Basis of <i>Escherichia coli</i> Pathoadaptation to Macrophages

<div><p>Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal <i>Escherichia coli</i> (<i>E. coli</i>) acquire this virulence trait. We show that when maintained <i>in vitro</i> under the selective pressure of host MΦ commensal <i>E. coli</i> can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing <i>in vitro</i>, while increasing their pathogenicity <i>in vivo</i>, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the <i>E. coli yrfF</i> gene. Moreover, transposition of the IS186 element into the promoter of <i>Lon</i> gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of <i>E. coli</i> evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity.</p></div

Loss of BRCC3 deubiquitinating enzyme leads to abnormal angiogenesis and is associated with syndromic moyamoya

Moyamoya is a cerebrovascular angiopathy characterized by a progressive stenosis of the terminal part of the intracranial carotid arteries and the compensatory development of abnormal and fragile collateral vessels, also called moyamoya vessels, leading to ischemic and hemorrhagic stroke. Moyamoya angiopathy can either be the sole manifestation of the disease (moyamoya disease) or be associated with various conditions, including neurofibromatosis, Down syndrome, TAAD (autosomal-dominant thoracic aortic aneurysm), and radiotherapy of head tumors (moyamoya syndromes). Its prevalence is ten times higher in Japan than in Europe, and an estimated 6%-12% of moyamoya disease is familial in Japan. The pathophysiological mechanisms of this condition remain obscure. Here, we report on three unrelated families affected with an X-linked moyamoya syndrome characterized by the association of a moyamoya angiopathy, short stature, and a stereotyped facial dysmorphism. Other symptoms include an hypergonadotropic hypogonadism, hypertension, dilated cardiomyopathy, premature coronary heart disease, premature hair graying, and early bilateral acquired cataract. We show that this syndromic moyamoya is caused by Xq28 deletions removing MTCP1/MTCP1NB and BRCC3. We also show that brcc3 morphant zebrafish display angiogenesis defects that are rescued by endothelium-specific expression of brcc3. Altogether, these data strongly suggest that BRCC3, a deubiquitinating enzyme that is part of the cellular BRCA1 and BRISC complexes, is an important player in angiogenesis and that BRCC3 loss-of-function mutations are associated with moyamoya angiopathy. © 2011 The American Society of Human Genetics

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

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

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

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