Philippe Chouvarine

Interclonal gradient of virulence in the Pseudomonas aeruginosa pangenome from disease and environment

The population genomics of Pseudomonas aeruginosa was analysed by genome sequencing of representative strains of the 15 most frequent clonal complexes in the P. aeruginosa population and of the five most common clones from the environment of which so far no isolate from a human infection has been detected. Gene annotation identified 5892-7187 open reading frame (ORFs; median 6381 ORFs) in the 20 6.4-7.4 Mbp large genomes. The P. aeruginosa pangenome consists of a conserved core of at least 4000 genes, a combinatorial accessory genome of a further 10 000 genes and 30 000 or more rare genes that are present in only a few strains or clonal complexes. Whole genome comparisons of single nucleotide polymorphism synteny indicated unrestricted gene flow between clonal complexes by recombination. Using standardized acute lettuce, Galleria mellonella and murine airway infection models the full spectrum of possible host responses to P. aeruginosa was observed with the 20 strains ranging from unimpaired health following infection to 100% lethality. Genome comparisons indicate that the differential genetic repertoire of clones maintains a habitat-independent gradient of virulence in the P. aeruginosa population.

The cystic fibrosis lower airways microbial metagenome

Chronic airway infections determine most morbidity in people with cystic fibrosis (CF). Herein, we present unbiased quantitative data about the frequency and abundance of DNA viruses, archaea, bacteria, moulds and fungi in CF lower airways. Induced sputa were collected on several occasions from children, adolescents and adults with CF. Deep sputum metagenome sequencing identified, on average, approximately 10 DNA viruses or fungi and several hundred bacterial taxa. The metagenome of a CF patient was typically found to be made up of an individual signature of multiple, lowly abundant species superimposed by few disease-associated pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, as major components. The host-associated signatures ranged from inconspicuous polymicrobial communities in healthy subjects to low-complexity microbiomes dominated by the typical CF pathogens in patients with advanced lung disease. The DNA virus community in CF lungs mainly consisted of phages and occasionally of human pathogens, such as adeno- and herpesviruses. The S. aureus and P. aeruginosa populations were composed of one major and numerous minor clone types. The rare clones constitute a low copy genetic resource that could rapidly expand as a response to habitat alterations, such as antimicrobial chemotherapy or invasion of novel microbes. The CF lung metagenome is composed of few viruses and fungi and hundreds of bacterial species, clones and subclones http://ow.ly/ZiqUE

Intraclonal genome diversity of the major Pseudomonas aeruginosa clones C and PA14

Summary Bacterial populations differentiate at the subspecies level into clonal complexes. Intraclonal genome diversity was studied in 100 isolates of the two dominant P seudomonas aeruginosa clones C and PA14 collected from the inanimate environment, acute and chronic infections. The core genome was highly conserved among clone members with a median pairwise within‐clone single nucleotide sequence diversity of 8 × 10−6 for clone C and 2 × 10−5 for clone PA14. The composition of the accessory genome was, on the other hand, as variable within the clone as between unrelated clones. Each strain carried a large cargo of unique genes. The two dominant worldwide distributed P. aeruginosa clones combine an almost invariant core with the flexible gain and loss of genetic elements that spread by horizontal transfer.

PPARγ agonist pioglitazone reverses pulmonary hypertension and prevents right heart failure via fatty acid oxidation

Oral pioglitazone reverses pulmonary hypertensive vascular disease and prevents right heart failure via epigenetic, transcriptional, and metabolic mechanisms. PPARsing the role of lipid metabolism in PAH During pulmonary hypertension, maladaptive right ventricular hypertrophy, altered mitochondrial metabolism, and occlusive pulmonary vascular remodeling can ultimately lead to heart failure. Here, Legchenko et al. show that activation of the peroxisome proliferator–activated receptor γ (PPARγ) via pioglitazone treatment protects against heart failure in the Sugen hypoxia rat model of pulmonary arterial hypertension. The differential expression of microRNAs in lung tissue and pulmonary vessels from patients with idiopathic pulmonary arterial hypertension was mirrored in the rodent model of heart failure, and cardiac lipid metabolism, genetic, and epigenetic changes associated with PAH were reversed with pioglitazone in the rodents. These findings suggest that targeting PPARγ activation to restore fatty acid oxidation could be therapeutic for pulmonary hypertension and other diseases with altered lipid metabolism. Right ventricular (RV) heart failure is the leading cause of death in pulmonary arterial hypertension (PAH). Peroxisome proliferator–activated receptor γ (PPARγ) acts as a vasoprotective metabolic regulator in smooth muscle and endothelial cells; however, its role in the heart is unclear. We report that deletion of PPARγ in cardiomyocytes leads to biventricular systolic dysfunction and intramyocellular lipid accumulation in mice. In the SU5416/hypoxia (SuHx) rat model, oral treatment with the PPARγ agonist pioglitazone completely reverses severe PAH and vascular remodeling and prevents RV failure. Failing RV cardiomyocytes exhibited mitochondrial disarray and increased intramyocellular lipids (lipotoxicity) in the SuHx heart, which was prevented by pioglitazone. Unbiased ventricular microRNA (miRNA) arrays, mRNA sequencing, and lipid metabolism studies revealed dysregulation of cardiac hypertrophy, fibrosis, myocardial contractility, fatty acid transport/oxidation (FAO), and transforming growth factor–β signaling in the failing RV. These epigenetic, transcriptional, and metabolic alterations were modulated by pioglitazone through miRNA/mRNA networks previously not associated with PAH/RV dysfunction. Consistently, pre-miR-197 and pre-miR-146b repressed genes that drive FAO (Cpt1b and Fabp4) in primary cardiomyocytes. We recapitulated our major pathogenic findings in human end-stage PAH: (i) in the pressure-overloaded failing RV (miR-197 and miR-146b up-regulated), (ii) in peripheral pulmonary arteries (miR-146b up-regulated, miR-133b down-regulated), and (iii) in plexiform vasculopathy (miR-133b up-regulated, miR-146b down-regulated). Together, PPARγ activation can normalize epigenetic and transcriptional regulation primarily related to disturbed lipid metabolism and mitochondrial morphology/function in the failing RV and the hypertensive pulmonary vasculature, representing a therapeutic approach for PAH and other cardiovascular/pulmonary diseases.

Filtration and Normalization of Sequencing Read Data in Whole-Metagenome Shotgun Samples

Ever-increasing affordability of next-generation sequencing makes whole-metagenome sequencing an attractive alternative to traditional 16S rDNA, RFLP, or culturing approaches for the analysis of microbiome samples. The advantage of whole-metagenome sequencing is that it allows direct inference of the metabolic capacity and physiological features of the studied metagenome without reliance on the knowledge of genotypes and phenotypes of the members of the bacterial community. It also makes it possible to overcome problems of 16S rDNA sequencing, such as unknown copy number of the 16S gene and lack of sufficient sequence similarity of the “universal” 16S primers to some of the target 16S genes. On the other hand, next-generation sequencing suffers from biases resulting in non-uniform coverage of the sequenced genomes. To overcome this difficulty, we present a model of GC-bias in sequencing metagenomic samples as well as filtration and normalization techniques necessary for accurate quantification of microbial organisms. While there has been substantial research in normalization and filtration of read-count data in such techniques as RNA-seq or Chip-seq, to our knowledge, this has not been the case for the field of whole-metagenome shotgun sequencing. The presented methods assume that complete genome references are available for most microorganisms of interest present in metagenomic samples. This is often a valid assumption in such fields as medical diagnostics of patient microbiota. Testing the model on two validation datasets showed four-fold reduction in root-mean-square error compared to non-normalized data in both cases. The presented methods can be applied to any pipeline for whole metagenome sequencing analysis relying on complete microbial genome references. We demonstrate that such pre-processing reduces the number of false positive hits and increases accuracy of abundance estimates.

98 The cystic fibrosis lower airways microbial metagenome

ABSTRACT Chronic airway infections determine most morbidity in people with cystic fibrosis (CF). Herein, we present unbiased quantitative data about the frequency and abundance of DNA viruses, archaea, bacteria, moulds and fungi in CF lower airways. Induced sputa were collected on several occasions from children, adolescents and adults with CF. Deep sputum metagenome sequencing identified, on average, approximately 10 DNA viruses or fungi and several hundred bacterial taxa. The metagenome of a CF patient was typically found to be made up of an individual signature of multiple, lowly abundant species superimposed by few disease-associated pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, as major components. The host-associated signatures ranged from inconspicuous polymicrobial communities in healthy subjects to low-complexity microbiomes dominated by the typical CF pathogens in patients with advanced lung disease. The DNA virus community in CF lungs mainly consisted of phages and occasionally of human pathogens, such as adenoand herpesviruses. The S. aureus and P. aeruginosa populations were composed of one major and numerous minor clone types. The rare clones constitute a low copy genetic resource that could rapidly expand as a response to habitat alterations, such as antimicrobial chemotherapy or invasion of novel microbes.

37 The microbial metagenome of cystic fibrosis lower airways

The use of culture-independent microbiome analysis is expanding our view of respiratory tract infection in Cystic fibrosis (CF). The lower airways of people with CF are colonized with polymicrobial communities of bacteria, viruses, fungi and molds. We investigated the microbial metagenome in 20 exocrine pancreatic insufficient and 10 exocrine pancreatic sufficient CF patients. One to five sputum samples were collected from each patient over a 2-year period. The isolated DNA was sequenced by random whole genome sequencing. The reads were aligned to the human, bacterial, virus, fungi and molds reference genomes. Sequences were normalized by GC content and length reference genome. Analysis of temporal series of specimens indicate that each patient carries a specific signature of microbes in his airways unless drastic interventions were undertaken to eradicate unpleasant pathogens such as Burkholderia spp. The spectrum of bacterial species ranged from metagenomes indistinguishable from a healthy non-CF control to metagenomes dominated by the typical CF pathogens Staphylococcus aureus or Pseudomonas aeruginosa . During exacerbations the relative and absolute abundance of species changed, but not the overall signature. On the average several hundred bacterial and viral species, but just a few fungal species were detected by our pipeline. Five to forty species made up 95% of the bacterial metagenome. Our paradigmatic pilot study demonstrates that whole genome sequencing provides deep insight into the microbial CF lung metagenome. Supported by Mukoviszidose e.V. and the German Center for Lung Research (DZL).

Three-base periodicity of sites of sequence variation in Pseudomonas aeruginosa and Staphylococcus aureus core genomes

The three‐base periodicity property is characteristic of protein‐coding sequences. Here, we report on three‐base periodicity of sequence variation in the core genome of bacteria. Single nucleotide polymorphism (SNP) syntenies were extracted from pairwise genome alignments of 41 Staphylococcus aureus or 20 Pseudomonas aeruginosa strains. The length of fragment pairs with identical nucleotides at all SNP positions showed a length‐dependent overrepresentation of multiples of three nucleotides at corresponding codon positions of the AT‐rich S. aureus and the GC‐rich P. aeruginosa. Three‐base SNP periodicity seems to be a characteristic feature of the tightly arranged bacterial core genome.