Transgenic laboratory animals in cystic fibrosis research

Cystic fibrosis (CF) is the most common fatal autosomal recessive genetic disorder in Caucasian populations. The incidence in Denmark is approximately 1:4500 and about 50.000 CF patients are registred in Europe and the Americas.The diseaseis characterized by malabsorption due to exocrine pancreatic insufficiency, chronic bacterial infections in the lower respiratory tract, increased salt loss in sweat, and male infertility due to absence or stenosis of the vas deferens. The CF gene wasidentified on Chromosome 7 in 1989. More than 450 different mutations have been detected, the most common being the AF508.This gene encodes for the cystic fibrosis transmembrane regulator protein (CFTR) which is a Cl' channel regulatedby protein kinase C and ATP. It facilitates transport of Cl' and other ions through the cell membrane. Two years ago. three different groups of scientists published articles describing three different variants of CF mice (>>knock—out<< mice).Two of these mice suffer from severe CF-like symptoms. especially in the intestine. and most of them die within 3 weeks. The last one has some residual CFTR activity and survives for several months. These mice allow investigations on genetherapy using different vectors and investigations on the pathogenesis of the chronic Pseudomonas aeruginosa lung infection to be carried out. Thus. the prospects for understanding CF seems promising

The Small RNA ErsA of Pseudomonas aeruginosa Contributes to Biofilm Development and Motility through Post-transcriptional Modulation of AmrZ

The small RNA ErsA of Pseudomonas aeruginosa was previously suggested to be involved in biofilm formation via negative post-transcriptional regulation of the algC gene that encodes the virulence-associated enzyme AlgC, which provides sugar precursors for the synthesis of several polysaccharides. In this study, we show that a knock-out ersA mutant strain forms a flat and uniform biofilm, not characterized by mushroom-multicellular structures typical of a mature biofilm. Conversely, the knock-out mutant strain showed enhanced swarming and twitching motilities. To assess the influence of ErsA on the P. aeruginosa transcriptome, we performed RNA-seq experiments comparing the knock-out mutant with the wild-type. More than 160 genes were found differentially expressed in the knock-out mutant. Parts of these genes, important for biofilm formation and motility regulation, are known to belong also to the AmrZ transcriptional regulator regulon. Here, we show that ErsA binds in vitro and positively regulates amrZ mRNA at post-transcriptional level in vivo suggesting an interesting contribution of the ErsA-amrZ mRNA interaction in biofilm development at several regulatory levels

Convergent Metabolic Specialization through Distinct Evolutionary Paths in <i>Pseudomonas aeruginosa</i>

ABSTRACT Evolution by natural selection under complex and dynamic environmental conditions occurs through intricate and often counterintuitive trajectories affecting many genes and metabolic solutions. To study short- and long-term evolution of bacteria in vivo, we used the natural model system of cystic fibrosis (CF) infection. In this work, we investigated how and through which trajectories evolution of Pseudomonas aeruginosa occurs when migrating from the environment to the airways of CF patients, and specifically, we determined reduction of growth rate and metabolic specialization as signatures of adaptive evolution. We show that central metabolic pathways of three distinct Pseudomonas aeruginosa lineages coevolving within the same environment become restructured at the cost of versatility during long-term colonization. Cell physiology changes from naive to adapted phenotypes resulted in (i) alteration of growth potential that particularly converged to a slow-growth phenotype, (ii) alteration of nutritional requirements due to auxotrophy, (iii) tailored preference for carbon source assimilation from CF sputum, (iv) reduced arginine and pyruvate fermentation processes, and (v) increased oxygen requirements. Interestingly, although convergence was evidenced at the phenotypic level of metabolic specialization, comparative genomics disclosed diverse mutational patterns underlying the different evolutionary trajectories. Therefore, distinct combinations of genetic and regulatory changes converge to common metabolic adaptive trajectories leading to within-host metabolic specialization. This study gives new insight into bacterial metabolic evolution during long-term colonization of a new environmental niche. IMPORTANCE Only a few examples of real-time evolutionary investigations in environments outside the laboratory are described in the scientific literature. Remembering that biological evolution, as it has progressed in nature, has not taken place in test tubes, it is not surprising that conclusions from our investigations of bacterial evolution in the CF model system are different from what has been concluded from laboratory experiments. The analysis presented here of the metabolic and regulatory driving forces leading to successful adaptation to a new environment provides an important insight into the role of metabolism and its regulatory mechanisms for successful adaptation of microorganisms in dynamic and complex environments. Understanding the trajectories of adaptation, as well as the mechanisms behind slow growth and rewiring of regulatory and metabolic networks, is a key element to understand the adaptive robustness and evolvability of bacteria in the process of increasing their in vivo fitness when conquering new territories

Genome Analysis of a Transmissible Lineage of Pseudomonas aeruginosa Reveals Pathoadaptive Mutations and Distinct Evolutionary Paths of Hypermutators.

Genome sequencing of bacterial pathogens has advanced our understanding of their evolution, epidemiology, and response to antibiotic therapy. However, we still have only a limited knowledge of the molecular changes in in vivo evolving bacterial populations in relation to long-term, chronic infections. For example, it remains unclear what genes are mutated to facilitate the establishment of long-term existence in the human host environment, and in which way acquisition of a hypermutator phenotype with enhanced rates of spontaneous mutations influences the evolutionary trajectory of the pathogen. Here we perform a retrospective study of the DK2 clone type of P. aeruginosa isolated from Danish patients suffering from cystic fibrosis (CF), and analyze the genomes of 55 bacterial isolates collected from 21 infected individuals over 38 years. Our phylogenetic analysis of 8,530 mutations in the DK2 genomes shows that the ancestral DK2 clone type spread among CF patients through several independent transmission events. Subsequent to transmission, sub-lineages evolved independently for years in separate hosts, creating a unique possibility to study parallel evolution and identification of genes targeted by mutations to optimize pathogen fitness (pathoadaptive mutations). These genes were related to antibiotic resistance, the cell envelope, or regulatory functions, and we find that the prevalence of pathoadaptive mutations correlates with evolutionary success of co-evolving sub-lineages. The long-term co-existence of both normal and hypermutator populations enabled comparative investigations of the mutation dynamics in homopolymeric sequences in which hypermutators are particularly prone to mutations. We find a positive exponential correlation between the length of the homopolymer and its likelihood to acquire mutations and identify two homopolymer-containing genes preferentially mutated in hypermutators. This homopolymer facilitated differential mutagenesis provides a novel genome-wide perspective on the different evolutionary trajectories of hypermutators, which may help explain their emergence in CF infections

Developmental stability in a cystic fibrosis mouse model

The aim of this study was to investigate the influence of cystic fibrosis (CF) and chronic experimental lung  infection with Pseudomonas aeruginosa on developmental instability and behaviour in the transgenic  Cftrtm1Unc -TgN(FABPCFTR) mouse compared to different heterozygote (CFTR+/-) and wildtype (CFTR+/+)  controls. Developmental instability measured as fluctuating asymmetry (FA), body weight and open-field  behaviour were assessed in CFTR-/-, CFTR+/- and CFTR+/+ mice. FA and different behavioural tests were  investigated in relation to tracheotomy and lung infection with P. aeruginosa. Body weight was in general  decreased in the CFTR-/- mice and increased in the CFTR+/- mice. CFTR-/- mice had a significantly higher  degree of FA (4%-5.5%) than all other groups (1%-3%) (P&lt;0.001), while having cystic fibrosis did not  seem to influence the behaviour of these mice indicating that the clinical impact from the model is rather  low, which is positive from a welfare point of view. FA and motor performance was influenced by neither  the lung infection nor the tracheotomy. Tracheotomy increased the level of fear in the light-dark box  (P&lt;0.05), and the lung infection decreased activity in the open field (P&lt;0.05). From this we may conclude  that well-being expressed as changed behaviour is a result of the lung infection more than a consequence  of the mutation.

Electrochemical sensing of biomarker for diagnostics of bacteria-specific infections

Aim: Pseudomonas aeruginosa is a pathogen that is prevalent in serious infections in compromised patients worldwide. A unique virulence factor of this bacterium is the redox-active molecule pyocyanin, which is a potential biomarker for the identification of P. aeruginosa infections. Here we report a direct, selective and rapid detection technique of pyocyanin. Materials &amp; methods: Pyocyanin was detected by amperometry at a relatively high potential where the pyocyanin signal was unaffected by background contributions. Results &amp; conclusion: Pyocyanin was detected at concentrations down to 125 nM in a 50 µM mixture of interfering compounds with a reproducibility of r2 = 0.999 (n = 5) within 200 s. The results document a step toward a point-of-care technique for diagnosis of P. aeruginosa infections. </jats:p