Úloha Gag polyproteínov pri zbaľovaní a pučaní retrovírusov

Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyPřírodovědecká fakultaFaculty of Scienc

Parallel Evolution in Pseudomonas aeruginosa over 39,000 Generations In Vivo

The Gram-negative bacterium Pseudomonas aeruginosa is a common cause of chronic airway infections in individuals with the heritable disease cystic fibrosis (CF). After prolonged colonization of the CF lung, P. aeruginosa becomes highly resistant to host clearance and antibiotic treatment; therefore, understanding how this bacterium evolves during chronic infection is important for identifying beneficial adaptations that could be targeted therapeutically. To identify potential adaptive traits of P. aeruginosa during chronic infection, we carried out global transcriptomic profiling of chronological clonal isolates obtained from 3 individuals with CF. Isolates were collected sequentially over periods ranging from 3 months to 8 years, representing up to 39,000 in vivo generations. We identified 24 genes that were commonly regulated by all 3 P. aeruginosa lineages, including several genes encoding traits previously shown to be important for in vivo growth. Our results reveal that parallel evolution occurs in the CF lung and that at least a proportion of the traits identified are beneficial for P. aeruginosa chronic colonization of the CF lung

Transcriptional adaptations during long-term persistence of Staphylococcus aureus in the airways of a cystic fibrosis patient

AbstractThe lungs of Cystic fibrosis (CF) patients are often colonized and/or infected by Staphylococcus aureus for years, mostly by one predominant clone. For long-term survival in this environment, S. aureus needs to adapt during its interactions with host factors, antibiotics, and other pathogens. Here, we study long-term transcriptional as well as genomic adaptations of an isogenic pair of S. aureus isolates from a single patient using RNA sequencing (RNA-Seq) and whole genome sequencing (WGS). Mimicking in vivo conditions, we cultivated the S. aureus isolates using artificial sputum medium before harvesting RNA for subsequent analysis. We confirmed our RNA-Seq data using quantitative real-time (qRT)-PCR and additionally investigated intermediate isolates from the same patient representing in total 13.2 years of persistence in the CF airways. Comparative RNA-Seq analysis of the first and the last (“late”) isolate revealed significant differences in the late isolate after 13.2 years of persistence. Of the 2545 genes expressed in both isolates that were cultivated aerobically, 256 genes were up- and 161 were down-regulated with a minimum 2-fold change (2f). Focusing on 25 highly (≥8f) up- (n=9) or down- (n=16) regulated genes, we identified several genes encoding for virulence factors involved in immune evasion, bacterial spread or secretion (e.g. spa, sak, and esxA). Moreover, these genes displayed similar expression trends under aerobic, microaerophilic and anaerobic conditions. Further qRT-PCR-experiments of highly up- or down-regulated genes within intermediate S. aureus isolates resulted in different gene expression patterns over the years. Using sequencing analysis of the differently expressed genes and their upstream regions in the late S. aureus isolate resulted in only few genomic alterations. Comparative transcriptomic analysis revealed adaptive changes affecting mainly genes involved in host-pathogen interaction. Although the underlying mechanisms were not known, our results suggest adaptive processes beyond genomic mutations triggered by local factors rather than by activation of global regulators

Proteomic profiling of Burkholderia cenocepacia clonal isolates with different virulence potential retrieved from a cystic fibrosis patient during chronic lung infection

Respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. In this work, we assessed the virulence potential of three B. cenocepacia clonal isolates obtained from a CF patient between the onset of infection (isolate IST439) and before death with cepacia syndrome 3.5 years later (isolate IST4113 followed by IST4134), based on their ability to invade epithelial cells and compromise epithelial monolayer integrity. The two clonal isolates retrieved during late-stage disease were significantly more virulent than IST439. Proteomic profiling by 2-D DIGE of the last isolate recovered before the patient's death, IST4134, and clonal isolate IST439, was performed and compared with a prior analysis of IST4113 vs. IST439. The cytoplasmic and membrane-associated enriched fractions were examined and 52 proteins were found to be similarly altered in the two last isolates compared with IST439. These proteins are involved in metabolic functions, nucleotide synthesis, translation and protein folding, cell envelope biogenesis and iron homeostasis. Results are suggestive of the important role played by metabolic reprogramming in the virulence potential and persistence of B. cenocepacia, in particular regarding bacterial adaptation to microaerophilic conditions. Also, the content of the virulence determinant AidA was higher in the last 2 isolates. Significant levels of siderophores were found to be secreted by the three clonal isolates in an iron-depleted environment, but the two late isolates were more tolerant to low iron concentrations than IST439, consistent with the relative abundance of proteins involved in iron uptake.This work was supported by FEDER and FCT – Fundação para a Ciência e a Tecnologia (contract PEst-OE/EQB/LA0023/2011_ research line: Systems and Synthetic Biology; PhD grant to A.M. – SFRH/BD/37012/2007, and PD grants to S.S. – SFRH/BPD/75483/2010 and C.C. – SFRH/BPD/ 81220/2011. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

Development of an ex vivo porcine lung model for studying growth, virulence, and signaling of Pseudomonas aeruginosa

Research into chronic infection by bacterial pathogens, such as Pseudomonas aeruginosa, uses various in vitro and live host models. While these have increased our understanding of pathogen growth, virulence, and evolution, each model has certain limitations. In vitro models cannot recapitulate the complex spatial structure of host organs, while experiments on live hosts are limited in terms of sample size and infection duration for ethical reasons; live mammal models also require specialized facilities which are costly to run. To address this, we have developed an ex vivo pig lung (EVPL) model for quantifying Pseudomonas aeruginosa growth, quorum sensing (QS), virulence factor production, and tissue damage in an environment that mimics a chronically infected cystic fibrosis (CF) lung. In a first test of our model, we show that lasR mutants, which do not respond to 3-oxo-C12-homoserine lactone (HSL)-mediated QS, exhibit reduced virulence factor production in EVPL. We also show that lasR mutants grow as well as or better than a corresponding wild-type strain in EVPL. lasR mutants frequently and repeatedly arise during chronic CF lung infection, but the evolutionary forces governing their appearance and spread are not clear. Our data are not consistent with the hypothesis that lasR mutants act as social “cheats” in the lung; rather, our results support the hypothesis that lasR mutants are more adapted to the lung environment. More generally, this model will facilitate improved studies of mi- crobial disease, especially studies of how cells of the same and different species interact in polymicrobial infections in a spatially structured environment

Genomic Expression Analysis Reveals Strategies of Burkholderia cenocepacia to Adapt to Cystic Fibrosis Patients' Airways and Antimicrobial Therapy

Pulmonary colonization of cystic fibrosis (CF) patients with Burkholderia cenocepacia or other bacteria of the Burkholderia cepacia complex (Bcc) is associated with worse prognosis and increased risk of death. During colonization, the bacteria may evolve under the stressing selection pressures exerted in the CF lung, in particular, those resulting from challenges of the host immune defenses, antimicrobial therapy, nutrient availability and oxygen limitation. Understanding the adaptive mechanisms that promote successful colonization and long-term survival of B. cenocepacia in the CF lung is essential for an improved therapeutic outcome of chronic infections. To get mechanistic insights into these adaptive strategies a transcriptomic analysis, based on DNA microarrays, was explored in this study. The genomic expression levels in two clonal variants isolated during long-term colonization of a CF patient who died from the cepacia syndrome were compared. One of the isolates examined, IST439, is the first B. cenocepacia isolate retrieved from the patient and the other isolate, IST4113, was obtained three years later and is more resistant to different classes of antimicrobials. Approximately 1000 genes were found to be differently expressed in the two clonal variants reflecting a marked reprogramming of genomic expression. The up-regulated genes in IST4113 include those involved in translation, iron uptake (in particular, in ornibactin biosynthesis), efflux of drugs and in adhesion to epithelial lung tissue and to mucin. Alterations related with adaptation to the nutritional environment of the CF lung and to an oxygen-limited environment are also suggested to be a key feature of transcriptional reprogramming occurring during long-term colonization, antibiotic therapy and the progression of the disease

Ultrafast Evolution and Loss of CRISPRs Following a Host Shift in a Novel Wildlife Pathogen, Mycoplasma gallisepticum

Measureable rates of genome evolution are well documented in human pathogens but are less well understood in bacterial pathogens in the wild, particularly during and after host switches. Mycoplasma gallisepticum (MG) is a pathogenic bacterium that has evolved predominantly in poultry and recently jumped to wild house finches (Carpodacus mexicanus), a common North American songbird. For the first time we characterize the genome and measure rates of genome evolution in House Finch isolates of MG, as well as in poultry outgroups. Using whole-genome sequences of 12 House Finch isolates across a 13-year serial sample and an additional four newly sequenced poultry strains, we estimate a nucleotide diversity in House Finch isolates of only ∼2% of ancestral poultry strains and a nucleotide substitution rate of 0.8−1.2×10−5 per site per year both in poultry and in House Finches, an exceptionally fast rate rivaling some of the highest estimates reported thus far for bacteria. We also found high diversity and complete turnover of CRISPR arrays in poultry MG strains prior to the switch to the House Finch host, but after the invasion of House Finches there is progressive loss of CRISPR repeat diversity, and recruitment of novel CRISPR repeats ceases. Recent (2007) House Finch MG strains retain only ∼50% of the CRISPR repertoire founding (1994–95) strains and have lost the CRISPR–associated genes required for CRISPR function. Our results suggest that genome evolution in bacterial pathogens of wild birds can be extremely rapid and in this case is accompanied by apparent functional loss of CRISPRs