Unravelling the genome-wide contributions of specific 2-alkyl-4-quinolones and PqsE to quorum sensing in Pseudomonas aeruginosa

The pqs quorum sensing (QS) system is crucial for Pseudomonas aeruginosa virulence both in vitro and in animal models of infection and is considered an ideal target for the development of anti-virulence agents. However, the precise role played by each individual component of this complex QS circuit in the control of virulence remains to be elucidated. Key components of the pqs QS system are 2-heptyl-4-hydroxyquinoline (HHQ), 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), the transcriptional regulator PqsR and the PQS-effector element PqsE. To define the individual contribution of each of these components to QS-mediated regulation, transcriptomic analyses were performed and validated on engineered P. aeruginosa strains in which the biosynthesis of 2-alkyl 4-quinolones (AQs) and expression of pqsE and pqsR have been uncoupled, facilitating the identification of the genes controlled by individual pqs system components. The results obtained demonstrate that i) the PQS biosynthetic precursor HHQ triggers a PqsR-dependent positive feedback loop that leads to the increased expression of only the pqsABCDE operon, ii) PqsE is involved in the regulation of diverse genes coding for key virulence determinants and biofilm development, iii) PQS promotes AQ biosynthesis, the expression of genes involved in the iron-starvation response and virulence factor production via PqsR-dependent and PqsR-independent pathways, and iv) HQNO does not influence transcription and hence does not function as a QS signal molecule. Overall this work has facilitated identification of the specific regulons controlled by individual pqs system components and uncovered the ability of PQS to contribute to gene regulation independent of both its ability to activate PqsR and to induce the iron-starvation response

Granulocyte-macrophage colony stimulatory factor enhances the pro-inflammatory response of interferon-γ-treated macrophages to pseudomonas aeruginosa infection

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN)-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human macrophages infected with P. aeruginosa: untreated < treated with GM-CSF < treated with IFN-γ < treated with GM-CSF and IFN-γ

Evolving cell models for systems and synthetic biology

This paper proposes a new methodology for the automated design of cell models for systems and synthetic biology. Our modelling framework is based on P systems, a discrete, stochastic and modular formal modelling language. The automated design of biological models comprising the optimization of the model structure and its stochastic kinetic constants is performed using an evolutionary algorithm. The evolutionary algorithm evolves model structures by combining different modules taken from a predefined module library and then it fine-tunes the associated stochastic kinetic constants. We investigate four alternative objective functions for the fitness calculation within the evolutionary algorithm: (1) equally weighted sum method, (2) normalization method, (3) randomly weighted sum method, and (4) equally weighted product method. The effectiveness of the methodology is tested on four case studies of increasing complexity including negative and positive autoregulation as well as two gene networks implementing a pulse generator and a bandwidth detector. We provide a systematic analysis of the evolutionary algorithm’s results as well as of the resulting evolved cell models

Alcohol consumption and the risk of morbidity and mortality for different stroke types - a systematic review and meta-analysis

<p>Abstract</p> <p>Background</p> <p>Observational studies have suggested a complex relationship between alcohol consumption and stroke, dependent on sex, type of stroke and outcome (morbidity vs. mortality). We undertook a systematic review and a meta-analysis of studies assessing the association between levels of average alcohol consumption and relative risks of ischemic and hemorrhagic strokes separately by sex and outcome. This meta-analysis is the first to explicitly separate morbidity and mortality of alcohol-attributable stroke and thus has implications for public health and prevention.</p> <p>Methods</p> <p>Using Medical Subject Headings (alcohol drinking, ethanol, cerebrovascular accident, cerebrovascular disorders, and intracranial embolism and thrombosis and the key word stroke), a literature search of MEDLINE, EMBASE, CINAHL, CABS, WHOlist, SIGLE, ETOH, and Web of Science databases between 1980 to June 2009 was performed followed by manual searches of bibliographies of key retrieved articles. From twenty-six observational studies (cohort or case-control) with ischemic or hemorrhagic strokes the relative risk or odds ratios or hazard ratios of stroke associated with alcohol consumption were reported; alcohol consumption was quantified; and life time abstention (manually estimated where data for current abstainers were given) was used as the reference group. Two reviewers independently extracted the information on study design, participant characteristics, level of alcohol consumption, stroke outcome, control for potential confounding factors, risk estimates and key criteria of study quality using a standardized protocol.</p> <p>Results</p> <p>The dose-response relationship for hemorrhagic stroke had monotonically increasing risk for increasing consumption, whereas ischemic stroke showed a curvilinear relationship, with a protective effect of alcohol for low to moderate consumption, and increased risk for higher exposure. For more than 3 drinks on average/day, in general women had higher risks than men, and the risks for mortality were higher compared to the risks for morbidity.</p> <p>Conclusions</p> <p>These results indicate that heavy alcohol consumption increases the relative risk of any stroke while light or moderate alcohol consumption may be protective against ischemic stroke. Preventive measures that should be initiated are discussed.</p

Optimised chronic infection models demonstrate that siderophore ‘cheating’ in Pseudomonas aeruginosa is context specific

The potential for siderophore mutants of Pseudomonas aeruginosa to attenuate virulence during infection, and the possibility of exploiting this for clinical ends, have attracted much discussion. This has largely been based on the results of in vitro experiments conducted in iron-limited growth medium, in which siderophore mutants act as social ‘cheats:’ increasing in frequency at the expense of the wild type to result in low-productivity, low-virulence populations dominated by mutants. We show that insights from in vitro experiments cannot necessarily be transferred to infection contexts. First, most published experiments use an undefined siderophore mutant. Whole-genome sequencing of this strain revealed a range of mutations affecting phenotypes other than siderophore production. Second, iron-limited medium provides a very different environment from that encountered in chronic infections. We conducted cheating assays using defined siderophore deletion mutants, in conditions designed to model infected fluids and tissue in cystic fibrosis lung infection and non-healing wounds. Depending on the environment, siderophore loss led to cheating, simple fitness defects, or no fitness effect at all. Our results show that it is crucial to develop defined in vitro models in order to predict whether siderophores are social, cheatable and suitable for clinical exploitation in specific infection contexts

Ecological character displacement in the face of gene flow: Evidence from two species of nightingales

<p>Abstract</p> <p>Background</p> <p>Ecological character displacement is a process of phenotypic differentiation of sympatric populations caused by interspecific competition. Such differentiation could facilitate speciation by enhancing reproductive isolation between incipient species, although empirical evidence for it at early stages of divergence when gene flow still occurs between the species is relatively scarce. Here we studied patterns of morphological variation in sympatric and allopatric populations of two hybridizing species of birds, the Common Nightingale (<it>Luscinia megarhynchos</it>) and the Thrush Nightingale (<it>L. luscinia</it>).</p> <p>Results</p> <p>We conducted principal component (PC) analysis of morphological traits and found that nightingale species converged in overall body size (PC1) and diverged in relative bill size (PC3) in sympatry. Closer analysis of morphological variation along geographical gradients revealed that the convergence in body size can be attributed largely to increasing body size with increasing latitude, a phenomenon known as Bergmann's rule. In contrast, interspecific interactions contributed significantly to the observed divergence in relative bill size, even after controlling for the effects of geographical gradients. We suggest that the divergence in bill size most likely reflects segregation of feeding niches between the species in sympatry.</p> <p>Conclusions</p> <p>Our results suggest that interspecific competition for food resources can drive species divergence even in the face of ongoing hybridization. Such divergence may enhance reproductive isolation between the species and thus contribute to speciation.</p

Antimicrobial resistance (AMR) nanomachines: mechanisms for fluoroquinolone and glycopeptide recognition, efflux and/or deactivation

In this review, we discuss mechanisms of resistance identified in bacterial agents Staphylococcus aureus and the enterococci towards two priority classes of antibiotics—the fluoroquinolones and the glycopeptides. Members of both classes interact with a number of components in the cells of these bacteria, so the cellular targets are also considered. Fluoroquinolone resistance mechanisms include efflux pumps (MepA, NorA, NorB, NorC, MdeA, LmrS or SdrM in S. aureus and EfmA or EfrAB in the enterococci) for removal of fluoroquinolone from the intracellular environment of bacterial cells and/or protection of the gyrase and topoisomerase IV target sites in Enterococcus faecalis by Qnr-like proteins. Expression of efflux systems is regulated by GntR-like (S. aureus NorG), MarR-like (MgrA, MepR) regulators or a two-component signal transduction system (TCS) (S. aureus ArlSR). Resistance to the glycopeptide antibiotic teicoplanin occurs via efflux regulated by the TcaR regulator in S. aureus. Resistance to vancomycin occurs through modification of the D-Ala-D-Ala target in the cell wall peptidoglycan and removal of high affinity precursors, or by target protection via cell wall thickening. Of the six Van resistance types (VanA-E, VanG), the VanA resistance type is considered in this review, including its regulation by the VanSR TCS. We describe the recent application of biophysical approaches such as the hydrodynamic technique of analytical ultracentrifugation and circular dichroism spectroscopy to identify the possible molecular effector of the VanS receptor that activates expression of the Van resistance genes; both approaches demonstrated that vancomycin interacts with VanS, suggesting that vancomycin itself (or vancomycin with an accessory factor) may be an effector of vancomycin resistance. With 16 and 19 proteins or protein complexes involved in fluoroquinolone and glycopeptide resistances, respectively, and the complexities of bacterial sensing mechanisms that trigger and regulate a wide variety of possible resistance mechanisms, we propose that these antimicrobial resistance mechanisms might be considered complex ‘nanomachines’ that drive survival of bacterial cells in antibiotic environments

Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells

Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins. We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization

Interspecific variation in the relationship between clutch size, laying date and intensity of urbanization in four species of hole-nesting birds

The increase in size of human populations in urban and agricultural areas has resulted in considerable habitat conversion globally. Such anthropogenic areas have specific environmental characteristics, which influence the physiology, life history, and population dynamics of plants and animals. For example, the date of bud burst is advanced in urban compared to nearby natural areas. In some birds, breeding success is determined by synchrony between timing of breeding and peak food abundance. Pertinently, caterpillars are an important food source for the nestlings of many bird species, and their abundance is influenced by environmental factors such as temperature and date of bud burst. Higher temperatures and advanced date of bud burst in urban areas could advance peak caterpillar abundance and thus affect breeding phenology of birds. In order to test whether laying date advance and clutch sizes decrease with the intensity of urbanization, we analyzed the timing of breeding and clutch size in relation to intensity of urbanization as a measure of human impact in 199 nest box plots across Europe, North Africa, and the Middle East (i.e., the Western Palearctic) for four species of hole-nesters: blue tits (Cyanistes caeruleus), great tits (Parus major), collared flycatchers (Ficedula albicollis), and pied flycatchers (Ficedula hypoleuca). Meanwhile, we estimated the intensity of urbanization as the density of buildings surrounding study plots measured on orthophotographs. For the four study species, the intensity of urbanization was not correlated with laying date. Clutch size in blue and great tits does not seem affected by the intensity of urbanization, while in collared and pied flycatchers it decreased with increasing intensity of urbanization. This is the first large-scale study showing a species-specific major correlation between intensity of urbanization and the ecology of breeding. The underlying mechanisms for the relationships between life history and urbanization remain to be determined. We propose that effects of food abundance or quality, temperature, noise, pollution, or disturbance by humans may on their own or in combination affect laying date and/or clutch size