Novel screening assay for in vivo selection of Klebsiella pneumoniae genes promoting gastrointestinal colonisation

ABSTRACT: BACKGROUND: Klebsiella pneumoniae is an important opportunistic pathogen causing pneumonia, sepsis and urinary tract infections. Colonisation of the gastrointestinal (GI) tract is a key step in the development of infections; yet the specific factors important for K. pneumoniae to colonize and reside in the GI tract of the host are largely unknown. To identify K. pneumoniae genes promoting GI colonisation, a novel genomic-library-based approach was employed. RESULTS: Screening of a K. pneumoniae C3091 genomic library, expressed in E. coli strain EPI100, in a mouse model of GI colonisation led to the positive selection of five clones containing genes promoting persistent colonisation of the mouse GI tract. These included genes encoding the global response regulator ArcA; GalET of the galactose operon; and a cluster of two putative membrane-associated proteins of unknown function. Both ArcA and GalET are known to be involved in metabolic pathways in Klebsiella but may have additional biological actions beneficial to the pathogen. In support of this, GalET was found to confer decreased bile salt sensitivity to EPI100. CONCLUSIONS: The present work establishes the use of genomic-library-based in vivo screening assays as a valuable tool for identification and characterization of virulence factors in K. pneumoniae and other bacterial pathogens.     

Increased responsiveness in feeding behaviour of Caenorhabditis elegans after experimental coevolution with its microparasite Bacillus thuringiensis

Immune responses, either constitutive or induced, are costly. An alternative defence strategy may be based on behavioural responses. For example, avoidance behaviour reduces contact with pathogens and thus the risk of infection as well as the requirement of immune system activation. Similarly, if pathogens are taken up orally, preferential feeding of pathogen-free food may be advantageous. Behavioural defences have been found in many animals, including the nematode Caenorhabditis elegans. We here tested nematodes from a laboratory based evolution experiment which had either coevolved with their microparasite Bacillus thuringiensis (BT) or evolved under control conditions. After 48 generations, coevolved populations were more sensitive to food conditions: in comparison with the controls, they reduced feeding activity in the presence of pathogenic BT strains while at the same time increasing it in the presence of non-pathogenic strains. We conclude that host-parasite coevolution can drive changes in the behavioural responsiveness to bacterial microbes, potentially leading to an increased defence against pathogens.     

MS Data Miner: A web-based software tool to analyze, compare, and share mass spectrometry protein identifications

Data processing and analysis of proteomics data are challenging and time consuming. In this paper, we present MS Data Miner (MDM) (http://sourceforge.net/p/msdataminer), a freely available web-based software solution aimed at minimizing the time required for the analysis, validation, data comparison, and presentation of data files generated in MS software, including Mascot (Matrix Science), Mascot Distiller (Matrix Science), and ProteinPilot (AB Sciex). The program was developed to significantly decrease the time required to process large proteomic data sets for publication. This open sourced system includes a spectra validation system and an automatic screenshot generation tool for Mascot-assigned spectra. In addition, a Gene Ontology term analysis function and a tool for generating comparative Excel data reports are included. We illustrate the benefits of MDM during a proteomics study comprised of more than 200 LC-MS/MS analyses recorded on an AB Sciex TripleTOF 5600, identifying more than 3000 unique proteins and 3.5 million peptides.     

Sulfur-36S stable isotope labeling of amino acids for quantification (SULAQ)

We introduce a universal metabolic labeling strategy using elemental heavy 36Sulfur (36S) called 36Sulfur stable isotope labeling of amino acids for quantification (SULAQ). In the proof of principle experiment, Pseudomonas putida KT2440 was grown in defined minimal medium with sodium benzoate or sodium succinate as the sole carbon and 32S- or 36S-sodium sulfate as the sole sulfur sources. Quantification using mass spectrometry resulted in 562 proteins with 1991 unique peptides. SULAQ technology can be a valuable alternative strategy for the quantitative comparisons in MS-based proteomics approaches characterizing bacterial and other biological samples in different growth conditions.     

Nongeminate Recombination in Planar and Bulk Heterojunction Organic Solar Cells

Nongeminate recombination in organic solar cells based on copper phthalocyanine (CuPc) and C₆₀ is investigated. Two device architectures, the planar heterojunction (PHJ) and the bulk heterojunction (BHJ), are directly compared in view of differences in charge carrier decay dynamics. A combination of transient photovoltage (TPV) experiments, yielding the small perturbation charge carrier lifetime, and charge extraction measurements, providing the charge carrier density is applied. In organic solar cells, charge photogeneration and recombination primarily occur at the donor–acceptor heterointerface. Whereas the BHJ can often be approximated by an effective medium due to rather small scale phase separation, the PHJ has a well defined two‐dimensional heterointerface. In order to study nongeminate recombination dynamics in PHJ devices the charge accumulation at this interface is most relavent. As only the spatially averaged carrier concentration can be determined from extraction techniques, the charge carrier density at the interface n_int is derived from the open circuit voltage. Comparing the experimental results with macroscopic device simulation, the differences of recombination and charge carrier densities in CuPc:C₆₀ PHJ and BHJ devices are discussed with respect to the device performance. The open circuit voltage of BHJ is larger than for PHJ at low light intensities, but at 0.3 sun the situation is reversed: here, the PHJ can finally take advantage of its generally longer charge carrier lifetimes, as the active recombination region is smaller.     

The Helicobacter pylori virulence effector CagA abrogates human β-defensin 3 expression via inactivation of EGFR signaling

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Highlights? H. pylori is highly susceptible to the bactericidal activity of hBD3 ? hBD3 is initially induced by H. pylori via activation of EGFR/MAPK/JAK signaling ? H. pylori evades killing by downregulating hBD3 via CagA-mediated SHP-2 activation ? CagA virulence factor is a potent inhibitor of an immune effector mechanism     

Nubp1 Is Required for Lung Branching Morphogenesis and Distal Progenitor Cell Survival in Mice

The lung is a complex system in biology and medicine alike. Whereas there is a good understanding of the anatomy and histology of the embryonic and adult lung, less is known about the molecular details and the cellular pathways that ultimately orchestrate lung formation and affect its health. From a forward genetic approach to identify novel genes involved in lung formation, we identified a mutated Nubp1 gene, which leads to syndactyly, eye cataract and lung hypoplasia. In the lung, Nubp1 is expressed in progenitor cells of the distal epithelium. Nubp1(m1Nisw) mutants show increased apoptosis accompanied by a loss of the distal progenitor markers Sftpc, Sox9 and Foxp2. In addition, Nubp1 mutation disrupts localization of the polarity protein Par3 and the mitosis relevant protein Numb. Using knock-down studies in lung epithelial cells, we also demonstrate a function of Nubp1 in regulating centrosome dynamics and microtubule organization. Together, Nubp1 represents an essential protein for lung progenitor survival by coordinating vital cellular processes including cell polarity and centrosomal dynamics.     

The stability of pseudopeptides bearing sulfoximines as chiral backbone modifying element towards proteinase K

Incorporation of sulfoximines as backbone modifying element results in two new pseudopeptide bonds which display enhanced (bond A) and strongly reduced reactivity (bond B) towards hydrolysis by Proteinase K.     

sacB-5-Fluoroorotic Acid-pyrE-Based Bidirectional Selection for Integration of Unmarked Alleles into the Chromosome of Rhodobacter capsulatus

The gram-negative, purple nonsulfur, facultative photosynthetic bacterium Rhodobacter capsulatus is a widely used model organism and has well-developed molecular genetics. In particular, interposon mutagenesis using selectable gene cartridges is frequently employed for construction of a variety of chromosomal knockout mutants. However, as the gene cartridges are often derived from antibiotic resistance-conferring genes, their numbers are limited, which restricts the construction of multiple knockout mutants. In this report, sacB—5-fluoroorotic acid (5FOA)—pyrE-based bidirectional selection that facilitates construction of unmarked chromosomal knockout mutations is described. The R. capsulatus pyrE gene encoding orotate phosphoribosyl transferase, a key enzyme of the de novo pyrimidine nucleotide biosynthesis pathway, was used as an interposon in a genetic background that is auxotrophic for uracil (Ura⁻) and hence resistant to 5FOA (5FOA^r). Although Ura⁺ selection readily yielded chromosomal allele replacements via homologous recombination, selection for 5FOA^r to replace pyrE with unmarked alleles was inefficient. To improve the latter step, 5FOA^r selection was combined with sucrose tolerance selection using a suicide plasmid carrying the Bacillus subtilis sacB gene encoding levansucrase that induces lethality upon exposure to 5% (wt/vol) sucrose in the growth medium. Sucrose-tolerant, 5FOA^r colonies that were obtained carried chromosomal unmarked mutant alleles of the target gene via double crossovers between the resident pyrE-marked and incoming unmarked alleles. The effectiveness of this double selection was proven by seeking insertion and deletion alleles of helC involved in R. capsulatus cytochrome c biogenesis, which illustrated the usefulness of this system as a genetic means for facile construction of R. capsulatus unmarked chromosomal mutants.     

Landscape perspectives on agricultural intensification and biodiversity – ecosystem service management

Understanding the negative and positive effects of agricultural land use for the conservation of biodiversity, and its relation to ecosystem services, needs a landscape perspective. Agriculture can contribute to the conservation of high‐diversity systems, which may provide important ecosystem services such as pollination and biological control via complementarity and sampling effects. Land‐use management is often focused on few species and local processes, but in dynamic, agricultural landscapes, only a diversity of insurance species may guarantee resilience (the capacity to reorganize after disturbance). Interacting species experience their surrounding landscape at different spatial scales, which influences trophic interactions. Structurally complex landscapes enhance local diversity in agroecosystems, which may compensate for local high‐intensity management. Organisms with high‐dispersal abilities appear to drive these biodiversity patterns and ecosystem services, because of their recolonization ability and larger resources experienced. Agri‐environment schemes (incentives for farmers to benefit the environment) need to broaden their perspective and to take the different responses to schemes in simple (high impact) and complex (low impact) agricultural landscapes into account. In simple landscapes, local allocation of habitat is more important than in complex landscapes, which are in total at risk. However, little knowledge of the relative importance of local and landscape management for biodiversity and its relation to ecosystem services make reliable recommendations difficult.