Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis

Recruiting complex metabolic reaction networks for chemical synthesis has attracted considerable attention but frequently requires optimization of network composition and dynamics to reach sufficient productivity. As a design framework to predict optimal levels for all enzymes in the network is currently not available, state-of-the-art pathway optimization relies on high-throughput phenotype screening. We present here the development and application of a new in vitro real-time analysis method for the comprehensive investigation and rational programming of enzyme networks for synthetic tasks. We used this first to rationally and rapidly derive an optimal blueprint for the production of the fine chemical building block dihydroxyacetone phosphate (DHAP) via Escherichia coli's highly evolved glycolysis. Second, the method guided the three-step genetic implementation of the blueprint, yielding a synthetic operon with the predicted 2.5-fold-increased glycolytic flux toward DHAP. The new analytical setup drastically accelerates rational optimization of synthetic multienzyme networks.     

Respiratory response of the deep-sea amphipod Stephonyx biscayensis indicates bathymetric range limitation by temperature and hydrostatic pressure

Depth zonation of fauna on continental margins is well documented. Whilst increasing hydrostatic pressure with depth has long been considered a factor contributing significantly to this pattern, discussion of the relative significance of decreasing temperature with depth has continued. This study investigates the physiological tolerances of fed and starved specimens of the bathyal lysianassoid amphipod Stephonyx biscayensis at varying temperature to acute pressure exposure by measuring the rate of oxygen consumption. Acclimation to atmospheric pressure is shown to have no significant interaction with temperature and/or pressure effects. Similarly, starvation is shown to have no significant effect on the interaction of temperature and pressure. Subsequently, the effect of pressure on respiration rate is revealed to be dependent on temperature: pressure equivalent to 2000 m depth was tolerated at 1 and 3°C; pressure equivalent to 2500 m depth was tolerated at 5.5°C; at 10°C pressure equivalent to 3000 m depth was tolerated. The variation in tolerance is consistent with the natural distribution range reported for this species. There are clear implications for hypotheses relating to the observed phenomenon of a biodiversity bottleneck between 2000 and 3000 metres, and for the potential for bathymetric range shifts in response to global climate change.     

Differential effects of TNF (TNFSF2) and IFN-γ on intestinal epithelial cell morphogenesis and barrier function in three-dimensional culture

Background

The cytokines TNF (TNFSF2) and IFNγ are important mediators of inflammatory bowel diseases and contribute to enhanced intestinal epithelial permeability by stimulating apoptosis and/or disrupting tight junctions. Apoptosis and tight junctions are also important for epithelial tissue morphogenesis, but the effect of TNF and IFNγ on the process of intestinal epithelial morphogenesis is unknown.

Methods/Principal Findings

We have employed a three-dimensional cell culture system, reproducing in vivo-like multicellular organization of intestinal epithelial cells, to study the effect of TNF on intestinal epithelial morphogenesis and permeability. We show that human intestinal epithelial cells in three-dimensional culture assembled into luminal spheres consisting of a single layer of cells with structural, internal, and planar cell polarity. Exposure of preformed luminal spheres to TNF or IFNγ enhanced paracellular permeability, but via distinctive mechanisms. Thus, while both TNF and IFNγ, albeit in a distinguishable manner, induced the displacement of selected tight junction proteins, only TNF increased paracellular permeability via caspase-driven apoptosis and cell shedding. Infliximab and adalumimab inhibited these effects of TNF. Moreover, we demonstrate that TNF via its stimulatory effect on apoptosis fundamentally alters the process of intestinal epithelial morphogenesis, which contributes to the de novo generation of intestinal epithelial monolayers with increased permeability. Also IFNγ contributes to the de novo formation of monolayers with increased permeability, but in a manner that does not involve apoptosis.

Conclusions

Our study provides an optimized 3D model system for the integrated analysis of (real-time) intestinal epithelial paracellular permeability and morphogenesis, and reveals apoptosis as a pivotal mechanism underlying the enhanced permeability and altered morphogenesis in response to TNF, but not IFNγ.     

Structural Reevaluation of Streptococcus pneumoniae Lipoteichoic Acid and New Insights into Its Immunostimulatory Potency

Streptococcus pneumoniae is a Gram-positive human pathogen with a complex lipoteichoic acid (pnLTA) structure. Because the current structural model for pnLTA shows substantial inconsistencies, we reinvestigated purified and, more importantly, O-deacylated pnLTA, which is most suitable for NMR spectroscopy and electrospray ionization-MS spectrometry. We analyzed pnLTA of nonencapsulated pneumococcal strains D39Δcps and TIGR4Δcps, respectively. The data obtained allowed us to (re)define (i) the position and linkage of the repeating unit, (ii) the putative α-GalpNAc substitution at the ribitiol 5-phosphate (Rib-ol-5-P), and (iii) the length of (i.e. the number of repeating units in) the pnLTA chain. We here also describe for the first time that the terminal sugar residues in the pnLTA (Forssman disaccharide; α-d-GalpNAc-(1→3)-β-d-GalpNAc-(1→)), responsible for the cross-reactivity with anti-Forssman antigen antibodies, can be heterogeneous with respect to its degree of phosphorylcholine substitution in both O-6-positions. To assess the proinflammatory potency of pnLTA, we generated a (lipopeptide-free) Δlgt mutant of strain D39Δcps, isolated its pnLTA, and showed that it is capable of inducing IL-6 release in human mononuclear cells, independent of TLR2 activation. This finding was quite in contrast to LTA of the Staphylococcus aureus SA113Δlgt mutant, which did not activate human mononuclear cells in our experiments. Remarkably, this is also contrary to various other reports showing a proinflammatory potency of S. aureus LTA. Taken together, our study refines the structure of pnLTA and indicates that pneumococcal and S. aureus LTAs differ not only in their structure but also in their bioactivity.     

Effects of spatial patterns on the pollination success of a less attractive species

Plant individuals rely on pollinator services for their reproduction and often have to share these services with co‐occurring neighbours, creating complex indirect plant–plant interactions. Many current theoretical models focus on the effect of floral resources’ density on the net outcome of these indirect plant–plant interactions, often neglecting the identity of plant species in the communities and especially the species’ spatial pattern. To fill this gap, we created a spatially explicit model whose goal was to study the interplay between relative densities and spatial distribution patterns of two plant species differing in their attractiveness for pollinators. Since theory predicts that pollinator behaviour strongly governs the outcome of pollination, we allowed the pollinators to systematically change their plant preferences based on their foraging experience. Thus the interplay between density and spatial patterns of plants was tested over a continuum of behaviours from specialists to generalists. Our most striking finding was that reproductive success of the less attractive species was affected in an opposite way by spatial patterns depending on whether the species had relatively low or high densities. Namely, when the less attractive species was highly abundant, its survival was higher when aggregated in large monospecific patches than when uniformly distributed. On the other hand, when the attractive species was more abundant, the less attractive species survived better when uniformly distributed. These results were consistent as long as the scale of the plant spatial aggregation was similar to or larger than the pollinators’ detection range. Our results suggest that aggregated plant spatial patterns manipulate pollinator behaviour by trapping them within monospecific patches. This effect was sufficiently strong to enhance the survival of a competitively inferior species and hence to act in a way similar to the more familiar niche or temporal separation among plant species.     

Organellar Na+/H+ exchangers: novel players in organelle pH regulation and their emerging functions

Mammalian Na+/H+ exchangers (NHEs) play a fundamental role in cellular ion homeostasis. NHEs exhibit an appreciable variation in expression, regulation, and physiological function, dictated by their dynamics in subcellular localization and/or interaction with regulatory proteins. In recent years, a subgroup of NHEs consisting of four isoforms has been identified, and its members predominantly localize to the membranes of the Golgi apparatus and endosomes. These organellar NHEs constitute a family of transporters with an emerging function in the regulation of luminal pH and in intracellular membrane trafficking as expressed, for example, in cell polarity development. Moreover, specific roles of a variety of cofactors, regulating the intracellular dynamics of these transporters, are also becoming apparent, thereby providing further insight into their mechanism of action and overall functioning. Interestingly, organellar NHEs have been related to mental disorders, implying a potential role in the brain, thus expanding the physiological significance of these transporters.