Flexibility in the receptor-binding domain of the enzymatic colicin E9 is required for toxicity against Escherichia coli cells

The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood. It has been suggested that unfolding of the coiled-coil Mull receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation. We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain. After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9. On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9. The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins. The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB

Immunity protein release from a cell-bound nuclease colicin complex requires global conformational rearrangement

Nuclease colicins bind their target receptor BtuB in the outer membrane of sensitive Escherichia coli cells in the form of a high-affinity complex with their cognate immunity proteins. The release of the immunity protein from the colicin complex is a prerequisite for cell entry of the colicin and occurs via a process that is still relatively poorly understood. We have previously shown that an energy input in the form of the cytoplasmic membrane proton motive force is required to promote immunity protein (Im9) release from the colicin E9/Im9 complex and colicin cell entry. We report here that engineering rigidity in the structured part of the colicin translocation domain via the introduction of disulfide bonds prevents immunity protein release from the colicin complex. Reduction of the disulfide bond by the addition of DTT leads to immunity protein release and resumption of activity. Similarly, the introduction of a disulfide bond in the DNase domain previously shown to abolish channel formation in planar bilayers also prevented immunity protein release. Importantly, all disulfide bonds, in the translocation as well as the DNase domain, also abolished the biological activity of the Im9-free colicin E9, the reduction of which led to a resumption of activity. Our results show, for the first time, that conformational flexibility in the structured translocation and DNase domains of a nuclease colicin is essential for immunity protein release, providing further evidence for the hypothesis that global structural rearrangement of the colicin molecule is required for disassembly of this high-affinity toxin-immunity protein complex prior to outer membrane translocation

Relative effectiveness of insulin pump treatment over multiple daily injections and structured education during flexible intensive insulin treatment for type 1 diabetes: cluster randomised trial (REPOSE).

Objective To compare the effectiveness of insulin pumps with multiple daily injections for adults with type 1 diabetes, with both groups receiving equivalent training in flexible insulin treatment.Design Pragmatic, multicentre, open label, parallel group, cluster randomised controlled trial (Relative Effectiveness of Pumps Over MDI and Structured Education (REPOSE) trial).Setting Eight secondary care centres in England and Scotland.Participants Adults with type 1 diabetes who were willing to undertake intensive insulin treatment, with no preference for pumps or multiple daily injections. Participants were allocated a place on established group training courses that taught flexible intensive insulin treatment ("dose adjustment for normal eating," DAFNE). The course groups (the clusters) were then randomly allocated in pairs to either pump or multiple daily injections.Interventions Participants attended training in flexible insulin treatment (using insulin analogues) structured around the use of pump or injections, followed for two years.Main outcome measures The primary outcomes were a change in glycated haemoglobin (HbA1c) values (%) at two years in participants with baseline HbA1c value of ≥7.5% (58 mmol/mol), and the proportion of participants achieving an HbA1c value of <7.5%. Secondary outcomes included body weight, insulin dose, and episodes of moderate and severe hypoglycaemia. Ancillary outcomes included quality of life and treatment satisfaction.Results 317 participants (46 courses) were randomised (156 pump and 161 injections). 267 attended courses and 260 were included in the intention to treat analysis, of which 235 (119 pump and 116 injection) had baseline HbA1c values of ≥7.5%. Glycaemic control and rates of severe hypoglycaemia improved in both groups. The mean change in HbA1c at two years was -0.85% with pump treatment and -0.42% with multiple daily injections. Adjusting for course, centre, age, sex, and accounting for missing values, the difference was -0.24% (-2.7 mmol/mol) in favour of pump users (95% confidence interval -0.53 to 0.05, P=0.10). Most psychosocial measures showed no difference, but pump users showed greater improvement in treatment satisfaction and some quality of life domains (dietary freedom and daily hassle) at 12 and 24 months.Conclusions Both groups showed clinically relevant and long lasting decreases in HbA1c, rates of severe hypoglycaemia, and improved psychological measures, although few participants achieved glucose levels currently recommended by national and international guidelines. Adding pump treatment to structured training in flexible intensive insulin treatment did not substantially enhance educational benefits on glycaemic control, hypoglycaemia, or psychosocial outcomes in adults with type 1 diabetes. These results do not support a policy of providing insulin pumps to adults with poor glycaemic control until the effects of training on participants' level of engagement in intensive self management have been determined.Trial registration Current Controlled Trials ISRCTN61215213.This research was funded by the UK Health Technology Assessment Programme (project No 08/107/01) and will be published in full in Health Technology Assessment. See the HTA programme website for further project information. (http://www.nets.nihr.ac.uk/). This report presents independent research commissioned by the National Institute for Health Research (NIHR). We also acknowledge financial support from the Research and Development Programmes of the Department of Health for England and the Scottish Health and Social Care Directorates who supported the costs of consumables, and of Medtronic UK, which provided the insulin pumps for the trial. These funders had no involvement in the design of the protocol; the collection, analysis, and interpretation of the data; the writing of this paper; or the decision to submit this article for publication. The views and opinions expressed herein are those of the authors and do not necessarily reflect those of the HTA, NIHR, NHS, the Department of Health, or Medtronic UK

Notch inhibits Yorkie activity in Drosophila wing discs.

During development, tissues and organs must coordinate growth and patterning so they reach the right size and shape. During larval stages, a dramatic increase in size and cell number of Drosophila wing imaginal discs is controlled by the action of several signaling pathways. Complex cross-talk between these pathways also pattern these discs to specify different regions with different fates and growth potentials. We show that the Notch signaling pathway is both required and sufficient to inhibit the activity of Yorkie (Yki), the Salvador/Warts/Hippo (SWH) pathway terminal transcription activator, but only in the central regions of the wing disc, where the TEAD factor and Yki partner Scalloped (Sd) is expressed. We show that this cross-talk between the Notch and SWH pathways is mediated, at least in part, by the Notch target and Sd partner Vestigial (Vg). We propose that, by altering the ratios between Yki, Sd and Vg, Notch pathway activation restricts the effects of Yki mediated transcription, therefore contributing to define a zone of low proliferation in the central wing discs

The type VII secretion system of <i>Staphylococcus aureus</i> secretes a nuclease toxin that targets competitor bacteria

The type VII protein secretion system (T7SS) plays a critical role in the virulence of human pathogens including Mycobacterium tuberculosis and Staphylococcus aureus. Here we report that the S. aureus T7SS secretes a large nuclease toxin, EsaD. The toxic activity of EsaD is neutralised during its biosynthesis through complex formation with an antitoxin, EsaG, which binds to its C-terminal nuclease domain. The secretion of EsaD is dependent upon a further accessory protein, EsaE, that does not interact with the nuclease domain, but instead binds to the EsaD N-terminal region. EsaE has a dual cytoplasmic/membrane localization and membrane-bound EsaE interacts with the T7SS secretion ATPase, EssC, implicating EsaE in targeting the EsaDG complex to the secretion apparatus. EsaD and EsaE are co-secreted whereas EsaG is found only in the cytoplasm and may be stripped off during the secretion process. Strain variants of S. aureus that lack esaD encode at least two copies of EsaG-like proteins most likely to protect themselves from the toxic activity of EsaD secreted by esaD(+) strains. In support of this, a strain overproducing EsaD elicits significant growth inhibition against a sensitive strain. We conclude that T7SSs may play unexpected and key roles in bacterial competitiveness

Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction

The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (Kd ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost

The association between diet quality, dietary patterns and depression in adults: a systematic review

BACKGROUND: Recent evidence suggests that diet modifies key biological factors associated with the development of depression; however, associations between diet quality and depression are not fully understood. We performed a systematic review to evaluate existing evidence regarding the association between diet quality and depression. METHOD: A computer-aided literature search was conducted using Medline, CINAHL, and PsycINFO, January 1965 to October 2011, and a best-evidence analysis performed. RESULTS: Twenty-five studies from nine countries met eligibility criteria. Our best-evidence analyses found limited evidence to support an association between traditional diets (Mediterranean or Norwegian diets) and depression. We also observed a conflicting level of evidence for associations between (i) a traditional Japanese diet and depression, (ii) a “healthy” diet and depression, (iii) a Western diet and depression, and (iv) individuals with depression and the likelihood of eating a less healthy diet. CONCLUSION: To our knowledge, this is the first review to synthesize and critically analyze evidence regarding diet quality, dietary patterns and depression. Further studies are urgently required to elucidate whether a true causal association exists

Cell entry mechanism of enzymatic bacterial colicins: Porin recruitment and the thermodynamics of receptor binding

Binding of enzymatic E colicins to the vitamin B-12 receptor, BtuB, is the first stage in a cascade of events that culminate in the translocation of the cytotoxic nuclease into the Escherichia coli cytoplasm and release of its tightly bound immunity protein. A dogma of colicin biology is that the toxin coiled-coil connecting its functional domains must unfold or unfurl to span the periplasm, with recent reports claiming this reaction is initiated by receptor binding. We report isothermal titration calorimetry data of BtuB binding the endonuclease toxin ColE9 and a disulfide form (CoIE9(S-S)) where unfolding of the coiled-coil is prevented and, as a consequence, the toxin is biologically inactive. Contrary to expectation, the thermodynamics of receptor binding, characterized by large negative values for T Delta S, are identical for the two colicins, arguing against any form of BtuB-induced unfolding. We go on to delineate key features of the "colicin translocon" that assembles at the cell surface after BtuB binding by using a complex of histidine-tagged Im9 bound to ColE9(S-S). First, we show that the porin OmpF is recruited directly to the BtuB-colicin complex to form the translocon. Second, recruitment is through the natively unfolded region of the colicin translocation domain, with this domain likely having two contact points for OmpF. Finally, the immunity protein is not released during its assembly. Our study demonstrates that although colicin unfolding is undoubtedly a prerequisite for E. coli cell death, it must occur after assembly of the translocon

Loss-of-function genetic tools for animal models: cross-species and cross-platform differences

Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method