Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection

The future decision support system, deliverable 8.5 of the H2020 project SafetyCube

The European Road Safety Decision Support System (DSS) is a comprehensive “one stop shop” designed to inform evidence based policy by providing state of the art scientific knowledge on road safety. A short promotion video is available here: www.youtube.com/watch?v=Y-mVUde3knU. The DSS (www.roadsafety-dss.eu) has a user friendly web-based interface allowing users access to compressive information about a large range of road safety risk (problems) and measures (solutions), and links between the two. In addition, users are presented with information about serious road injuries, accident scenario fact sheets and an Economic Efficiency Evaluation (E3) tool. The E3 tool allows users to evaluate the cost effectiveness of road safety measures as well as providing a selection of worked examples. The European Road Safety DSS was developed by the European Commission supported Horizon 2020 project Safety CaUsation, Benefits and Efficiency (SafetyCube). The object of SafetyCube was to develop an innovative road safety Decision Support System (DSS) that will enable policy-makers and stakeholders to select and implement the most appropriate strategies, measures and cost-effective approaches to reduce casualties of all road user types and all severities. Detailed information about the development and DSS status at the end of the SafetyCube project are available in Yannis & Papadimitriou (2018). An overview of the DSS scientific content and a summary of the methodology used to develop the DSS can be found in the SafetyCube Final Project Report (Thomas & Talbot, 2018). The present Deliverable (8.5) gives a brief overview of the current state of the art DSS, describes the future enhanced version of the DSS and provides information for potential funder(s). Opportunity is available for new funders to support the European road safety DSS as it is developed and enhanced for future users. Through supporting the DSS, the future funder(s) will be contributing the Global UN Sustainable Development Goals on road safety by taking a leading position to actively promote effective solutions to road safety’s most pressing challenges. Aspirations for the future DSS will make the scientific content more accessible through translation of content in to local languages and filtering information into the manner most appropriate for low and middle income countries. The content will be expanded to include more topics and more detail about existing topics. Expansion of knowledge will include knowledge about implementing measures and a focus on the interdependences of road safety measures considering the impacts of implementing measures in combination. In addition to the future visions for content there are also aspirations for the future web based interface. A key enhancement will be to give users the ability to customise the display and select the information they would like to see for each individual coded study. The best case future for DSS operation is that of extended growth supported by considerable external funding. The exact structure, legal entity and governance of the future enhanced DSS will be decided in collaboration between the SafetyCube consortia and the future funder(s). In this cooperative way funding partners will have the chance to influence the development process in the manner most appropriate to meet their stakeholder needs. It is envisaged that the future DSS will be financed by several Organisations, therefore, the governance, time schedule and strategy for extended growth will be mutually decided. Within the SafetyCube project activities have been undertaken to advertise the DSS and provide information for potential funders. The European road safety DSS is the first integrated road safety support system developed in Europe. It aims to be the “go to tool”for road safety knowledge. The next funder(s) of the DSS have the exciting opportunity to take the DSS to the next level in facilitating the future of evidence based road safety policy making, ensuring safe roads for all

Temporal transcriptomic analysis of the Listeria monocytogenes EGD-e σB regulon

<p>Abstract</p> <p>Background</p> <p>The opportunistic food-borne gram-positive pathogen <it>Listeria monocytogenes </it>can exist as a free-living microorganism in the environment and grow in the cytoplasm of vertebrate and invertebrate cells following infection. The general stress response, controlled by the alternative sigma factor, σ^B, has an important role for bacterial survival both in the environment and during infection. We used quantitative real-time PCR analysis and immuno-blot analysis to examine σ^Bexpression during growth of <it>L. monocytogenes </it>EGD-e. Whole genome-based transcriptional profiling was used to identify σ^B-dependent genes at different growth phases.</p> <p>Results</p> <p>We detected 105 σ^B-positively regulated genes and 111 genes which appeared to be under negative control of σ^Band validated 36 σ^B-positively regulated genes <it>in vivo </it>using a reporter gene fusion system.</p> <p>Conclusion</p> <p>Genes comprising the σ^Bregulon encode solute transporters, novel cell-wall proteins, universal stress proteins, transcriptional regulators and include those involved in osmoregulation, carbon metabolism, ribosome- and envelope-function, as well as virulence and niche-specific survival genes such as those involved in bile resistance and exclusion. Ten of the σ^B-positively regulated genes of <it>L. monocytogenes </it>are absent in <it>L. innocua</it>. A total of 75 σ^B-positively regulated listerial genes had homologs in <it>B. subtilis</it>, but only 33 have been previously described as being σ^B-regulated in <it>B. subtilis </it>even though both species share a highly conserved σ^B-dependent consensus sequence. A low overlap of genes may reflects adaptation of these bacteria to their respective environmental conditions.</p

The application of systems approach for road safety policy making, Deliverable 8.1 of the H2020 project SafetyCube

The present Deliverable (D8.1) describes the co-ordination of the analysis of risks and measures using a systems framework within the SafetyCube project. It outlines the results of Task 8.1 of Work Package (WP) 8 of SafetyCube. This has involved (i) defining the systems approach to be used within SafetyCube, (ii) developing a taxonomy of risks and measures, (iii) identifying a common set of accident scenarios and (iv) initiating work on the Decision Support System (DSS) development. WP8 of the SafetyCube project has a number of specific aims, including developing the European DSS for supporting evidence-based policy making. It also aims to co-ordinate analysis undertaken in other WPs ensuring integrated research outputs, compilation of the project outputs into a suitable form to be incorporated within the DSS and the European Road Safety Observatory, and finally to develop tools to enable the continued support of evidence based road safety policies beyond SafetyCube. Evidence-based policy making enables policy makers to make justified decisions in the complex reality of road safety interventions. It refers to the use of objective, scientifically-based evidence in all stages of the policy making process. Two important pillars for evidence-based road safety policy making are road safety data and statistics and scientific knowledge (Wegman et al, 2015). This type of policy making can be beneficial (e.g. helps to identify road safety problems and select most appropriate interventions) but also has it’s challenges (e.g. a lot of information at varying levels of detail is required to inform decisions). The DSS that is being developed within SafetyCube aims to support decision makers as well as other stakeholders in their evidence-based policy making. In addition to evidence-based policy making, SafetyCube and in particular the DSS is grounded in the systems approach. The systems approach aims to steer away from the more traditionally ‘human error’ blame focussed approach to road safety, and instead takes into account all ‘components’ in a system (i.e. road users, vehicles, roads) which contribute to a risk of an accident occurring. In SafetyCube, the systems approach is being integrated in the DSS in two main ways. First, the risk factors which relate to the road user, the road or the vehicle will be linked to measures in any or all of these areas if appropriate. Second, to clarify the added value of complementary measures rather than measures in isolation, where appropriate, a description of a measure will pay special attention to and link to supporting measures. The SafetyCube DSS is underpinned by four taxonomies; Road User Behaviour (WP4), Infrastructure (WP5), Vehicles (WP6) and Post Impact Care (WP7). The taxonomy is a main structural part of the DSS system, it can be used as a search option in the DSS, it creates a uniform structure over all work packages and it can be used as a basis for linking risk factors with their corresponding measures. The structure consists of three levels, which are topic, subtopic and specific topic. Thirteen main topics were identified for Road User Behaviour (WP4), 10 main topics for Infrastructure (WP5) and six main topics for Vehicle (WP6). Four topics (based on the DaCoTA webtext on Post Impact Care, 2012), were included in WP7 (Post Impact Care). As expected, there was found to be some overlap between risk factors in one taxonomy and risk factors in another (e.g. is poor vehicle maintenance a Vehicle or Road User-related risk factor?), and some overlaps where a topic could be a risk factor or a countermeasure. Discussions between WPs ensured decisions could be made about how to overcome these ambiguities. Accident scenarios are used within SafetyCube. These are considered to be a classification system for crashes whereby crash types may be grouped according to similar characteristics under a particular scenario heading, creating specific clusters. In total, nine high level accident scenarios will form an entry point to the DSS. Each high level has multiple sub-levels which provide more detailed information about the conflict situation (before the crash). A total of 63 sub-level scenarios are considered. The task of linking risks and measures is currently underway within the SafetyCube project. The accident scenarios will provide a useful and systematic way by which to link risks and measures. They will be used, in order to generate a meaningful set of links, between risks related to specific situations, and measures to address them. The primary objective of the DSS is to provide the European and Global road safety community a user friendly, web-based, interactive Decision Support Tool which will enable policy-makers and stakeholders to select and implement the most appropriate strategies, measures and cost-effective approaches to reduce casualties and crash severity for all road users. It consists of information such as risk factors, road safety measures, cost-benefit, casualty reduction effectiveness estimates. In order to develop the DSS, a review of current existing Decision Support Systems was carried out to provide a first insight into such tools (e.g. Crash Modification Factors Clearinghouse, PRACT Repository, Road Safety Engineering Kit, iRAP). No European DSS were found in the search and of the DSS reviewed, the majority focussed on infrastructure and no risk factors were included. The SafetyCube DSS addresses these gaps. To understand user needs better, three stakeholders workshops were carried out, which allowed participants to comment on the proposed DSS and suggest ‘hot topics’ (i.e. important risk factors) to address in SafetyCube, and the findings of these workshops found that the DSS should be suitable for use by a wide range of users, should be impartial, include robust data and access to all studies used and generated results. A comprehensive common SafetyCube methodology was designed, which included: a complete taxonomy of human behaviour, infrastructure and vehicle; a detailed and recorded literature review and the development of a template for coding research studies and existing results to be stored in a database linked to the DSS. The DSS is being created on the basis of a number of design principles (e.g. modern web-based tool, ergonomic interface, simple, easily updated…). As well as a consistent layout the content itself is also of high importance (e.g. quantitative results over qualitative, methodologically sound, clarity). The DSS itself consists of the backend (relational database), the front end (website) and the way they integrate (queries). The heart of the DSS consists of the searchable/dynamic and static aspects, which consists of five entry points and three levels. The design principles of the DSS ensure a smooth integration of the Work Packages in two ways, firstly that the SafetyCube common methodology is applied and secondly that the fully linked search allows the end user to better perceive the interactions between various components in road safety. There are five entry points into DSS: ‘text search’, ‘risk factors’, ‘road safety measures’, ‘road user groups’ and ‘accident scenarios’. Once a search has been undertaken using one of these five entry points, a results page is shown to the user, which consists of a table listing the available synopses1 (overview of the topic created by synthesising findings from the coding of existing studies), meta-analysis and other studies in the database. From this, the user can then also access the individual study pages for each study listed in the results. Finally, a Tools page allows the user to access other SafetyCube tools (e.g. cost-benefit calculator, methodology information, glossary). 1 More details about the synopses can be found in the Milestone M3.1 (Martensen 2016). So far, more than 500 studies have been analysed in the area of road risks with more than 3,500 risk estimates, summarised in more than 60 synopses (including approximately 10 meta-analyses), and the related measures analyses are in progress. This wealth of information will all be incorporated into the DSS and become its core outputs. The overall design of the DSS is finalised and is currently available, with the next stage being the DSS development, including all risk factors and measures. The DSS Pilot Operation will occur later in the project, followed by the final opening of the DSS, with continual updates from the end of the project onwards. The SafetyCube DSS is intended to have a life well beyond the end of the SafetyCube research project

The European road safety decision support system on risks and measures

The European Road Safety Decision Support System (roadsafety-dss.eu) is an innovative system providing the available evidence on a broad range of road risks and possible countermeasures. This paper describes the scientific basis of the DSS. The structure underlying the DSS consists of (1) a taxonomy identifying risk factors and measures and linking them to each other, (2) a repository of studies, and (3) synopses summarizing the effects estimated in the literature for each risk factor and measure, and (4) an economic efficiency evaluation instrument (E3-calculator). The DSS is implemented in a modern web-based tool with a highly ergonomic interface, allowing users to get a quick overview or go deeper into the results of single studies according to their own needs

The European road safety decision support system. A clearinghouse of road safety risks and measures, Deliverable 8.3 of the H2020 project SafetyCube

Safety CaUsation, Benefits and Efficiency (SafetyCube) is a European Commission supported Horizon 2020 project with the objective of developing an innovative road safety Decision Support System (DSS) that will enable policy-makers and stakeholders to select and implement the most appropriate strategies, measures and cost-effective approaches to reduce casualties of all road user types and all severities. The core of the SafetyCube project is a comprehensive analysis of accident risks and the effectiveness and cost-benefit of safety measures, focusing on road users, infrastructure, vehicles and post-impace care, framed within a Safe System approach ,with road safety stakeholders at the national level, EU and beyond having involvement at all stages. The present Deliverable (8.3) outlines the methods and outputs of SafetyCube Task 8.3 - ‘Decision Support System of road safety risks and measures’. A Glossary of the SafetyCube DSS is available to the Appendix of this report. The identification and assessment of user needs for a road safety DSS was conducted on the basis of a broad stakeholders’ consultation. Dedicated stakeholder workshops yielded comments and input on the SafetyCube methodology, the structure of the DSS and identification of road safety "hot topics" for human behaviour, infrastructure and vehicles. Additionally, a review of existing decision support systems, was carried out; their functions and contents were assessed, indicating that despite their usefulness they are of relatively narrow scope.... continue

SafetyCube: Building a decision support system on risks and measures

The EU research project SafetyCube (Safety CaUsation, Benefits and Efficiency) is developing an innovative road safety Decision Support System (DSS) collecting the available evidence on a broad range of road risks and possible countermeasures. The structure underlying the DSS consists of (1) a taxonomy identifying risk factors and measures and linking them to each other, (2) a repository of studies, and (3) synopses summarizing the effects estimated in the literature for each risk factor and measure, and (4) an economic efficiency evaluation (E3-calculator). The DSS is implemented in a modern web-based tool with a highly ergonomic interface, allowing users to get a quick overview or go deeper into the results of single studies according to their own needs

Identification of safety effects of infrastructure related measures, Deliverable 5.2 of the H2020 project SafetyCube

Identification of safety effects of infrastructure related measures, Deliverable 5.2 of the H2020 project SafetyCub

Group B Streptococcus GAPDH Is Released upon Cell Lysis, Associates with Bacterial Surface, and Induces Apoptosis in Murine Macrophages

Glyceraldehyde 3-phosphate dehydrogenases (GAPDH) are cytoplasmic glycolytic enzymes that, despite lacking identifiable secretion signals, have been detected at the surface of several prokaryotic and eukaryotic organisms where they exhibit non-glycolytic functions including adhesion to host components. Group B Streptococcus (GBS) is a human commensal bacterium that has the capacity to cause life-threatening meningitis and septicemia in newborns. Electron microscopy and fluorescence-activated cell sorter (FACS) analysis demonstrated the surface localization of GAPDH in GBS. By addressing the question of GAPDH export to the cell surface of GBS strain NEM316 and isogenic mutant derivatives of our collection, we found that impaired GAPDH presence in the surface and supernatant of GBS was associated with a lower level of bacterial lysis. We also found that following GBS lysis, GAPDH can associate to the surface of many living bacteria. Finally, we provide evidence for a novel function of the secreted GAPDH as an inducer of apoptosis of murine macrophages