Monoclonal antibodies against Yersinia pestis lipopolysaccharide detect bacteria cultured at 28 degrees C or 37 degrees C.

Four monoclonal antibodies were generated against Yersinia pestis lipopolysaccharide by immunising mice with a cell surface preparation from Y. pestis strain 1255. In an ELISA the monoclonal antibodies reacted with live whole cells of Y. pestis strain GB cultured at 28 degrees C or 37 degrees C. The lowest detection threshold for Y. pestis strain GB cultured at 28 degrees C was 4 x 10(5) cfu ml(-1) and for bacteria cultured at 37 degrees C was 1 x 10(4) cfu ml(-1). The monoclonal antibodies did not cross react with other pathogenic Yersinia in an ELISA, but showed some cross reactivity in an immuno-blot. The monoclonal antibodies could be used for the detection of Y. pestis cultured at different temperatures and with varying plasmid profiles as the lipopolysaccharide molecule is not temperature regulated and the genes encoding its biosynthesis are located on the bacterial chromosome

Burkholderia thailandensis strain E555 is a surrogate for the investigation of Burkholderia pseudomallei replication and survival in macrophages

This is the final version. Available on open access from BMC via the DOI in this recordBackground: Burkholderia pseudomallei is a human pathogen causing severe infections in tropical and subtropical regions and is classified as a bio-threat agent. B. thailandensis strain E264 has been proposed as less pathogenic surrogate for understanding the interactions of B. pseudomallei with host cells. Results: We show that, unlike B. thailandensis strain E264, the pattern of growth of B. thailandensis strain E555 in macrophages is similar to that of B. pseudomallei. We have genome sequenced B. thailandensis strain E555 and using the annotated sequence identified genes and proteins up-regulated during infection. Changes in gene expression identified more of the known B. pseudomallei virulence factors than changes in protein levels and used together we identified 16% of the currently known B. pseudomallei virulence factors. These findings demonstrate the utility of B. thailandensis strain E555 to study virulence of B. pseudomallei. Conclusions: A weakness of studies using B. thailandensis as a surrogate for B. pseudomallei is that the strains used replicate at a slower rate in infected cells. We show that the pattern of growth of B. thailandensis strain E555 in macrophages closely mirrors that of B. pseudomallei. Using this infection model we have shown that virulence factors of B. pseudomallei can be identified as genes or proteins whose expression is elevated on the infection of macrophages. This finding confirms the utility of B. thailandensis strain E555 as a surrogate for B. pseudomallei and this strain should be used for future studies on virulence mechanisms.United Kingdom Ministry of Defens

Zoonoses under our noses.

This is the final version of the article. Available from Elsevier via the DOI in this record.One Health is an effective approach for the management of zoonotic disease in humans, animals and environments. Examples of the management of bacterial zoonoses in Europe and across the globe demonstrate that One Health approaches of international surveillance, information-sharing and appropriate intervention methods are required to successfully prevent and control disease outbreaks in both endemic and non-endemic regions. Additionally, a One Health approach enables effective preparation and response to bioterrorism threats.A.R.C. is supported by a BBSRC iCASE Studentship in partnership with the University of Exeter and the Defence Science and Technology Laboratory (Dstl) (Grand no.BB/M016404/1). S.R. is supported by the BBSRC grant number BB/N001591/1

Unraveling the B. pseudomallei Heptokinase WcbL: from structure to drug discovery

Journal ArticleOpen Access funded by Biotechnology and Biological Sciences Research Council under a Creative Commons Attribution 4.0 International Public LicenseGram-negative bacteria utilize heptoses as part of their repertoire of extracellular polysaccharide virulence determinants. Disruption of heptose biosynthesis offers an attractive target for novel antimicrobials. A critical step in the synthesis of heptoses is their 1-O phosphorylation, mediated by kinases such as HldE or WcbL. Here, we present the structure of WcbL from Burkholderia pseudomallei. We report that WcbL operates through a sequential ordered Bi-Bi mechanism, loading the heptose first and then ATP. We show that dimeric WcbL binds ATP anti-cooperatively in the absence of heptose, and cooperatively in its presence. Modeling of WcbL suggests that heptose binding causes an elegant switch in the hydrogen-bonding network, facilitating the binding of a second ATP molecule. Finally, we screened a library of drug-like fragments, identifying hits that potently inhibit WcbL. Our results provide a novel mechanism for control of substrate binding and emphasize WcbL as an attractive anti-microbial target for Gram-negative bacteria.Biotechnology and Biological Sciences Research Counci

Structural characterisation of the capsular polysaccharide expressed by Burkholderia thailandensis strain E555:: wbiI (pKnock-KmR) and assessment of the significance of the 2-O-acetyl group in immune protection

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.Burkholderia pseudomallei and its close relative B. mallei are human pathogens that are classified as Tier 1 bio-threat agents. Both organisms have previously been shown to constitutively produce a capsular polysaccharide (CPS) that is both a virulence determinant and protective antigen. Extraction and purification of CPS for use as a potential vaccine candidate requires containment level 3 laboratories which is expensive and time-consuming. B. thailandensis strain E555 is closely related to B. pseudomallei and B. mallei, but is non-pathogenic to humans and based on immunological cross-reactivity has previously been shown to express a B. pseudomallei-like CPS. In this study, capsular polysaccharide isolated from an O-antigen deficient strain of B. thailandensis E555 was identified by 1H and 13C NMR spectroscopy as -3-)-2-O-acetyl-6-deoxy-β-d-manno-heptopyranose-(-1, and identical to that produced by B. pseudomallei. This was further substantiated by anti-CPS monoclonal antibody binding. In connection with the production of CPS fragments for use in glycoconjugate vaccines, we set out to assess the importance or otherwise of the CPS 2-OAc groups in immune protection. To this end conjugates of the native and de-O-acetylated CPS with the Hc fragment of tetanus toxin (TetHc) were used as vaccines in a mouse model of melioidosis. The level of protection provided by deacetylated CPS was significantly lower than that from native, acetylated CPS. In addition, sera from mice vaccinated with the deacetylated CPS conjugate did not recognise native CPS. This suggests that CPS extracted from B. thailandensis can be used as antigen and that the acetyl group is essential for protection.This research was funded by the US Defence Threat Reduction Agency (DTRA), grant CBBAA12-VAXBT2-1-0032 and the United Kingdom Ministry of Defence. Studies at the JIC were supported by the UK BBSRC Institute Strategic Programme on Understanding and Exploiting Metabolism (MET) [BB/J004561/1] and the John Innes Foundation. MID was supported by a BBSRC Industrial CASE award with Mologic Ltd

What Types of Evidence are Available for Translating Health Evidence into Planning Strategies for Higher Density Living? A Review of the Literature

Despite abundant knowledge and research highlighting the significance of urban planning for improving health outcomes, there remains an absence of understanding of how health evidence can be translated into planning policy and practice, particularly for higher density urban development. This paper presents the findings of combined systematic and narrative review of academic literature pertaining to urban planning for higher density living. The study examined: 1) What types of health-related evidence have been translated into current planning strategies; 2) What types of health-related evidence have been used to critique existing planning strategies; and 3) What types of health evidence are proposed for translation into planning policy and practice? The findings reveal that while health evidence is regularly used to critique existing planning strategies, it is rarely applied to the practice of planning and constructing higher density developments. This indicates there is a need to improve integration of health evidence within the planning stages of higher density development. Our review also exposes an extensive range of suggestions for embedding health evidence in future planning strategies to improve human and environmental health outcomes in higher density environments. We conclude that targeted transdisciplinary research is required to apply, test, and evaluate the implementation of health evidence within specific local higher density contexts. This is essential for ensuring that urban planning strategies can successfully enhance the health and wellbeing of the growing population predicted to be living in higher density urban environments in the future

Protection against Experimental Melioidosis with a Synthetic manno-Heptopyranose Hexasaccharide Glycoconjugate

This is the final version of the article. Available from the publisher via the DOI in this record.Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1→3 linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease.This work was funded by the United Kingdom Ministry of Defence. The mass spectral data described here were acquired on an Orbitrap Fusion mass spectrometer funded by National Institutes of Health grant 1S10OD010645-01A1

Profiling of external metabolites during production of hantavirus nucleocapsid protein with recombinant Saccharomyces cerevisiae

Recombinant strains of Saccharomyces cerevisiae, producing hantavirus Puumala nucleocapsid protein for diagnostics and as a candidate vaccine were analyzed for uptake and excretion of intermediary metabolites during process optimization studies of fed-batch bioreactor cultures. Concentrations of glucose, maltose, galactose, pyruvate, acetaldehyde, ethanol, acetate, succinate and formaldehyde (used as a selection agent) were measured in the culture medium in order to find a metabolite pattern, indicative for the physiological state of the producer culture. When the inducer galactose was employed as a growth substrate, the metabolite profile of recombinant yeast cells was different from those of the non-recombinant original strain which excreted considerable amounts of metabolites with this substrate. In contrast, galactose-induced heterologous gene expression was indicated by the absence of excreted intermediary metabolites, except succinate. A model strain expressing a GFP fusion of hantavirus nucleocapsid protein differed in the excretion of metabolites from strains without GFP. In addition, the influence of alkali ions, employed for pH control is also demonstrated

Using technology to deliver cancer follow-up : a systematic review

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