Integrated human-virus metabolic stoichiometric modelling predicts host-based antiviral targets against Chikungunya, Dengue and Zika viruses

Current and reoccurring viral epidemic outbreaks such as those caused by the Zika virus illustrate the need for rapid development of antivirals. Such development would be facilitated by computational approaches that can provide experimentally testable predictions for possible antiviral strategies. To this end, we focus here on the fact that viruses are directly dependent on their host metabolism for reproduction. We develop a stoichiometric, genome-scale metabolic model that integrates human macrophage cell metabolism with the biochemical demands arising from virus production and use it to determine the virus impact on host metabolism and vice versa. While this approach applies to any host–virus pair, we first apply it to currently epidemic viruses Chikungunya, Dengue and Zika in this study. We find that each of these viruses causes specific alterations in the host metabolic flux towards fulfilling their biochemical demands as predicted by their genome and capsid structure. Subsequent analysis of this integrated model allows us to predict a set of host reactions, which, when constrained, inhibit virus production. We show that this prediction recovers known targets of existing antiviral drugs, specifically those targeting nucleotide production, while highlighting a set of hitherto unexplored reactions involving both amino acid and nucleotide metabolic pathways, with either broad or virus-specific antiviral potential. Thus, this computational approach allows rapid generation of experimentally testable hypotheses for novel antiviral targets within a host

Genome-wide saturation mutagenesis of Burkholderia pseudomallei K96243 predicts essential genes and novel targets for antimicrobial development.

UNLABELLED: Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 10(6) transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. IMPORTANCE: Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive

A Noise Trimming and Positional Significance of Transposon Insertion System to Identify Essential Genes in Yersinia pestis

This is the final version of the article. Available from Springer Nature via the DOI in this record.Massively parallel sequencing technology coupled with saturation mutagenesis has provided new and global insights into gene functions and roles. At a simplistic level, the frequency of mutations within genes can indicate the degree of essentiality. However, this approach neglects to take account of the positional significance of mutations - the function of a gene is less likely to be disrupted by a mutation close to the distal ends. Therefore, a systematic bioinformatics approach to improve the reliability of essential gene identification is desirable. We report here a parametric model which introduces a novel mutation feature together with a noise trimming approach to predict the biological significance of Tn5 mutations. We show improved performance of essential gene prediction in the bacterium Yersinia pestis, the causative agent of plague. This method would have broad applicability to other organisms and to the identification of genes which are essential for competitiveness or survival under a broad range of stresses.This work was supported by the Defence Science and Technology Laboratory under contract DSTLX-1000060221 (WP1)

Genome-wide saturation mutagenesis of Burkholderia pseudomallei K96243 predicts essential genes and novel targets for antimicrobial development

This is the final version of the article. Available from the publisher via the DOI in this record.Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 10(6) transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. IMPORTANCE: Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.This work was partially funded by the Defence Science and Technology Laboratories (DSTL)

Characterization of secreted sphingosine-1-phosphate lyases required for virulence and intracellular survival of <i>Burkholderia pseudomallei</i>

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in the orchestration of immune responses. S1P levels within the mammalian host are tightly regulated, in part through the activity of S1P lyase (S1PL) which catalyses its irreversible degradation. Herein we describe the identification and characterization of secreted S1PL orthologues encoded by the facultative intracellular bacteria Burkholderia pseudomallei and Burkholderia thailandensis. These bacterial orthologues exhibited S1PL enzymatic activity, functionally complemented an S1PL-deficient yeast strain, and conferred resistance to the antimicrobial sphingolipid D-erythro-sphingosine. We report that secretion of these bacterial S1PLs is pH-dependent, and is observed during intracellular infection. S1PL-deficient mutants displayed impaired intracellular replication in murine macrophages (associated with an inability to evade the maturing phagosome) and were significantly attenuated in murine and larval infection models. Furthermore, treatment of Burkholderia-infected macrophages with either S1P or a selective agonist of S1P receptor 1 enhanced bacterial colocalisation with LAMP-1 and reduced their intracellular survival. In summary, our studies confirm bacterial-encoded S1PL as a critical virulence determinant of B. pseudomallei and B. thailandensis, further highlighting the pivotal role of S1P in host-pathogen interactions. In addition, our data suggest that S1P pathway modulators have potential for the treatment of intracellular infection.We thank HL Ho & K Haynes (University of Exeter) for provision of strains and relevant vectors for yeast complementation studies. This work was supported by the Defence Science 26 and Technology Laboratory under contract DSTLX-1000060221 (WP1). CJM was funded by the EASTBIO Doctoral Training Partnership. The funders had no role in study design, data collection and analysis, or preparation of the manuscript

Extended Zee model for Neutrino Mass, Leptogenesis and Sterile Neutrino like Dark Matter

We propose an extension of the standard model with a $U(1)_{\rm B-L}$ global symmetry that accommodates radiative neutrino masses along with dark matter and leptogenesis. The observed matter antimatter asymmetry of the universe is generated through the leptogenesis route keeping the $U(1)_{\rm B-L}$ symmetry intact. The $B-L$ global symmetry is then softly broken, providing the sub-eV neutrino masses. The model then incorporates a MeV scale sterile neutrino like dark matter.Comment: Significantly revised, accepted for publication in PR

An integrated computational-experimental approach reveals Yersinia pestis genes essential across a narrow or a broad range of environmental conditions

This is the final version. Available from BMC via the DOI in this recordAvailability of data and materials: The datasets supporting the conclusions of this article are available at the NCBI GEO website https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE100226.BACKGROUND: The World Health Organization has categorized plague as a re-emerging disease and the potential for Yersinia pestis to also be used as a bioweapon makes the identification of new drug targets against this pathogen a priority. Environmental temperature is a key signal which regulates virulence of the bacterium. The bacterium normally grows outside the human host at 28 °C. Therefore, understanding the mechanisms that the bacterium used to adapt to a mammalian host at 37 °C is central to the development of vaccines or drugs for the prevention or treatment of human disease. RESULTS: Using a library of over 1 million Y. pestis CO92 random mutants and transposon-directed insertion site sequencing, we identified 530 essential genes when the bacteria were cultured at 28 °C. When the library of mutants was subsequently cultured at 37 °C we identified 19 genes that were essential at 37 °C but not at 28 °C, including genes which encode proteins that play a role in enabling functioning of the type III secretion and in DNA replication and maintenance. Using genome-scale metabolic network reconstruction we showed that growth conditions profoundly influence the physiology of the bacterium, and by combining computational and experimental approaches we were able to identify 54 genes that are essential under a broad range of conditions. CONCLUSIONS: Using an integrated computational-experimental approach we identify genes which are required for growth at 37 °C and under a broad range of environments may be the best targets for the development of new interventions to prevent or treat plague in humans.This work was funded by the Defence Science and Technology Laboratory, award DSTLX-1000060221 (WP1)

Constraining dark matter halo properties using lensed SNLS supernovae

This paper exploits the gravitational magnification of SNe Ia to measure properties of dark matter haloes. The magnification of individual SNe Ia can be computed using observed properties of foreground galaxies and dark matter halo models. We model the dark matter haloes of the galaxies as truncated singular isothermal spheres with velocity dispersion and truncation radius obeying luminosity dependent scaling laws. A homogeneously selected sample of 175 SNe Ia from the first 3-years of the Supernova Legacy Survey (SNLS) in the redshift range 0.2 < z < 1 is used to constrain models of the dark matter haloes associated with foreground galaxies. The best-fitting velocity dispersion scaling law agrees well with galaxy-galaxy lensing measurements. We further find that the normalisation of the velocity dispersion of passive and star forming galaxies are consistent with empirical Faber-Jackson and Tully-Fisher relations, respectively. If we make no assumption on the normalisation of these relations, we find that the data prefer gravitational lensing at the 92 per cent confidence level. Using recent models of dust extinction we deduce that the impact of this effect on our results is very small. We also investigate the brightness scatter of SNe Ia due to gravitational lensing. The gravitational lensing scatter is approximately proportional to the SN Ia redshift. We find the constant of proportionality to be B = 0.055 +0.039 -0.041 mag (B < 0.12 mag at the 95 per cent confidence level). If this model is correct, the contribution from lensing to the intrinsic brightness scatter of SNe Ia is small for the SNLS sample.Comment: 11 pages, 7 figures, accepted for publication in MNRA

An integrated computational-experimental approach reveals Yersinia pestis genes essential across a narrow or a broad range of environmental conditions

Background The World Health Organization has categorized plague as a re-emerging disease and the potential for Yersinia pestis to also be used as a bioweapon makes the identification of new drug targets against this pathogen a priority. Environmental temperature is a key signal which regulates virulence of the bacterium. The bacterium normally grows outside the human host at 28 °C. Therefore, understanding the mechanisms that the bacterium used to adapt to a mammalian host at 37 °C is central to the development of vaccines or drugs for the prevention or treatment of human disease. Results Using a library of over 1 million Y. pestis CO92 random mutants and transposon-directed insertion site sequencing, we identified 530 essential genes when the bacteria were cultured at 28 °C. When the library of mutants was subsequently cultured at 37 °C we identified 19 genes that were essential at 37 °C but not at 28 °C, including genes which encode proteins that play a role in enabling functioning of the type III secretion and in DNA replication and maintenance. Using genome-scale metabolic network reconstruction we showed that growth conditions profoundly influence the physiology of the bacterium, and by combining computational and experimental approaches we were able to identify 54 genes that are essential under a broad range of conditions. Conclusions Using an integrated computational-experimental approach we identify genes which are required for growth at 37 °C and under a broad range of environments may be the best targets for the development of new interventions to prevent or treat plague in humans

Melioidosis Vaccines: A Systematic Review and Appraisal of the Potential to Exploit Biodefense Vaccines for Public Health Purposes

The designation of Burkholderia pseudomallei as a category B select agent has resulted in considerable research funding to develop a protective vaccine. This bacterium also causes a naturally occurring disease (melioidosis), an important cause of death in many countries including Thailand and Australia. In this study, we explored whether a vaccine could be used to provide protection from melioidosis. An economic evaluation based on its use in Thailand indicated that a vaccine could be a cost-effective intervention if used in high-risk populations such as diabetics and those with chronic kidney or lung disease. A literature search of vaccine studies in animal models identified the current candidates, but noted that models failed to take account of the common routes of infection in natural melioidosis and major risk factors for infection, primarily diabetes. This review highlights important areas for future research if biodefence-driven vaccines are to play a role in reducing the global incidence of melioidosis