Rapid bacterial Virulence Annotation for the post genomic era

Lead Research Organisation: University of Bath
Department Name: Biology and Biochemistry

Abstract

Enormous amounts of money have been and are continuing to be spent on the genome sequencing of many different pathogenic bacteria. Despite this most of this sequence remains with little or no biological annotation. In order to achieve this biological annotation it would require extensive animal experimentation, which is both expensive and ethically problematic. Indeed it is widely recognized that there is a need to reduce the amount of animal experimentation currently conducted. With this in mind we have developed a technology for whole-genome mapping of bacterial virulence factors using invertebrate hosts that negates the need for mammalian testing. Simply we challenge three different invertebrate animals (insect, neamtode and amoeba) with cosmid libraries from a fully sequenced pathogen. End sequencing of cosmids that adversly effect any of these hosts are aligned with the genome sequence thus identifying pathogenic genes as bounded by clusters of cosmids. This fundamentally new approach allows us to rapidly and inexpensively bridge the enormous knowledge gap that has arisen through intensive genome sequencing. The current grant does not propose to follow-up on each of the virulence factors identified in detail but to provide access to the results as a community wide resource for workers studying each of the three bacteria. Details of the screens will be posted on the web with open access and other researchers will be able to choose cosmids for further research. Alternatively, we will also enter into collaborations with such workers to investigate genes in specific cosmids using the mutants generated.

Technical Summary

This project plans to bridge the gap between bacterial genome sequences and their functional annotation. We have set up a number of parallel screens against different organisms that allow for the rapid identification of putative virulence factors from end-sequenced cosmid libraries. Briefly, we will prepare cosmid libraries from three important pathogenic bacteria; Yersinia pseudotuberculosis strain IP32953, Francisella tularensis subspecies tularensis strain SCHU S4 and Campylobacter jejuni strain 81-176 and end-sequence these entire libraries. We will then ordinate these end sequences onto the published genomes to validate the quality and coverage of the libraries. The cosmid screens rely on the recovery of individual comids (when screened individually, for example via insect injection) or groups of cosmids (when screened as pools of clones or whole libraries) that map together in different screens. Preliminary data from the emerging human pathogen Photorhabdus asymbiotica shows that cosmids cluster in different screens depending on the apparent mode of action of the different virulence factors they contain. For example, cell toxicity or the promotion of intracellular survival. We will analyse candidates from these clusters of cosmids via transposon mutatagenesis in order to identifiy the genes responsible and post this functional annotation on a web site. This will allow other workers in the field to access the data and request specific cosmids. Alternatively, we will enter into specific collaborations to further test a range of cosmids and their associated transposon mutants with groups interested in a specific virulence factor. In this manner we will develop a community based resource designed to reduce the use of costly animal testing and to promote the functional annotation of novel virulence factors in bacterial genomes.

Publications

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Ahantarig A (2009) PirAB toxin from Photorhabdus asymbiotica as a larvicide against dengue vectors. in Applied and environmental microbiology

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Dowling AJ (2014) An unbiased method for clustering bacterial effectors using host cellular phenotypes. in Applied and environmental microbiology

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Fuchs SW (2013) Formation of 1,3-cyclohexanediones and resorcinols catalyzed by a widely occurring ketosynthase. in Angewandte Chemie (International ed. in English)

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Hapeshi A (2017) Photorhabdus asymbiotica as an Insect and Human Pathogen. in Current topics in microbiology and immunology

 
Description RVA: Rapid bacterial Virulence Annotation for the post genomic era; allowed us to develop a new technique for discovery and mapping of virulence genes in bacterial pathogens. This technique was well received and adopted a screening technique my several other labs. The technique uses insects and other invertebrates to screen so contributes to the replacement of mammalian testing for bacterial virulence studies. Furthermore the technique also identified may novel toxins for the insect pathogen Photorhabdus that could be used in crop-protection and insect vector control if required. This work also allowed us to access small molecule natural products from insect pathogens, facilitating further funding from the EU FP7 call in a large consortium award called GAMEXP. This allowed us to identify over 500 novel small molecules for drug discovery.
Exploitation Route Novel protein toxins and small molecules discovered could be adapted for use in crop protection and human/animal health applications. The use of insects as animal model hosts developed in this grant provides an alternative to mammalian innate immune studies.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The findings of this RVA grant enabled us to secure a large EU FP7 consortium grant called GAMEXP funding several research groups in labs in UK, Germany, Thailand and Vietnam. We are continuing to analyse the small molecule library we defined as a result of this for future exploitation. Collaborators continue to study various toxins identified by this work including the "Toxin Complex", the Mcf1 toxin, PirAB toxins and the PVC toxin delivery system and many small molecules products. Several publications were possible because of this funding and also outreach activities. Fo example we discussed this work on "Material World" a live Radio 4 broadcast.
Sector Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description EU FP7 (HEALTH-2007-2.3.4-3)
Amount € 468,857 (EUR)
Funding ID Contract No.223328 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 03/2009 
End 08/2012
 
Title Rapid Virulence Annotation 
Description We developed a screening system to map toxins onto the genomes of bacterial pathogens using insect, nematode, amoeba and mammalian macrophage screens. This also allowed to to assign biological activities to small molecule biosynthetic genes. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Isolation of over 500 novel small molecules by recombinant expression in E. coli and testing in invertebrate models. 
 
Description Development and analysis of polymer based multifunctional bactericidal materials 
Organisation Max Planck Society
Country Germany 
Sector Charity/Non Profit 
PI Contribution These are the three main collaborators in an EU FP7 consortium award. My collaboration was experimental in addition to meetings and network events. Joint publications were produced as a result of collaborations.
Collaborator Contribution Collaboration was experimental in addition to meetings and network events. Joint publications were produced as a result of collaborations.
Impact Joint publications including: Pereira Morais MP, Marshall D, Flower SE, Caunt CJ, James TD, Williams RJ, Waterfield NR, van den Elsen JM. Sci Rep. 2013;3:1437. Poulter N, Donaldson M, Mulley G, Duque L, Waterfield N, Shard AG, Spencer S, Jenkins ATA, Johnson AL (2011) New Journal of Chemistry 35: 1477-1484. Amos MR, Sanchez-Contreras M, Jackson RW, Muñoz-Berbel X, Ciche TA, Yang G, Cooper RM, Waterfield NR. Appl Environ Microbiol. 2011 Feb;77(3):776-85. Jones RT, Sanchez-Contreras M, Vlisidou I, Amos MR, Yang G, Muñoz-Berbel X, Upadhyay A, Potter UJ, Joyce SA, Ciche TA, Jenkins AT, Bagby S, Ffrench-Constant RH, Waterfield NR. BMC Microbiol. 2010 May 12;10:141. Poulter N, Munoz-Berbel X, Johnson AL, Dowling AJ, Waterfield N, Jenkins AT. Chem Commun (Camb). 2009 Dec 21;(47):7312-4.
Start Year 2008
 
Description Development and analysis of polymer based multifunctional bactericidal materials 
Organisation University of Bath
Country United Kingdom 
Sector Academic/University 
PI Contribution These are the three main collaborators in an EU FP7 consortium award. My collaboration was experimental in addition to meetings and network events. Joint publications were produced as a result of collaborations.
Collaborator Contribution Collaboration was experimental in addition to meetings and network events. Joint publications were produced as a result of collaborations.
Impact Joint publications including: Pereira Morais MP, Marshall D, Flower SE, Caunt CJ, James TD, Williams RJ, Waterfield NR, van den Elsen JM. Sci Rep. 2013;3:1437. Poulter N, Donaldson M, Mulley G, Duque L, Waterfield N, Shard AG, Spencer S, Jenkins ATA, Johnson AL (2011) New Journal of Chemistry 35: 1477-1484. Amos MR, Sanchez-Contreras M, Jackson RW, Muñoz-Berbel X, Ciche TA, Yang G, Cooper RM, Waterfield NR. Appl Environ Microbiol. 2011 Feb;77(3):776-85. Jones RT, Sanchez-Contreras M, Vlisidou I, Amos MR, Yang G, Muñoz-Berbel X, Upadhyay A, Potter UJ, Joyce SA, Ciche TA, Jenkins AT, Bagby S, Ffrench-Constant RH, Waterfield NR. BMC Microbiol. 2010 May 12;10:141. Poulter N, Munoz-Berbel X, Johnson AL, Dowling AJ, Waterfield N, Jenkins AT. Chem Commun (Camb). 2009 Dec 21;(47):7312-4.
Start Year 2008
 
Description Development and analysis of polymer based multifunctional bactericidal materials 
Organisation University of Exeter
Country United Kingdom 
Sector Academic/University 
PI Contribution These are the three main collaborators in an EU FP7 consortium award. My collaboration was experimental in addition to meetings and network events. Joint publications were produced as a result of collaborations.
Collaborator Contribution Collaboration was experimental in addition to meetings and network events. Joint publications were produced as a result of collaborations.
Impact Joint publications including: Pereira Morais MP, Marshall D, Flower SE, Caunt CJ, James TD, Williams RJ, Waterfield NR, van den Elsen JM. Sci Rep. 2013;3:1437. Poulter N, Donaldson M, Mulley G, Duque L, Waterfield N, Shard AG, Spencer S, Jenkins ATA, Johnson AL (2011) New Journal of Chemistry 35: 1477-1484. Amos MR, Sanchez-Contreras M, Jackson RW, Muñoz-Berbel X, Ciche TA, Yang G, Cooper RM, Waterfield NR. Appl Environ Microbiol. 2011 Feb;77(3):776-85. Jones RT, Sanchez-Contreras M, Vlisidou I, Amos MR, Yang G, Muñoz-Berbel X, Upadhyay A, Potter UJ, Joyce SA, Ciche TA, Jenkins AT, Bagby S, Ffrench-Constant RH, Waterfield NR. BMC Microbiol. 2010 May 12;10:141. Poulter N, Munoz-Berbel X, Johnson AL, Dowling AJ, Waterfield N, Jenkins AT. Chem Commun (Camb). 2009 Dec 21;(47):7312-4.
Start Year 2008
 
Description Natural Product drug discovery in Photorhabdus and Xenorhabdus 
Organisation Goethe University Frankfurt
Country Germany 
Sector Academic/University 
PI Contribution The collaborations added here are from an EU FP7 consortium award named GAMEXP. My collaboration included experimental contributions in addition to meetings and networking events. Joint efforts by consortium members gave rise to joint publications and generated data which is still being used for follow on research.
Collaborator Contribution The collaborations added here are from an EU FP7 consortium award named GAMEXP. Their collaborations included experimental contributions in addition to meetings and networking events. Joint efforts by consortium members gave rise to joint publications and generated data which is still being used for follow on research.
Impact Several publications in preparation from ongoing collaboration. Previous directly relevant publications include: Fuchs SW, Bozhüyük KA, Kresovic D, Grundmann F, Dill V, Brachmann AO, Waterfield NR, Bode HB. Angew Chem Int Ed Engl. 2013 Apr 8;52(15):4108-12. Epub 2013 Feb 19. Thanwisai A, Tandhavanant S, Saiprom N, Waterfield NR, Ke Long P, Bode HB, Peacock SJ, Chantratita N. PLoS One. 2012;7(9). Dowling AJ, Wilkinson PA, Holden MT, Quail MA, Bentley SD, Reger J, Waterfield NR, Titball RW, Ffrench-Constant RH. PLoS One. 2010 Dec 22;5(12) Wilkinson P, Paszkiewicz K, Moorhouse A, Szubert JM, Beatson S, Gerrard J, Waterfield NR, Ffrench-Constant RH. FEMS Microbiol Lett. 2010 Aug 1;309(2):136-43. Waterfield N. Curr Biol. 2010 Jan 12;20(1):R13-5
Start Year 2009
 
Description Natural Product drug discovery in Photorhabdus and Xenorhabdus 
Organisation Mahidol University
Country Thailand 
Sector Academic/University 
PI Contribution The collaborations added here are from an EU FP7 consortium award named GAMEXP. My collaboration included experimental contributions in addition to meetings and networking events. Joint efforts by consortium members gave rise to joint publications and generated data which is still being used for follow on research.
Collaborator Contribution The collaborations added here are from an EU FP7 consortium award named GAMEXP. Their collaborations included experimental contributions in addition to meetings and networking events. Joint efforts by consortium members gave rise to joint publications and generated data which is still being used for follow on research.
Impact Several publications in preparation from ongoing collaboration. Previous directly relevant publications include: Fuchs SW, Bozhüyük KA, Kresovic D, Grundmann F, Dill V, Brachmann AO, Waterfield NR, Bode HB. Angew Chem Int Ed Engl. 2013 Apr 8;52(15):4108-12. Epub 2013 Feb 19. Thanwisai A, Tandhavanant S, Saiprom N, Waterfield NR, Ke Long P, Bode HB, Peacock SJ, Chantratita N. PLoS One. 2012;7(9). Dowling AJ, Wilkinson PA, Holden MT, Quail MA, Bentley SD, Reger J, Waterfield NR, Titball RW, Ffrench-Constant RH. PLoS One. 2010 Dec 22;5(12) Wilkinson P, Paszkiewicz K, Moorhouse A, Szubert JM, Beatson S, Gerrard J, Waterfield NR, Ffrench-Constant RH. FEMS Microbiol Lett. 2010 Aug 1;309(2):136-43. Waterfield N. Curr Biol. 2010 Jan 12;20(1):R13-5
Start Year 2009
 
Description Natural Product drug discovery in Photorhabdus and Xenorhabdus 
Organisation University of Exeter
Country United Kingdom 
Sector Academic/University 
PI Contribution The collaborations added here are from an EU FP7 consortium award named GAMEXP. My collaboration included experimental contributions in addition to meetings and networking events. Joint efforts by consortium members gave rise to joint publications and generated data which is still being used for follow on research.
Collaborator Contribution The collaborations added here are from an EU FP7 consortium award named GAMEXP. Their collaborations included experimental contributions in addition to meetings and networking events. Joint efforts by consortium members gave rise to joint publications and generated data which is still being used for follow on research.
Impact Several publications in preparation from ongoing collaboration. Previous directly relevant publications include: Fuchs SW, Bozhüyük KA, Kresovic D, Grundmann F, Dill V, Brachmann AO, Waterfield NR, Bode HB. Angew Chem Int Ed Engl. 2013 Apr 8;52(15):4108-12. Epub 2013 Feb 19. Thanwisai A, Tandhavanant S, Saiprom N, Waterfield NR, Ke Long P, Bode HB, Peacock SJ, Chantratita N. PLoS One. 2012;7(9). Dowling AJ, Wilkinson PA, Holden MT, Quail MA, Bentley SD, Reger J, Waterfield NR, Titball RW, Ffrench-Constant RH. PLoS One. 2010 Dec 22;5(12) Wilkinson P, Paszkiewicz K, Moorhouse A, Szubert JM, Beatson S, Gerrard J, Waterfield NR, Ffrench-Constant RH. FEMS Microbiol Lett. 2010 Aug 1;309(2):136-43. Waterfield N. Curr Biol. 2010 Jan 12;20(1):R13-5
Start Year 2009