The interplay of sRNAs Hfq and RNase E in the control of gene expression; a novel mechanism linked to pathogenic bacterial virulence

Lead Research Organisation: University of Portsmouth
Department Name: Inst of Biomedical and Biomolecular Sc

Abstract

With antibiotic resistance on the rise, research into understanding the workings of bacterial organisms is crucially important, as are new approaches to combating the infections they cause. When bacterial cells bring about infection, one of the first steps is that they must gain entry to the host cell. Recently scientists have found that an interlinked sequence of events occurs at the molecular level which aids the process of bacterial invasion into a host cell. They found that within the bacteria, messenger molecules (mRNA) played an important role in the invasion, but that these molecules were either degraded or stabilized by destruction (RNase E) or protection (Hfq) molecules respectively. It is also known that bacteria use signal molecules (sRNAs) to trigger either the destruction or the protection of the messenger molecules (mRNA). During the life of a bacterial cell, it is now understood that a complex sequence of interactions continually occurs between these molecules. Recent advances have taken the first steps to understanding this complex sequence of interactions, but quite how the events are communicated and regulated within the bacterial cell is still unknown. How does the destruction pathway work and how does the protector molecule prevent it? Are different signal molecules (sRNAs) treated differently? Can the protection pathway be interrupted in order to prevent the bacteria invading the host cell, and thereby preventing infection? Current data are lacking to answer these most fundamental questions. The aim of this research proposal is therefore to understand the interactions between the signal, messenger, protector and destructor molecules (sRNA, mRNA, Hfq and RNase E) found within a model bacterial cell. Only with this knowledge will it be possible to accurately inhibit the appropriate interactions to develop novel antibacterial approaches.

Technical Summary

The aim of this project is to increase our understanding of a newly discovered mechanism of genetic regulation with potential applications in the field of antibacterial research. The essential ribonuclease RNase E has a critical role in initiating mRNA decay, and therefore serves an important role in the post-transcriptional control of gene expression. Recently, non-coding small RNAs (sRNAs) have been identified that can program RNase E to target specific mRNA transcripts for destruction. This targeting is mediated through interaction with the RNA chaperone Hfq. However, certain sRNAs have been shown to have entirely the opposite effect, in that they and their mRNA targets are stabilized by Hfq against cleavage by RNase E. This protective mode has been shown to be critical for the transcription of major virulence factors in various pathogenic bacteria, with Hfq deletion mutants displaying attenuation of invasive virulence. The interplay of sRNAs, their mRNA targets and Hfq results in a finely balanced mechanism of communication with RNase E to bring about either the destruction or the stabilization and subsequent translation of specific transcripts. The fundamental questions in this area are how this communication occurs and whether this mechanism, with a direct impact on pathogenic bacterial virulence, can be exploited in the search for novel antibacterial approaches and/or targets. The proposed research uses a toolbox of biochemical, biophysical and structural characterization techniques, initially to investigate and understand isolated interactions (e.g. sRNA-RNase E and sRNA-Hfq) and subsequently to analyse the key steps in the pathway as a whole (e.g. efficiency of sRNA-mRNA duplex formation in the presence and absence of Hfq and RNase E).
 
Title 3D Printed Molecular Structures 
Description Working with Dr Darren Gowers at the University of Portsmouth, who runs the 3D molecular models printing service, the molecular structures of a number of molecules of interest to our research were generated. By incorporating tiny magnets into the structures it was possible to generate molecular models of protomers (single units) that could be assembled into their native oligomeric states; thus yielding flexible molecular models that could be disassembled and reassembled to appreciate the interactions involved in creating multi-biomolecule complexes. 
Type Of Art Artwork 
Year Produced 2014 
Impact These models provide hands-on aids when presenting our research findings to both scientific and general public audiences. This has allowed potentially complex molecular details to be communicated clearly and easily and has supported my research team in maximising the accessibility of our research to the wider scientific and lay communities. 
 
Description With antibiotic resistance on the rise, research into understanding the workings of bacterial organisms is crucially important, as are new approaches to combating the infections they cause. When bacterial cells bring about infection, one of the first steps is that they must gain entry to the host cell. Recently scientists have found that an interlinked sequence of events occurs at the molecular level which aids the process of bacterial invasion into a host cell. They found that within the bacteria, messenger molecules (mRNA) played an important role in the invasion, but that these molecules were either degraded or stabilized by destruction (RNase E) or protection (Hfq) molecules respectively. It is also known that bacteria use signal molecules (sRNAs) to trigger either the destruction or the protection of the messenger molecules (mRNA). During the life of a bacterial cell, it is now understood that a complex sequence of interactions continually occurs between these molecules.

Our studies have taken significant steps to understand this complex sequence of interactions between the signal, messenger, protector and destructor molecules (sRNA, mRNA, Hfq and RNase E) found within a model bacterial cell (E. coli) and, in some instances, a bacterial pathogen (V. cholerae). Specifically, we have identified the strength of the interactions between these key players to understand interaction preferences. We have unravelled the molecular structural details of Hfq-sRNA complexes and identified that Hfq does not change shape upon complex formation whereas the sRNA does. This demonstrated that Hfq has a shape-moulding role towards the sRNAs. We have also shown that RNase E and Hfq can work together in a sRNA-dependent manner to enhance the effect of the sRNA signal. We have discovered that a novel metal-ion dependent regulation of sRNA activity exists and recognized important molecular differences between E. coli and V. cholerae Hfq. In addition, we successfully identified a number of small molecules capable of inhibiting RNase E activity. Collectively, the knowledge gained on the interplay of sRNA, mRNA, Hfq and RNase E takes us closer to being able to accurately inhibit the appropriate interactions to develop novel antibacterial approaches whilst the identification of RNase E inhibitors takes the first steps towards achieving this aim.

Finally, whilst conducting this research, we discovered a novel technology which holds great potential for impacting RNA research in the future. This discovery provided the basis for both BBSRC follow-on-funding and Higher Education Innovation Funding investment from Portsmouth University to pursue this work. A patent has been filed (UK Patent Application Number 1108041.3) to protect the intellectual property and support the translation of this discovery to the market place. While the development of this technology is ongoing, this clearly demonstrates a commitment to successfully translating and maximising the impact of the fundamental research conducted as part of this grant.
Exploitation Route Antibacterials are of major importance in infection control and have the potential to significantly improve quality of life. The results in this work provide important information to support the development of a novel antibacterial approach. While follow on work may initially be exploited within the academic community, this work provides the potential basis for future pharmaceutical drug development. Economically, there is a long term possibility of pursuing any potential applications as an industry partnerships. With respect to the discovery of a novel technology, which holds great potential for impacting RNA research in the future, it is envisaged that this will have impacts within fields such as transcriptomics.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description The findings of this grant have resulted in a number of publications and the filing of a patent application. This has also supported the leverage of further funding to explore development of the novel technology discovered as part of this research.
 
Description BBSRC Follow on Funding Pathfinder
Amount £8,000 (GBP)
Funding ID BB/FOF/PF/3/10 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 06/2010 
End 12/2010
 
Description BBSRC responsive mode - APP Project
Amount £360,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 12/2015 
End 11/2018
 
Description BBSRC responsive mode - PNPase Project
Amount £360,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 03/2013 
End 08/2016
 
Description Biochemical Society Vacation Studentship
Amount £1,600 (GBP)
Organisation Biochemical Society 
Sector Learned Society
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 06/2013 
End 08/2013
 
Description Biochemical Society Vacation Studentship 2014
Amount £1,600 (GBP)
Organisation Biochemical Society 
Sector Learned Society
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 06/2014 
End 08/2014
 
Description DSTL PhD Studentship
Amount £152,000 (GBP)
Organisation Defence Science & Technology Laboratory (DSTL) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2014 
End 09/2018
 
Description Daphne Jackson Fellowship for a returner to science to join my team
Amount £55,000 (GBP)
Organisation University of Surrey 
Department Daphne Jackson Trust
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 08/2015 
End 07/2017
 
Description Follow on Funding - 'Printing' RNA Arrays
Amount £152,000 (GBP)
Funding ID BB/I532988/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 04/2011 
End 02/2013
 
Description Nuffield Foundation Science Bursary - Vacation Studentship
Amount £1,440 (GBP)
Organisation Nuffield Foundation 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 06/2009 
End 08/2009
 
Description Royal Society Research Grant
Amount £15,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 09/2009 
End 08/2010
 
Description University of Portsmouth IBBS PhD studentship
Amount £55,000 (GBP)
Organisation University of Portsmouth 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2008 
End 09/2011
 
Description University of Portsmouth Strategic Research Fund
Amount £11,000 (GBP)
Organisation University of Portsmouth 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 09/2011 
End 01/2012
 
Description University of Portsmouth, Higher Education Innovation Fund
Amount £17,000 (GBP)
Organisation University of Portsmouth 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 04/2010 
End 04/2011
 
Title Novel RNA Array Technology 
Description Proof-of-concept for a method for generating RNA arrays has been devised. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact A patent on the method has been filed. 
 
Title RNA-Tagging 
Description A novel method for tagging RNA for surface immobilisation has been devised. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact The RNA-tagging approach has been published to allow others to use the method. 
 
Description Collaboration with Ben Luisi, Cambridge University 
Organisation University of Cambridge
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Research collaboration resulting in a publication.
Start Year 2008
 
Description Collaboration with Dr Helen Atkins at DSTL 
Organisation Defence Science & Technology Laboratory (DSTL)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Researchers in my team are undertaking experiments to support the goals of DSTL in developing and exploring novel antibacterial approaches.
Collaborator Contribution Dr Atkins at DSTL provides project input and guidance, as well as access to facilities if required in the future.
Impact Poster and oral presentations have been undertaken as part of this collaboration.
Start Year 2013
 
Description Collaboration with Dr Jon Watts at the RNA Therapeutics Institute, University of Massachusetts Medical School. 
Organisation University of Massachusetts (UMass)
Department University of Massachusetts Medical School
Country United States of America 
Sector Academic/University 
PI Contribution Researchers in my team have been testing RNA inhibitors synthesised by researchers in Dr Watts' team on a collaborative project.
Collaborator Contribution Researchers in Dr Watts' team synthesised potential RNA inhibitors which they provided to my team for testing.
Impact Seed corn funding has been leveraged from the University of Portsmouth to support preliminary data collection on this project.
Start Year 2012
 
Description Collaboration with Dr Kenny McDowall at Leeds University 
Organisation University of Leeds
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution The collaboration involved researchers in my team conducting in vitro tests of potential inhibitor molecules identified and provided by Dr McDowall's group following computational analysis.
Collaborator Contribution Potential inhibitor molecules were identified and provided by Dr McDowall's group for interaction testing by researchers within my team.
Impact A paper of the findings resulted from this collaboration. My team provided in vitro molecular interaction and activity testing expertise whilst Dr McDowall's team also provided expertise in activity testing as well as computational analysis.
Start Year 2009
 
Description Collaboration with Frank Sobott, University of Antwerp 
Organisation University of Antwerp
Country Belgium, Kingdom of 
Sector Academic/University 
PI Contribution Research collaboration resulting in publications.
Start Year 2009
 
Description Collaboration with Marc Malfois, Diamond Light Source 
Organisation DIAMOND Light Source Ltd
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Research collaboration with Dr Marc Malfois resulting in a publication.
Start Year 2010
 
Description Collaboration with Paul Discoll and members of his research group at NIMR 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Research collaboration resulting in a publication.
Start Year 2009
 
Description Collaboration with Prof. Denise Bazzolli at the Universidade Federal de Viçosa 
Organisation Universidade Federal de Viçosa
Country Brazil, Federative Republic of 
Sector Academic/University 
PI Contribution My research team are undertaking molecular interaction studies guided by microbiology data provided by Prof. Bazzolli's team.
Collaborator Contribution Prof. Bazzolli's team are providing microbiology data and expertise.
Impact One of Prof. Bazzolli's team has secured funding to undertake a year-long placement in my research group to support this collaboration.
Start Year 2015
 
Description Collaboration with Prof. Paul Langford at Imperial College London 
Organisation Imperial College London (ICL)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution My research team are conducting in vitro molecular interaction studies, and exploring the applicability of utilising our patented novel array technology, to explore cellular mechanisms within bacteria.
Collaborator Contribution Prof. Langford's team provide microbiology expertise and access to in vivo testing studies.
Impact A joint BBSRC grant was secured to support this collaborative research.
Start Year 2015
 
Description Collaboration with colleagues at Portsmouth University, Institute of Biomedical and Biomolecular Sciences 
Organisation University of Portsmouth
Department Institute of Biomedical and Biomolecular Sciences
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Research collaboration with Drs Pete Cary and Darren Gowers resulting in publications.
Start Year 2010
 
Title METHOD OF IMMOBILISING RNA ONTO A SURFACE 
Description A novel method to generate RNA arrays was developed. 
IP Reference WO/2012/156718, WO2012156718 
Protection Patent application published
Year Protection Granted 2012
Licensed No
Impact - Discussions, under NDA, are on-going with interested companies. - Academic collaborations are supporting the further development of the technology.
 
Description DSTL-DGA meetings and visits 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presented research at meetings hosted jointly by the Defence Science & Technology Laboratory (DSTL) and the Ministère de la Défense (DGA, France). Alignment of research agendas was clearly evidence and plans for collaborative working were highlighted.
Year(s) Of Engagement Activity 2014,2015
 
Description Discussion meeting and presentation at DSTL 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Industry/Business
Results and Impact Discussion meeting, including a presentation on my 'Research & Capabilities of Potential Interest to DSTL'.


The subsequent collaboration that resulted led to me securing a DSTL-funded PhD studentship.
Year(s) Of Engagement Activity 2012
 
Description Invited seminar on the Molecular Control of RNA Metabolism - Kent 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Invited seminar speaker at the University of Kent, April 2014.

Effective networking, building collaborative relationships.
Year(s) Of Engagement Activity 2014
 
Description Invited seminar on the Molecular Control of RNA Metabolism - Southampton 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Invited seminar speaker, University of Southampton, April 2014.

Effective networking, building collaborative relationships.
Year(s) Of Engagement Activity 2014
 
Description Invited seminar presentation on the Molecular Control of RNA Metabolism - Bath 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Invited seminar speaker at Bath University, April 2011.

Effective networking, building collaborative relationships.
Year(s) Of Engagement Activity 2011
 
Description Maintaining an Active Online Presence 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact My group has an active Twitter account with around 200 followers. We publish highlights from our research, outreach and engagement activities.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
 
Description Promoting PG study 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Presentation by members of my research team at various departmental events for undergraduates to promote engagement in postgraduate study. this involved the individuals highlighting their research work, including their day to day work, opportunities for collaboration and engagement as well as their outputs and impact.
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description Science Fairs 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Myself and my team have participated in supporting a number of Science Fairs in the region, engaging with attendees to promote science and the research we undertake.
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description University Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Myself and my team regularly support University Open days. Activities can be many and varied, including giving talks, presenting posters, running hands-on laboratory demonstrations and engaging in question and answer sessions. There are usually a number of these events per year, with over 100 participants (schools and college students, sometimes accompanied by a parent/guardian) attending each event. Feedback from such events has highlighted our success in inspiring the next generation of scientists and has been specifically linked to an increase in the number of students applying to study Biochemistry over the last few years.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013