Bacterial toxin-antitoxin system functionality and bacteriophage abortive infection: structure function and biology

Lead Research Organisation: University of Cambridge
Department Name: Biochemistry

Abstract

Bacteria are attacked and killed by specific viruses called bacteriophages (phages). Phages are the most abundant biological entities on Earth and they outnumber their bacterial hosts by 10-1. Despite this superabundance of phages, the bacteria are still ubiquitous and this is because they have evolved remarkable systems to prevent the potentially lethal effects of viral infection. Some of these protective anti-viral systems are activated by the virus after infection in a process called abortive infection (Abi). There are over 20 known Abi systems which are thought to act at different stages in the viral infection and replication process. We discovered a new Abi system encoded by a plasmid in the bacterial plant pathogen, Erwinia - the causative agent of commercially significant potato soft rot disease. This new Abi system works via two components; one a protein (ToxN) that is toxic to the bacterial cell, and the other an RNA molecule (ToxI) that suppresses the toxin under uninfected conditions, but, when inactivated, leads to activation of the ToxN toxin, leading eventually to programmed death of the bacterial host cell. In this way, infected bacterial cells commit 'suicide' and an important net effect is that the virus is trapped and cannot be released to infect the other sibling members of the bacterial population. The group thus survives viral infection of some individual cells in a behavior akin to bacterial altruism. We have shown that this novel Abi system operates in different bacteria and in response to many different phages. We think that this Toxin-Antitoxin (TA) based system is likely to be important in controlling viral propagation in the environment, and for both bacterial and phage evolution. In this project we are trying to dissect the mechanism of action of the TA system. We will determine the structure of the toxin (ToxN) that can kill the bacteria and try to determine how the ToxI (RNA) molecule can suppress the function of the toxin. We will also study the relationship between the structure of the toxin and its biological function (in aborting phage infection and killing bacterial cells). We will investigate the nature of the cellular target(s) of the toxin. We will study mutant viruses that are able to get around the lethal effects of the ToxIN system and, by studying these mutants, we hope to be able to work out what components of the phage act to activate the ToxIN system. We will then try expressing the specific viral components artificially to see if we can switch on the antiviral system or altruistic suicide, to kill bacteria. Developing a deeper structural and mechanistic understanding of the interactions between phages, bacteria and the antiviral defence systems is important in our appreciation of evolution and adaptation of bacteria (particularly to viral attack). However, in addition, this research could lead eventually to the development of novel chemicals that have uses in antibacterial chemotherapy - new antibiotics.

Technical Summary

This project will investigate a new class of Toxin-Antitoxin (TA) system from the potato blackleg pathogen, Erwinia, that operates as a phage abortive infection (Abi) system. TA systems drive the host cell into prokaryotic apoptosis - in this case, post-viral infection - thereby limiting phage spread in bacterial populations. Although discovered in Erwinia, this new system (ToxIN) is functional in E. coli and other bacteria and against a wide spectrum of phages. The ToxIN module is comprised of a two gene operon: toxI, toxN with toxI encoding a regulatory RNA and toxN encoding the protein toxin. The toxicity of the ToxN protein to bacteria is suppressed by interaction with ToxI RNA and ToxN may has nuclease activity, although the precise cellular targets are not known. Neither do we know how infecting phages activate the system but we think it possible that phage products act directly (or indirectly) to allow the ToxN protein to be activated e.g. by displacing the ToxI RNA or by alternative routes. Predicted homologues of this new TA system from Erwinia have been identified in diverse bacterial genera. The main themes of this proposal are to solve the structure of the ToxN toxin of Erwinia and some of the homologues from other bacteria possessing similar systems. We intend to analyse how the function of the ToxN protein is affected by ToxI in a mutagenesis programme on both the the ToxN protein and cognate regulatory ToxI RNAs. We will also investigate the physiological effects on the host of artificial and 'natural' (phage-induced) ToxN activation, and identify cellular targets of ToxN activity. We will determine the molecular bases of viral escape from the ToxIN system and we will examine the cellular physiology impacts of ToxIN when specific phage products are artificially expressed.

Publications

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Blower TR (2011) A processed noncoding RNA regulates an altruistic bacterial antiviral system. in Nature structural & molecular biology

 
Description Although there are 10 times more bacterial viruses than bacteria on Earth, clearly the bacteria are still around! The reason for this is that bacteria have evolved anti-viral resistance strategies that can circumvent potentially lethal viral infection.

This research investigated how a bacterial plant pathogen (Pectobacterium), and other bacteria that infect insects (Bacillus) avoid lethal infection by their own viral predators. The bacteria make a complex called ToxIN. This is comprised of a toxic protein (ToxN) that can kill the bacteria carrying it, but it's toxicity is suppressed by an anti-toxin (ToxI) and this is an RNA molecule that takes up a special shape called a pseudoknot. The ToxI and ToxN fit together like a lock and key such that the ToxIN complex is not lethal to the bacteria that carry it.

However, this is like a time bomb that can be activated, causing bacterial suicide. Some bacterial viruses, on infection of bacteria carrying the ToxIN complex, destabilise the ToxIN complex and this leads to release of the ToxN toxin that kills the bacterium. Because bacterial viruses can only reproduce inside living bacteria, if the bacterial cell commits suicide soon after viral infection (through ToxN activation) then the virus will not be able to replicate and go on to produce progeny that could then go on to infect and kill other bacteria in the bacterial population. Consequently, this bacterial behaviour can be viewed as an altruistic suicide.

In this research we have determined the molecular structure of the ToxIN complex (protein toxin and RNA antitoxin together) for both bacterial pathogens and they show many similarities, even though the bacteria involved are very different.

We showed that the ToxI:ToxN pairs are extremely specific because the Pectobacterium and Bacillus versions cannot be mixed-and-matched to make functional heterologous ToxIN pairs. We isolated some new bacterial viruses that infect the plant pathogen, Pectobacterium, from the natural environment and we studied two of these viruses in detail. Both of these viruses could activate the ToxIN system on infection of Pectobacterium, causing the induction of suicide in the infected bacteria. However, we saw the evolution of very rare virus mutants by spontaneous mutation that allowed the virus to escape the induction of the ToxIN system so that they were able to replicate unhindered by altruistic suicide. The escape mutants of the two viruses evolved totally different strategies. In one, the virus made a defective protein that then failed to induce the ToxIN system. In another virus, it evolved the ability to make its own versions of ToxI - essentially mimicking the normal ToxI antitoxin of the bacterial host. In effect, the virus evolved a way of continuing to suppress the ToxN of the infected cell such that it kept the bacterial host alive to enable viral replication. In this way the virus suppressed altruistic suicide in the bacterium and thereby ensured its own survival. Study of such curious bacterial and viral systems might inform us about novel targets for potential development of new antibiotics.
Exploitation Route This work has implications for the study of the phenomenon of persistence with significant impacts in antibiotic resistance. The ToxIN system has capabilities for the development of novel gene cloning and selection systems. The work also has implications for the practical application of phage therapy. It also has ramifications in viral ecology and viral evolution.
The emergence of viral escape mutants in our system provided an insight into the molecular mechanisms of how bacterial viruses evolve to circumvent the lethal impacts of TA systems operating through altruistic suicide. In particular, the discovery of an RNA based molecular mimicry system in the virus is an exciting development. This discovery, coupled with our data on alternative modes of evolution in the bacterial virus has revealed the remarkable plasticity of co-evolution between bacteria and their viral parasites.
This work has potential applications in microbial ecology, drug resistance, persistence, vector development and phage therapy.
Sectors Agriculture, Food and Drink,Education,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.bbsrc.ac.uk/news/fundamental-bioscience/2012/121109-n-viruses-evolve-bacterial-hosts-suicide.aspx
 
Description We had considered non-academic uses of the Type III TA system. But our experience suggests there may be little industrial interest. We previously lodged a patent application on the Erwinia (and related) Type III TA system because of potential applications in cloning and genetic engineering, anti-phage strategies for industrial fermentations and for potential therapeutics. This patent was allowed to lapse after a year, in the absence of industrial interest.
First Year Of Impact 2014
Sector Agriculture, Food and Drink,Education,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic,Policy & public services

 
Description Scottish Science Advisory Council
Geographic Reach National 
Policy Influence Type Citation in other policy documents
 
Description Cambridge International Student Scholarship (CISS)
Amount £109,947 (GBP)
Organisation University of Cambridge 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 09/2017
 
Title Generalised transducing phages for Dickeya species 
Description Isolation of transducing phages that infect Dickeya solani, the potato pathogen. 
Type Of Material Biological samples 
Year Produced 2015 
Provided To Others? Yes  
Impact This allows the engineering of the plant pathogen by phage-medicated strain construction 
 
Title Pseudomonas aeruginosa generalised transducing phage phiPA3 
Description An environmental phage isolated and genomically characterised (full genome sequence). This phage is a generalised transducer with wide host range among environmental and clinical isolates of Pseudomonas aeruginosa. The phage allows horizontal gene transfer for strain construction and analysis. 
Type Of Material Biological samples 
Year Produced 2011 
Provided To Others? Yes  
Impact bacterial strain engineering 
 
Title Transducing phage for Serratia strains from the rhizosphere and clinical isolates 
Description Isolation of the phage phiMAM1 that is a generalised transducer for environmental and clinical strains of Serratia and Kluyvera. This allows strain engineering. 
Type Of Material Biological samples 
Year Produced 2015 
Provided To Others? Yes  
Impact The phage has a wide host range (in terms of susceptible strains) and enables strain engineering. This has been useful to study virulence of some Serratia strains and the production of secondary metabolites (including antibiotics) 
 
Title Genome sequence of a transducing phage that infects biocontrol Serratia strain that makes antibiotics 
Description ncbi deposit of viral genome sequence 
Type Of Material Database/Collection of data 
Year Produced 2012 
Provided To Others? Yes  
Impact The phage genome has proved useful in the study of viral evolution and host range 
 
Title Genome sequence of gas vesicle-producing Serratia strain 
Description deposited in the ncbi database 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact This genome has been a resource for multiple projects in different labs. Scientific themes include secondary metabolite gene clusters (prodigiosin and a carbapenem antibiotic) plus the gas vesicle-producing cluster. 
 
Description Structural biology of bacterial Type III toxin-antitoxin systems 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution The postdoctoral researcher and graduate students provided the expressed proteins for crystallographic analyses in the project. They were actively involved also in the crystallographic analyses to help determine structures of several Type III toxin-antitoxin systems
Collaborator Contribution Provision of a expertise and technical know-how in structural biology; assistance in analysis of data and interpretation of outcomes; assistance with biochemical purification and crystallographic tools. A strong collaborative approach.
Impact The collaboration has produced multiple papers on the structures of several Type III bacterial toxin-antitoxin systems and structure-function relationships plus comparative interpretations of the roles of pseudoknot RNA in quaternary structural assembly, maintenance, stability and endonucleolytic catalysis. These outputs are reported in the relevant sections of the return.
Start Year 2010
 
Description A processed non-coding RNA regulates a dual abortive infection and toxin-antitoxin system 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited Talk at the meeting on Molecular Genetics of bacteria and phages, Cold Spring Harbor Meeting, Madison, Wi, USA, 2011.



The overabundance of bacteriophages on Earth drives the adaptive evolution of phage-resistance mechanisms within host bacteria. Abortive infection, during which an infected bacterial cell commits altruistic suicide to protect the clonal population, can be mediated by the toxin-antitoxin pair, ToxIN. This system was found upon a cryptic plasmid of Pectobacterium atrosepticum, and is a homologue of AbiQ from Lactococcus lactis. We tested suites of phages and showed that the ToxIN system could very effectively abort infections by multiple phages, within Pectobacterium, Escherichia and Serratia host backgrounds. It currently remains unclear what links the ToxIN-susceptible phages, but through phage genomics and sequencing spontaneous phage mutants that are resistant to ToxIN, we hope to identify factors involved in this lethal process.

ToxIN, though an abortive infection system, is also the defining member of the new Type III toxin-antitoxin family, in which antitoxic RNA, ToxI, directly inhibits the activity of the toxic protein, ToxN. To better understand the interaction between these two components, we solved the structure of the ToxIN complex, determined to 2.75 Å by X-ray crystallography (Nat Struct Mol Biol 18:185-190 (2011)). The striking triangular complex shows ToxI RNA forming pseudoknots, binding to ToxN protein and caging the toxic protein prior to activation, in a heterohexameric assembly formed solely through protein-RNA interfaces. ToxN itself was shown to be an endoribonuclease, processing the antitoxic ToxI non-coding RNA. This structure suggests multiple routes by which a phage can induce abortive infection and illuminates how infected cells commit suicide and destroy the pathogen.

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description A processed non-coding RNA regulates an altruistic bacterial antiviral system 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited talk at the American Society for Microbiology Conference on RNA, Puerto Rico, March 2011.



Invitation to George Salmond, who was unable to attend. Lecture given by Tim Blower (BBSRC-funded, PDRA)

no actual impacts realised to date
Year(s) Of Engagement Activity 2014
 
Description A processed non-coding RNA regulates an altruistic bacterial antiviral system 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach regional
Primary Audience Participants in your research or patient groups
Results and Impact Invited talk at the Cambridge RNA Club 2010.

no actual impacts realised to date
Year(s) Of Engagement Activity 2010
 
Description American Society of Microbiology Annual Conference, San Francisco, Invited Plenary 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk to a very large audience on our work on bacterial toxin-antitoxin systems and the role of the Type III system in aborting viral infection A 20 minute talk with an open Q&A session
Year(s) Of Engagement Activity 2012
 
Description Bacteriophage 2015. Chair and Talk, O2, London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Chaired a 1 day session and gave introductory talk and summing up. Attended other talks and poster sessions, judged posters of graduate students, Q&A session and networking.
Year(s) Of Engagement Activity 2015
 
Description Chair and invited talk at Euroscicon meeting on Bacteriophages in Bioscience, Biotechnology and Medicine, O2, London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Chaired sessions, gave introductory talk and summing up. Other talks, posters and poster judging, networking sessions. A mix of practicing scientists, industrial representatives and company representatives concerned about domestic and EU policy regarding policy affecting phage therapy applications
Year(s) Of Engagement Activity 2014
 
Description Europhages 2012 Conference, Oxford, Invited talk and Chair 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Chaired the meeting on the diverse nature and applications of bacterial viruses. Gave a talk and had a Q&A session plus posters plus networking. There were company representatives and EU policy representatives present.
Year(s) Of Engagement Activity 2012
 
Description Harden Conference on RNA Biology, invited talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk on Type III toxin-antitoxin systems in bacteria and the role of pseudoknot RNA in the structure and evolution of the antiviral complex. Talk plus Q&A session plus posters and networking
Year(s) Of Engagement Activity 2012
 
Description Invited lectures at several UK universities 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Invited lectures at various universities in 2010 and 2011: Leicester, York, Aberdeen, Warwick & Sheffield

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Invited talk at The Holyrood Conference "New Frontiers in Infection Control", Surgeons Hall, Edinburgh 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The conference involved diverse members including scientists, medical practitioners, nurses, infection control experts, policymakers. I gave a talk on phages and phage therapy prospects and attended other talks plus Q&A sessions.
Year(s) Of Engagement Activity 2014
 
Description Invited talk at the Society for General Microbiology Meeting, Nottingham, 2010 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited talk at the Society for General Microbiology. Meeting on "New Insights into Secondary Metabolism", University of Nottingham, UK, 2010

no actual impacts realised to date
Year(s) Of Engagement Activity 2010
 
Description Invited talk at the Society for General Microbiology meeting on "Impact of Bacteriophages", Sussex University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Gave an invited talk on bacteriophage abortive infection through altruistic suicide. Multiple talks were presented, plus posters and networking with many microbiologists working on very diverse topics.
Year(s) Of Engagement Activity 2013
 
Description Invited talk. Society for General Microbiology meeting on RNA and riboswitches in bacterial regulation., Liverpool. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave a talk to a diverse audience, followed by Q&A session, plus other talks, posters and networking.
Year(s) Of Engagement Activity 2014
 
Description Keynote Lecture: Meeting on Phage therapy for control of plant and tree diseases, Reading 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave the Keynote talk and contributed to other presentations. Although a small meeting it was attended by diverse contributors from academic and industrial backgrounds.
Year(s) Of Engagement Activity 2014
 
Description Plenary Lecture at the 20th Biennial Evergreen International Phage Meeting, Olympia, Washington State, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave the invited Plenary Talk followed by a Q&A session, poster sessions and networking. This international meeting is held every two years.
Year(s) Of Engagement Activity 2013
 
Description Talk at Cold Spring Harbor Meeting, Madision, Wi, USA, 2011 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Talk at Cold Spring Harbor Meeting on Molecular Genetics of Bacteria and Phages, Madison, Wisconsin, USA, 2011.

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Talk at the ASM Conference, Miami, USA, 2011. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach international
Primary Audience Participants in your research or patient groups
Results and Impact Talk at the American Society for Microbiology Conference on Bacterial Cell-Cell Communication, Miami, Florida, USA, 2011

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description The Bacteriophage 2016, O2, London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave a talk on anti-viral abortive infection systems in bacteria, followed by Q&A, posters and networking.
Year(s) Of Engagement Activity 2016