Development of intergrative technology for gene inactivation in Clostridium difficile

Lead Research Organisation: University of Nottingham
Department Name: Inst of Infections and Immunity

Abstract

There are currently heightened public concerns over infection rates in UK hospitals, and in particular those caused by so called superbugs that have become resistant to available antibiotics. The bacterium Clostridium difficile is a highly resistant bug that is a major cause of infections in hospitalised patients. It causes debilitating diarrhoea, which in extreme cases can cause death. Currently, only two antibiotics are available for treating the disease, metranidizole and vancomycin. The disease mainly affects old people. Thus 80 per cent of cases occur in the people who are over the age of 65. It follows, that as the proportion of the UK population that is over 65 is increasing, the disease is becoming more common. Aside from the human suffering it causes, outbreaks of C. difficile cost the National Health Service considerable sums of money. This is mainly because infected patients need to stay in hospital for extended periods of time. With 28,819 cases in the UK last year, it can be estimated to be costing the NHS over 115 million pounds per year. Moreover, there is a danger that the situation could escalate out of control should strains resistant to metranidizole and vancomycin emerge. More effective ways of preventing the disease are required. To control infections, it is crucial that medical science understands how an organism causes disease. Under Wellcome Trust sponsorship, the complete genome sequence of the organism (i.e. its genetic blueprint) has now been determined at the Sanger Institute, Cambridge. However, whilst we now know the precise sequences of every gene in the C. difficile chromosome, in the majority of cases we do not understand their precise function. The most effective method of working out what individual genes do, is to mutate them (make them non-functional) and assess the consequences. Such an approach is not currently possible with this bacterium because the genetic tools necessary to bring about the specific mutation of target genes are not available to the research scientist. The development of these mutational tools is the overall goal of this proposal. Their availability should allow the identification of those genes which are required for infection, which should eventually lead to more effective ways of controlling the disease.

Technical Summary

Despite its impact on public health, Clostridium difficile remains poorly characterised with regard to pathogenesis, gene regulation, cellular metabolism, and sporulation/germination. The genome sequence of C. difficile CD630 has now been completed. In other bacteria such a wealth of data may be combined with functional genomic approaches to better understand the organisms biology. Pivotally, the generation of mutants is employed to ascribe function to individual genes, and gene sets. However, there are currently no effective integration vectors for mutational studies in C. difficile. Indeed, there is a lack of such systems in the genus as a whole. The body of evidence amassed to date suggest that whilst recombination in clostridia is possible, it is a relatively inefficient process. It follows that in order to detect such rare events the DNA to be integrated needs to be introduced at high frequencies. Thus, the species in which gene integration is most easily achieved (C. perfringens) has the highest transformation frequency. In C. acetobutylicum the relatively lower frequencies obtained are compensated for by using 1-2 mg of DNA per transformation. The frequencies of transfer in C. difficile are relatively poor (between 10 to the power of 6 and 10 to the power of 7 transconjugants per donor cell), and most likely provide a rational explanation as to why homologous recombination is difficult to demonstrate. The most obvious solutions to this bottleneck are to either (1) increase the frequency of DNA transfer in C. difficile, thereby increasing the likelihood of the detection of rare recombination events, or (2) integrate the DNA by a process which is largely independent of host recombination factors. As the ultimate goal would be to transform every cell within the population, this can be most easily achieved through the use of a vector that is conditional for replication. We have developed a fac promoter (the C. pasteurianum ferredoxin promoter derivatised to include a lac operator, lacO), which in conjunction with lacI, is ITPG-inducible in C. beijerinckii. This system does not function in C. difficile. It does, however, provide the opportunity to undertake conditional vector, proof of principle studies in C. beijerinckii, while an equivalent system is developed for C. difficile. This will allow a more rational selection of the route to be taken in C. difficile once an inducible promoter is available for this organism. Our strategy will therefore be to explore various means of using a lac0-based system to impose IPTG-mediated control of plasmid maintenance in C. beijerinckii, while at the same time developing an equivalent inducible promoter system for use in C. difficile. A number of different promoter systems and strategies will be evaluated, including inducible and temperature sensitive control of plasmid maintenance (replicative ability and integrity). In parallel, we will test the utility of non-host based systems for mediating gene inactivation. Our priority will be to evaluate the use of the L1.LtrB Group II intron. Dependent on progress, we may also test the utility of lambda Red. The difficulties of undertaking gene inactivation in clostridia, and in particular C. difficile cannot be overstated. There is, therefore, a pressing need to develop such technology, particularly if the opportunities presented by genome sequence data are to be fully exploited. The project will therefore focus exclusively on this goal. If successful at an early stage, the technology will be exploited to analyse a putative homologue discovered in the genome of the Staphylococcus aureus Agr quorum sensing system.

Publications

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Cartman ST (2010) The emergence of 'hypervirulence' in Clostridium difficile. in International journal of medical microbiology : IJMM

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Heap JT (2010) The ClosTron: Mutagenesis in Clostridium refined and streamlined. in Journal of microbiological methods

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Heap JT (2007) The ClosTron: a universal gene knock-out system for the genus Clostridium. in Journal of microbiological methods

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Heap JT (2009) A modular system for Clostridium shuttle plasmids. in Journal of microbiological methods

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Heap JT (2010) ClosTron-targeted mutagenesis. in Methods in molecular biology (Clifton, N.J.)

 
Description This project set out to develop reproducible methods for making directed mutants of Clostridium difficile, a prerequisite for the assignment of function to the various encoded products and eventually rational formulation of more effective countermeasures. This objective was entirely met through:-

[1] the formulation and exploitation of a novel method for the insertion of group II intron-encoding DNA into the genome at specifically targeted sites within coding regions. The 'ClosTron' (www.clostron.com) has been deployed at Nottingham and in laboratories around the globe to create over 150 clostridial mutants to date (2009). Its significance cannot be overstated. It represents a step-change in clostridial molecular biology.

[2] The project contributed (along with BB/D522797/1) to the development of a standardized modular (18 components) vector system (the pMTL80000 series) for Clostridium, to facilitate the rapid assembly of plasmids tailored to need. We propose that this system is adopted as a standard for vector development by the clostridial scientific community. To aid this task we have established an online facility at http://www.clostron.com/pMTL80000.php where any of the 400 plasmid variants may be assembled in silico and the predicted plasmid sequence downloaded as a '*.gb' file.
Exploitation Route Our ClosTron mutagenesis method is universally applicable, not only to Clostridium difficile, but potentially to any Clostridium species, and indeed any bacteria in which the components function. It can be used to identify intervention targets in the pathogens (C. difficile, C. perfringens, C. tetanus and C. botulinum), as well as in those benign clostridial species useful in the production of chemicals and fuels, or indeed in those species being pursued as tumour delivery vehicles for cancer therapy. The ClosTron has become the most widely used mutagen in the clostridial scientific community.
Sectors Aerospace, Defence and Marine,Agriculture, Food and Drink,Energy,Healthcare,Pharmaceuticals and Medical Biotechnology,Other

URL http://www.clostron.com
 
Description There are currently heightened public concerns over infection rates in UK hospitals, and in particular those caused by so called superbugs that have become resistant to available antibiotics. The bacterium Clostridium difficile is a highly resistant bug that is a major cause of infections in hospitalised patients. It causes debilitating diarrhoea, which in extreme cases can cause death. Currently, only two antibiotics are available for treating the disease, metranidizole and vancomycin. The disease mainly affects old people. Thus 80 per cent of cases occur in the people who are over the age of 65. It follows, that as the proportion of the UK population that is over 65 is increasing, the disease is becoming more common. C. difficile-associated deaths in England and Wales in 2008 were 5391, meaning that it caused almost four times as many deaths as MRSA. Aside from the human suffering it causes, outbreaks of C. difficile cost the National Health Service considerable sums of money. This is mainly because infected patients need to stay in hospital for extended periods of time. Its financial impact on the healthcare system has recently been estimated to cost at least 2 billion dollars in Europe and 3.2 billion dollars per year in the US. Moreover, there is a danger that the situation could escalate out of control should strains resistant to metranidizole and vancomycin emerge. More effective ways of preventing the disease are required. To control infections, it is crucial that medical science understands how an organism causes disease. Under Wellcome Trust sponsorship, the complete genome sequence of the organism (i.e. its genetic blueprint) has now been determined at the Sanger Institute, Cambridge. However, whilst we now know the precise sequences of every gene in the C. difficile chromosome, in the majority of cases we do not understand their precise function. The most effective method of working out what individual genes do, is to mutate them (make them non-functional) and assess the consequences. Such an approach was not possible with this bacterium at the inception of this project, because the genetic tools necessary to bring about the specific mutation of target genes were not available to the research scientist. The development of these mutational tools was the overall goal of this proposal. Our overall goal was achieved, and a novel tool, the ClosTron, was successfully created which can be used to mutate any gene within the C. difficile genome. The mechanism by which the ClosTron mutates genes is complex. In essence, by making very small, specific changes to the DNA sequence of the ClosTron, the element is able to jump from the plasmid delivery vehicle introduced into the Clostridium cell, and insert itself into the target gene, causing its inactivation. The nature of the small changes made make this targeting process very specific, such that the ClosTron only inserts itself into the intended gene. The process has proven to be highly effective and reproducible. Thus, at the beginning of the project just 3 genes in Clostridium difficile had been mutated. By the projects end, between our laboratory and those of collaborators, over 40 genes have been inactivated. The availability of the ClosTron will now allow the identification of those genes which are required for infection, which should eventually lead to more effective ways of controlling the disease.
First Year Of Impact 2007
Sector Agriculture, Food and Drink,Chemicals,Energy,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Astellas: Analysis of the Effects of Fidaxomicin on Spores
Amount £217,000 (GBP)
Funding ID CDS-FDX-2012-MINTON-study 
Organisation Astellas Pharma 
Sector Private
Country Japan
Start 02/2012 
End 12/2014
 
Description BBSRC Responsive Mode
Amount £643,519 (GBP)
Funding ID BB/E021271/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2007 
End 03/2011
 
Description ERANET SysMO1
Amount £364,436 (GBP)
Funding ID BB/F003390/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2007 
End 06/2010
 
Description FP7 Collaborative Project (Theme HEALTH) HYPERDIFF (3m euros across 7 participants)
Amount £350,513 (GBP)
Funding ID 223585 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 11/2008 
End 10/2011
 
Description HORIZON2020 Marie Curie ETN, CLOSPORE
Amount € 3,946,605 (EUR)
Funding ID 642068 
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 03/2015 
End 02/2018
 
Description MRC Grant
Amount £1,590,252 (GBP)
Funding ID G0601176 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2007 
End 03/2013
 
Description MRC Pilot Industry Collaboration Award (PICA)
Amount £139,997 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 12/2009 
End 05/2011
 
Description Marie Curie Initial Training Network (ITN)
Amount £4,111,621 (GBP)
Funding ID 215697-2 
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start 08/2009 
End 08/2013
 
Description Research Contract (Summit Plc)
Amount £49,206 (GBP)
Organisation Summit Plc 
Sector Private
Country United Kingdom
Start 10/2013 
End 12/2014
 
Description SNSF Sinergia Programme (In vivo germination of Clostridium difficile endospores)
Amount £207,000 (GBP)
Funding ID DECISION CRSII3_147603/1 
Organisation Swiss National Science Foundation 
Sector Public
Country Switzerland
Start 10/2013 
End 09/2016