Development of intergrative technology for gene inactivation in Clostridium difficile

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.

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.