How is V. cholerae lifestyle switching controlled?

Vibrio cholerae is a bacterium. It is the biological agent that causes Cholera, a severe form of diarrhoea. Worldwide, approximately 4 million people are infected by V. cholerae every year. This results in hundreds of thousands of fatalities. Many cases are attributable to an ongoing global pandemic, the 7th to sweep our planet in recorded history. However, disease is also associated with local epidemics; the upsurge of Cholera cases in Haiti, following the 2010 earthquake, being of note. The annual financial cost associated with V. cholerae infections exceeds $3 billion annually. Hence, there is significant commercial interest in solving the "Cholera problem". At present, epidemics may be controlled by vaccinating the population in affected areas. However, currently available Cholera vaccines are poorly effective. Consequently, industrial operations worldwide are identifying strategies to develop better vaccines. The growing problem of resistance to antimicrobial agents is also fueling this trend.

The ability of V. cholerae to persist in the environment, and cause disease, is dependent on exploitation of two different environmental niches. Principally, V. cholerae is an aquatic organism that colonises the surface of crustaceans. In this situation, the bacterium's primary source of nutrition is chitin; a component of the shellfish exoskeleton. Thus, V. cholerae forms biofilms on such surfaces. Conversely, when V. cholerae is ingested by fish, or humans, the bacterium's lifestyle is "reprogrammed" so that it can colonise the host. This is beneficial to the bacterium since it can multiply rapidly in hosts before being shed back into the environment. In humans this process is associated with the disease known as Cholera. Understanding how V. cholerae adapts to its different environments is therefore of great interest.

In recent work we have shown how applying the next generation of experimental tools can rapidly progress our understanding of bacterial pathogens. We propose to apply such tools to understand lifestyle switching in V. cholerae. Ultimately, by picking apart the control of lifestyle switching, and redesigning V. cholerae to regulate this process differently, we hope to stimulate a new era of vaccine design. We expect that this approach will be applicable to many diseases caused by bacteria. We have identified UK based biopharma likely to benefit from these advances and will liaise closely with them throughout the project. The purpose of this interaction is to maximise the impact of our work and to exploit opportunities that arise for commercialisation.

In summary, we will study a globally important pathogen and develop strategies applicable to the control of all bacteria. Importantly, this application comes at a time when alternatives to antibiotics are being sought and the window of opportunity to act is open. We have already fostered links with important "end-users" of our data to maximise the economic impact of our work.

Understanding bacterial lifestyle switching offers opportunities to disrupt problematic environmental niches and improve vaccine design. We propose to couple genomic, environmental and synthetic biology to reinvigorate interest in such therapeutic strategies. We will work with the bacterium Vibrio cholerae, a common inhabitant of aquatic environments that can i) form biofilms on crustaceans ii) colonise fish and iii) cause severe disease in humans. The switch between different V. cholerae lifestyles is controlled by changes in quorum sensing and the production of pathogenicity factors. We seek to understand, modify and exploit this regulatory process.

The V. cholerae El Tor biotype, with which we will work, is responsible for the ongoing global Cholera pandemic. For many years this organism has been difficult to study; genomic and biochemical tools have been lacking. However, this situation has changed. First, approaches such as Chromatin Immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) can quickly map gene regulatory systems in V. cholerae. Second, an in vitro transcription system, which we have developed, can be used to monitor V. cholerae gene regulation. Third, a technique called MuGENT, developed by Andrew Camilli's lab, can be used to rapidly redesign V. cholerae genomes.

In preliminary work we have exploited technological advances to identify a transcription factor that couples the control of V. cholerae lifestyle and pathogenicity. We propose to build on this initial observation to identify i) when key quorum sensing and pathogenicity genes are turned on and off ii) how the transcription factor exerts its effect at the molecular level iii) modifications that can be made to the V. cholerae genome to control lifestyle.

The end users that will benefit from this research are:

1. Prokarium LTD (a UK based company set up to produce bacterial vaccines) and UK biopharma in general.
2. World Heath Organisation (WHO).
3. The UK general public.
4. Academia (Biologists, Medics and bioinformaticians internationally).

We have expanded on how these different groups will benefit below.

THE PRIVATE SECTOR (PROKARIUM LTD, UK BIOPHARMA, Immediate Impact): Numerous private initiatives are actively funding research into developing new vaccines for bacterial disease. There is a particular focus on generating vaccines for common enteric diseases given the rapid rise in the number of bacteria that are resistant to antimicrobials. This marketplace currently includes TDVaccines (Denmark), Sanofi Pasteur (France), PaxVax (USA) and Prokarium LTD (UK). The Institute of Microbiology and Infection (IMI) has existing links with Prokarium's vaccine development programme via a joint BBSRC/TSB grant. Hence, there is clear potential for the proposed project to "add value" to the existing collaboration and vice-versa. The particular benefit to Prokarium LTD will be the increased economic competitiveness that will result from i) better understanding pathogenic bacteria and ii) knowing how to exploit synthetic biology approaches to generate better vaccines. In broader terms, bacteria can be redesigned for many industrial applications. For example, strains may be generated that over-express proteins more efficiently, grow better at defined temperatures or grow faster in a defined media. This will be of general interest to UK Biopharma.

POLICY-MAKERS AND GOVERNMENT AGENCIES (WHO; Immediate to medium term impact): The WHO is a global heath advisory group that disseminates information to the public and governments worldwide. The WHO will benefit from i) better understanding V. cholerae ecology and persistence in the environment and ii) the identification of new strategies for developing vaccines for bacterial disease. This will in turn benefit policy makers and government agencies globally who rely on advice from the WHO.

THE WIDER PUBLIC (SCHOOLS, STUDENTS; Immediate to long term impact): Our work will raise general awareness of bacterial disease and intervention stratergies that can be used. We will also use these interactions with schools and students to raise the issue of antimicrobial resistance and the need for alternative tools to control bacteria.

ACADEMIA: Academics will benefit from i) new approaches to study bacterial systems ii) the availability of genomic datasets that can be mined in order to understand other bacterial systems.