Mycobacterial determinants of survival and fitness within the bovine host

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Bovine tuberculosis (BTB) is a persistent problem in certain areas of the UK. The current control strategy is to test herds for the presence of BTB at timely intervals and slaughter test-positive animals. However, the observation that BTB is spreading in both geographical area and prevalence suggests that the existing control strategy is not working effectively. The cost of the control programme is £100 million annually, therefore not only is the policy ineffective, it is also a significant economic burden. In addition, BTB can cause tuberculosis in humans, and while this is currently rare, the escalating prevalence of BTB has the potential to develop into a zoonotic (the passage of disease from animals to humans) risk.BTB is caused by a bacterium called Mycobacterium bovis. This is very closely related to Mycobacterium tuberculosis, a bacterium that commonly causes tuberculosis. Tuberculosis in cattle and humans show great similarities and, like the human disease, vaccination is an alternative strategy to control BTB. The vaccine against human tuberculosis is a strain of Mycobacterium bovis called Mycobacterium bovis BCG, however this provides limited protection against the disease in both cattle and humans. Mycobacterium bovis is transmitted by aerosol, once within the animal it enters into cells called macrophages. These cells, normally dedicated to the killing and removal of bacterial pathogens, are unable to kill the bacteria. Instead, Mycobacterium bovis adapts and survives within the lungs in structures called granulomas. However, we do not know much about the genes that allow the bacteria to survive and cause disease in the host. We will knock-out the function of every single gene in the genome of Mycobacterium bovis (approximately 4000) and determine those genes that are required for survival in bovine macrophages and in the whole cow. We will achieve this by using a technique called Transposon Directed Insertion Seqeuncing (TraDIS). This technique has been applied successfully to the study of other bacterial pathogens of medical and veterinary importance, but this will be the first time it has been used to study the genetic determinants of disease in Mycobacterium bovis. We will make libraries of mutants where the function of every gene in the genome has been knocked-out. We will then put the libraries into "screens". Mutants that are recovered after being through a "screen" are compared to the original pre-screened mutant pool by DNA sequencing. Mutants that are unable to survive the screen are identified and as a result we will identify all the genes necessary for survival in the host. Those mutants that are unable to survive in the host represent potential vaccine candidates. We will be able to assess the function of essential genes using a combination of computational analysis, literature searching and comparisons to screens performed on different types of bacteriological media (in vitro). The functions that are essential for survival in the host also reflects the conditions within the host. At the end of this project we will have filled in key gaps in the knowledge of BTB in the area of host pathogen interactions and have identified several potential vaccine candidates to be considered for future development.