Thesis: The Role of Endotoxin Phosphoforms in Sepsis and Meningitis caused by Neisseria meningitidis.
The main aim of the project is to use protein crystallography to determine how the endotoxin produced by the bacterial form of meningitis binds to two proteins found on the surface of immune cells. The endotoxin, also known as the lipid A group has varying phosphoform groups formed by different side chains being attached to the central lipid structure.
The two proteins that interact to bind to the endotoxin are known as Toll-like receptor-4 (TLR-4) and myloid differentiation factor protein 2 (MD-2). The binding of the endotoxin to MD-2 allows the two proteins to form a complex and dimerise, leading to a signaling cascade pathway being initiated.
To determine binding of the endotoxin, the TLR-4/MD-2 protein complex is to be co-expressed in a Baculovirus expression system, and then purified on a Nickel affinity column. Lipid groups with varying phosphoforms will then to be soaked with protein crystals before sending crystals to the Australian Synchrotron in Melbourne for screening and collection of xray diffraction data.
Why my research is important
Nisseria meningitidis (N. meningitidis) is the largest cause of bacterial meningitis and fatal septic shock on a worldwide scale causing major epidemics, particularly though Africa along the Meningitis Belt, a region in the sub-Sahara stretching from Ethiopia to Senegal (east to west) (1-3). More than 500,000 cases occur annually (4) with infection rates as high as 1 in 100 (5) and mortality rates of approximately 10%, or 50% if left untreated (6). This disease is therefore one of the top 10 causes of worldwide death (3). N. meningitidis is seen to be more prevelant among infants without immunity to the disease and young adults (1). The disease can be carried asymptomatically by 5-10% of healthy individuals in which the bacterial strains are non pathogenic (7, 8). N. meningitidis can cause two types of infection; meningococcal meningitis where bacteria are localized to the cerebral spinal fluid in the brain, and septic shock where bacteria are present in the bloodstream. In meningococcal meningitis the proliferation of bacteria is gradual compared to the rapid proliferation that causes septic shock. The exact mechanism of how N. meningitidis causes the two forms of disease is unknown, however susceptibility can depend on the immune complement system of individuals for protection against invasive disease particularly in septic shock (3). Understanding how the protein complex binds to the endotoxin produced by N. meningitidis allows for potential drug targets to be designed to significantly decrease the potency of the bacterium.
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