MICA: Does phase variation contribute to virulence and immune evasion of Neisseria meningitidis?

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Many bacterial pathogens colonise and persist in our bodies as harmless 'commensals' that do not cause disease. These bacteria persist even though our bodies produce antibodies against exposed structures on their surface. In this proposal, we aim to improve our understanding of how bacteria avoid these antibodies and whether these avoidance mechanisms influence the ability of bacteria to cause disease.Neisseria meningitidis is a bacterium that is a major cause of meningitis and blood-poisoning but is found in 10-30% of people (i.e. 'carriers') as a harmless commensal of the upper respiratory tract. This bacterial species is present on nasal tissues and at the back of the throat where it can persist for up to twelve months despite the generation of antibodies against surface antigens of the bacteria. One way these bacteria avoid deleterious effects of antibodies is by changing the structure or amounts of these surface antigens. These changes are controlled by repetitive DNA tracts, which mutate at high frequencies resulting in alterations in the expression of genes that make these surface antigens. We have shown that persistence of N. meningitidis in the upper respiratory tract is facilitated by high frequencies of mutations in these repetitive DNA tracts causing reductions in expression of some of the surface antigens. As many of these surface antigens help the bacteria to invade tissues and to survive killing by cells and molecules present in blood, this finding has led us to speculate that reductions in expression of surface antigens, due to mutations in repetitive DNA, affects the ability of N. meningitidis to cause blood poisoning and meningitis. We will test this hypothesis by comparing the repetitive DNA tracts in bacterial isolates from patients and carriers. We will also test whether reductions in expression of specific antigens during persistent carriage of N. meningitidis is correlated with the presence of antibodies against that surface antigen.We will use samples from previous studies in volunteers from whom bacterial isolates, serum and saliva extracts were obtained at three or more time points over a six to twelve month time period. Similarly we will use pre-existing collections of N. meningitidis isolates from disease cases. The repetitive DNA tracts of multiple genes will be analysed by sizing of DNA fragments spanning the repetitive region so that we can count the numbers of repeats and then use these numbers to determine whether a gene is expressed. We will examine if a gene changes as the bacteria persist within a carrier and if the genes are in different states in the disease isolates as compared to those of carriers. The amounts of antibodies will be measured using a microassay. For this, protein products of genes will be purified, linked to fluorescent microspheres and incubated with serum. This will result in specific binding of antibodies to the proteins on the microspheres. Antibodies will then be detected with a different fluorescent tag and quantified in a fluorescence detection machine.We anticipate two major outcomes. Firstly, evidence of whether repetitive DNA produces a particular pattern of gene expression that enables N. meningitidis to cause disease. Identification of a 'virulence-associated' pattern would lead to production of novel preventive measures (i.e. vaccines containing these virulence determinants) or management of disease-cases (i.e. identification of sources of infections and rational decisions on when to provide preventive therapeutic treatment to contacts). Secondly, a demonstration that antibodies are driving reductions in expression of N. meningitidis surface antigens in carriers. This finding will indicate that vaccines containing surface antigens could reduce persistence of disease-causing N. meningitidis strains on mucosal surfaces of carriers and would be important for assessing whether Bexsero (the new MenB vaccine) can prevent spread of disease-causing strain.