Since it has no animal or environmental reservoir, M. tuberculosis (MTB) must be aerosolized by diseased individuals in order to propagate. The organism accomplishes this most effectively by forming pulmonary cavities. One of us (PMS) has recently observed genetic deficiencies in MTB strains with reduced capacity to cause pulmonary cavities in humans. Another of us (RH) has developed a mouse model that suggests the toxicity of cord factor (trehalose 6,6"""""""" dimycolate (TDM)) contributes to the formation of cavities. Together, we now propose to identify and characterize MTB genes that control the formation of pulmonary cavities. We will use: 1) two different DNA microarrays to determine, on a genome wide scale, the presence and expression profile of all MTB genes; 2) a new mouse model that manifests the type of caseating granuloma that gives rise to cavities; and 3) conventional genetic approaches for gene knock-outs and complementation. We will build a matrix database of well characterized clinical isolates that records clinical cavity formation, results of assays of toxic lipids and whole genome DNA microarray identification of gene deletion and expression. Analysis of this database will generate specific hypotheses as to which mycobacterial genes are associated with the production of toxic lipids and/or are responsible for pulmonary cavities. These hypotheses will be tested using conventional genetic approaches and by further application of our mouse model. Specifically, we will use three existing NIH funded collections to identify 30 mycobacterial clones that do and 30 that do not cause cavitation. We will grow these isolates as pellicles and assay the amount, distribution, structure and toxicity of TDM. Next, we will use a Bayesian statistical approach to identify genes whose presence or expression is associated with loss of cavity production in humans and/or with the production of toxic TDM. In parallel, we will validate the relevance of our animal model using strains that do or do not produce cavities in humans. Finally, we will use conventional genetic approaches to knock out or insert genes associated with cavity formation and will test the capacity of these modified strains to produce TDM (or other relevant parameters) and induce cavities in our mouse model. If successful, new insights into the pathogenesis of pulmonary cavities may lead to specific interventions to prevent this highly infectious disease manifestation.
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