Many pathogens adopt an intravesicular lifestyle, but for most a detailed molecular description of their habitat and status once inside a host cell is lacking. A molecular understanding however would greatly advance fundamental knowledge of this lifestyle, allow directed functional characterization and facilitate the identification of novel targets for strategies of diagnosis and/or intervention. In many cases, our lack of knowledge is due to the currently inadequate technologies to purify infected compart ments to enable global, high resolution analysis e.g. by proteomics. The objective of this project is to:I) Overcome this technical limitation by developing a a novel, coloured approach to purify pathogen habitats based on fluorescence activated sorting. I I) Use this technology to characterize the habitat of the human pathogen Leishmania donovani to decipher the principles of its intracellular life. III) Apply this knowledge to develop an affordable live vaccine for clinical testing against the etiologic ag ent of visceral leishmaniasis that causes thousands of deaths each year.Transgenic mice will be generated that exhibit a green fluorescence-tagged endocytic pathway. The technology has the potential for a broad application in cell biology in general. The t ransgenic mice will provide primary host cells for infection with diverse, red fluorescence-coded pathogens. Here, these will be dendritic cells infected with red Leishmania donovani for characterization of the habitat and parasite molecular composition by proteomics. Parasite-derived molecules identified by this novel approach will be classified based on their topology and proteins secreted or surface exposed will be selected as candidate molecules for vaccine development. A live vaccine based on recombina nt Salmonella enterica serovar Typhi strain Ty21a (a licensed typhoid fever vaccine; Typhoral) expresssing these molecules will be constructed for testing in clinical studies.
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