Solute provision to the chlamydial inclusion

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Chlamydiales comprise obligate intracellular pathogens and endosymbionts that possess a highly restricted metabolic capacity and thus essentially rely on the import of missing metabolites from the host cell. Interestingly, Chlamydiales reside in a special compartment of the host cell, the so called inclusion and accordingly are surrounded by an additional membrane. An important question that is not fully clarified yet is how the required metabolites enter the inclusion or particularly cross the inclusion membrane. It was shown that fusion of host cell derived vesicles delivers lipids to the inclusion membrane. This probably also leads to an unspecific provision of solutes but most likely not to a highly controlled supply of essentially required metabolites in accordance to the specific bacterial demands. In the proposed project we will establish a protocol for the enrichment of inclusion membranes. Determination of the inclusion membrane proteome will allow important insights into the protein composition and function of this organelle. Furthermore, we will analyze metabolite fluxes, like that of amino acids, nucleotides or cofactors after reconstitution of the membrane proteins into artificial lipid vesicles. Additionally, biochemical characterization of recombinant inclusion proteins and selected candidate carriers allows us to identify whether these proteins are involved in transport or directly catalyze metabolite trafficking across the inclusion membrane.