Solute provision to the chlamydial inclusion

衣原菌包括专性细胞内病原体和内共生体，它们具有高度受限的代谢能力，因此基本上依赖于从宿主细胞输入缺失的代谢物。有趣的是，衣原体存在于宿主细胞的一个特殊隔间中，即所谓的包涵体，因此被一层额外的膜包围。一个尚未完全澄清的重要问题是所需的代谢物如何进入包涵体，特别是如何穿过包涵体膜。结果表明，宿主细胞源性囊泡的融合可将脂质传递到包涵膜。这也可能导致溶质的非特异性供应，但很可能不会根据细菌的特定需求高度控制基本所需代谢物的供应。在提议的项目中，我们将建立一个包合膜富集的方案。包涵膜蛋白质组的测定将对该细胞器的蛋白质组成和功能提供重要的见解。此外，我们将分析代谢产物通量，如氨基酸、核苷酸或辅助因子在膜蛋白重组成人工脂质囊泡后的通量。此外，重组包涵蛋白和选择的候选载体的生化表征使我们能够确定这些蛋白质是否参与运输或直接催化代谢物通过包涵膜运输。

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.