Membranous environments are colonized by various strains of A. baumannii, which raises the question on how A. baumannii manages to adapt its metabolism to the skin environment. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant phospholipids found in human cell membranes and make them good candidates as carbon and energy source. Initial degradation of PC or PE is facilitated by a set of different phospholipases whose substrate specificity is determined by the acyl side chains and the hydrophilic group. A. baumannii ATCC 19606 encodes six phospholipases belonging to the phospholipase superfamilies PLA, PLC and PLD. Two of them are known from the literature as virulence factors. To elucidate the function of all phospholipases they will be produced in E. coli, purified and their enzymatic properties will be determined. Transcriptional and immunological analyses will identify conditions under which the encoding genes are expressed. Phospholipase mutants will be generated using the established markerless mutagenesis strategy and mutant studies will unravel the role of the phospholipases in phospholipid degradation, survival of in cell culture, adhesion to eukaryotic cells and complement resistance as well as their role in cytotoxicity on prokaryotic and eukaryotic cells. Furthermore, we aim to identify and characterize phospholipase exporters by genetic and biochemical techniques. Our own preparatory work demonstrated that A. baumannii takes up the PC cleavage product choline and oxidizes it to glycine betaine. The genome of A. baumannii 19606 predicts a two-step oxidation pathway, a transcriptional regulator and two choline transporters (BetT1 and BetT2). BetT1 in A. baylyi was found to facilitate osmolarity-independent choline transport, most likely by an uniport mechanism. Analogously BetT1 in A. baumannii might play a role in metabolic adaptation to choline rich environments. To analyze the role of the choline transporters and choline oxidation in metabolic adaptation and pathogenicity of A. baumannii the encoding genes will be deleted. Mutant studies will be performed to elucidate the biochemistry of choline transport and to determine the role in virulence in cell cultures.
Another focus will be to identify the substrate of the transcriptional regulator BetI and study its mode of interaction with the DNA. To unravel the molecular transport mechanism of H+-coupled choline symport and proton-independent choline uniport in the BCCT family we will purify both choline transporters and attempt to solve their atomic structures by X-ray crystallography on 3D crystals in a high-throughput process. Furthermore, we will investigate their interactions with specific lipids by 2D crystallization and cryo-electron microscopy. To elucidate the transport mechanism we will perform transport experiments in vivo and in vitro in cells proteoliposomes, respectively, as well as binding studies by ITC and TRP-fluorescence.