University thesis:
Dissertation, Universität Freiburg, 2020
Footnote:
Description:
Abstract: ATP-binding cassette (ABC) transporters have been described as one of the essential transport systems used by Mycobacterium tuberculosis (M. tuberculosis), the major causative agent of tuberculosis, for the uptake of a variety of necesseray nutrients (lipids, ions, amino acids, iron, carbohydrates, etc). Therefore, inhibitory studies targeting these transporters might represent a general strategy for killing mycobacteria under limited nutrient conditions. In this thesis, a simple and rapid lipoproteomics protocol was constructed to identify ABC transporters of carbohydrates in M. smegmatis mc2155. The constructed protocol is based on three conditions: (1) the identified transport systems have a high-affinity periplasmic substrate binding protein (SBP); (2) SBPs could be significantly induced by a respective carbohydrate; (3) SBPs belong to the class of lipoproteins, which can be extracted via using the detergent Triton X-114. The constructed workflow is time-saving and easier to handle. With the help of the constructed protocol, the ABC transporter of L-arabinose from M. smegmatis mc2155 could be identified, which was verified by biochemical and microbiological experiments. Theoretically, the constructed lipoproteomics protocol is also applicable to other carbohydrates or nutrients.<br>In order to survive and adapt under a variety of environmental stimuli and changes, M. tuberculosis requires two-component regulatory systems which have been identified as a potential target for new drug discovery based on their vital functions in mycobacterial viability, virulence, and their lack in human. In this work, the potential three-component system containing a heme binding protein MSMEG_0243 and one PrrA/PrrB two-component regulatory system (MSMEG_0244/0246) was studied in M. smegmatis mc2155. Lipid profiles revealed the accumulations of diacyl phosphatidylinositol dimannoside (Ac2PIM2) and diacyl phosphatidylinositol hexamannoside (Ac2PIM6) in the absence of msmeg_0243-PrrA/PrrB resulting in changes of cell morphology, biofilm formation, and sliding motility. The results indicated that a heme binding protein MSMEG_0243 (a potential PrrA/PrrB accessory protein) and a PrrA/PrrB two-component regulatory system (MSMEG_0244/0246) are potentially involved in the regulation of biosynthesis of phosphatidylinositol mannosides (PIMs), especially in the regulation of acylation of PIMs. Further investigation could explore the interaction between MSMEG_0243 and PrrA/PrrB and the regulated mechanism in PIMs biosynthesis, especially on the level of acylation