Production and functional characterization of the L-lysine exporters from bacterial pathogens ABSTRACT We will study the functional mechanism(s) of L-lysine membrane exporters (LysE) from Mycobacterium tuberculosis, the causative agent of tuberculosis, which is a severe disease affecting millions of people each year with often lethal outcome. To gain broader understanding of this family of membrane transporters, and particularly their roles in the physiology of pathogenic bacteria, we will expand the study to include also LysE proteins from Vibrio cholerae and Helicobacter pylori. Acquired knowledge about these exporters would help to understand how to inhibit their function. It will also expand the understanding of the basic molecular mechanisms in these dangerous microbes. LysE exporters, found in diverse bacteria, are responsible for the removal of basic amino acids, such as the metabolic products L-lysine and L-arginine, from the cytosol. LysE deficiency leads to elevated levels of L- lysine in the cell and the suppression of bacterial growth. Therefore, in pathogenic bacteria, LysE exporters are a potential target for drug development and inhibition. Pharmacological targeting of LysEs requires a detailed understanding of the structure-function relationship in these proteins. However, thus far, the studies on the LysE exporters have been very limited, and those targeted in the current proposal have not been studied at all. To overcome this deficiency, we will conduct a detailed functional study on the LysE proteins from the proposed bacteria. First, we will develop strategies for large-scale production of the LysE exporters in E. coli cells. Then, we will develop protocols for efficient protein purification. Next, we will develop substrate transport assays on liposome-reconstituted proteins to establish the substrate translocation rate and specificity. Based on data found in relevant literature, we hypothesize that the transport activity of LysE is allosterically regulated by substrate concentration. Through these assays, we will also examine the functional roles of conserved amino acids in LysE polypeptides by mutating them to other amino acids with different properties. We further hypothesize that LysE functional units might be homo-oligomers, similar to other transporters. To establish LysE quaternary organization in lipid membranes, we will employ chemical cross-linking and pulse electron paramagnetic resonance spectroscopy. Thus, our study will advance the knowledge about the functional mechanism(s) of LysE exporters and will lay the foundation for a more comprehensive characterization of these proteins through functional approaches and higher resolution structural methods.
/RELEVANCE The proposed studies will provide functional and structural information about the molecular mechanisms of LysE membrane exporters from the highly contagious and resilient pathogenic bacterium Mycobacterium tuberculosis (Mtb), which in infected individuals leads to the very serious disease tuberculosis (TB). Mtb often acquires drug- and multidrug-resistance, making treatment of TB extremely difficult and even impossible, and placing the disease among the major causes of death worldwide. The project will also include LysE exporters from the bacteria Vibrio cholerae (V. cholerae) and Helicobacter pylori (H. pylori), which can cause fatal diseases, such as cholera and gastric cancer, respectively.
| Georgieva, Elka R (2018) Non-Structural Proteins from Human T-cell Leukemia Virus Type 1 in Cellular Membranes-Mechanisms for Viral Survivability and Proliferation. Int J Mol Sci 19: |
