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Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling
Published on: June 25, 2015
Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens
Hyungjin Eoh1, Prabagaran Narayanasamy, Amanda C Brown
1Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
Abstract:
Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.
Insights
Researchers developed methods to study IspE, a vital bacterial enzyme in the isoprenoid biosynthesis pathway. This work paves the way for new antibacterial drug development targeting this essential pathway.
Area of Science:
- Biochemistry
- Microbiology
- Drug Discovery
Background:
- The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is crucial for bacterial survival, producing isoprenoids essential for cell function.
- This pathway is absent in humans, making it an attractive target for novel antibacterial therapies.
- Characterizing key enzymes like 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) is vital for drug development but has been challenging.
Purpose of the Study:
- To overcome challenges in obtaining pure IspE and enantiopure CDP-ME for detailed study.
- To characterize the essentiality and biochemical properties of IspE in bacterial pathogens.
- To lay the groundwork for developing high-throughput screening assays and structural studies of IspE.
Main Methods:
- Chemical synthesis of enantiopure CDP-ME.
- Purification and characterization of recombinant IspE from bacterial pathogens.
- Investigating IspE essentiality through gene disruption attempts in Mycobacterium tuberculosis and Mycobacterium smegmatis.
Main Results:
- Enantiopure CDP-ME was successfully synthesized.
- Recombinant IspE proteins were purified and characterized.
- IspE was confirmed as essential in Mycobacterium smegmatis, although gene disruption was not feasible in Mycobacterium tuberculosis.
- Biochemical and kinetic data for IspE were obtained.
Conclusions:
- The successful synthesis of CDP-ME and purification of IspE enable further investigation of this critical enzyme.
- IspE's essentiality in M. smegmatis highlights its potential as a drug target.
- The obtained biochemical and kinetic data are foundational for developing new antibacterial strategies targeting the MEP pathway.
