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Method for the Isolation of Francisella tularensis Outer Membranes
Published on: June 29, 2010
Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella
Laura E Batten1, Alice E Parnell1, Neil J Wells1
1Department of Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K.
Abstract:
The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using (31)P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1 nm) resolution. The structure consists of a six-stranded parallel β-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors.
Insights
Polyphosphate kinase 2 (PPK2) from Francisella tularensis is crucial for bacterial virulence and intracellular growth. Its biochemical and structural characterization reveals potential as a novel antibacterial target.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Polyphosphate metabolism is vital for pathogen virulence and represents a potential antibacterial target.
- The gene FTT1564 in Francisella tularensis encodes a polyphosphate kinase 2 (PPK2) family enzyme, FtPPK2.
- A mutant lacking FtPPK2 shows impaired intracellular growth and reduced virulence in mice.
Purpose of the Study:
- To biochemically and structurally characterize Francisella tularensis polyphosphate kinase (FtPPK2).
- To evaluate FtPPK2 as a potential novel target for antibacterial inhibitors.
Main Methods:
- HPLC-based activity assays and (31)P-NMR were used to determine substrate specificity.
- X-ray crystallography was employed to determine the 3D structure of FtPPK2 to 2.23 Å resolution.
- Microbiological studies assessed the virulence and antibiotic sensitivity of a FTT1564 deletion mutant.
Main Results:
- FtPPK2 utilizes purine nucleotides but not pyrimidine nucleotides as substrates.
- The crystal structure revealed high similarity to other PPK2 family members and thymidylate kinase superfamily, with identified key residues for substrate binding and catalysis.
- The FTT1564 deletion mutant exhibited increased sensitivity to various antibiotics.
Conclusions:
- FtPPK2 plays a significant role in Francisella tularensis virulence.
- The biochemical and structural data provide a basis for developing PPK2-specific small molecule inhibitors.
- Targeting FtPPK2 may offer a novel strategy for combating Francisella infections.
