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Acquisition of thymidylate by the obligate intracytoplasmic bacterium Rickettsia prowazekii
1Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile 36688.
Abstract:
The pathway for the acquisition of thymidylate in the obligate bacterial parasite Rickettsia prowazekii was determined. R. prowazekii growing in host cells with or without thymidine kinase failed to incorporate into its DNA the [3H]thymidine added to the culture. In the thymidine kinase-negative host cells, the label available to the rickettsiae in the host cell cytoplasm would have been thymidine, and in the thymidine kinase-positive host cells, it would have been both thymidine and TMP. Further support for the inability to utilize thymidine was the lack of thymidine kinase activity in extracts of R. prowazekii. However, [3H]uridine incorporation into the DNA of R. prowazekii was demonstrable (973 +/- 57 dpm/3 x 10(8) rickettsiae). This labeling of rickettsial DNA suggests the transport of uracil, uridine, uridine phosphates (UXP), or 2'-deoxyuridine phosphates, the conversion of the labeled precursor to thymidylate, and subsequent incorporation into DNA. This is supported by the demonstration of thymidylate synthase activity in extracts of R. prowazekii. The enzyme was determined to have a specific activity of 310 +/- 40 pmol/min/mg of protein and was inhibited greater than or equal to 70% by 5-fluoro-dUMP. The inability of R. prowazekii to utilize uracil was suggested by undetectable uracil phosphoribosyltransferase activity and by its inability to grow (less than 10% of control) in a uridine-starved mutant cell line (Urd-A) supplemented with 50 microM to 1 mM uracil. In contrast, the rickettsiae were able to grow in Urd-A cells that were uridine starved and supplemented with 20 microM uridine (117% of control). However, no measurable uridine kinase activity could be measured in extracts of R. prowazekii. Normal rickettsial growth (92% of control) was observed when the host cell was blocked with thymidine so that the host cell's dUXP pool was depressed to a level inadequate for growth and DNA synthesis in the host cell. Taken together, these data strongly suggest that rickettsiae transport UXP from the host cell's cytoplasm and that they synthesize TTP from UXP.
Insights
Rickettsia prowazekii cannot use thymidine directly. Instead, this bacterial parasite acquires thymidylate by transporting uridine phosphates (UXP) from host cells and synthesizing thymidylate internally.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Obligate intracellular bacteria like Rickettsia prowazekii have unique metabolic pathways.
- Understanding nutrient acquisition is crucial for targeting these pathogens.
Purpose of the Study:
- To elucidate the pathway for thymidylate acquisition in Rickettsia prowazekii.
- To determine if R. prowazekii can utilize host-derived thymidine or must synthesize thymidylate.
Main Methods:
- Utilized radiolabeled thymidine and uridine to trace incorporation into R. prowazekii DNA.
- Assayed for thymidine kinase and thymidylate synthase activities in R. prowazekii extracts.
- Grew R. prowazekii in engineered host cell lines with manipulated nucleotide pools.
Main Results:
- R. prowazekii failed to incorporate exogenous [3H]thymidine into its DNA.
- Demonstrated [3H]uridine incorporation into R. prowazekii DNA, indicating de novo synthesis or salvage pathway.
- Detected significant thymidylate synthase activity but no detectable thymidine kinase activity in R. prowazekii.
- R. prowazekii could not utilize uracil but could utilize uridine from host cells.
- R. prowazekii growth was supported by host cell-derived uridine phosphates (UXP).
Conclusions:
- R. prowazekii cannot salvage thymidine from the host.
- R. prowazekii possesses the enzymatic machinery to synthesize thymidylate from UXP.
- R. prowazekii likely acquires thymidylate by transporting UXP from the host cell and converting it to TTP.