Related Experiment Videos
Entry of methotrexate into Streptococcus pneumoniae: a study on a wild-type strain and a methotrexate resistant
Abstract:
Entry of methotrexate (MTX) into the folate prototrophic bacterium Streptococcus pneumoniae was poorly inhibited by folate or its natural derivative folinic acid, suggesting that if MTX is transported via a folate transporter, the affinity of that transporter for MTX is higher than for folate. In the range of concentrations tested, MTX uptake was non-concentrative and decreased in ATP-depleted bacteria. When the external concentration of MTX was increased from 1 X 10(-7) M to 1 X 10(-6) M, uptake became saturated and was insensitive to ionophores. However when external MTX concentrations were increased to 1 X 10(-5) M, uptake increased linearly, and was inhibited by the ionophores carbonyl cyanide m-chlorophenylhydrazone (CCCP) and valinomycin, suggesting that the process was energized by the protonmotive force (delta p) at this concentration. A model for MTX entry in S. pneumoniae is proposed with respect to these results. The high level of resistance to MTX of the nonsense mutant amiA9 cannot be entirely explained by a decrease in MTX uptake.
Insights
Methotrexate (MTX) enters Streptococcus pneumoniae via a transporter with high affinity for MTX. MTX uptake is energy-dependent and influenced by proton motive force at higher concentrations.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Methotrexate (MTX) is a key chemotherapy drug.
- Understanding MTX transport in bacteria like Streptococcus pneumoniae is crucial for developing new antimicrobial strategies.
- The folate pathway is essential for bacterial growth and is a target for MTX.
Purpose of the Study:
- To investigate the mechanism of methotrexate (MTX) entry into Streptococcus pneumoniae.
- To determine the role of folate transporters in MTX uptake.
- To elucidate the energy dependence of MTX transport.
Main Methods:
- Bacterial growth and uptake assays using radiolabeled MTX.
- Inhibition studies with folate, folinic acid, and ionophores (CCCP, valinomycin).
- Experiments with ATP-depleted bacteria to assess energy dependency.
- Analysis of MTX transport kinetics at various concentrations.
Main Results:
- MTX uptake in S. pneumoniae showed poor inhibition by folate or folinic acid, suggesting high MTX affinity for its transporter.
- MTX uptake was non-concentrative and decreased in ATP-depleted bacteria.
- At high MTX concentrations (1 X 10(-5) M), uptake was linear and energized by the protonmotive force (delta p), indicated by inhibition with ionophores.
- The high MTX resistance in the amiA9 mutant was not fully explained by reduced MTX uptake.
Conclusions:
- MTX transport in S. pneumoniae involves a high-affinity transporter, distinct from folate transport.
- MTX uptake is an active process dependent on cellular energy, likely utilizing the protonmotive force at higher concentrations.
- Further research is needed to fully understand the mechanisms of MTX resistance in S. pneumoniae.