Rifampicin Mediated Cell Death in Staphylococcus aureus
Creators: name: Alexander, Seth
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 2018

description: Antibiotic tolerance and resistance create significant barriers to pharmacologic treatment of bacterial infections. Rifampicin, a commonly used rifamycin, is a bactericidal antibiotic that binds to RNA polymerase in the cell and inhibits transcription elongation after the synthesis of a strand of two or three nucleotides. It is not understood how this ultimately leads to cell death as opposed to stasis. Previous studies have shown that the metabolic state of S. aureus can alter the degree of tolerance to antibiotics but have not provided information regarding how this relates to the mechanism by which rifampicin induces cell death. Rifampicin mediated cell death is potentially the result of a futile cycle (continued synthesis of short strands of nucleotides), which could exhaust the cell of available nucleotides. Alternatively, DNA damage could be caused by the inhibition of transcriptional enzymes causing irreversible collapse of the cell’s nucleoid and/or double stranded DNA breaks. 
To test the concept of a futile elongation cycle, the bactericidal effect of rifampicin on mid-exponential phase cells was compared to that of rifabutin (a transcription initiation inhibitor) which served as a negative control. There was no significant difference in cell death when comparing the bactericidal effects of rifampicin and rifabutin. This suggests that cell death is not the result of a futile cycle. To determine if the nucleoid is enlarged in rifampicin treated cells, TEM images were analyzed to quantify the average nucleoid area to cell area ratio. A dps overexpression strain and perR mutant were created to increase nucleoid compaction. Analysis revealed that nucleoids are significantly larger in rifampicin treated cells. However, there was no significant difference in rifampicin’s bactericidal effects on the genetically altered strains when compared to control cultures. These data indicate that although rifampicin treated cells have a more diffuse nucleoid, this effect is not the mechanism of rifampicin mediated cell death. With regards to the metabolic state of the cell, it was found that S. aureus cultured in glucose containing media exhibits an increase in resistance to rifampicin when compared to media containing amino acids as an alternative carbon source.
These results suggest that rifampicin mediated cell death is not the result of a futile cycle nor improper nucleoid condensation. These findings leave open the possibility that lethal, double-stranded DNA breaks are being caused by collision between inhibited transcriptional enzymes and replication enzymes. This possibility should be the subject of future study along with studies pertaining to how metabolism of glucose may alter the state of the cell so as to increase resistance to rifampicin. Determination of the mechanisms leading to rifampicin mediated cell death could aid in the development of more robust antibiotics and treatments which could more effectively combat tolerant and resistant strains of bacteria.