(175g) Therapeutically Exploring Persister Metabolism in Bacteria

Bacterial persisters represent a small subpopulation of phenotypic variants in isogenic cell populations that are temporarily tolerant to high concentrations of antibiotics. The rise of antibiotic tolerance is one of the most critical global public threats of our century. Bacterial persistence contributes to this problem because these cells are thought to facilitate the re-occurrence of chronic infections (1, 2). Although persister eradication holds the potential to cure these infections, effective anti-persister methods remain scarce. We previously showed that persister cells were mostly obtained from stationary-phase-cell subpopulations with increased redox activities (3). These redox activities were largely maintained by degradation of endogenous proteins and RNA. This intracellular degradation yielded cell subpopulations that were less fit to produce protein and resume growth upon exposure to fresh nutrients, while providing temporary protection against antibiotics. Our study further demonstrated that inhibiting stationary-phase respiratory activities could prevent intracellular degradation and reduce persister formation.

In our current study, when we treated the stationary-phase cells with an ATP synthase inhibitor, chlorpromazine hydrochloride (CPZ) (4), we were able to reduce (more than 200-fold) both ampicillin and ofloxacin persistence in Escherichia coli (Fig. 1A). This pretreatment decreased stationary-phase-redox activities (Fig. 1B) and protein degradation, and gave rise to cells that succumbed to death upon exposure to antibiotics in fresh media. We note that CPZ is an FDA-approved, anti-psychotic drug that is effective, safe and listed as an essential medicine by the World Health Organization. To further identify additional anti-persister therapeutics that are medicinally relevant, we developed a rapid and straightforward chemical screening strategy using a degradable fluorescent protein and a small chemical library containing FDA-approved drugs and antibiotics among ~360 known chemical compounds. Our screening strategy identified several chemical inhibitors such as, polymyxin B, poly-l-lysine and phenothiazine anti-psychotic drugs, that were able to significantly reduce stationary-phase-redox activities and persistence in E. coli (Fig. 1A and B). Using fluorescent protein dilution method, these pretreatments also reduced viable but non-culturable cell (VBNC) formation during stationary phase. Due to their non-proliferating state, VBNC cells can also tolerate antibiotic treatments. However, unlike persisters, VBNC cells are rarely recolonize in standard culture medium in the absence of antibiotics. Pretreatment of stationary-phase Pseudomonas aeruginosa cultures with these chemicals also substantially reduced persistence, confirming the presence of a persistence mechanism similar to that found in E. coli (Fig. C). P. aeruginosa is involved in many hospital-related biofilm infections; it is the predominant cause of morbidity and mortality in cystic fibrosis patients with compromised immune systems (5).

Overall, our results demonstrate that our method can identify medically relevant chemicals. Our study further verifies that persister-cell metabolism is a rich source of therapeutic strategies to eliminate antibiotic-tolerant cells. This study has been published in “Frontiers in Microbiology” (6).

References

1. Lewis, K. (2007). Persister cells, dormancy and infectious disease. Nature Reviews Microbiology, 5(1), 48-56.
2. Lewis, K. (2010). Persister cells. Annual review of microbiology, 64, 357-372.
3. Orman, M. A., & Brynildsen, M. P. (2015). Inhibition of stationary phase respiration impairs persister formation in E. coli. Nature communications, 6, 7983.
4. Bullough, D. A., Kwan, M., Laikind, P. K., Yoshida, M., & Allison, W. S. (1985). The varied responses of different F1-ATPases to chlorpromazine. Archives of biochemistry and biophysics, 236(2), 567-575.
5. Mulcahy, L. R., Burns, J. L., Lory, S., & Lewis, K. (2010). Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis. Journal of bacteriology, 192(23), 6191-6199.
6. Mohiuddin, S. G., Hoang, T., Saba, A., Karki, P., & Orman, M. (2020). Identifying Metabolic Inhibitors to Reduce Bacterial Persistence. Frontiers in Microbiology, 11, 472.