Pleiotropic Actions of Phenothiazine Drugs are Detrimental to Bacterial Persister Cells



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Bacterial persister cells are temporarily tolerant to bactericidal antibiotics but are not necessarily dormant and may exhibit physiological activities leading to cell damage. Persistence is a nonheritable trait by which normal growing cells switch phenotypically to antibiotic tolerant persister cells. This transient state enables persister cells to recover and grow into an antibiotic-sensitive population. Persister cells have been observed in many pathogenic and nonpathogenic bacteria and ascribed as the reason behind the recurrent infections. Although persister eradication holds the potential to cure chronic infections, effective antipersister methods remain scarce. Therefore, we exploited the link between fluoroquinolone-mediated SOS responses and persister cell recovery, and screened chemicals that target fluoroquinolone persisters in a high-throughput manner. Metabolic inhibitors (e.g., phenothiazines) combined with ofloxacin (OFX) perturbed persister levels in metabolically active cell populations. When metabolically stimulated, intrinsically tolerant stationary phase cells also became OFX-sensitive in the presence of phenothiazines. The effects of phenothiazines on cell metabolism and physiology are highly pleiotropic: at sublethal concentrations, phenothiazines reduce cellular metabolic, transcriptional, and translational activities; impair cell repair and recovery mechanisms; transiently perturb membrane integrity; and disrupt proton motive force by dissipating the proton concentration gradient across the cell membrane. Screening a subset of mutant strains lacking membrane-bound proteins revealed the pleiotropic effects of phenothiazines potentially rely on their ability to inhibit a wide range of critical metabolic proteins. Altogether, our study further highlights the complex roles of metabolism in persister cell formation, survival, and recovery, and suggests metabolic inhibitors such as phenothiazines can be selectively detrimental to persister cells. In the final project, we explored the effectiveness of proton motive force (PMF) inhibitors as a new treatment strategy to eliminate antibiotic-tolerant cells. Previous studies have shown the usefulness of proton motive force (PMF) inhibitors at killing bacterial cells. Utilizing this knowledge, we used known PMF inhibitors and two different Methicillin-resistant Staphylococcus aureus (MRSA) isolates, and showed that the bactericidal potency of PMF inhibitors seemed to correlate with their ability to disrupt PMF and permeabilize cell membranes. By screening a small chemical library to verify this correlation, we identified a subset of chemicals (including nordihydroguaiaretic acid, gossypol, trifluoperazine, and amitriptyline) that strongly disrupted PMF in MRSA cells by dissipating either the transmembrane electric potential (ΔΨ) or the proton gradient (ΔpH). These drugs robustly permeabilized cell membranes and reduced MRSA cell levels below the limit of detection. Overall, our study further highlights the importance of cellular PMF as a target for designing new bactericidal therapeutics for pathogens.



Persister cells, Phenothiazine drugs, Persister metabolism, Repair mechanism, Proton motive force, Metabolomics, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, SOS response, Staphylococcus aureus, High-throughput screening, PMF inhibitors, Metabolic inhibitors


Portions of this document appear in: Mohiuddin, Sayed Golam, Thuy Hoang, Adesola Saba, Prashant Karki, and Mehmet A. Orman. "Identifying metabolic inhibitors to reduce bacterial persistence." Frontiers in microbiology 11 (2020): 472; and in: Mohiuddin, Sayed Golam, Sreyashi Ghosh, Han G. Ngo, Shayne Sensenbach, Prashant Karki, Narendra K. Dewangan, Vahideh Angardi, and Mehmet A. Orman. "Cellular Self-Digestion and Persistence in Bacteria." Microorganisms 9, no. 11 (2021): 2269; and in: Mohiuddin, Sayed Golam, Sreyashi Ghosh, Pouria Kavousi, and Mehmet A. Orman. "Proton Motive Force Inhibitors Are Detrimental to Methicillin-Resistant Staphylococcus aureus Strains." Microbiology Spectrum 10, no. 4 (2022): e02024-22.