Proton Motive Force Inhibitors Are Detrimental to Methicillin-Resistant Staphylococcus aureus Strains

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) strains are tolerant of conventional antibiotics, making them extremely dangerous. Previous studies have shown the effectiveness of proton motive force (PMF) inhibitors at killing bacterial cells; however, whether these agents can launch a new treatment strategy to eliminate antibiotic-tolerant cells mandates further investigation. Here, using known PMF inhibitors and two different MRSA isolates, we 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. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) emerged as a major hypervirulent pathogen that causes severe health care-acquired infections. These pathogens can be multidrug-tolerant cells, which can facilitate the recurrence of chronic infections and the emergence of diverse antibiotic-resistant mutants. In this study, we aimed to investigate whether proton motive force (PMF) inhibitors can launch a new treatment strategy to eliminate MRSA cells. Our in-depth analysis showed that PMF inhibitors that strongly dissipate either the transmembrane electric potential or the proton gradient can robustly permeabilize cell membranes and reduce MRSA cell levels below the limit of detection.

Keywords: PMF inhibitors; high-throughput drug screening; membrane permeabilization; methicillin-resistant Staphylococcus aureus; proton motive force; tolerant cells.

FIG 1

Proton motive force (PMF) inhibitors increased membrane permeability, disrupted cellular PMF, and reduced cell survival levels in strain MRSA BAA-41. (A) MRSA BAA-41 cells were grown to the exponential phase (optical density at 600 nm [OD₆₀₀] of ~0.1) in Mueller-Hinton broth and treated with polymyxin B, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), or thioridazine at concentrations of 5× and 10× MIC (Table S1A). After 1 h treatment, the cells were collected and stained with propidium iodide (PI) (20 μM) dye for flow cytometry analysis. Live and ethanol-treated (70%, vol/vol) dead cells were used as negative (–) and positive (+) controls (Fig. S2). A representative flow cytometry diagram is shown here; all independent biological replicates produced similar results. (B) Cells grown to the exponential phase (OD₆₀₀ of ~0.1) were transferred to 3,3′-dipropylthiadicarbocyanine iodide (DiSC₃[5]) assay buffer (50 mM HEPES, 300 mM KCl, and 0.1% glucose) and stained with DiSC₃(5). When the cells reached an equilibrium state (t = 30 min), they were treated with polymyxin B, CCCP, or thioridazine at the indicated concentrations. The fluorescence levels were measured with a plate reader at the designated time points. Cultures stained with the DiSC₃(5) but not treated with PMF inhibitors were used as control. (C) Cells at the exponential phase (OD₆₀₀ of ~0.1) were treated with the drugs at the indicated concentrations for 6 h. At designated time points during treatments, cells were collected, washed to remove the chemicals, and spotted on Mueller-Hinton agar plates to enumerate the colony forming units (CFU). The dashed lines in panel C indicate the limit of detection. The number of biological replicates (n) = 3. The data points represent means ± SD. FSC-H, Forward scatter.

FIG 2

PMF inhibitors increased membrane permeability, disrupted cellular PMF, and reduced cell survival levels in strain MRSA 700699. Effects of polymyxin B, CCCP, and thioridazine treatments on cell membranes (A), PMF (B), and cell survival levels (C) of MRSA 700699 cells were determined as described in the legend to Fig. 1. A representative flow cytometry diagram is shown here; all independent biological replicates (n = 3) produced similar results. The dashed lines in panel C indicate the limit of detection. The data points represent means ± SD.

FIG 3

Simple multivariable regression analysis correlates the disruption of PMF and membrane permeability to cell survival levels. (A, B) Two- and three-dimensional scatterplots including all data points for PMF inhibitors and conventional antibiotics for all concentrations and strains tested. In panel A, the red oval indicates cluster I, and the blue oval indicates cluster II. The cell survival levels corresponding to each cluster are presented in the inset. A Student’s t test with unequal variance was used to find the statistical significance between the cell survival levels of clusters I and II. ***, P < 0.0001. (C) Multivariable linear regression analysis without an interaction between the independent variables. (D) Multivariable linear regression with a two-way interaction between the independent variables. P_L = cell survival level; P_D = PMF disruption; P_M = membrane permeabilization; β₀ = the estimate of the model intercept; β₁ = the estimate of the model coefficient of PMF disruption; β₂ = the estimate of the model coefficient of membrane permeability; β₃ = the estimate of the model coefficient of the interaction term. F statistics were used for the statistical analysis with the threshold value set to P = 0.01.

FIG 4

The identified drugs increased membrane permeability, disrupted cellular PMF, and reduced cell survival levels in strain MRSA BAA-41. The effects of nordihydroguaiaretic acid (NDGA), gossypol, trifluoperazine, and amitriptyline treatments on cell membranes (A), PMF (B), and cell survival levels (C) of MRSA BAA-41 cells were determined as described in the legend to Fig. 1. A representative flow cytometry diagram is shown here; all independent biological replicates (n = 3) produced similar results. The dashed lines in panel C indicate the limit of detection. The data points represent means ± SD.

FIG 5

The identified drugs increased membrane permeability, disrupted cellular PMF, and reduced cell survival levels in strain MRSA 700699. The effects of nordihydroguaiaretic acid (NDGA), gossypol, trifluoperazine, and amitriptyline treatments on cell membranes (A), PMF (B), and cell survival levels (C) of MRSA 700699 cells were determined as described in the legend to Fig. 1. A representative flow cytometry diagram is shown here; all independent biological replicates (n = 3) produced similar results. The dashed lines in panel C indicate the limit of detection. The data points represent means ± SD.