Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 1 (4)
eCollection

Antibiotic Bactericidal Activity Is Countered by Maintaining pH Homeostasis in Mycobacterium Smegmatis

Affiliations

Antibiotic Bactericidal Activity Is Countered by Maintaining pH Homeostasis in Mycobacterium Smegmatis

I L Bartek et al. mSphere.

Abstract

Antibiotics target specific biosynthetic processes essential for bacterial growth. It is intriguing that several commonalities connect the bactericidal activity of seemingly disparate antibiotics, such as the numerous conditions that confer broad-spectrum antibiotic tolerance. Whether antibiotics kill in a manner unique to their specific targets or by a universal mechanism is a critical and contested subject. Herein, we demonstrate that the bactericidal activity of diverse antibiotics against Mycobacterium smegmatis and four evolutionarily divergent bacterial pathogens was blocked by conditions that worked to maintain intracellular pH homeostasis. Single-cell pH analysis demonstrated that antibiotics increased the cytosolic pH of M. smegmatis, while conditions that promoted proton entry into the cytosol prevented intracellular alkalization and antibiotic killing. These findings led to a hypothesis that posits antibiotic lethality occurs when antibiotics obstruct ATP-consuming biosynthetic processes while metabolically driven proton efflux is sustained despite the loss of proton influx via ATP synthase. Consequently, without a concomitant reduction in respiratory proton efflux, cell death occurs due to intracellular alkalization. Our findings indicate the effects of antibiotics on pH homeostasis should be considered a potential mechanism contributing to antibiotic lethality. IMPORTANCE Since the discovery of antibiotics, mortality due to bacterial infection has decreased dramatically. However, infections from difficult to treat bacteria such as Mycobacterium tuberculosis and multidrug-resistant pathogens have been on the rise. An understanding of the cascade of events that leads to cell death downstream of specific drug-target interactions is not well understood. We have discovered that killing by several classes of antibiotics was stopped by maintaining pH balance within the bacterial cell, consistent with a shared mechanism of antibiotic killing. Our findings suggest a mechanism of antibiotic killing that stems from the antibiotic's ability to increase the pH within bacterial cells by disrupting proton entry without affecting proton pumping out of cells. Knowledge of the core mechanism necessary for antibiotic killing could have a significant impact on the development of new lethal antibiotics and for the treatment of recalcitrant and drug-resistant pathogens.

Keywords: Antibiotics; bactericidal activity; mycobacteria; pH homeostasis.

Figures

FIG 1
FIG 1
Conditions that countered intracellular alkalization increased M. smegmatis antibiotic tolerance. M. smegmatis was challenged with antibiotics over a pH range or in combination with DNP, CCCP, or DCCD at pH 6.6. Antibiotics were assayed in the presence of DMSO or methanol to control for vehicle effects. Antibiotics were administered at the concentrations listed in Table 3, and CFU were determined at 0 h (no pattern), 3 h (checkered), 12 h (gridded), 24 h (diagonal up), and 48 h (latticed). The data shown are means ± standard deviations from at least 3 replicates. All conditions were compared to the respective pH 6.6 time point for statistical t test analysis (*, P < 0.05; **, P < 0.01).
FIG 2
FIG 2
The protonophore concentration required for rescue was proportional to the antibiotic concentration. (A) M. smegmatis cultures were grown in the presence of 32 µg/ml norfloxacin (4× the calculated MBC) both with and without 5 mM DNP. CFU were determined at 0 h (no pattern), 12 h (gridded), and 24 h (diagonal up). (B) A second set of cultures were exposed to 6 µg/ml norfloxacin across a range of DNP concentrations, and CFU were determined after 24 h of exposure. For statistical analysis on panel A, a t test was used. For statistical analysis on panel B, a Pearson’s correlation coefficient was determined (***, P < 0.001).
FIG 3
FIG 3
Loss of antibiotic lethality in acidic pH was not due to antibiotic degradation. Antibiotics were preincubated for 24 h at 37°C in DTA medium at pHs 5.6, 6.6, and 7.6. The pH was then adjusted to 6.6, and M. smegmatis was added to assay the drug efficacy post-drug incubation at each pH. The data shown are means ± standard deviations.
FIG 4
FIG 4
Single-cell flow cytometry analysis of pHIN. Cultures of M. smegmatis containing a ratiometric pH-sensitive GFP (20) were exposed to antibiotics (Cpz, chlorpromazine; Kan, kanamycin; Rif, rifampin; Nor, norfloxacin) with or without DNP, and individual cells were analyzed by flow cytometry. (A) The average pHIN of the entire M. smegmatis population was determined as described previously (46). (B) Number of cells in the high-pH-gated population (pH of >8.6 [gate 1]) after cells were counted based on ratiometric fluorescence intensities. Samples were compared to the untreated control for statistical t test analysis (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG 5
FIG 5
The rate of antibiotic killing was directly proportional to the efficiency of intracellular alkalization. (A) The percentage of survival was calculated for each antibiotic used with M. smegmatis (Fig. 1) at the 24-h time point. This was plotted against the proportion of cells observed in the high-pH population (Fig. 2), and a trend line was calculated with a Pearson’s correlation coefficient of R2 = 0.752. Kan, kanamycin; Rif, rifampin; Cpz, chlorpromazine; Nor, norfloxacin. (B) The same analysis was performed excluding rifampin, and a trend line was calculated with a Pearson’s correlation coefficient of R2 = 0.999.
FIG 6
FIG 6
Alkalization alone caused cell death. M. smegmatis was exposed to an pHEX of 6.6, 7.6, 8.6, or 9.6 with or without the protonophores DNP and CCCP, and samples were plated at 0 h (no pattern), 24 h (diagonal up), and 48 h (latticed). A t test was used for statistical analyses, and all conditions were compared to the same time point at pH 7.6 (approximate pHIN).
FIG 7
FIG 7
Shifting pH homeostasis toward intracellular acidification inhibited antibiotic killing of Gram-negative, Gram-positive, and acid-fast pathogens. S. aureus, E. coli, P. aeruginosa, and M. tuberculosis were challenged with antibiotics at concentrations listed in Table 3 over a pH range or at the mid-pH in combination with DNP. CFU were determined at 0 h (no pattern), 30 min (brick), 1.5 h (diagonal down), 3 h (checkered), 12 h (gridded), 24 h (diagonal up), and 72 h (light horizontal). A t test was used for statistical analysis, and all conditions were compared to the same time point at the standard mid-pH (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG 8
FIG 8
Anoxic antibiotic killing was inhibited by conditions that promoted intracellular acidification. Antibiotics (listed in Table 3) were added to anoxic cultures of E. coli in the presence or absence of DNP. CFU were determined at 0 h (no pattern), 3 h (checkered), or 6 h (heavy horizontal) for plating to determine survival by CFU. Kanamycin, an aminoglycoside, did not readily kill E. coli as a strong ΔΨ is required for aminoglycoside uptake (25). A t test was used for statistical analysis, and survival under each condition was compared to that at the same time point at pH 7 (*, P < 0.05; **, P < 0.01).
FIG 9
FIG 9
Antibiotic uptake was not decreased by acidic pHEX or protonophore. [3H]norfloxacin (Nor) was added to the M. smegmatis, E. coli, or S. aureus culture, [3H]vancomycin (Vanc) was added to the S. aureus culture, and [3H]tetracycline (Tet) was added to the P. aeruginosa culture, and intracellular antibiotic concentrations were determined over time. The amount of added antibiotic was normalized to the intracellular protein concentration. For E. coli, S. aureus, and P. aeruginosa, pH 6 is in pink, pH 7 is in white, pH 8 is in blue, and pH 7 with DNP is in yellow. For M. smegmatis, pH 5.6 is in pink, pH 6.6 is in white, pH 7.6 is in blue, and pH 6.6 with DNP is in yellow. For statistical t test analyses, each condition was compared to the same time point at the mid-pH (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG 10
FIG 10
pH homeostatic hypothesis of antibiotic lethality. In the absence of antibiotics, nutrient-replete conditions sustain reduction of electron carriers and drive proton translocation via the ETS. Protons return to the cytosol through ATP synthesis. Bactericidal antibiotics target ATP-consuming reactions in a manner that fails to coordinately reduce the rate of proton translocation. Intracellular alkalization follows as the rate of ATP synthesis abates from an overall reduction in all biosynthetic reactions. Bacteria die as countermeasures to intracellular alkalization fail and pHIN increases beyond viable limits.
FIG 11
FIG 11
Flow cytometry analysis of DNP effect on proton gradient. (A) A sigmoidal standard curve was generated in the presence of CCCP similar to that observed previously (46). (B) DNP at 0, 0.5, 1, 2, or 4 mM DNP was added to M. smegmatis culture containing the ratiometric pH-sensitive GFP for 20 min, and cells were subsequently analyzed via flow cytometry. (C) pHIN was calculated from the experiments shown in panel B. Even at the highest dose of 4,000 µM DNP (4-fold higher than the concentration used for M. smegmatis experiments), the pHIN never reached the pHEX of the medium. In contrast, 500 µM CCCP, a much stronger protonophore, equalized pHIN to that of the medium within 20 min. (D) For flow cytometric gate placement, the mean fluorescence ratio of the untreated control and the standard deviation were calculated using Summit5.3 software. Gate 2 was set as the mean − 1.5 standard deviations from the mean. Gate 3 encompassed the mean + 1.5 standard deviations from the mean. Gate 1 included all cells with a lower fluorescence intensity ratio (higher pHIN) than gate 2, and gate 4 included all cells with a higher fluorescence intensity ratio (lower pHIN) than gate 3.

Similar articles

See all similar articles

Cited by 9 articles

See all "Cited by" articles

References

    1. Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S. 2004. Bacterial persistence as a phenotypic switch. Science 305:1622–1625. doi:10.1126/science.1099390. - DOI - PubMed
    1. Maisonneuve E, Gerdes K. 2014. Molecular mechanisms underlying bacterial persisters. Cell 157:539–548. doi:10.1016/j.cell.2014.02.050. - DOI - PubMed
    1. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. 2007. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 130:797–810. doi:10.1016/j.cell.2007.06.049. - DOI - PubMed
    1. Gusarov I, Shatalin K, Starodubtseva M, Nudler E. 2009. Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics. Science 325:1380–1384. doi:10.1126/science.1175439. - DOI - PMC - PubMed
    1. Shatalin K, Shatalina E, Mironov A, Nudler E. 2011. H2S: a universal defense against antibiotics in bacteria. Science 334:986–990. doi:10.1126/science.1209855. - DOI - PubMed
Feedback