Carbonic Anhydrase Inhibition as a Target for Antibiotic Synergy in Enterococci

Abstract

Enterococcus faecalis is a hospital-associated opportunistic pathogen that can cause infections with high mortality, such as infective endocarditis. With an increasing occurrence of multidrug-resistant enterococci, there is a need for alternative strategies to treat enterococcal infections. We isolated a gentamicin-hypersusceptible E. faecalis strain from a patient with infective endocarditis that carried a mutation in the alpha-carbonic anhydrase (α-CA) and investigated how disruption of α-CA sensitized E. faecalis to killing with gentamicin. The gentamicin-hypersusceptible α-CA mutant strain showed increased intracellular gentamicin uptake in comparison to an isogenic strain encoding full-length, wild-type α-CA. We hypothesized that increased gentamicin uptake could be due to increased proton motive force (PMF), increased membrane permeability, or both. We observed increased intracellular ATP production in the α-CA mutant strain, suggesting increased PMF-driven gentamicin uptake contributed to the strain's gentamicin susceptibility. We also analyzed the membrane permeability and fatty acid composition of isogenic wild-type and α-CA mutant strains and found that the mutant displayed a membrane composition that was consistent with increased membrane permeability. Finally, we observed that exposure to the FDA-approved α-CA inhibitor acetazolamide lowered the gentamicin MIC of eight genetically diverse E. faecalis strains with intact α-CA but did not change the MIC of the α-CA mutant strain. These results suggest that α-CA mutation or inhibition increases PMF and alters membrane permeability, leading to increased uptake of gentamicin into E. faecalis. This connection could be exploited clinically to provide new combination therapies for patients with enterococcal infections. IMPORTANCE Enterococcal infections can be difficult to treat, and new therapeutic approaches are needed. In studying an E. faecalis clinical strain from an infected patient, we found that the bacteria were rendered hypersusceptible to aminoglycoside antibiotics through a mutation that disrupted the α-CA. Our follow-on work suggested two different ways that α-CA disruption causes increased gentamicin accumulation in E. faecalis: increased proton motive force-powered uptake and increased membrane permeability. We also found that a mammalian CA inhibitor could sensitize a variety of E. faecalis strains to killing with gentamicin. Given that mammalian CA inhibitors are frequently used to treat conditions such as glaucoma, hypertension, and epilepsy, our findings suggest that these "off-the-shelf" inhibitors could also be useful partner antibiotics for the treatment of E. faecalis infections.

Keywords: Enterococcus faecalis; antibiotic synergy; carbonic anhydrase.

FIG 1

Alpha-carbonic anhydrase (α-CA) disruption is associated with increased gentamicin-Texas Red (GTTR) uptake in E. faecalis. (A) Surface (top) and ribbon (bottom) homology model views of the E. faecalis α-CA. Residue E209 is colored red, and residues that sit C-terminal to E209—which are predicted to be missing in the DVT809 α-CA mutant strain—are colored salmon. (B) GTTR uptake into DVT1134 α-CA wild-type (WT) or DVT809 α-CA mutant (MUT) strain. Bars show mean fluorescence intensity values (arbitrary units, a.u.) per CFU, and error bars denote standard error of the mean. CFU present at the beginning of the experiment was used to normalize fluorescence intensities for each strain. P value is from a two-tailed t test.

FIG 2

Increased intracellular ATP accumulation and increased PMF are associated with gentamicin susceptibility in E. faecalis. (A) Intracellular ATP measured in mid-log-phase cultures of DVT1134 (wild type, WT), DVT1134 grown in the presence of 10 μM heme, and DVT809 (α-CA mutant, MUT). Bars show mean picomoles of ATP per million CFU, and error bars denote standard error of the mean. CFU present at the beginning of the experiment was used to normalize ATP abundance. P values are from two-tailed t tests. (B) Growth of strains shown in panel A in various concentrations of gentamicin. Optical densities measured at 600 nm (OD₆₀₀) versus gentamicin concentration are shown. Data points show the averages from three biological replicates, and error bars represent the standard error of the mean.

FIG 3

Differences in membrane permeability and fatty acid composition associated with α-CA disruption in E. faecalis. (A) Propidium iodide (PI) uptake into mid-log-phase cultures of DVT1134 (wild type, WT) and DVT809 (α-CA mutant, MUT) strains. Bars show mean fluorescence intensity values (arbitrary units, a.u.) of six biological replicates and three technical replicates, normalized to the optical density of the culture measured at 600 nm (OD₆₀₀). Error bars denote standard error of the mean. (B) Fatty acid methyl ester (FAME) abundance of fatty acids comprising more than 10% of the total membrane fatty acid content of the DVT1134 and DVT809 strains. Bars show mean percentages from four biological replicates, and error bars denote standard error of the mean. All P values are from two-tailed t tests.