Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics

Abstract

Bacterial nitric oxide synthases (bNOS) are present in many Gram-positive species and have been demonstrated to synthesize NO from arginine in vitro and in vivo. However, the physiological role of bNOS remains largely unknown. We show that NO generated by bNOS increases the resistance of bacteria to a broad spectrum of antibiotics, enabling the bacteria to survive and share habitats with antibiotic-producing microorganisms. NO-mediated resistance is achieved through both the chemical modification of toxic compounds and the alleviation of the oxidative stress imposed by many antibiotics. Our results suggest that the inhibition of NOS activity may increase the effectiveness of antimicrobial therapy.

Fig. 1

Mechanisms of bNOS protection against acriflavine. (A) Proposed chemistry of NO-mediated detoxification of acriflavine (ACR). (B) Optical density (OD) growth curves of B. anthracis wt (Sterne) and Δnos in the presence of ACR (8 µg/ml). (C) Changes in absorbance spectra of ACR upon interaction with NO. (D) OD growth curves of B. subtilis in the presence of MAHMA (NO) and ACR, or acridine orange (AO). Data are shown as the mean ± SE from three experiments. (E) NO-dependent degradation of ACR in vivo. The plot shows intracellular ACR concentration normalized per milligram of total protein of E. coli harboring either empty vector (pBad) or pNOS_Ban grown in the presence or absence of ACR. Data are shown as the mean ± SE from three experiments. (F) NO protects against ACR-induced ROSs. B. subtilis were pretreated with 0.5 mM bipyridyl (By) or 100 µM NO donor (NO) followed by ACR. The percentage of surviving cells was determined by counting colony-forming units (CFU), and is shown as the mean ± SD from three experiments.

Fig. 2

NO-mediated defense against P. aeruginosa and its mechanism. (A) Chemical structure of the PYO toxin. (B) B. subtilis-generated NO allows growth in the presence of PYO. Growth curves of B. subtilis 6051 (wt) and Δnos strains after the addition of 25 µM PYO (time 0) are shown. After 1 hour, an NO donor (green triangles) or glucose (blue triangles) was added to Δnos cells. Data are shown as the mean ± SE from three experiments. The inset shows tubes with wt (left) and Δnos (right) cultures after a 4-hour incubation with PYO. (C) bNOS expression renders cells resistant to PYO. A paper disk saturated with 10 mM PYO was placed on the B. subtilis bacterial lawn. baNOS indicates B. subtilis expressing nos from B. anthracis. Zone borders are marked with dashed lines. (D) Deletion of the nos gene sensitizes B. anthracis to PYO. Growth curves of B. anthracis Sterne (squares) and Δnos (circles) strains grown in 100 µM PYO are shown. The inset shows tubes with the Sterne (left) and Δnos (right) strains after an 8-hour incubation with PYO. (E) Endogenous NO protects B. subtilis and B. anthracis from P. aeruginosa. Five microliters of a P. aeruginosa PA14 overnight culture was placed on the Bacilli lawns on P agar plates. Lysis zone borders are marked with dashed lines. (F) SodA is critical for bacterial defense against PYO. Experimental conditions were as in (B), except that wt B. subtilis 168 was used as a background strain for all the mutants. Values are the means ± SD from three independent experiments. (G) bNOS controls SodA expression. The lacZ reporter was placed under a chromosomal copy of the sodA promoter (pMsod) in B. subtilis 168 (wt) and Δnos strains. Cultures were sampled to measure the growth [optical density at 600 nm (OD₆₀₀), open symbols] and β-galactosidase activity (solid symbols). Means ± SD from three experiments are given.

Fig. 3

The mechanism of bNOS protection against cefuroxime. (A) Chemical structure of cefuroxime (CEF). (B) bNOS-dependent growth of S. aureus in the presence of CEF. Overnight cultures of S. aureus 4220 and its Δnos derivative were diluted into fresh LB media containing 0.4 µg/ml CEF. Cells were grown in triplicate at 37°C with aeration using a Bioscreen C automated growth analysis system. (C) nos deletion renders B. subtilis more sensitive to cefuroxime. B. subtilis 6051 and Δnos strains were grown to OD₆₀₀ ~1.0, followed by the addition of cefuroxime (25 µg/ml) (time 0). The survival was determined by counting CFU and is shown as the mean ± SD from three independent experiments. (D) Stimulation of bNOS activity by antibiotic treatment. Conditions were the same as in (C). The graph demonstrates the changes in the total nitrate/nitrite concentration in wt and Δnos cultures before and after the addition of CEF (50 µg/ml). (E) NO protects B. subtilis against ROS-mediated CEF toxicity. Conditions were the same as in (C). Cells were pretreated with 0.5 mM bipyridyl (an iron chelator) or 100 µM NO donor or 150 mM thiourea (a ROS scavenger) for 3 min, followed by CEF (50 µg/ml). The percentage of surviving cells was determined by colony formation and is shown as the mean ± SD from four experiments.