Artesunate potentiates antibiotics by inactivating heme-harbouring bacterial nitric oxide synthase and catalase
© Zeng et al; licensee BioMed Central Ltd. 2011
Received: 20 February 2011
Accepted: 30 June 2011
Published: 30 June 2011
A current challenge of coping with bacterial infection is that bacterial pathogens are becoming less susceptible to or more tolerant of commonly used antibiotics. It is urgent to work out a practical solution to combat the multidrug resistant bacterial pathogens.
Oxidative stress-acclimatized bacteria thrive in rifampicin by generating antibiotic-detoxifying nitric oxide (NO), which can be repressed by artesunate or an inhibitor of nitric oxide synthase (NOS). Suppressed bacterial proliferation correlates with mitigated NO production upon the combined treatment of bacteria by artesunate with antibiotics. Detection of the heme-artesunate conjugate and accordingly declined activities of heme-harbouring bacterial NOS and catalase indicates that artesunate renders bacteria susceptible to antibiotics by alkylating the prosthetic heme group of hemo-enzymes.
By compromising NO-mediated protection from antibiotics and triggering harmful hydrogen peroxide burst, artesunate may serve as a promising antibiotic synergist for killing the multidrug resistant pathogenic bacteria.
A current challenge of coping with bacterial infection is that bacterial pathogens are becoming less susceptible to or more tolerant of commonly used antibiotics. For example, globally endemic tuberculosis caused by multidrug resistant strains of Mycobacterium tuberculosis remains a formidable threat to human health. Although the development of more potent antibiotics may prohibit the lethal pathogens from worldwide transmission, it has proved to be costly, time-consuming and technically difficult . Otherwise, if an antibiotic synergist like artesunate can benefit to fight against the antibiotic resistant bacteria, it will minimise the dosage of antibiotics in antibacterial therapy and diminish the heavy incidence and rapid transmission of multidrug resistant bacterial pathogens.
Bacterial culture and NO content estimation
A single colony of B. licheniformis BL20386 or E. coli DH5α was inoculated in LB broth and cultured overnight at 37°C. A 1% aliquot of pre-cultured bacteria was inoculated in LB broth and cultured at 37°C until absorbance at 600 nm (A600) to 0.6. For oxidative stress acclimatization, triangle bottles with the bacterial culture were placed without agitation for 3 d at room temperature (the hypoxia group) or in a 4°C refrigerator (the hypoxia + cold group). For antibiotic exposure, a 1% aliquot of the overnight bacterial culture was inoculated in LB broth supplementing with different concentrations of rifampicin, cefotaxime, or ampicillin. For inhibitor treatment, a 1% aliquot of the overnight bacterial culture was inoculated in LB broth supplementing with 60 μg/ml artesunate or 1 mM NG-monomethyl-L-arginine monoacetate (L-NMMA). On each day, 1 ml of the bacterial culture was taken out for estimation of NO content using a commercially available kit and according to the manufacturer's instruction. The content of NO is represented by the amount of nitrate (NO3-)/nitrite (NO2-). For plotting a standard curve of nitrate/nitrite, 1 M NaNO3 was dissolved to a series of dilutions (1, 2, 5, 10, 20, 30, 40, 60 μM) by LB broth for measurement of A540, from which a regression equation and a determinant coefficient were calculated.
Bacterial growth assay
For monitoring the growth rate of oxidative stress-acclimatized bacteria, a 1% aliquot of the bacterial culture standing at room temperature for 3 d or that without oxidative stress treatment was inoculated in LB broth supplementing with rifampicin and cultured at 37°C with agitation. After cultured for 3, 6, 9 and 12 h, 1 ml of the bacterial culture was taken out and diluted with 9 ml of fresh LB broth to measure A600 for plotting the growth rate curve. For assaying the bacterial growth rate following treatment by rifampicin or artesunate + rifampicin, a 1% aliquot of the overnight bacterial culture was inoculated in LB broth supplementing with 60 μg/ml artesunate and different concentration of rifampicin, and cultured at 37°C. After cultured for 3, 6, 9, 12 and 24 h, 1 ml of the bacterial culture was diluted with 9 ml of fresh LB broth to measure A600 for plotting the growth rate curve.
Detection of heme and heme-artesunate conjugate
A 1% overnight culture of B. licheniformis BL20386 was inoculated in LB broth supplementing with 60 μg/ml artesunate and cultured at 37°C for 2 d. The triangle bottle with a pre-culture was first placed at ambient temperature for 1 d and subsequently at a 4°C refrigerator for 1 d. After supplementing with fresh LB broth and 60 μg/ml artesunate, the bacterial strain was continuously cultured overnight at 37°C. The culture was taken out on 3, 6 and 9 h for collecting cells by centrifugation and lysing them through repeated cycles of frozen-thaw, and finally the lysate was applied to measure A415 and A476.
Determination of catalase activity
A 1% overnight culture of B. licheniformis BL20386 was inoculated in LB broth supplementing with 60 μg/ml artesunate in the treatment group or without artesunate in the control group, and cultured at 37°C for 2 d. The triangle bottle with a pre-culture was first placed at ambient temperature for 1 d and subsequently at a 4°C refrigerator for 1 d. After supplementing with fresh LB broth containing 60 μg/ml artesunate or 60 μg/ml artesunate + different concentrations of rifampicin, the bacterial strain was continuously cultured overnight at 37°C. The culture was taken out from each group on 9, 12 and 24 h for measurement of A405 using a commercially available kit. The activity of catalase (U/ml) was calculated according the formula of (A405 control group - A405 treatment group) × 271 × 1/6.
This work was supported by Natural Science Foundation of Guangdong China (No. 9145624536-4000003) and Scientific Development Project of Guangdong China (No. 2007B031404008).
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