Interaction of chromosomal stains with DNA

Multidrug resistance pumps (MDRs) protect microbial cells from both synthetic and natural antimicrobials. Amphipathic cations are preferred substrates of MDRs. Berberine alkaloids, which are cationic antimicrobials produced by a variety of plants, are readily extruded by MDRs. Several Berberis medicinal plants producing berberine were found also to synthesize an inhibitor of the NorA MDR pump of a human pathogen Staphylococcus aureus. The inhibitor was identified as 5-methoxyhydnocarpin (5-MHC), previously reported as a minor component of chaulmoogra oil, a traditional therapy for leprosy. 5-MHC is an amphipathic weak acid and is distinctly different from the cationic substrates of NorA. 5-MHC had no antimicrobial activity alone but strongly potentiated the action of berberine and other NorA substrates against S. aureus. MDR-dependent efflux of ethidium bromide and berberine from S. aureus cells was completely inhibited by 5-MHC. The level of accumulation of berberine in the cells was increased strongly in the presence of 5-MHC, indicating that this plant compound effectively disabled the bacterial resistance mechanism against the berberine antimicrobial.multidrug resistance ͉ efflux inhibitor B acteria have evolved numerous defenses against antimicrobial agents, and drug-resistant pathogens are on the rise (1). A general and effective defense is conferred by ubiquitous multidrug resistance pumps (MDRs), membrane translocases that extrude structurally unrelated toxins from the cell (2-5). Preferred substrates of most MDRs are synthetic hydrophobic cations such as quaternary ammonium antiseptics (6, 7). We have identified a group of cationic berberine alkaloids as natural substrates of MDR pumps (6). We suggested that berberine alkaloids represent a possibly larger group of cationic toxins that fueled the evolution of MDRs (7). Considering that microbial MDRs can render berberine alkaloids essentially ineffective, we reasoned that plants would benefit from making an MDR inhibitor. Here we show that Berberis fremontii, a berberine producer (8) used in Native American traditional medicine (9, 10), synthesizes a potent MDR inhibitor. Structural determination identified the substance as 5Ј-methoxyhydnocarpin (5Ј-MHC). Efflux of berberine from pathogenic Staphylococcus aureus expressing the NorA MDR pump that confers resistance to quinolones and antiseptics (6,11,12) was inhibited completely by 5Ј-MHC. This is a clear example of synergy between components of a medicinal plant described at a molecular level. Materials and MethodsCell Culturing and Susceptibility Testing. S. aureus 4222 parent strain and the norA mutant KLE 820 (6) were cultured in Mueller-Hinton (MH) broth. Cells (10 5 ͞ml) were inoculated into MH broth and dispensed at 0.2 ml͞well in microtiter plates. All tests were done in triplicate by following National Center for Clinical Laboratory Standards recommendations. Briefly, minimal inhibitory concentrations (MIC) were determined by serial 2-fold dilution of test compounds. MIC was defined as a concentr...

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“…1) that resembles EtdBr and binds to DNA (25). The DNA binding apparently contributes to the antimicrobial activity of berberine.…”

Section: Resultsmentioning

confidence: 99%

Synergy in a medicinal plant: Antimicrobial action of berberine potentiated by 5′-methoxyhydnocarpin, a multidrug pump inhibitor

Stermitz

Lorenz

Tawara

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

646

461

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“…It is an amphipathic cation that resembles quaternary ammonium antiseptics in its chemical properties and possibly in its mechanism of action as well. The likely targets of berberine are the cytoplasmic membrane and DNA, into which it intercalates (20). Amphipathic cations are the preferred substrates of most MDRs (26), and we reasoned that the low level of activity of berberine might result from effective efflux.…”

Section: Discussionmentioning

confidence: 99%

Multidrug Pump Inhibitors Uncover Remarkable Activity of Plant Antimicrobials

Tegos

Stermitz

Lomovskaya

et al. 2002

Antimicrob Agents Chemother

426

317

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Plant antimicrobials are not used as systemic antibiotics at present. The main reason for this is their low level of activity, especially against gram-negative bacteria. The reported MIC is often in the range of 100 to 1,000 g/ml, orders of magnitude higher than those of common broad-spectrum antibiotics from bacteria or fungi. Major plant pathogens belong to the gram-negative bacteria, which makes the low level of activity of plant antimicrobials against this group of microorganisms puzzling. Gram-negative bacteria have an effective permeability barrier, comprised of the outer membrane, which restricts the penetration of amphipathic compounds, and multidrug resistance pumps (MDRs), which extrude toxins across this barrier. It is possible that the apparent ineffectiveness of plant antimicrobials is largely due to the permeability barrier. We tested this hypothesis in the present study by applying a combination of MDR mutants and MDR inhibitors. A panel of plant antimicrobials was tested by using a set of bacteria representing the main groups of plant pathogens. The human pathogens Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica serovar Typhimurium were also tested. The results show that the activities of the majority of plant antimicrobials were considerably greater against the gram-positive bacteria Staphylococcus aureus and Bacillus megaterium and that disabling of the MDRs in gram-negative species leads to a striking increase in antimicrobial activity. Thus, the activity of rhein, the principal antimicrobial from rhubarb, was potentiated 100-to 2,000-fold (depending on the bacterial species) by disabling the MDRs. Comparable potentiation of activity was observed with plumbagin, resveratrol, gossypol, coumestrol, and berberine. Direct measurement of the uptake of berberine, a model plant antimicrobial, confirmed that disabling of the MDRs strongly increases the level of penetration of berberine into the cells of gram-negative bacteria. These results suggest that plants might have developed means of delivering their antimicrobials into bacterial cells. These findings also suggest that plant antimicrobials might be developed into effective, broad-spectrum antibiotics in combination with inhibitors of MDRs.

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“…The author indicates on affinity of QM to guanine reflected by quenching of QM fluorescence upon binding to polynucleotides with high GC content. Using electroluminescence method Jennings and Ridler [29] found that QM intercalates into DNA in a similar manner as ethidium bromide, which is considered a classical model of intercalation. However, we were unable to find in the literature the spectral data and interaction constants of QM binding to DNA.…”

Section: Intercalator-dna Systemmentioning

confidence: 99%

The “interceptor” properties of chlorophyllin measured within the three-component system: Intercalator–DNA–chlorophyllin

Pietrzak

Wieczorek

et al. 2006

Biophysical Chemistry

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In aqueous solutions, in the presence of double-stranded DNA, chlorophyllin (CHL) forms complexes with each of the three DNA intercalators: acridine orange (AO), quinacrine mustard (QM), and doxorubicin (DOX). The evidence for these interactions was obtained by measurement changes in the absorption and fluorescence spectra of the mixtures containing DNA and intercalators during titration with CHL. A model of simple competition between DNA and CHL for the intercalator was used to define the measured interactions. The concentrations of the complexes estimated based on this model were consistent with the concentrations obtained by actual measurement of the absorption spectra.The present data provide further support for the role of chlorophyllin as an "interceptor" that may neutralize biological activity of aromatic compounds including mutagens and antitumor drugs.

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Interaction of chromosomal stains with DNA

Cited by 33 publications

References 14 publications

Synergy in a medicinal plant: Antimicrobial action of berberine potentiated by 5′-methoxyhydnocarpin, a multidrug pump inhibitor

Synergy in a medicinal plant: Antimicrobial action of berberine potentiated by 5′-methoxyhydnocarpin, a multidrug pump inhibitor

Multidrug Pump Inhibitors Uncover Remarkable Activity of Plant Antimicrobials

The “interceptor” properties of chlorophyllin measured within the three-component system: Intercalator–DNA–chlorophyllin

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