Frank R. Stermitz

Multidrug Pump Inhibitors Uncover Remarkable Activity of Plant Antimicrobials

ABSTRACT 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 μg/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.

Enantiomeric-Dependent Phytotoxic and Antimicrobial Activity of (±)-Catechin. A Rhizosecreted Racemic Mixture from Spotted Knapweed

In this communication, we unravel part of the mystery surrounding the allelopathic capability of the noxious weed spotted knapweed ( Centaurea maculosa ). We have found that (−)-catechin is a root-secreted phytotoxin that undoubtedly contributes to spotted knapweeds invasive behavior in the

Acute hepatitis associated with the Chinese herbal product Jin Bu Huan

Estimates of the percentage of patients using alternative medications worldwide range from 4% to 50% [1]. According to a recent U.S. survey, 34% of adult respondents used unconventional therapy and 3% used herbal medicines [2]. In 1990, Americans made 425 million visits to providers of unconventional therapy, which exceeded the number of visits to all primary care physicians, and they spent

Altered Profile of Secondary Metabolites in the Root Exudates of Arabidopsis ATP-Binding Cassette Transporter Mutants

13.7 billion, which exceeded the cost of all hospitalizations in the United States [2]. Herbal products are rapidly gaining popularity in North America as remedies for various medical conditions. Jin Bu Huan Anodyne Tablets (Lycopodium serratum), a traditional Chinese herbal remedy, has been used for more than 1000 years as a sedative and analgesic but has only been available in the United States for 10 years [3]. The alkaloid levo-tetrahydropalmatine is responsible for the morphine-like properties of Jin Bu Huan [4]. A recent study [5] described three children who had taken unintentional overdoses of Jin Bu Huan tablets and who developed central nervous system and respiratory depression with bradycardia. We subsequently identified three adult patients with acute hepatitis associated with Jin Bu Huan ingestion and reported this information to the Centers for Disease Control and Prevention and to the Food and Drug Administration [6]. In the present report, we describe the clinical and laboratory features of seven adult patients (including the previously described patients) who ingested Jin Bu Huan and discuss possible mechanisms for Jin Bu Huan hepatotoxicity. Methods Patients All seven patients were white and had no history of hepatic disease, obesity, diabetes mellitus, or atopy. Six of seven patients were women. All denied a history of excessive alcohol or hepatotoxic drug intake. Risk factors for viral hepatitis were absent in all patients. Five patients resided in Los Angeles, California; three patients had purchased Jin Bu Huan Anodyne Tablets (Kwangsi Pai Se Pharmaceutical/Bose Drug Manufactory, Kwangsi, China) at the same drug store. Two other patients resided in Hawaii and Toronto, Canada, respectively. All patients developed symptoms between March 1993 and March 1994 with the exception of patient 7, whose symptoms began in 1991. Ultrasound examinations of the liver and biliary tract were normal in each patient. Serologic test results in all seven patients were negative for antinuclear, anti-smooth muscle, and antimitochondrial antibodies. Prothrombin times were normal throughout the course of illness of each patient. Case Reports Patient 1 A 66-year-old woman presented to her physician with symptoms of fever, nausea, and fatigue for 5 weeks. She was anicteric with a palpable, nontender liver. Stigmata indicating chronic liver disease were absent. She had taken 2 tablets of Jin Bu Huan at night, 2 to 3 times a week for the previous 3 months, for back pain and insomnia. Her medical history included osteoarthritis. She had used nonsteroidal anti-inflammatory drugs during the previous 2 years without adverse effects. Results of serologic tests showed convalescent antibodies for viral hepatitis A and B. A second-generation enzyme-linked immunosorbent assay for anti-hepatitis C virus was negative. Table 1. Liver Test Results in Patients with Jin Bu Huan Toxicity* Two weeks after she stopped taking Jin Bu Huan, maximal levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were 9.4 kat/L and 5.2 kat/L, respectively, and were near normal 3 weeks later Table 1 and Figure 1 A. At this time, the patient resumed use of Jin Bu Huan for insomnia (2 tablets each night for 7 days). Two weeks after she resumed taking Jin Bu Huan, symptoms returned and enzyme levels were again increased (ALT, 16.0 kat/L; AST, 9.9 kat/L). Liver test results returned to normal 3 weeks later. A second-generation enzyme-linked immunosorbent assay for anti-hepatitis C virus was repeated, and the result was again negative. Twelve weeks later, she was asymptomatic and liver test results were normal. Figure 1. Effect of Jin Bu Huan Anodyne Tablets on aminotransferase levels. Patient 2 A 24-year-old woman presented to her physician with symptoms of fever, nausea, vomiting, fatigue, and pruritus for 3 weeks. She had deep jaundice, excoriations of her extremities, and tender hepatomegaly. Stigmata indicating chronic liver disease were absent. She was hospitalized for 5 days. She had taken 4 tablets of Jin Bu Huan at night for insomnia, 4 times a week for the previous 2 months. She stopped taking Jin Bu Huan 1 week after the onset of symptoms. She had used an oral contraceptive pill daily for the previous 8 years. No other medical illness was present. In-hospital results of liver tests showed the following maximal levels: ALT, 24.5 kat/L; AST, 14.9 kat/L; alkaline phosphatase, 2.2 kat/L; and total bilirubin, 479 mol/L. Results from ultrasound examination of the liver and biliary tract were normal. Results of serologic tests for viral hepatitis A, B, and C; cytomegalovirus; and Epstein-Barr virus were negative. The level of serum ceruloplasmin was normal. The peripheral leukocyte count increased to 10.8 109/L with 7% eosinophils (normal, <3% eosinophils). The liver biopsy specimen showed acute hepatitis with focal necrosis, cholestasis, and inflammation with numerous eosinophils in the portal tracts, results consistent with a drug reaction (Figure 2). Pruritus improved with cholestyramine treatment. Nine weeks after discontinuing Jin Bu Huan use, she was asymptomatic and anicteric, showed resolution of eosinophilia, and had normal liver test results (Table 1). Figure 2. Liver biopsy specimen from patient 2. arrow arrows arrows arrows Patient 3 A 45-year-old woman, a friend of patient 2, presented to her physician with symptoms of nausea, anorexia, fatigue, pruritus, and right upper quadrant abdominal pain. Physical examination showed tender hepatomegaly. Stigmata indicating chronic liver disease were absent. She had taken 4 tablets of Jin Bu Huan at night for insomnia, 3 to 4 times a week for the previous 12 weeks. During the previous 6 months, she had intermittently used another Chinese herbal product, Ma Huang (active ingredients are ephedrine and pseudoephedrine), without adverse effects. She had used no other medications and had no medical illness. She stopped taking both herbal products because of her illness. Two weeks later, she noted jaundice. Results from liver tests showed the following maximal levels: ALT, 21.8 kat/L; AST, 16.7 kat/L; alkaline phosphatase, 3.8 kat/L; and total bilirubin, 58 mol/L. Results of serologic tests for viral hepatitis A, B, and C and Epstein-Barr virus were negative. During the next 4 weeks, symptoms resolved and liver tests showed decreased levels of enzymes (Table 1). However, 12 weeks after she stopped taking the herbal product, she developed an unidentified illness and increased aminotransferase levels. She denied using Jin Bu Huan, alcohol, or other hepatotoxic drugs. Tests for viral hepatitis A, B, and C and Epstein-Barr virus were repeated, and the results were negative for these viruses. Results from liver tests were maximal 7 weeks later (ALT, 10.2 kat/L; AST, 6.5 kat/L) and were normal by 19 weeks (a total of 30 weeks after cessation of Jin Bu Huan). Patient 4 A 48-year-old woman residing in Hawaii presented to her physician with fatigue and a temperature of 40.6 C. The physical examination was normal, and stigmata indicating chronic liver disease were absent. She had taken 3 tablets of Jin Bu Huan nightly for 7 weeks for insomnia. She had used no other medications and had no other medical illness. Liver test results were abnormal (ALT, 3.2 kat/L; AST, 1.2 kat/L; and alkaline phosphatase, 9.2 kat/L). Results of serologic tests for viral hepatitis A, B, and C and Epstein-Barr virus were negative, and the serum ceruloplasmin level was normal. The peripheral leukocyte count was 9.3 109/L with 6% eosinophils. After cessation of Jin Bu Huan, she became asymptomatic, her eosinophilia resolved, and liver test results returned to normal within 4 weeks (Table 1). Patient 5 A 46-year-old man presented to his physician with a temperature of 40.6 C, headaches, fatigue, and tender hepatomegaly. Stigmata indicating chronic liver disease were absent. He had taken 3 tablets of Jin Bu Huan for insomnia, 3 times a week intermittently for 6 months. He had used no other medications and had no other medical illness. Aminotransferase levels were abnormal 2 weeks after stopping Jin Bu Huan (ALT, 6.7 kat/L; AST, 4.7 kat/L). Results of serologic tests for viral hepatitis A, B, and C were negative, and the serum ceruloplasmin level was normal. Five weeks after he stopped taking Jin Bu Huan, symptoms improved and liver test results were normal Table 1 and Figure 1 B. After reuse of Jin Bu Huan for 4 weeks, the ALT level increased to 1.7 kat/L. A second-generation enzyme-linked immunosorbent assay for anti-hepatitis C virus was negative. After Jin Bu Huan use was discontinued, liver test results returned to normal in 4 weeks. Patient 6 A 31-year-old woman presented to her physician with nausea, vomiting, malaise, and deep jaundice. The physical examination was normal, and stigmata indicating chronic liver disease were absent. She had taken 6 tablets of Jin Bu Huan for insomnia, 4 to 6 times a week intermittently for 10 months and then nightly for 8 weeks. She had used no other medications and had no other medical illness. Liver test results were abnormal (ALT, 35.8 kat/L; AST, 17.4 kat/L; alkaline phosphatase, 2.8 kat/L; and total bilirubin, 262 mol/L). Results of serologic tests for viral hepatitis A, B, and C were negative, and the serum ceruloplasmin level was normal. Two weeks after she stopped taking Jin Bu Huan, liver test results showed that the enzyme levels were decreasing (Table 1). She is presently asymptomatic. Patient 7 A 53-year old woman residing in Toronto, Canada, presented to her physician with

Host plant utilization and iridoid glycoside sequestration byEuphydryas anicia (Lepidoptera: Nymphalidae)

Following recent indirect evidence suggesting a role for ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transporter genes highly expressed in the root cells most likely to be involved in secretion processes. Of these 25 genes, we also selected six full-length ABC transporters and a half-size transporter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of these seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals missing in various ABC transporter mutants compared to the wild type when the samples were analyzed by high-performance liquid chromatography-mass spectrometry. These data suggest that more than one ABC transporter can be involved in the secretion of a given phytochemical and that a transporter can be involved in the secretion of more than one secondary metabolite. The primary and secondary metabolites present in the root exudates of the mutants were also analyzed by gas chromatography-mass spectrometry, which allowed for the identification of groups of compounds differentially found in some of the mutants compared to the wild type. For instance, the mutant Atpdr6 secreted a lower level of organic acids and Atmrp2 secreted a higher level of amino acids as compared to the wild type. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters.

DNA cross-linking in mammalian cells by pyrrolizidine alkaloids: Structure-activity relationships

The iridoid glycoside content of individual adultEuphydryas anicia butterflies from two Colorado populations was quantitatively determined. At one site (Red Hill), larval host plants wereCastilleja integra andBesseya plantaginea, while at the other site (Cumberland Pass) a single host plant,B. alpina, was used. At Red Hill, macfadienoside and catalpol were sequestered, while at Cumberland Pass, catalpol and aucubin were sequestered. Artificial diet studies showed that larvae hydrolyzed a major iridoid ofB. plantaginea, 6-isovanilIylcatalpol, to catalpol (which was sequestered) and isovanillic acid (which was excreted). Large year-to-year and individual variation in butterfly iridoid content was established as was a female-male difference in macfadienoside vs. catalpol content. Larval host plant distributions and numbers were determined at Red Hill for two years and compared with changes in butterfly populations and sequestered iridoids.

Inhibitors of Bacterial Multidrug Efflux Pumps Potentiate Antimicrobial Photoinactivation

Pyrrolizidine alkaloids (PAs) are common constituents of many species of flowering plants which possess carcinogenic as well as anticarcinogenic activity in vivo. Pyrrolizidine alkaloids are genotoxic in various short-term assays. The mechanisms by which these compounds exert these effects is still unclear. In this study, we characterized the ability of eight bifunctional PAs, with differing stereochemistry and functional groups, to cross-link cellular DNA in cultured bovine kidney epithelial cells. PAs representative of three major structural classes, the macrocycles (seneciphylline, riddelline, retrorsine, senecionine, monocrotaline), the open diesters (heliosupine, latifoline), and pyrrolizidine base (retronecine) were cultured for 2 hr with cells and an external metabolizing system. Every PA induced DNA cross-links which consisted primarily of proteinase-sensitive cross-links (DPC), but also to a smaller extent, DNA interstrand cross-links (ISC). None of the PAs induced detectable amounts of DNA single-strand breaks. The PAs which produced DPC and/or ISC (ranked from most potent to least) were: seneciphylline (DPC greater than ISC); riddelline (DPC greater than ISC); retrorsine (DPC greater than ISC); senecionine (DPC greater than ISC); heliosupine (DPC greater than ISC); monocrotaline (ISC = DPC); latifoline (DPC greater than ISC); and retronecine (ISC greater than DPC). Although the PAs induced DNA cross-linking to varying degrees, cell viabilities for all treatment groups were greater than 90% as determined by trypan blue dye exclusion. Since the cross-linking ability of these PAs paralleled their ability to inhibit colony formation, cross-link formation may be involved in the biological activity of these compounds. Two structural determinants of biological activity appear to be the presence of both a macrocyclic necic acid ester and an alpha,beta-unsaturated ester function since the cross-linking ability of seneciphylline, riddelline, retrorsine, and senecionine far exceeded that of monocrotaline, heliosupine, latifoline, and retronecine. In addition, the stereochemical orientation of the ester linkage was found to have no effect on biological activity.

Oxalate contributes to the resistance of Gaillardia grandiflora and Lupinus sericeus to a phytotoxin produced by Centaurea maculosa

ABSTRACT Antimicrobial photodynamic inactivation (APDI) combines a nontoxic photoactivatable dye or photosensitizer (PS) with harmless visible light to generate singlet oxygen and reactive oxygen species that kill microbial cells. Cationic phenothiazinium dyes, such as toluidine blue O (TBO), are the only PS used clinically for APDI, and we recently reported that this class of PS are substrates of multidrug efflux pumps in both gram-positive and gram-negative bacteria. We now report that APDI can be significantly potentiated by combining the PS with an efflux pump inhibitor (EPI). Killing of Staphylococcus aureus mediated by TBO and red light is greatly increased by coincubation with known inhibitors of the major facilitator pump (NorA): the diphenyl urea INF271, reserpine, 5′-methoxyhydnocarpin, and the polyacylated neohesperidoside, ADH7. The potentiation effect is greatest in the case of S. aureus mutants that overexpress NorA and least in NorA null cells. Addition of the EPI before TBO has a bigger effect than addition of the EPI after TBO. Cellular uptake of TBO is increased by EPI. EPI increased photodynamic inactivation killing mediated by other phenothiazinium dyes, such as methylene blue and dimethylmethylene blue, but not that mediated by nonphenothiazinium PS, such as Rose Bengal and benzoporphyrin derivative. Killing of Pseudomonas aeruginosa mediated by TBO and light was also potentiated by the resistance nodulation division pump (MexAB-OprM) inhibitor phenylalanine-arginine beta-naphthylamide but to a lesser extent than for S. aureus. These data suggest that EPI could be used in combination with phenothiazinium salts and light to enhance their antimicrobial effect against localized infections.

Transcriptome analysis of Arabidopsis roots treated with signaling compounds: a focus on signal transduction, metabolic regulation and secretion

Centaurea maculosa Lam. is a noxious weed in western North America that produces a phytotoxin, (±)-catechin, which is thought to contribute to its invasiveness. Areas invaded by C. maculosa often result in monocultures of the weed, however; in some areas, North American natives stand their ground against C. maculosa and show varying degrees of resistance to its phytotoxin. Two of these resistant native species, Lupinus sericeus Pursh and Gaillardiagrandiflora Van Houtte, were found to secrete increased amounts of oxalate in response to catechin exposure. Mechanistically, we found that oxalate works exogenously by blocking generation of reactive oxygen species in susceptible plants and reducing oxidative damage generated in response to catechin. Furthermore, field experiments show that L. sericeus indirectly facilitates native grasses in grasslands invaded by C. maculosa, and this facilitation can be correlated with the presence of oxalate in soil. Addition of exogenous oxalate to native grasses and Arabidopsis thaliana (L.) Heynh grown in vitro alleviated the phytotoxic effects of catechin, supporting the field experiments and suggesting that root-secreted oxalate may also act as a chemical facilitator for plant species that do not secrete the compound.

Two phytoalexins from sugarbeet (Beta vulgaris) leaves

Gene expression in response to signaling molecules has been well studied in the leaves of the model plant species Arabidopsis thaliana. However, knowledge of gene expression and metabolic regulation at the root level is limited. Here, the signaling compounds salicylic acid (SA), methyl jasmonate (MeJA) and nitric oxide (NO) were applied exogenously to induce various defense responses in roots, and their effect was studied using a combination of genomic, molecular and biochemical approaches. Genes involved in defense signaling/activation, cellular redox state, metabolism, transcription factors and membrane transport were altered in expression following treatment with SA, MeJA and NO. In addition, it was found that SA-, MeJA- and NO-elicited roots increased the root exudation of phytochemicals compared with the roots of nontreated control plants. Transport systems likely to be involved in the root exudation of phytochemicals, including the MATE, ABC, MFS, amino acid, sugar and inorganic solute transporters, showed altered expression profiles in response to treatments. Overall, significant differences were found in the signaling compound-elicited expression profiles of genes in roots vs those in leaves. These differences could be correlated to the underground nature of roots and their exposure to higher microbial inoculum rates under natural conditions.