plik

Modulation of the antifungal activity of new medicinal plant extracts active on

Candida glabrata by the major transporters and regulatorsof the pleiotropic drug-

resistance network in Saccharomyces cerevisiae.

By Kolaczkowski, Marcin

Introduction

TWO YEAST SPECIES, Candida albicans and Candida glabrata, cause the majority of human

fungal infections, for which a limited number of effective treatments is available. Systemic infections,

associated with high mortality rates especially in immunocompromised patients, areparticularly difficult to

cure. This often results from the increased tolerance to the most commonly used azole antifungals, including

fluconazole and ketoconazole, generally observed for C. glabrata and the rise in azole-resistant C. albicans

infections. (9,26,38) Exposure of yeast to azoles promotes the development of resistance, which is most often

associated with alterations in their target, Erg11, as wellas overproduction of multidrug efflux pumps, such

as CaCdr1, CaCdr2,and CaMdr1 of C. albicans (10,29,27) or CgCdr1, CgCdr2, and CgSng2 of C. glabrata.

(19,28,37) New treatment strategies, especially those active against azole-resistant isolates, are urgently

needed.

The molecular mechanisms of multidrug-resistance development are best understood in

Saccharomyces cerevisiae, which is evolutionarily related to C. glabrata. In baker's yeast, prolonged

exposure to many toxic compounds, including azoles, leads to the acquisition of point mutations leading to

an overproduction of multidrug efflux pumps in plasma membranes. These gain-of-function mutations

cluster in two homologous transcriptional regulators of the [Zn.sub.2][Cys.sub.6] family,Pdr1p and Pdr3p,

largely increasing their activatory capabilities. (13,24) The ATP-binding cassette superfamily multidrug

transporters PDR5, SNQ2, and YOR1 are among the most highly induced targets, next toother genes

affecting different cellular functions, including lipid metabolism. (7,8,11,16) The substrate profile of the

major contributor to baker's yeast's pleiotropic drug resistance, Pdr5p, overlaps to some extent with profiles

of Sng2 and Yorlp and strikingly resembles those of CaCdr1 and CaCdr2. (15,29) Although a number of

medicinal plants had long been used to treat infections, relatively few extracts showing activity against fungi,

mostly at relatively high concentrations, were identified. Another complication in the process of isolation of

small molecule antifungal components of plants is the loss of activity during further fractionation steps.

Medicinal plants are, however, a rich source of novel, complex, and diverse chemical structures, which

warrants their more thorough investigation as a potential source of novel antifungal agents. The extent to

which multidrug efflux proteins interfere with the antifungal action of the newly identified extracts, active on

C. glabrata,was explored here using the related model yeast species S. cerevisiae.

Materials and Methods Reagents

The following reagents were purchased from the respective suppliers: yeast extract, tryptone,

peptone, and agar (Becton Dickinson, Warsaw, Poland); glucose and sodium chloride (Standard, Lublin,

Poland); ketoconazole and RPMI1640 (Sigma, Poznan, Poland); and MOPS (Amresco,Solon, OH). Strains

and growth conditions The C. albicans CCM8320 and C. glabrata CCM8270 strains were from the Czech

Collection of Microorganisms. The clinical C. albicans isolates 15 and 28 (39) were kindly provided by

Theodore White. The clinical C. glabrata isolates BPY112 and 126 (30) were kindly provided by Maurizio

Sanguinetti. The bacterial strains of Escherichia coli K12 and Staphylococcus aureus F137 were kindly

provided by Jacek Bania. The isogenic S. cerevisiae strains are described in Table 1. Strains FYMK 26/8-4D

and -9C are segregants from FYMKD 26/8. Disruption of YOR1in FYMK 26/8-4D, -9C, -5A, -5C was

performed as previously described. (15) The minimal inhibitory concentrations (MICs) for the Candida

strains were determined by the microdilution assay in MOPS-buffered RPMI 1640 medium as previously

described. (6) MIC values for S. cerevisiae were determined by the microdilution assay essentially as

previously described. (17) Growth was monitored by [OD.sub.550] and [OD.sub.600] measurements in a

microplate reader as well as by visual inspection after 24 and 48h. The extracts and the purified compounds

were added as dimethylsulfoxide stock solutions. This solvent did not affectgrowth at the applied

concentrations. The MIC values represent the values observed in three independent measurements, in which

the error did not exceed the window determined by the serial dilution factor. The growth not exceeding 20%

of the solvent control was used for MIC breakpoint determination. The MIC values for bacterial strains were

determined by the microdilution procedure in the Mueller-Hinton II medium (cation adjusted) according to

the previously published guidelines. (22) Plant material Dried, ground plant material was extracted

sequentially with hexane, chloroform [(CHCl.sub.3)], ethyl acetate (EtOAc), and methanol (MeOH), and the

solvent was removed in vacuo. The growth inhibition assays were conducted on the dried solvent residues. In

the case of Artemisia annua, the EtOAc residue was then chromatographed on silica gel and the fractions

listed in Table 5 correspond to those described in Stermitz et al. (35) Mahonia fortunei was a gift from Dr.

Xihuang Ji.(12) Berberis fendleri was described in Stermitz et al. (33) Pinus edulis was described in Stermitz

et al. (36) Hydrastis canadensis was field grown and a gift from Dean Gray (Herb Pharm, Williams, OR).

Dalea formosa was collected near Spring Canyon Rockbound State Park SE of Deming, New Mexico

(voucher FRS 540). Zanthoxylum zanthoxyuloides was a gift from Abayomi Sofowora, University of Ife,

Nigeria. A. annuawas a gift from Hauser Research Laboratories, Boulder, C0. (35) Results Identification of

new medicinal plant extracts active on pathogenic yeast In the search for new antifungal medicinal plant

extracts, we identified several fractions showing growth inhibitory activity against C. glabrata with MIC

values below 120 [micro]g/ml (Table 2). A generally lower susceptibility was observed for C. albicans

(Table 2). The antifungal activity of extracts was dependent on the solvent used for fractionation (Table 2).

As development of resistance to the most commonly used azole antifungals becomes an increasingly

important problem in the chemotherapy of fungal infections, the effectiveness of the most potent extracts of

D. formosa, the EtOAc extract of A. annua, and the methanolic extract of H. canadensis leaves against azole-

resistant clinical isolates of C. albicans and C. glabrata was also verified. A comparable activity to that

reported in Table 2, of the H. canadensis and A. annua extracts (MIC of 160 and 240[micro]g/ml,

respectively) against both of the previously characterized azole-resistant C. albicans isolates 15

(overexpressing CDR1 and CDR2) and 28 (overexpressing CDR2), (39) was observed. These isolates,

similarly to the azolesensitive reference strain (Table 2), were not inhibited by the D. formosa extract. The

latter was active, however, with a similar potency (MIC of 10[micro]g/ml) against a matched pair of the

azole-sensitive clinical isolate of C. glabrata BPY112 and resistant, overexpressing CgCDR1, CgCDR2, and

CgSNQ2, BPY 126. (30) Both isolates also showeda similarity to that reported in Table 21evel of sensitivity

to H. canadensis (MIC of 20 [micro]g/ml) and A. annua (MIC of 60 [micro]g/ml) extracts. To verify the

pathogen selectivity, the antibacterial activity of the most potent extracts against the representatives of Gram-

negative(E. coli) and Gram-positive species (S. aureus) was measured. The extracts of D. formosa and A.

annua did not show any antibacterial activity, at the highest concentrations used, whereas the methanolic

extract of H. canadensis leaves was only active against S. aureus with the MIC of 80 [micro]g/ml, which was

within the range of its anticandidal activity. The pleiotropic drug-resistance network of genes differentially

affects the antifungal properties of active crude extracts The therapeutic effect of plants that contain complex

mixtures of secondary metabolites often results from the synergistic action of several compounds. Such a

synergistic strategy has recently been shown to potentiate the antibacterial activity of the alkaloid berberine

bythe efflux pump inhibitor 5-methoxyhydnocarpin. (34) The latter compound has no growth inhibitory

properties when applied alone, but potentiates berberine activity against S. aureus through inhibition of the

Nor A multidrug efflux transporter. Because separation of growth inhibitory and efflux modulatory activities

may lead to the often observed loss of activity during fractionation, we have further characterized the

identified extracts using the related to C. glabrata model yeast S. cerevisiae. This system has the advantage

of having a well-characterized network of multidrug-resistance genes, and can easily be manipulated

genetically, in contrast to the clinical Candida isolates,in which resistance is often mulifactorial and less well

understood.(30,39) The effect of the extracts on the growth of single and multiple knockouts in the multidrug

transporter genes PDR5, SNQ2, and YOR1 in the overexpressing PDR1-3 regulatory mutant or the strain

bearing the wild-type PDR1 allele was measured. Double disruption of the regulators PDR1 and PDR3 was

also included. In the case of the A. annua EtOAc extract, the increased sensitivity to growth inhibition in the

hyperactivating PDR1-3 regulatory mutant background upon disruption of PDR5, SNQ2, and YOR1 was

observed (Table 3). Disruptions not affecting growth were omitted for clarity. A similar response to this

extract wasseen for the corresponding knockouts in the PDR1 wild-type background. The double disruption

of PDR1 and PDR3 had a similar effect on theactivity of the A. annua EtOAc extract as the inactivation of

the three transporters (Table 4). In contrast, disruption of both regulators had a profound effect on growth

inhibition by the methanolic extract of P. edulis needles (Table 4). The triple knockout of PDR5, SNQ2, and

YOR1 showed increased sensitivity to other extracts, excluding Z.zanthoxyloides, for which disruption of

the analyzed drug-resistancegenes did not increase its growth inhibitory effect (Table 4). Fractionation of A.

annua extract reveals the separation of growthinhibitory and azole-sensitizing activities Further analysis of

fractions of the extract of A. annua after silica gel chromatography revealed the presence of two kinds of

activities. The growth inhibitory activity against the reference strain of S.cerevisiae was only seen in

fractions 5 and 6. This activity was strongly enhanced by deletion of the major multidrug transporters PDR5,

SNQ2, and YOR1 and extended to fraction 11, peaking in fraction 8. Another activity that was observed was

the potentiation of the antifungal effect of a subinhibitory dose of ketoconazole. It showed a different

distribution profile from the growth inhibitory activity and was present in fractions 5 and 9 to 12 as well as

18 (Table 5). Artemisinin shows a toxicity profile different from that of the crude extract and affects

ketoconazole tolerance Artemisinin is an important active ingredient that was isolated from A. annua and is

well known for its antimalarial activity. Analysisof its growth inhibitory properties against a set of isogenic

S. cerevisiae strains bearing disruptions of the major multidrug ABC transporter genes in the

multidrugresistant PDR1-3 background revealed a dependence on PDR5 expression with no further influence

of the inactivation of YOR1 and SNQ2 (Table 6 and data not shown). In addition, artemisinin potentiated the

antifungal effect of ketoconazole. This effect was much stronger in the presence of the wild-type PDR1 allele

as compared with the multidrugresistant PDR1-3 mutant highly overproducing Pdr5p, Sng2p, and Yorlp

(Table 6).

Discussion

The success of antifungal chemotherapy is often compromised due toa limited number of effective

drugs and the development of resistance. C. glabrata, which generally shows a low sensitivity to the most

commonly used azole drugs, is rarely used for natural products screening. In this study several crude

medicinal plant extracts mainly inhibiting the growth of this second most frequent human fungal pathogen

were identified. These include the most active extract of D. formosa, the medicinalherb native to the

southwestern United States and northern Mexico, used as a remedy for influenza and viral infections as well

as to relieve aches and growing pains. (4) Its antifungal activity had not beenreported previously. The

antimicrobial properties of berberine-containing plants, including Berberis/Mahonia species as well as the

widely used goldenseal (H. canadensis) that is native to southeastern Canada and northeasternUnited States,

have long been attributed to the presence of this alkaloid. (3,21) The observed anticandidal effect of

Berberis/Mahonia extracts corresponded to the distribution of berberine, which is predominantly found in the

roots of Berberis species, whereas high amounts of berberine are present in M. fortunei leaves. (33) A similar

growthinhibitory effect of another berberine-containing medicinal herb Coptis chinensis against C. glabrata,

with reduced sensitivity of C. albicans, was reported recently. (31) The growth inhibitory effect of

goldenseal (H. canadensis) leaves, however, is probably related to components other than berberine, which is

located in the roots and rhi zomes of this plant. Hydrastine, a possible candidate present in both rhizomes and

leaves, proved inactive (data not shown). Z. zanthoxyloides is an important African herb used to treat several

kinds of infections, especially those associated with dental problems. (5) Although we could not detect its

growth inhibition of C. albicans, a similar observation was made by others with the aqueous-ethanolic

extracts of the roots and stem bark of this plant, which showed weak activity only at very high

concentrations, with MIC values of 1,000 [micro]g/ml. (23) However, for the first time, we demonstrate its

potent growth inhibitory effect against C. glabrata. Further analysis of the most active extracts of A. annua,

H. canadensis, and D. formosa revealed their similar growth inhibitory activity against the azole-sensitive

and azoleresistant Candida isolates. This could result from the synergistic effects of complex mixtures of

plant secondary metabolites. Interestingly, compounds isolated from species related to D. formosa, such as

the arylbenzofuran derivatives from Dalea spinosa (2) and a chalcone from Dalea versicolor, (1)

werereported to potentiate antibiotic activities against S. aureus. To further characterize the identified

extracts, the well-defined isogenic strains of the model yeast, S. cerevisiae, expressing different levels of

their most important endogenous drug efflux pumps and their regulators were used. This approach revealed

three types of responses. Whereas the toxicity of a crude EtOAc extract of A. annua was dependent on the

combined presence of the major ABC MDR transporter genes PDR5, SNQ2, and YOR1 (Tables 3 and 4), the

sensitivity of the methanolic extract of P. edulis needles only slightly increased in their absence. In this case,

however, deletion of the master regulators of the pleiotropic drugresistance network PDR1 and PDR3 had a

much stronger effect (Table 4). In contrast, the EtOAc extract of Z. zanthoxyloides was not detoxified by the

pleiotropic drug-resistance network (Table 4), suggesting the possible presence of antifungal components

escaping the activity of the major drug efflux pumps. The presence of Pdr5p, Yorlp, and Sng2p substrates in

the crude extract of A. annua suggested that it may also contain modulators of antifungal drug resistance.

This was further supported by the analysis of the chromatographic fractions of the extract in which a

different distribution of the antifungal and ketoconazole-sensitizing activities was observed, thanks to the use

of the well-defined strains of the model yeast S. cerevisiae. Several reports suggest a synergistic activity

between artemisinin and other constituents of A. annua crude extract against Plasmodium. (20,25) In

particular, the flavones chrysosplenol-D and chrysosplenetin from A. annua were reported to potentiatethe

antimalarial activity of artemisinin against Plasmodium falciparum. (18) These two flavonols, showing only

very weak growth inhibitory action, have also been shown to potentiate the activity of berberine and

norfloxacin against a drug-resistant strain of S. aureus. (35) In contrast to the crude extract, artemisinin was

specifically detoxified by Pdr5p, suggesting that it is a transported substrate. Interestingly, artemisinin also

potentiated the antifungal activity of ketoconazole. This may at least in part be related to the competition for

transport by Pdr5p with ketoconazole. This would be in line with the stronger effect observed in the wild-

type strain than in the PDR1-3 mutant, which highly overproduces Pdr5p. The identification of the medicinal

plant extracts active against Candida isolates of reduced azole susceptibility and showing pathogenselectivity

is important from a practical view point, especially as A. annua is promoted in the United States for the

treatment of yeast infections. (32) Our study suggests a similar use for the identified extracts of D. formosa

and the leaves of the commonly used goldensealH. canadensis. Our observations supporting the view that the

often observed decrease of antifungal activity upon crude medicinal plant extract fractionation may result

from the separation of synergistically acting modulators of efflux pumps and growth inhibitory compounds

have practical implications for the development of new antifungals and reversing agents. Acknowledgments

We are grateful to Aleksander Sikorski, Jan Szopa, and Antoni Polanowski for sharing laboratory equipment;

Xihuang Ji, Dean Gray, and Abayomi Sofowora for sharing plant material; and Theodore White, Maurizio

Sanguinetti, and Jacek Bania for the kind gift of strains. This work was supported by the Ministry of Science

and Higher Education (funds for the Wroclaw Medical University). Disclosure Statement No competing

financial interests exist.

References

(1.) Belofsky, G., R. Carreno, K. Lewis, A. Ball, G. Casadei, and G.P. Tegos. 2006. Metabolites of

the

"Smoke

Tree"

Dalea spinosa,

potentiate

antibiotic

activity

against

multidrug-resistant

Staphylococcusaureus. J. Nat. Prod. 69:261-264.

(2.) Belofsky, G., D. Percivill, K. Lewis, G.P. Tegos, and J. Ekart. 2004. Phenolic metabolites of

Dalea versicolor that enhance activity against model pathogenic bacteria. J. Nat. Prod. 67.481-484.

(3.) Birdsall, T.C., and G.S. Kelly. 1997. Berberine: therapeutic potential of an alkaloid found in

several medicinal plants. Alt. Med.Rev. 2:94-103.

(4.) Carter, J.L. 1997. Trees and Shrubs of New Mexico. Johnson Books, Boulder, CO.

(5.) Chukwuka, K.S., and F.T. Agulanna. 2005. Laboratory assay of the active ingredients in

Zanthoxylum zanthoxyloides (Lam) Zepern andTimler and its remedy in dental problems. Int. J. Biomed.

Health. Sci. 1:37-40.

(6.) Cuenca-Estrella, M., W. Lee-Yang, M.A. Ciblak, B.A. Arthington-Skaggs, E. Mellado, D.W.

Warnock, and J.L. Rodriguez-Tudela. 2002. Comparative evaluation of NCCLS M27-A and EUCAST broth

microdilution procedures for antifungal susceptibility testing of Candida species. Antimicrob. Agents

Chemother. 46:3644-3647.

(7.) Delaveau, T., A. Delahodde, E. Carvajal, J. Subik, and C. Jacq. 1994. PDR3, a new yeast

regulatory gene, is homologous to PDR1 andcontrols the multidrug resistance phenomenon. Mol. Gen.

Genet. 244:501-511.

(8.) DeRisi J., H.B. van den Hazel, P. Marc, E. Balzi, P. Brown, C. Jacq, and A. Goffeau. 2000.

Genome microarray analysis of transcriptional activation in multidrug resistance yeast mutants. FEBS Lett.

470:156-160.

(9.) Ellis, M. 2002. Invasive fungal infections: evolving challenges for diagnosis and therapeutics.

Mol. Immunol. 38:947-957.

(10.) Fidel, P.L., Jr., J.A. Vazquez, and J.D. Sobel. 1999. Candida glabrata: review of epidemiology,

pathogenesis, and clinical disease with comparison to C. albicans. Clin. Microbiol. Rev. 12:80-96.

(11.) Hallstrom, T.C., L. Lambert, S. Schorling, E. Balzi, A. Goffeau, and W.S. Moye-Rowley. 2001.

Coordinate control of sphingolipid biosynthesis and multidrug resistance in Saccharomyces cerevisiae. J.

Biol. Chem. 276:23674-23680.

(12.) Ji, X., Y. Li, H. Liu, Y. Yan, and J. Li. 2000. Determination of the alkaloid content in different

parts of some Mahonia plants by HPCE. Pharm. Acta Helv. 74:387-391.

(13.) Kolaczkowska, A., M. Kolaczkowski, A. Delahodde, and A. Goffeau. 2002. Functional

dissection of Pdrlp, a regulator of multidrug resistance in Saccharomyces cerevisiae. Mol. Genet. Genomics

267:96-106.

(14.) Kolaczkowska, A., M. Kolaczkowski, A. Goffeau, and W.S. Moye-Rowley. 2008.

Compensatory activation of the multidrug transporters Pdrlp, Sng2p, and Yorlp by Pdrlp in Saccharomyces

cerevisiae. FEBS Lett. 582:977-983.

(15.) Kolaczkowski, M., A. Cybularz-Kolaczkowska, J. Luczyfiski, S. Witek, and A. Goffeau. 1998.

In vivo characterization of the drug resistance profile of the major ABC transporters and other components

of the yeast ple iotropic drug resistance network. Microb. Drug Resist. 4:143-158.

(16.) Kolaczkowski, M., A. Kolaczkowska, B. Gaigg, R. Schneiter, and W.S. Moye-Rowley. 2004.

Differential regulation of ceramide synthase components LAC1 and LAG1 in Saccharomyces cerevisiae.

Eukaryot. Cell 3:880-892.

(17.) Kolaczkowski, M., K. Michalak, and N.R. Motohashi. 2003. Phenothiazines as potent

modulators of yeast multidrug resistance. Int. J. Antimicrob. Agents 22:279-283.

(18.) Liu, K.C.-S., S. L. Yang, M.F. Roberts, B.C. Elford, and J.D. Phillipson. 1992. Antimalarial

activity of Artemisia annua flavonoids from whole plants and cell cultures. Plant Cell Rep. 11:637-640.

(19.) Miyazaki, H., Y. Miyazaki, A. Geber, T. Parkinson, C. Hitchcock, D.J. Falconer, D.J. Ward, K.

Marsden, and J.E. Bennett. 1998. Fluconazole resistance associated with drug efflux and increased

transcription of a drug transporter gene, PDH1, in Candida glabrata. Antimicrob. Agents Chemother.

42:1695-1701.

(20.) Mueller, M.S., I.B. Karhagomba, H.M. Hirt, and E. Wemakor. 2000. The potential of Artemisia

annua L. as a locally produced remedyfor malaria in the tropics: agricultural, chemical and clinical aspects. J.

Ethnopharmacol. 73:487-493.

(21.) Nakamoto, K., S. Sadamori, and T. Hamada. 1990. Effects of crude drugs and berberine

hydrochloride on the activities of fungi. J.Prosthet. Dent. 64:691-694.

(22.) National Committee for Clinical Laboratory Standards. 2000. Approved standard: M7-A5.

Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, fifth edition.

NCCLS, Wayne, PA.

(23.) Ngono Ngane, A., L. Biyiti, P.H. Amvam Zollo, and P. Bouchet. 2000. Evaluation of antifungal

activity of extracts of two Cameroonian Rutaceae: Zanthoxylum leprieurii Guill. et Perr. and Zanthoxylum

xanthoxyloides Waterm. J. Ethnopharmacol. 70:335-342.

(24.) Nourani, A., D. Papajova, A. Delahodde, C. Jacq, and J. Subik. 1997. Clustered amino acid

substitutions in the yeast transcription regulator Pdrlp increase pleiotropic drug resistance and identify anew

central regulatory domain. Mol. Gen. Genet. 256:3971105.

(25.) Phillipson, J.D., C.W. Wright, G.C. Kirby, and D.C. Warhurst. 1995. Phytochemistry of some

plants used in traditional medicine for the treatment of protozoal disease. In K. Hostettman, A. Marston, M.

Maillard, and M. Hamburger (eds.), Phytochemistry of plants used intraditional medicine. Clarendon Press,

Oxford, pp. 95-135.

(26.) Ruhnke, M. 2006. Epidemiology of Candida albicans infectionsand role of non-Candida-

albicans yeasts. Curr. Drug Targets 7:495-504.

(27.) Sanglard, D., F. Ischer, D. Calabrese, M. de Michell, and J.Bille. 1998. Multiple resistance

mechanisms to azole antifungals in yeast clinical isolates. Drug Resist. Updates 1:255-265.

(28.) Sanglard, D., F. Ischer, D. Calabrese, P.A. Majcherczyk, andJ. Bille.1999. The ATP binding

cassette transporter gene CgCDR1 fromCandida glabrata is involved in the resistance of clinical isolates to

azole antifungal agents. Antimicrob. Agents Chemother. 43:2753-2765. (29.) Sanglard, D., F. Ischer, M.

Monod, and J. Bille. 1997. Cloning of Candida albicans genes conferring resistance to azole antifungal

agents: characterization of CDR2, a new multidrug ABC transporter gene. Microbiology 143:405-416.

(30.) Sanguinetti, M., B. Posteraro, B. Fiori, S. Ranno, R. Torelli, and G. Fadda. 2005. Mechanisms

of azole resistance in clinical isolates of Candida glabrata collected during a hospital survey of antifungal

resistance. Antimicrob. Agents Chemother. 49:668-679.

(31.) Seneviratne, C.J., R.W.K. Wong, and L.P. Samaranayake. 2007.Potent anti-microbial activity

of traditional Chinese medicine herbs against Candida species. Mycoses 51:30-34. (32.) Spelman, K., J.A.

Duke, and M.J. Bogenschutz-Godwin. 2006. The synergy principle at work with plants, pathogens, insects,

herbivores and humans. In L.J. Cseke, A. Kirakosyan, P.B. Kaufman, S.L. Warber, J.A. Duke, and H.L.

Brielmann (eds.), Natural Products from Plants. Taylor & Francis, Boca Raton, pp. 475-502.

(33.) Stermitz, F.R., T.D. Beeson, P.J. Mueller, J.F. Hsiang, and K. Lewis. 2001. Staphylococcus

aureus MDR efflux pump inhibitors froma Berberis and a Mahonia (sensu strictu) species. Biochem. Syst.

Ecol. 29:793-798.

(34.) Stermitz, F.R., P. Lorenz, J.N. Tawara, L.A. Zenewicz, and K. Lewis. 2000. Synergy in a

medicinal plant: antimicrobial action of berberine potentiated by 5'-methoxyhydnocarpin, a multidrug pump

inhibitor. Proc. Nad. Acad. Sci. USA 97: 1433-1437.

(35.) Stermitz, F.R., L.N. Scriven, G. Tegos, and K. Lewis. 2002. Two flavonols from Artemisia

annua which potentiate the activity of berberine and norfloxacin against a resistant strain of

Staphylococcusaureus. Planta Med. 68:1140-1141.

(36.) Stermitz, F.R., J.N. Tawara, M. Boeckl, M. Pomeroy, T.A. Foderaro, and F.G. Todd. 1994.

Piperidine alkaloid content of Picea (spruce) and Pinus (pine). Phytochemistry 35:451-453.

(37.) Torelli, R., B. Posteraro, S. Ferrari, M. La Sorda, G. Fadda, D. Sanglard, and M. Sanguinetti.

2008. The ATP-binding cassette transporter-encoding gene CgSNQ2 is contributing to the CgPDR1-

dependentazole resistance of Candida glabrata. Mol. Microbiol. 68:186-201.

(38.) Wenzel, R.P. 1995. Nosocomial candidemia: risk factors and attributable mortality. Clin.

Infect. Dis. 20:1531-1534.

(39.) White, T.C., S. Holleman, F. Dy, L.F. Mirels, and D.A. Stevens. 2002. Resistance mechanisms

in clinical isolates of Candida albicans. Antimicrob. Agents Chemother. 46:17041713.

Marcin Kolaczkowski, [1] Anna Kolaczkowska, [2] and Frank R. Stermitz [3]

[1] Department of Biophysics, Wroclaw Medical University, Wroclaw,Poland. [2] Faculty of

Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland. [3]

Department of Chemistry, Colorado State University, Fort Collins, Colorado. Address reprint requests to: Dr.

Marcin Kolaczkowski Department of Biophysics Wroclaw Medical University ul. Chalubinskiego 10 50-368

Wroclaw Poland E-mail: mkolacz2Cpoczta.onet.pl TABLE 1. SACCHAROMYCES CEREVISIAE

STRAINS USED IN THIS STUDY Strain Genotype Reference /source FY1679-28C (MATa, ura3-52,

trp1[Delta]63, (C. leu2AI, his3A200, GAL2+) Fairhead, Paris, France) FY1679-1D (MATa, ura3-52,

trp1[Delta]63, (C. his3A200, GAL2+) Fairhead) FYMK-1/1 (MATa, ura3-52, trp1[Delta]63, 15 leu2AI,

his3A200, GAL2+, pdr5-Al::hisG) FYMK 23/2 (MATa, ura3-52, trp1[Delta]63, 15 leu2AI, his3A200,

GAL2+, sng2-Al::hisG) FYAK 4 (MATa, ura3-52, trp1[Delta]63, 15 leu2AI, his3A200, GAL2+, yorl-

l::hisG) FYMK 26/8-10B (MATa, ura3-52, trp1[Delta]63, 15 leu2AI, his3A200, GAL2+, pdr5-Al::hisG,

sng2-Al::hisG) FYAK 1/1 (MATa, ura3-52, trp1[Delta]63, 14 leu2AI, his3A200, GAL2+, pdr5-Al::hisG,

yorl-1::hisG) FYAK 23/2 (MATa, ura3-52, trp1[Delta]63, 14 leu2AI, his3A200, GAL2+, sng2-Al::hisG,

yorl-1::hisG) FYAK 26/8-10B1 (MATa, ura3-52, trp1[Delta]63, 15 leu2AI, his3A200, GAL2+, pdr5-

Al::hisG, sng2-Al::hisG, yorl-1::hisG) FY1679/TDEC (MATa, ura3-52, trp1[Delta]63, 7 leu2AI, his3A200,

GAL2+, pdrl-A2::TRP1, pdr3-A::HIS3) FYMK 26/8-5C (MATa, ura3-52, trp1[Delta]63, 15 leu2AI,

his3A200, GAL2+, PDR1-3) FYMK 10/1-27D1 (MATa, ura3-52, trp1[Delta]63, 15 leu2AI, his3A200,

GAL2+, PDR1-3, pdr5-Al::hisG) FYMK 26/8-5A (MATa, ura3-52, trp1[Delta]63, 15 leu2AI, his3A200,

GAL2+, PDR1-3, pdr5-Al::hisG) FYMK 26/8-4D (MATa, ura3-52, trp1[Delta]63, This study leu2AI,

his3A200, GAL2+, PDR1-3, sng2-Al::hisG) FYAK 26/8-5C4 (MATa, ura3-52, trp1[Delta]63, This study

leu2AI, his3A200, GAL2+, PDR1-3, yorl-l::hisG) FYMK 26/8-9C (MATa, ura3-52, trp1[Delta]63, This

study leu2AI, his3A200, GAL2+, PDR1-3, pdr5-Al::hisG, sng2-Al::hisG) FYAK 26/8-5A2 (MATa, ura3-52,

trp1[Delta]63, This study leu2AI, his3A200, GAL2+, PDR1-3, pdr5-Al::hisG, yorl-1::hisG) FYAK 26/8-

4D2 (MATa, ura3-52, trp1[Delta]63, This study leu2AI, his3A200, GAL2+, PDR1-3, sng2-Al::hisG, yorl-

1::hisG) FYAK 26/8-9C1 (MATa, ura3-52, trp1[Delta]63, This study leu2AI, his3A200, GAL2+, PDR1-3,

pdr5-Al::hisG, sng2-Al::hisG, yorl-1::hisG) The strains are isogenic derivatives of the FY1679 parental

strain. TABLE 2. INHIBITION OF PATHOGENIC YEAST GROWTH BY MEDICINAL PLANT

EXTRACTS MIC ([micro]g/ml) Plant species Solvent Candida Candida (plant part) glabrata albicans

Mahonia (Berberis) CH[Cl.sub.3] >40 >40 fortunei (leaves) M. fortunei McOH (leaves) 40 320 Berberis

fendleri McOH (roots) 60 240 Pinus edulis McOH (needles) 120 >480 Zanthoxylum EtOAc (a) 80 >320

zanthoxyloides Hydrastis Hexane (leaves) >80 >80 canadensis H. canadensis EtOAc (leaves) >80 >80 H.

canadensis McOH (leaves) 40 160 Dalea formosa EtOAc (twigs, 20 >80 leaves) Artemisia annua EtOAc (a)

60 120 A. annua McOH (a) >120 >120 Fluconazole 8 0.25 (a) The whole above-ground plant material was

used for extraction. MIC, minimum inhibitory concentration; CHC13, chloroform; McOH, methanol;

EtOAc, ethyl acetate. TABLE 3. DELETIONS IN MAJOR ABC MDR TRANSPORTERS GENES

AFFECTING ARTEMISIA ANNUA EXTRACT SENSITIVITY Genotype PDR1-3 [Delta]5 [Delta]1

[Delta]5 MIC ([micro]g/m1) >240 >240 240 Genotype [Delta]2 [Delta]1 [Delta]5 [Delta]2 [Delta]5 MIC

([micro]g/m1) 120 120 The control PDR1-3 mutant and isogenic single and multiple knockouts in YOR1,

SNQ2, and PDR5 are marked [Delta]1, [Delta]2, and [Delta]5, respectively. MIC, minimum inhibitory

concentration. TABLE 4. GROWTH INHIBITION OF SACCHAROMYCES CEREVISIAE MUTANTS IN

THE MAJOR TRANSPORTERS AND REGULATORS OF THE PLEIOTROPIC DRUG-RESISTANCE

NETWORK BY MEDICINAL PLANT EXTRACTS ACTIVE ON PATHOGENIC YEAST MIC

([micro]g/ml) Plant species Solvent (plant part) PDR1 Mahonia fortunei MeOH (leaves) 360 Berberis

fendleri MeOH (roots) 240 Pinus edulis MeOH (needles) >600 Zanthoxylum zanthoxyloides EtOAc (a) 90

Hydrastis canadensis MeOH (leaves) 180 Dalea formosa EtOAc (twigs, leaves) >40 Artemisia annua EtOAc

(a) 240 A. annua MeOH (a) >120 MIC ([micro]g/ml) Plant species [Delta]1 pdr1[Delta], [Delta]2

pdr3[Delta] [Delta]5 Mahonia fortunei 90 90 Berberis fendleri 120 120 Pinus edulis 300 75 Zanthoxylum

zanthoxyloides 90 90 Hydrastis canadensis 90 180 Dalea formosa 40 40 Artemisia annua 60 60 A. annua 120

>120 (a) The whole above-ground plant material was used for extraction. The control PDR1 wild-type strain

is marked. Isogenic knockouts of YOR1, SNQ2, and PDR5 as well as in PDR1 and PDR3 are marked

[Delta]1[Delta]2[Delta]5 and pdr1[Delta] and pdr3[Delta], respectively. MIC, minimum inhibitory

concentration; McOH, methanol; EtOAc, ethyl acetate. TABLE 5. SACCHAROMYCES CEREVISIAE

GROWTH INHIBITION AND MODULATION OF KETOCONAZOLE TOLERANCE BY SELECTED

FRACTIONS FROM SILICA GEL CHROMATOGRAPHY OF ARTEMISIA ANNUA ETHYL ACETATE

EXTRACT Fraction Wt MIC ([micro]g Wt +keto- number (a) /ml) [Delta]1 conazole [Delta]2 [Delta]5 5

240 60 <5 6 360 90 360 7 >360 60 >360 8 >120 30 >120 9 >60 60 <5 10 >120 120 <5 11 >120 120 <5 12

NA NA <5 13 >60 >60 60 14 >120 >120 >120 15 >480 >480 >480 16 >360 >360 >360 17 >60 >60 >60 18

NA NA <5 The triple knockout of YOR1, SNQ2, and PDR5 ([Delta]1[Delta]2[Delta]5) and the isogenic

wild-type (wt) strains were used. MIC values in the presence of a subinhibitory ketoconazole concentration

are indicated +ketoconazole. (a) Fraction numbers correspond to those described in reference 35. MIC,

minimum inhibitory concentration; NA, no activity detected due to trace amounts of material. TABLE 6.

EFFECT OF ARTEMISININ ON GROWTH OF SACCHAROMYCES CEREVISIAE MUTANTS OF

MAJOR ABC MDR TRANSPORTERS AND ON MODULATION OF KETOCONAZOLE RESISTANCE

Genotype PDR1-3 [Delta]5 [Delta]l PDR1 [Delta]2 [Delta]5 MIC >200 50 50 >200 ([micro]g/ml Genotype

PDR1-3 PDR1 +keto +keto MIC 25 3 ([micro]g/ml