Ž .Immunopharmacology 41 1999 227–234

Human immunodeficiency virus type 1 protease inhibitorattenuates Candida albicans virulence properties in vitro 1

Andreas Gruber a,), Cornelia Speth a, Elisabeth Lukasser-Vogl a, Robert Zangerle b,Margarete Borg-von Zepelin c, Manfred P. Dierich a, Reinhard Wurzner a¨a Ludwig Boltzmann-Institut fur AIDS-Forschung and Institut fur Hygiene, UniÕersity of Innsbruck, Innsbruck, Austria¨ ¨

b Institut fur Dermatologie und Venerologie, UniÕersity of Innsbruck, Innsbruck, Austria¨c Institut fur Hygiene, UniÕersity of Gottingen, Gottingen, Germany¨ ¨ ¨

Accepted 24 February 1999

Abstract

Ž .The putative virulence factor secreted aspartyl proteinase SAP of Candida albicans and the human immunodeficiencyŽ .virus type 1 HIV-1 protease both belong to the aspartyl proteinase family. The present study demonstrates that the HIV-1

protease inhibitor Indinavir is a weak but specific inhibitor of SAP. In addition, Indinavir reduces the amount of cell boundas well as released SAP antigen from C. albicans. Furthermore, viability and growth of C. albicans are markedly reduced byIndinavir. These findings indicate that HIV-1 protease inhibitors may possess antifungal activity and we speculate that invivo SAP inhibition may add to the resolution of mucosal candidiasis in HIV-1 infected subjects. q 1999 Elsevier ScienceB.V. All rights reserved.

Keywords: Candida albicans; Indinavir; HIV-1

1. Introduction

Yeasts of the genus Candida, especially C. albi-cans, are a major cause of the increasing number of

AbbreÕiations: BSA, bovine serum albumin; HIV-1, humanimmunodeficiency virus type 1; PI, propidium iodide; SAP, se-creted aspartyl proteinase

) Corresponding author. CFAR-Center for AIDS Research,University of California, San Diego, Department of Medicine,Clinical Science Building, 9500 Gilman Drive, La Jolla, CA92093-0665, USA. Tel.: q1-619-534-7957; fax: q1-619-534-7743; e-mail: agruber@ucsd.edu

1 Presented in part at the 29th Meeting of the German SocietyŽof Immunology, Freiburg, Germany, 23–26 September 1998 Im-

Ž . .munobiology 199 1998 647 .

opportunistic fungal infections associated with mor-bidity and mortality in immunocompromised sub-

Ž .jects Wade, 1993 . A key virulence factor producedby C. albicans is the secreted aspartyl proteinasesŽ .SAP which may assist the fungus to colonize andinvade host tissues, to evade the host’s antimicrobialdefense mechanisms and to assimilate nitrogen from

Ž .proteinaceous sources Cutler, 1991 . C. albicansproduces at least nine highly conserved SAP isoen-

Ž .zymes SAP1-9 which act under distinct environ-Ž .mental conditions White and Agabian, 1995 . The

most dominant SAP isoenzyme in vitro and probablyŽalso in vivo is SAP2 Ollert et al., 1995; White and

.Agabian, 1995 .The C. albicans SAP isoenzymes belong to the

broad aspartyl proteinase family which includes fun-

0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0162-3109 99 00035-1

( )A. Gruber et al.r Immunopharmacology 41 1999 227–234228

gal as well as plant, mammalian and viral enzymes.A prominent member thereof is the human immuno-

Ž .deficiency virus type 1 HIV-1 protease which playsa central role during the maturation of HIV-1 as it is

Žrequired for the production of infectious virions Kohl.et al., 1988 . The currently used HIV-1 protease

inhibitors Saquinavir, Ritonavir, Indinavir and Nelfi-navir lead to a significant reduction in viral loadŽ .Flexner, 1998 and a change in immune statusŽ .Powderly et al., 1998 . Aside from these effects,brief clinical reports suggest that HIV-1 proteaseinhibitors may prevent or even resolve opportunisticinfections occurring during HIV-1 pathogenesisŽ .Sepkowitz, 1998 . Oral candidiasis, mainly causedby C. albicans, represents one of the main HIV-re-

Ž .lated opportunistic infections Klein et al., 1984 andresolves after treatment with HIV-1 protease in-

Ž .hibitors Zingman, 1996; Hood et al., 1998 . Whetherthe improvement of HIV-associated opportunistic in-fections is related to the reduced HIV-1 load, to thepartial restoration of the immune system or to adirect drug effect still needs to be elucidated. Thelatter possibility has previously been controversially

Ždiscussed in the case of human herpesvirus 8 HHV-. Ž .8 Rizzieri et al., 1997 and is addressed here for the

resolution of mucosal candidiasis after protease in-hibitor therapy in HIV-1 infected subjects.

2. Materials and methods

2.1. C. albicans cultiÕation

ŽC. albicans CBS 5982 Centraal Bureau voor.Schimmelcultures, Baarn, the Netherlands was used

throughout the study. The strain was initially grownŽon Sabouraud dextrose agar SDA; Oxoid, Bas-

.ingstoke, UK plates and then transferred into RPMIŽ .1640 medium Hyclone, Cramlington, UK without

any supplements. This cell suspension was used asstock solution and was kept for 1 week at 48C. Allexperiments were performed under sterile conditions.

2.2. Reagents

ŽThe HIV-1 protease inhibitor Indinavir trademarkw .Crixivan ; MK-639 or L-735,524 , kindly provided

by Merck, Whitehouse Station, NJ, was dissolved in

Žthe appropriate assay buffer protease induction.medium, pH 4.0, described below . No additivesr

carriers were required to ensure solubility at theconcentrations employed in this study. Proteinase K

Ž .from Tritirachium album EC 3.4.21.64 and pepsinŽ .from porcine stomach mucosa EC 3.4.23.1 were

purchased from Sigma, St. Louis, MO.

2.3. Induction and purification of C. albicans SAP

The induction of C. albicans SAP was performedŽas described previously Samaranayake and Holm-

.strup, 1989 , with minor modifications. Briefly, C.albicans, at a concentration of 1=107 yeast cellsrml, was inoculated into proteinase induction medium

Žfor 7 days, as recommended Macdonald and Odds,.1980 , at 258C. The proteinase induction medium

Ž .consisted of 0.2% wrv bovine serum albuminŽ . Ž .BSA, fraction V; Sigma , 2% wrv glucose, 0.1%Ž . Ž . Ž .wrv KH PO , 0.05% wrv MgSO and 1% vrv2 4 4

Ž .100= minimum essential medium vitamins Sigma ,pH 4.0.

Subsequent purification of candidal proteinasefrom 250 ml-cultures was performed as described

Ž .previously Macdonald and Odds, 1980 , with minormodifications. Briefly, after proteinase induction, theyeast suspension was centrifuged at 5000=g for 30min, the supernatant filtered through a 0.8 mm mem-

Ž .brane Sartorius, Gottingen, Germany and finally¨adjusted to pH 6.5 using 4 M NaOH. The enzymecontaining supernatant was incubated with 5 g DEAE

Ž .Sephadex A-25 Pharmacia, Uppsala, Swedenovernight at 48C. The DEAE Sephadex was recol-lected by using a Buchner funnel and finally trans-ferred to a column. Elution of proteinase from thecolumn was performed by using a linear gradient of0.01 to 0.1 M sodium citrate, pH 6.5. SAP contain-ing fractions, identified via their proteolytic activityas described below, or by SDS PAGE and Western

Ž .blot as described previously Ollert et al., 1995 ,were used for inhibitory studies.

2.4. Drug effect on C. albicans SAP production

To assess drug effects on C. albicans SAP pro-duction, 1=107 yeast cellsrml were grown in thepresence or absence of drug using the conditionsdescribed above but in a total volume of 1 ml

( )A. Gruber et al.r Immunopharmacology 41 1999 227–234 229

proteinase induction medium. Subsequent to cen-trifugation at 5000=g for 30 min, the supernatantwas passed through a 0.8 mm membrane filter andassayed for proteinase activity as described below.

2.5. Assay for C. albicans secreted proteinase actiÕ-ity

Purified SAP as well as culture supernatants of C.albicans grown in the presence or absence of drug

Žwere assessed for proteinase activity Macdonald and.Odds, 1980 . For this purpose 0.5 ml of the sample

Ž . Ž .was incubated with 2 ml of 1% wrv BSA SigmaŽ .in 0.05 M sodium citrate–HCl buffer pH 3.2 for 60

min at 378C under gentle agitation. Subsequently,Ž . Ž20% wrv trichloroacetic acid Merck, Darmstadt,

.Germany was added to stop the reaction. The pre-cipitated protein was removed by filtration through a0.8 mm membrane. The amount of proteolysis was

Ž .determined by measuring the optical density OD ofŽthe filtrate at 280 nm UV-160, Shimadzu, Vienna,

.Austria . A background control consisted of BSAincubated with buffer alone and to which trichloro-acetic acid was added before the enzyme-containingsupernatant.

2.6. FACS analyses for quantitatiÕe determination ofC. albicans SAP antigen

Induction of C. albicans SAP was performed asdescribed above. The amount of cell-bound SAP onthe surface of C. albicans was analyzed by FACSŽ .FACScan; Becton Dickinson, Heidelberg, Germanyusing the mouse monoclonal IgG FX 7-10, which is

Žgenerated against an epitope of SAP2 Borg et al.,.1988 , and FITC-conjugated goat anti-mouse im-

Ž .munoglobulin Dako, Glostrup, Denmark as a sec-ondary antibody. Per sample, 5=103 events wererecorded, and analyzed using the software Lysis IIŽ .Becton Dickinson .

( )2.7. Propidium iodide PI staining of killed C.albicans

Cell death due to a loss in cell membrane integrityof drug-treated C. albicans was assessed by flowcytometry measuring the level of PI uptake into

Ž .damaged cells Bjerknes, 1984 . The yeast cells used

in this procedure, as well as in MTT-assay andŽ .hyphal elongation assessment see below , were taken

from 7-day cultures of C. albicans in which pro-teinase induction medium was initially supplementedwith or without Indinavir as described above.

2.8. MTT-assay for testing Õiability

To assess the viability of C. albicans, 5=104

yeast cellsr100 mlrwell were incubated in a 96-wellŽ .cell culture plate Costar, Cambridge, UK with 0.8

Ž .mgrml 3- 4,5-dimethylthiazol-2-yl -2,5-diphenyl te-Ž .trazolium bromide dye MTT; Sigma , for 2 h at

378C. Finally, cells were lysed by adding 0.6%Ž . Ž .vrv acidic acid and 10% wrv SDS in dimethylsulfoxide, to release and dissolve the formed for-mazan salt. The amount of incorporated formazanwas determined spectrophotometrically at 550 nmŽDigiscan Microplate Reader, Asys, Eugendorf, Aus-

.tria .

2.9. Hyphal elongation assessment

The morphology and growth of C. albicans treatedwith or without drug was investigated by assessinghyphal elongation. Therefore, 2=105 yeast cellsrmlof RPMI 1640 medium were inoculated into microw-

Ž .ell plates Greiner, Kremsmunster, Austria and incu-¨bated at 378C. The morphology of the organisms wasdetermined microscopically after 6 h using a mi-crometer for length measurement. Each sample wasassessed in triplicate measuring 50 organisms persample.

2.10. Sequence comparison

Sequence alignments were performed using theŽ .program CLUSTAL W Thompson et al., 1994 .

Amino acid similarity groups are LVIMFWHYAC,Ž .PGAST, DEQN and RKH Stoiber et al., 1995 . All

the sequences were extracted from the protein se-Žquence data base SWISS-PROT Bairoch and Ap-

. Žweiler, 1997 , and are as follows: HIV-1 BH-10. Ž .isolate protease P03366 , Rous sarcoma virus pro-Ž . Žtease P03322 , SAP1-7 P28872; P28871; P43092

. Žto P43096 , human and porcine pepsin P00790;. Ž .P00791 , human renin P00797 , human cathepsin DŽ .and E P07339; P14091 .

( )A. Gruber et al.r Immunopharmacology 41 1999 227–234230

2.11. Statistics

Statistical significance was determined by usingStudent’s t-test analysis. All comparisons were two-tailed, and a P value of less than 0.05 was consid-ered significant.

3. Results

3.1. Sequence comparison between aspartyl pro-teinases

C. albicans SAP and HIV-1 protease both belongto the family of aspartyl proteinases. The eukaryoticaspartyl proteinases are made of a single chain, morethan 300 amino acids in length, which can be dividedinto a N- and C-terminal domain, each possessing anactive site region. The active HIV-1 protease is ahomodimer consisting of two identical monomers,harboring in each the catalytically important activesite and being 99 amino acids in length. Sequencealignment of the active site regions of HIV-1 pro-tease and of C. albicans SAP isoenzymes 1 and 2,revealed moderate similarity on their N-terminal do-

Ž .main 68.4%, both and high similarity at their C-Žterminal domain 84.2% and 78.9%, respectively;

.Table 1 . Porcine pepsin, the closest structural rela-Žtive of SAP within the mammalian proteases Abad-

.Zapatero et al., 1996 and different human aspartylproteinases, which may be affected by Indinaviruptake, share also moderate similarity with HIV-1

Žprotease in their N-terminal domain ranging from.63.2% to 68.4% but only remote similarity to the

Žviral protease in their C-terminal domain ranging.from 42.1% to 52.6%; Table 1 . The similarity be-

tween SAP3-7 and the HIV-1 protease was in theŽrange of mammalian aspartyl proteinases between

.52.6% and 68.4%; data not shown . The replacementof one DTG- by a DSG-motif in the active siteregion, a characteristic of SAP isoenzymes, exceptSAP7, is also found among other yeast and plant but

Žnot among mammalian aspartyl proteinases Cutfield.et al., 1995 , and does not influence the geometry of

Ž .the catalytic center of SAP Cutfield et al., 1995 .Furthermore, a region in the N-terminal domain ofSAP2 which is shared also by SAP1-6, forms a

Table 1Sequence comparison between different SAP isoenzymes, mam-malian aspartyl proteinases and HIV-1 protease in the active site

Ž . Ž .region A and the flap region of the HIV-1 protease B

HIV-1 active site region % similarity

( )AHIV-1 GQLKEALLDTGADDTVLEERSVP SvYitALLDSGADiTIIsE 73.7SAP1-N kQkfNVIVDTGSsDlWVpD 68.4SAP1-C GNI-DVLLDSGTtiTYLQQ 84.2SAP2-N nQklNVIVDTGSsDlWVpD 68.4SAP2-C TDnvDVLLDSGTtiTYLQQ 78.9pPep-N AQdftVIFDTGSsNlWVps 63.2pPep-C SggcQAIVDTGTslltgpt 47.4hPep-N AQdftVVFDTGSsNlWVps 63.2hPep-C AEgcQAIVDTGTslltgpt 52.6hRenin-N PQtfkVVFDTGSsNvWVps 63.2hRenin-C eDgclALVDTGAsyisgst 42.1hCathD-N PQCftVVFDTGSsNlWVps 68.4hCathD-C kEgcEAIVDTGTslmVgpv 52.6hCathE-N PQnftVIFDTGSsNlWVps 63.2hCathE-C SEgcQAIVDTGTslitgps 52.6

( )BHIV-1 PKMIGGIGGFIKRSVP npqIhGIGGgIp 50.0SAP1-N kprpGqsAdFCK 41.7SAP2-N vtYsdqtAdFCK 41.7SAP3-N qagqGqdPnFCK 41.7SAP4-N PKWpGdrGdFCK 66.7SAP5-N PKWrGdkGdFCK 66.7SAP6-N PKWrGdCGdFCK 75.0

RSVP, retroviral Rous sarcoma virus protease; prhPep, porcineand human pepsin, respectively; hRenin, human renin; hCathDrE,human cathepsin D and E, respectively. The letters N and Cfollowing these abbreviations indicate the N- and the C-terminal

Ž .domains of the respective enzymes. A gap – was inserted toŽ .facilitate sequence alignment A . Bold capital and normal capital

letters mark identical and similar residues, respectively. Aminoacid residues lacking similarity are written as lowercase letters.

Žbroad flap that extends towards the active site Cut-.field et al., 1995; Abad-Zapatero et al., 1996 . This

flap region of SAP, whose structure is not shared byŽmammalian aspartyl proteinases Abad-Zapatero et

.al., 1996 , showed moderate sequence similarity toŽ .the flap region of HIV-1 protease Table 1 . The

latter region has been shown to be critical for sub-Žstrate and inhibitor binding for both SAP Abad-

. ŽZapatero et al., 1996 and HIV-1 protease Shao et.al., 1997 . Interestingly, SAP4-6 contain each a

RGD-motif which is generally believed to promote

( )A. Gruber et al.r Immunopharmacology 41 1999 227–234 231

Ž .adhesive interactions D’Souza et al., 1991 and mayfurther hint to the importance of this loop for sub-straterinhibitor binding.

3.2. Direct effect of IndinaÕir on C. albicans SAPactiÕity in Õitro

To investigate the influence of Indinavir on SAP,proteinase activity assays were carried out usingpurified C. albicans SAP and different concentra-

Ž .tions of Indinavir Fig. 1 . Indinavir inhibited SAPactivity in a dose dependent manner. A concentrationof 0.5 mgrml Indinavir reduced the SAP activity by

Ž .more than 50% p-0.01 . Increasing the inhibitorconcentration to 5.0 mgrml abolished SAP activitycompletely. The concentration of Indinavir requiredto reduce enzyme activity by 50% is 3.9=10y4 MŽ . y90.28 mgrml for SAP compared to 0.5=10 M

Ž .for HIV-1 protease Dorsey et al., 1994 . Thus,Indinavir is a weak inhibitor of SAP, comparable tothe inhibitory efficacy of pepstatin A against HIV-1

Ž y4 . Ž .protease 2.5=10 M Seelmeier et al., 1988 .

3.3. Specificity of IndinaÕir for C. albicans SAP

To evaluate the specificity of Indinavir for SAP,the effect of Indinavir on equimolar amounts of SAP,

Fig. 1. Effect of Indinavir on C. albicans SAP activity. PurifiedSAP was incubated with BSA as substrate and various concentra-tions of Indinavir for 60 min at 378C. The reaction was stopped byadding trichloroacetic acid and enzyme activity was determinedspectro-photometrically by measuring the amount of trichloro-acetic acid soluble protein at 280 nm. Data shown representmeans"standard deviations of three experiments.

Fig. 2. Specificity of Indinavir for SAP. The same assay asdescribed in the legend to Fig. 1 was performed with purifiedSAP, the fungal serine proteinase K and the mammalian aspartylproteinase pepsin. The OD at 280 nm measured in the absence ofprotease inhibitor was set to 100% proteinase activity. Data are

ŽUU .means of three experiments, significance is indicated p-0.01 .

of the fungal serine proteinase K and of the mam-Ž .malian aspartyl proteinase pepsin was tested Fig. 2 .

Compared to the enzyme concentration, the substratewas added in a nearly 90-fold molar excess; Indi-navir was used in an approximately 40- to 4000-foldmolar excess, by adding 0.05 to 5.0 mgrml in-

Žhibitor, respectively. Under the conditions used pH.3.2 , pepsin retained full and proteinase K about

40% of their enzyme activities when compared to pH2.0 and pH 7.4, respectively. The activity of pro-teinase K was apparently unchanged after the addi-tion of Indinavir when compared to control treatmentwithout inhibitor. The activity of pepsin, an enzymestructurally most related to C. albicans SAP2 among

Ž .mammalian proteases Abad-Zapatero et al., 1996 ,was inhibited by Indinavir. However, while 0.5mgrml Indinavir inhibited SAP activity by more

Ž .than 50% p-0.01 , this concentration exerted nosignificant effect on pepsin. Furthermore, at a con-centration of 5 mgrml Indinavir, which completely

( )A. Gruber et al.r Immunopharmacology 41 1999 227–234232

inhibited SAP activity, pepsin still retained morethan 25% of its initial activity.

In addition, C. albicans, induced for SAP produc-tion by BSA and grown in various concentrations ofIndinavir, showed a dose dependent decrease of

Ž .cell-bound SAP antigen by Indinavir Fig. 3 . Al-ready at a concentration of 0.05 mgrml, Indinavirreduced the amount of cell bound SAP by approxi-mately 27%.

3.4. Impact of IndinaÕir on C. albicans Õiability

Finally, we investigated the effect of Indinavir onthe viability of the fungus. Treatment of C. albicanswith the HIV-1 protease inhibitor, Indinavir, inSAP-inducing medium caused a loss of cell mem-brane integrity only at the highest concentration ofIndinavir used and only in about 11% of the total

Ž .cell population Table 2 . The viability of C. albi-cans as measured by the MTT-assay, an alternative

Žmethod to the standard macrodilution procedure Jahn.et al., 1995 , decreased dose dependently after treat-

Ž .ment with Indinavir Table 2 . In addition, while C.albicans yeast cells treated with or without Indinavirretained their ability to form hyphae, hyphal elonga-

Fig. 3. Influence of Indinavir on surface expressed SAP. C.albicans yeast cells were incubated in proteinase induction mediuminitially supplemented with or without Indinavir for 7 days at258C. Subsequently, the amount of cell bound SAP antigen wasdetermined by FACS analysis using the monoclonal antibody FX

Ž7-10 generated against SAP2 Borg et al., 1988; Ollert et al.,.1995 . Data are from one representative experiment out of two.

Table 2Impact of Indinavir on C. albicans viability

Indinavir PI-staining MTT-assay HyphalŽ . Ž Ž . Ž .mgrml % of total % of control elongation mm

.population

0 20.7%"3.4 100%"10.5 18.2"5.20.05 22.1%"2.9 96.7%"12.1 16.9"4.60.5 21.7%"2.6 85.0%"12.9 10.3"3.1

U UU UU5.0 31.5%"3.4 48.3%"13.4 4.1"1.0

C. albicans yeast cells, treated as described in the legend to Fig.3, were assayed for cell membrane integrity and viability by

Ž .propidium iodide PI staining and MTT-assay, respectively. Inthe MTT-test the color reaction of C. albicans without Indinavirwas arbitrary set to 100%. Furthermore, yeast cells treated asstated above were washed and finally transferred into RPMI 1640medium to assess hyphal elongation after 6 h at 378C. Data aremeans"standard deviations of three experiments, significance is

ŽU UU .indicated p-0.05; p-0.01 .

tion of drug treated cells was delayed after incuba-Ž .tion with Indinavir Table 2 .

4. Discussion

The present study demonstrates that the HIV-1protease inhibitor Indinavir is a weak but specificinhibitor of C. albicans SAP enzyme activity. Thephenomenon that surface expressed antigen is alsoreduced may be explained by the fact that degrada-tion of BSA by exogenous SAP yields peptide se-quences not exposed on intact BSA, that are more

Žpotent and effective inducers of SAP Lerner and.Goldman, 1993 . Thus, inhibition of exogenous SAP

by Indinavir may reduce or prevent the subsequentproduction of SAP antigen by C. albicans.

Interestingly, Indinavir given as a single dose of0.5 mgrml, which is only one to two magnitudeshigher than the maximum plasma concentrationreached by Indinavir during a dosing intervalŽ .Flexner, 1998 , showed a marked and highly signifi-cant reduction in both, cell bound SAP and solubleSAP activity even after an incubation period of 7days. Therefore, it has to be investigated furtherwhether multiple dosing of Indinavir may reduce theinhibitor concentration required to cause the effectsobserved in this study. In addition, Indinavir treat-ment of C. albicans reduced viability and growth ofthe fungus, probably caused by interference of Indi-

( )A. Gruber et al.r Immunopharmacology 41 1999 227–234 233

navir with SAP assisted nitrogen assimilation. Thesefindings are in accordance with previous studies on

ŽSAP inhibitors Tsuobi et al., 1985; Lerner and.Goldman, 1993 , which have shown promising re-

Žsults in animal models of candidiasis Ruchel et al.,¨1990; Abad-Zapatero et al., 1996; Fallon et al.,

.1997 . However, only a minor percentage of Indi-navir treated C. albicans displayed severe cell mem-brane damage, characteristic of dead cells. In addi-tion, C. albicans yeast cells incubated with Indinavirretained the ability to form hyphae, even if at areduced rate when compared to untreated cells, whentransferred into inhibitor free medium. Therefore,Indinavir may act fungistatically rather than fungi-toxically on C. albicans. On the basis of amino acidsequence alignments, some regions within SAP showsignificant differences to mammalian aspartyl pro-

Žteinases Cutfield et al., 1995; Abad-Zapatero et al.,.1996 but share moderate to high similarity with the

HIV-1 protease. These differences, with their conse-quent influence on substraterinhibitor bindingŽ .Abad-Zapatero et al., 1996 , may also be responsi-ble for about 10-fold more potent inhibition of SAPover pepsin by Indinavir.

Whether multiple dosing or a combination ofHIV-1 protease inhibitors with other medications,e.g., antifungal drugs used in HIV-1 infected sub-jects, may enhance the antifungal activity of thesedrugs, thereby lowering the concentrations needed toresult in the effects presented in this study, stillneeds to be elucidated. Furthermore, the gastrointes-tinal and possibly also the oropharyngeal tract, mainreservoirs of C. albicans during mucosal candidiasisin HIV-infected subjects, may also be exposed tohigh concentrations of the orally administered HIV-1protease inhibitor, since a reflux of gastric fluid may

Žoccur as a known side effect of the drug Flexner,.1998 . Furthermore, on some occasions, e.g., in the

treatment of children, Indinavir is administered as aŽsuspension instead of intact capsules Hoetelmans et

.al., 1997 .Treatment with Saquinavir, another HIV-1 pro-

tease inhibitor, has recently been shown to even leadto the resolution of mucosal candidiasis that is re-

Žfractory to treatment with fluconazole Zingman,.1996 . Similar results were obtained after the treat-

ment of HIV-infected patients with IndinavirŽ .Zangerle, unpublished observation . Besides Indi-

navir, other HIV-1 protease inhibitors also inhibitŽSAP activity with varying specificities preliminary

.data and further studies are underway to investigatewhether or not HIV-1 protease inhibitors may inaddition prove effective in the treatment of mucosalcandidiasis insensitive to the currently available an-timycotics.

In conclusion, the direct action of Indinavir on C.albicans attenuates the virulence of the fungus invitro and although an indirect effect of the drugtreatment on the patient’s immune system cannot beexcluded at present we speculate that in vivo SAPinhibition may ease the resolution of mucosal can-didiasis in HIV-1 infected subjects by the partiallyrestored immune system.

Acknowledgements

Research was funded by grants from AustrianMinistry of Science and Research, European UnionŽ .BIOMED 2 aBMH4-CT96-1005 , Austrian Fondszur Forderung der wissenschaftlichen Forschung¨Ž .aP12551-Med , and Ludwig Boltzmann-Gesell-schaft, Austria. The authors express their apprecia-

Žtion to Prof. Dr. W.E. Trommer University of.Kaiserslautern, Germany for his kind support of this

study and to Merck and Whitehouse Station, NJ, forsupplying Indinavir.

References

Abad-Zapatero, C., Goldman, R., Muchmore, S.W. et al., 1996.Structure of a secreted aspartic protease from C. albicanscomplexed with a potent inhibitor: implications for the designof antifungal agents. Protein Sci. 5, 640.

Bairoch, A., Apweiler, R., 1997. The SWISS-PROT protein se-quence data bank and its supplement TrEMBL. Nucleic AcidsRes. 25, 31.

Bjerknes, R., 1984. Flow cytometric assay for combined measure-ment of phagocytosis and intracellular killing of Candidaalbicans. J. Immunol. Methods 72, 229.

Borg, M., Watters, D., Reich, B., Ruchel, R., 1988. Production¨and characterization of monoclonal antibodies against secre-tory proteinase of Candida albicans CBS 2730. Zentralbl.Bakteriol. Mikrobiol. Hyg. A 268, 62.

Cutfield, S.M., Dodson, E.J., Anderson, B.F., Moody, P.C., Mar-shall, C.J., Sullivan, P.A., Cutfield, J.F., 1995. The crystalstructure of a major secreted aspartic proteinase from Candidaalbicans in complexes with two inhibitors. Structure 3, 1261.

( )A. Gruber et al.r Immunopharmacology 41 1999 227–234234

Cutler, J.E., 1991. Putative virulence factors of Candida albicans.Annu. Rev. Microbiol. 45, 187.

Dorsey, B.D., Levin, R.B., McDaniel, S.L. et al., 1994. L-735,524:the design of a potent and orally bioavailable HIV proteaseinhibitor. J. Med. Chem. 37, 3443.

D’Souza, S.E., Ginsberg, M.H., Plow, E.F., 1991. Arginyl–Ž .glycyl–aspartic acid RGD : a cell adhesion motif. Trends

Biochem. Sci. 16, 246.Fallon, K., Bausch, K., Noonan, J., Huguenel, E., Tamburini, P.,

1997. Role of aspartic proteases in disseminated Candidaalbicans infection in mice. Infect. Immun. 65, 551.

Flexner, C., 1998. HIV-protease inhibitors. N. Engl. J. Med. 338,1281.

Hoetelmans, R.M., Meenhorst, P.L., Mulder, J.W., Burger, D.M.,Koks, C.H., Beijnen, J.H., 1997. Clinical pharmacology ofHIV protease inhibitors: focus on saquinavir, indinavir, andritonavir. Pharm. World Sci. 19, 159.

Hood, S., Bonington, A., Evans, J., Denning, D., 1998. Reductionin oropharyngeal candidiasis following introduction of pro-tease inhibitors. AIDS 12, 447.

Jahn, B., Martin, E., Stueben, A., Bhakdi, S., 1995. Susceptibilitytesting of Candida albicans and Aspergillus species by a

Žsimple microtiter menadione-augmented 3- 4,5-dimethyl-2-.thiazolyl -2,5-diphenyl-2 H-tetrazolium bromide assay. J. Clin.

Microbiol. 33, 661.Klein, R.S., Harris, C.A., Small, C.B., Moll, B., Lesser, M.,

Friedland, G.H., 1984. Oral candidiasis in high-risk patients asthe initial manifestation of the acquired immunodeficiencysyndrome. N. Engl. J. Med. 311, 354.

Kohl, N.E., Emini, E.A., Schleif, W.A. et al., 1988. Active humanimmunodeficiency virus protease is required for viral infectiv-ity. Proc. Natl. Acad. Sci. USA 85, 4686.

Lerner, C.G., Goldman, R.C., 1993. Stimuli that induce produc-tion of Candida albicans extracellular aspartyl proteinase. J.Gen. Microbiol. 139, 1643.

Macdonald, F., Odds, F.C., 1980. Inducible proteinase of Candidaalbicans in diagnostic serology and in the pathogenesis ofsystemic candidosis. J. Med. Microbiol. 13, 423.

Ollert, M.W., Wende, C., Gorlich, M., McMullan-Vogel, C.G.,Borg-von Zepelin, M., Vogel, C.W., Korting, H.C., 1995.Increased expression of Candida albicans secretory pro-teinase, a putative virulence factor, in isolates from humanimmunodeficiency virus-positive patients. J. Clin. Microbiol.33, 2543.

Powderly, W.G., Landay, A., Lederman, M.M., 1998. Recovery

of the immune system with antiretroviral therapy: the end ofopportunism?. JAMA 280, 72.

Rizzieri, D.A., Liu, J., Traweek, S.T., Miralles, G.D., 1997.Clearance of HHV-8 from peripheral blood mononuclear cellswith a protease inhibitor. Lancet 349, 775.

Ruchel, R., Ritter, B., Schaffrinski, M., 1990. Modulation of¨experimental systemic murine candidosis by intravenous pep-statin. Int. J. Med. Microbiol. 273, 391.

Samaranayake, L.P., Holmstrup, P., 1989. Oral candidiasis andhuman immunodeficiency virus infection. J. Oral Pathol. Med.18, 554.

Seelmeier, S., Schmidt, H., Turk, V., von der Helm, K., 1988.Human immunodeficiency virus has an aspartic-type proteasethat can be inhibited by pepstatin A. Proc. Natl. Acad. Sci.USA 85, 6612.

Sepkowitz, K.A., 1998. Effect of HAART on natural history ofAIDS-related opportunistic disorders. Lancet 351, 228.

Shao, W., Everitt, L., Manchester, M., Loeb, D.D., Hutchison,C.A., Swanstrom, R., 1997. Sequence requirements of theHIV-1 protease flap region determined by saturation mutagen-esis and kinetic analysis of flap mutants. Proc. Natl. Acad. Sci.USA 94, 2243.

Stoiber, H., Ebenbichler, C., Schneider, R., Janatova, J., Dierich,M.P., 1995. Interaction of several complement proteins withgp120 and gp41, the two envelope glycoproteins of HIV-1.AIDS 9, 19.

Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTALW: improving the sensitivity of progressive multiple sequencealignment through sequence weighting, position-specific gappenalties and weight matrix choice. Nucleic Acids Res. 22,4673.

Tsuobi, R., Kurita, Y., Negi, M., Ogawa, H., 1985. A specificinhibitor of keratinolytic proteinase from Candida albicanscould inhibit the cell growth of C. albicans. J. Invest. Derma-tol. 85, 438.

Wade, J.C., 1993. Candidiasis—Pathogenesis, Diagnosis andTreatment. Raven Press, New York, p. 85.

White, T.C., Agabian, N., 1995. Candida albicans secreted as-partyl proteinases: isoenzyme pattern is determined by celltype, and levels are determined by environmental factors. J.Bacteriol. 177, 5215.

Zingman, B.S., 1996. Resolution of refractory AIDS-related mu-cosal candidiasis after initiation of didanosine plus saquinavir.N. Engl. J. Med. 334, 1674–1675.