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In Vivo Expression and Localization of Candida albicansSecreted Aspartyl Proteinases during Oral Candidiasis inHIV-Infected Patients

Martin Schaller, Bernhard Hube,* Markus W. Ollert,† Wilhelm Schafer,* Margarete Borg-von Zepelin,‡Eva Thoma-Greber, and Hans C. KortingDepartment of Dermatology, Ludwig-Maximilians-University Munich, Munich, Germany; * Institute for General Botany, Applied Molecular Biology III,University of Hamburg, Hamburg, Germany; †Department of Dermatology and Allergy, Technical University Munich, Munich, Germany;‡Institute of Hygiene, Georg-August-University Gottingen, Gottingen, Germany

Isoforms of aspartyl proteinase (Sap), which areencoded by at least nine related SAP genes, have beenimplicated to be a major virulence factor of theopportunistic yeast Candida albicans in experimentalinfections. Although it is generally assumed thatproteinases are important for infections, detailedinformation on the pathogenetic role of Saps is stilllacking. The same applies to the question whether thegenes and corresponding isoforms of the enzyme areexpressed during oral infection. For in vivo investi-gations, parts of the lesional oral epithelium werecollected from three HIV-infected patients with oro-pharyngeal candidiasis. Immunoelectron microscopywas performed (pre- and post-embedding goldlabeling with silver enhancement) using an anti-Sapmurine monoclonal antibody directed against the gene

The opportunistic yeast Candida albicans, the majorcause of cutaneous and mucosal candidiasis, possessesa panel of putative virulence factors that are thoughtto enable the fungus to invade host tissue (Hommaet al, 1992; Hoegl et al, 1996). Among these virulence

attributes, many studies have focused on secreted aspartyl proteinases(Saps). Sap enzymatic activities have received considerable attentionin several in vitro (Borg-von Zepelin and Ruchel, 1988; DeBernardis et al, 1992; Homma et al, 1992; Ollert et al, 1993) andanimal studies (MacDonald and Odds, 1983; Kwon-Chung et al,1985; Ghannoum and Abu Elteen, 1986; Ray and Payne, 1988;Fallon et al, 1997). In these studies various biologic functions havebeen attributed to the Saps. For example, it has been suggestedthat Saps may enhance attachment to mucosal surfaces (Borg-von Zepelin and Ruchel, 1988), help to escape phagocytosisby granulocytes and macrophages (MacDonald and Odds, 1983;Ghannoum and Abu Elteen, 1986; Borg-von Zepelin et al, 1998),

Manuscript received October 7, 1998; revised November 13, 1998;accepted for publication November 16, 1998.

Reprint requests to: Dr. M. Schaller, Department of Dermatology,Ludwig-Maximilians-Universitat, Frauenlobstr. 9-11, D-80337 Munchen,Germany.

Abbrevations: SAP, secreted aspartyl proteinase (gene); Sap, secretedaspartyl proteinase (protein).

0022-202X/99/$10.50 · Copyright © 1999 by The Society for Investigative Dermatology, Inc.

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products Sap1–3. It was possible to demonstrateexpression of Sap antigens in each of the three samplesof human oral candidiasis. This suggests that at leastone of the genes SAP1–3 was expressed at the time ofsample collection. Furthermore, a possible role of theenzymes during the interaction of yeast cells andmucosal cells is suggested: the majority of Sap antigensis secreted by those C. albicans cells that adhere directlyto the epithelial surface. Sap immunoreactivity can bedetected in particular at the site of close contactbetween C. albicans and epithelial cells, suggesting apathogenetic role of the Saps in host–fungal inter-action. Thus, inhibition of the enzyme might proveto be an important alternative in the prevention andtreatment of candidiasis. Key words: HIV-infectedpatients/immunoelectron microscopy/SAP/silver enhance-ment. J Invest Dermatol 112:383–386, 1999

or hydrolyze immunoglobulins such as secretory IgA (Hoegl et al,1996; Hube, 1996). In previous electronmicroscopic investigationsof the interaction of C. albicans cells with human keratinocytes,the influence of keratinolytic enzymes on the initiation of diseasewas postulated (Borg-von Zepelin and Ruchel, 1988). Using surfaceelectron microscopy it has been shown that the addition of theaspartyl proteinase inhibitor pepstatin A reduced the number ofCandida elements adhering to epithelia in experimental infections(Borg-von Zepelin and Ruchel, 1988), scanning electron micro-scopic investigations also demonstrated that inhibition of Saps bypepstatin reduces a cavitation process of C. albicans on mousecorneocyte surfaces (Ray and Payne, 1988). Antibodies against Sapsin systemic candidiasis as indirect evidence for expression in manwere demonstrated (Ruchel, 1983). Until now at least nine differentSAP genes have been identified (Monod et al, 1998). Northernanalysis of the SAP gene family showed a differential regulation ofthese genes in response to environmental factors and cell morpho-logy (Hube et al, 1994; White and Agabian, 1995; Hube, 1996;Monod et al, 1998). This suggests that distinct SAP genes may playdifferent roles during the infection process (Hube et al, 1994).Western blot analysis of C. albicans cell culture supernatants experi-ments showed that Sap isoenzymes were predominantly regulatedon the transcriptional level (White and Agabian, 1995). Theexpression of SAP1 and SAP2 has been demonstrated by northernblot analysis by means of an experimental rat vaginitis model (DeBernardis et al, 1995). Animal experiments with SAP null mutants

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indirectly showed that SAP1, 2, 3 and at least one of the closelyrelated SAP4–6 genes are expressed during disseminated infections,and presumably contribute to the overall virulence of C. albicans(Hube et al, 1997; Sanglard et al, 1997). The regulation of SAPmRNA levels was recently investigated in an in vitro model of oralcandidiasis based on reconstituted human epithelium. In theseexperimental infections, the expression of SAP1, SAP3, and SAP6correlated chronologically with severe histologic alterations of theepithelium. Furthermore, immunoelectron microscopical investig-ations of the infected tissue demonstrated Sap antigen within theC. albicans and the epithelial cells, suggesting an important role ofSap isoenzymes during mucosal infections (Schaller et al, 1998). Asimilar gene expression pattern (SAP1, SAP2, SAP3, SAP6) wasalso detected by reverse transcriptase-polymerase chain reaction inclinical samples from patients suffering from oral candidiasis (Schalleret al, 1998). In this report we demonstrate the localization andexpression of Sap antigen during human oropharyngeal candidiasisfor the first time, using a monoclonal antibody directed against thesecreted isoforms of Sap1–3. The localization of the enzymesin vivo was studied by pre- and post-embedding immunoelectronmicroscopy.

MATERIALS AND METHODS

Patients and samples Samples of pseudomembrane were removed fromthe tongue of three volunteer male HIV-infected patients (A, B, C). Theywere 37, 56, and 44 y old and had been suffering from pseudomembraneousoropharyngeal candidiasis for at least 1 y. In spite of continuous treatmentwith systemic anti-mycotic drugs at the time the specimens were obtained,no improvement of the oropharyngeal candidiasis had been achieved. Partsof the clinical material of all three patients were used for microbiologicculture, biochemical characterization, and immunoelectron microscopicalinvestigations.

Mycologic examination The specimens obtained were inoculated onKimmig’s Agar (Merck, Darmstadt, Germany) and incubated for 72 h at37°C. Biochemical identification of C. albicans was based on the use ofthe Ready-Made System ATB 32 C (API System, bio Merieux, LaBalme-les-Grottes, Montalieu Vercieu, France).

Immunoelectron microscopy Post-embedding immunogold labelingwas carried out for intracellular detection of antigen. Scrapings of thetongue were fixed in Karnovsky solution for 30 min at room temperatureand embedded in glycide ether. Sections of 80–100 nm thickness weremounted on nickel grids. The grids were rinsed on drops of dH2O for10 min, and floated on drops of phosphate-buffered saline (PBS), containing5% (vol/vol) normal goat serum, for 2 3 10 min. Grids were thenincubated with the anti-Sap 1–3 monoclonal mouse IgG antibody FX 7–10 (Ollert et al, 1995; Schaller et al, 1998) diluted in PBS supplementedwith 0.1% bovine serum albumin and 0.05% Tween 20 (PBS-BT) at aratio of 1:50 for 14 h at 4°C. As demonstrated by enzyme-linkedimmunosorbent assay, FX 7–10 was shown to react strongly with Sap1,Sap2, and Sap3, but not with Sap4, Sap5, and Sap6. Cross-reactivity withpepsin and cathepsin D was not detected (Borg-von Zepelin, personalcommunication). The exact experimental system for testing the specificityof the antibody has been described previously (Borg-von Zepelin andGruness, 1993). Grids were washed repeatedly with PBS-BT and incubatedwith the 10 nm gold-conjugated goat anti-mouse IgG (Auroprobe EMImmunogold reagents, Amersham, Buckinghamshire, U.K.) diluted 1:25in TBS (0.02 M Tris hydrochloride acid buffer, 0.15 M NaCl, 0.015 MNa-azide, 0.1% bovine serum albumin, 0.05% Tween 20, adjusted topH 8.2) for 1 h at room temperature. In control samples the anti-Sap1–3monoclonal antibody was omitted. After several washing steps with TBS-BT, grids were fixed with 2% glutaraldehyde and washed again in dH2O.They were stained with 0.5% uranyl acetate for 10 min, and 2.7% leadcitrate for 5 min (Ultrastainer, Leica, Bensheim, Germany) at 20°C. Gridswere examined with a Zeiss EM 902 transmission electron microscope(Zeiss, Oberkochen, Germany) operating at 80 kV, at magnificationsbetween 33000 and 385,000.

Pre-embedding immunogold labeling was carried out for studyingantigen distribution on the cell surfaces. The unfixed scrapings from thetongue were preincubated with 5% normal goat serum (Amersham) inPBS for 20 min at room temperature and then incubated with the anti-Sap1–3 monoclonal mouse IgG antibody FX 7–10 (Ollert et al, 1995;Schaller et al, 1998) diluted in PBS supplemented with 0.1% bovine serumalbumin and 0.05% Tween 20 (PBS-BT) at a ratio of 1:10 for 12 h at

Figure 1. Post-embedding immunogold labeling using the FX 7–10 Sap1–3 specific antibody with 10 nm gold particles in a sampleof C. albicans from oral candidiasis of patient C. Strong and regulardeposition of Sap antigens within the cytoplasm of a C. albicans cell. Onlya few electron dense deposits are seen within the cell wall and the epithelialcell (EC). Scale bar: 1 µm.

4°C. After washing with PBS, tissue samples were incubated with 5 nmor the 10 nm gold-conjugated goat anti-mouse IgG (Auroprobe EMImmunogold reagents, Amersham), diluted 1:5 in TBS for 3 h at roomtemperature. In control samples the anti-Sap1–3 monoclonal antibody wasomitted. Tissue samples were washed with TBS, supplemented with 1%bovine serum albumin, fixed in Karnovsky solution for 1 h at roomtemperature, and repeatedly washed with PBS. For higher detectionefficiency silver enhancement (IntenSE M Silver Enhancement System,Amersham) was performed in a subset of the samples, which were labeledwith 5 nm gold particles. The other subset was post-fixed in Daltonsolution for 30 min at room temperature. Both subsets were dehydratedin graded series of ethanol, and embedded in glycide ether. Sections of60–90 nm thickness were mounted on uncoated copper grids and stainedwith 0.5% uranyl acetate for 10 min and 2.7% lead citrate for 5 min(Ultrastainer, Leica, Germany) at 20°C, and examined with the Zeiss EM902 transmission electron microscope operating at 80 kV, at magnificationsbetween 33000 and 385,000.

RESULTS AND DISCUSSION

All three samples investigated were from HIV-infected patientsbecause higher proteolytic activity in strains isolated from thosepatients was demonstrated (Ollert et al, 1995). Candida albicans wasidentified microbiologically in all three patients. In all patientsamples bacteria (not shown) and different morphologic types ofC. albicans cells (Figs 1–3) could be observed on the ultrastructurallevel. The diameters of the C. albicans cells ranged from 1.5 µm to3 µm. Often yeast elements were attached to keratinocytes (Fig 2),hyphal cells were penetrating parakeratotic keratinocytes or corneo-cytes (Fig 3) and were also found within these cells (Fig 1).Figures 1 and 2 demonstrate Sap immunoreactivity after post-embedding immunogold labeling, whereas Sap immunoreactivityafter pre-embedding immunogold labeling is shown in Fig 3. Twodifferent labeling methods were performed in all samples in orderto detect intra- and extracellularly localized Sap antigens with amonoclonal antibody directed against Sap1–3. Five nanometer goldparticles were used to improve sensitivity. Silver enhancement wasperformed to intensify the labeling of the 5 nm gold particles so

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Figure 2. Post-embedding immunogold labeling with 10 nm goldparticles in a sample of C. albicans from oral candidiasis of patientB. Deposition of Saps within the invaginations of the cytoplasmic membrane(arrow), the cell wall of a C. albicans cell (double arrow), and the epithelialcell (EC). Note the vesicles (arrowheads) within the Candida cell and at theouter surface of the cell wall. Scale bar: 0.5 µm.

that detection of the immunoreactivity could occur more easily.Sap immunoreactivity was seen in all three cases of oral infectionsand findings were similar in each patient. Sap antigens were detectedwithin and in close vicinity to different morphologic types of C.albicans cells, e.g., within yeast cells attached to keratinocytes orwith hyphal elements penetrating parakeratotic keratinocytes orcorneocytes. For detection of Sap immunoreactivity within the C.albicans cells or the keratinocytes, post-embedding labeling with10 nm gold particles showed two different patterns of expression.In a subset of cells, intensive gold labeling, representing theenzymes, could be detected in a regular distribution within thecytoplasm of the Candida cells, whereas only a few gold particleswere seen within the cell wall and the epithelial cells near by(Fig 1). In another subset the majority of Sap immunoreactivitywas identified in the outer area of the Candida cells, mainlyconcentrated at the site of direct contact between Candida and thehost cell. Gold labeling was detected especially in close proximityto the invaginations lying between the plasma membrane and thecell wall, and within the adjoining epithelial cells (Fig 2). It istempting to speculate that the intracellular distribution of Sap1–3antigen in the cytoplasm of Candida cells might represent a firststep during the infection process of a particular cell. Furthersteps comprised localization of the Saps in the space betweeninvaginations of the cytoplasmic membrane and the Candida cellwall and, after secretion, within the epithelial cells. The mainconcentration of intracellular gold labeling was often detected atthe site of direct interaction of the Candida and the host cells,indicating a role of Saps in this location of infection. We did notfind large pools of internal Sap antigen. In contrast the greatestpart of total gold labeling was located outside the Candida cells,supporting the view that Saps are secreted immediately aftertranslation (Homma et al, 1993). Membrane bound vesicles wereseen within the Candida cells at the inner side of the cell wall.Vesicle transport through the C. albicans cell wall was obvious. The

Figure 3. Pre-embedding immunogold labeling with 5 nm goldparticles and silver enhancement in a sample of C. albicans fromoral candidiasis of patient A. Antibody deposition, indicating thepresence of Saps at the site of penetration of an epithelial cell representinga possible microniche (arrows). Note the proteolysis of the epithelial outercell membrane and the keratin (stars) at the site of penetration. CM, intactcell membrane; arrowheads, exocytosis of vesicles from the Candida cell.Silver enhanced labeling is sometimes associated with the presence ofvesicles (arrows and arrowheads). Scale bar: 0.5 µm.

interior of the vesicles found was not labeled. In contrast to thesecretion of phospholipase within vesicles through the cell wall(Pugh and Cawson, 1975), such a pathway may not be relevant inthe case of secretion of Sap1–3 in vivo. Release of enzymes intothe periplasmatic and extracellular space in yeast cells is directedby the secretory pathway system. Transport of proteins occurs fromthe endoplasmatic reticulum via the Golgi apparatus and secretoryvesicles to the plasma membrane. Secretion to the cell surface isachieved by fusion of the vesicles with the plasma membrane. Thefusion event results in invaginations of the plasma membrane atthe inner side of the cell wall (Reid, 1991). This later stage ofprotein transport was frequently observed in our samples, asdemonstrated by gold labeling of the invagination in Fig 2,indicating that Saps are released into the periplasmatic space.

Minute inspection of Candida cells directly attached to epithelialcells gave evidence of Sap antigens at the site of penetration, andclearly showed destruction of the outer cell membrane and thekeratin of the parakeratotic epithelial cells (Fig 3). At a lowerdegree Sap immunoreactivity was also seen on the outer surface ofthe epithelial cells in the close vicinity, but without alterations oftheir outer membranes (not shown). Sap immunoreactivity wasalso associated with the presence of vesicles, but was never seen

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within the vesicles (Fig 3). Exocytosis of these vesicles from C.albicans cells was clearly demonstrated (Fig 3). In control experi-ments under identical conditions, yet without addition of mono-clonal antibody, no gold marker could be detected. The substratespecifity of C. albicans Sap2 and probably most other Sap isoenzymesis very broad. Host proteins, such as keratin, collagen, and mucin,were considered to be possible targets for Saps during infection(Borg-von Zepelin and Ruchel, 1988; Colina et al, 1996; Hube,1996). According to our results, degradation of parakeratotickeratinocytes or corneocytes is obviously demonstrated in thepresence of Sap immunoreactivity at the site of hyphal penetration(Fig 3). A part of the clinical material from one patient (patientC) was previously used to show a distinct SAP gene expression(Schaller et al, 1998). In this sample SAP1, SAP2, SAP3, and SAP6transcripts were detected, suggesting a role of these genes duringoral candidiasis (Schaller et al, 1998). The in vivo detection of SAPgene expression and Sap immunoreactivity in the same clinicalsample of patient C indicates the presence of secretory aspartylproteinases in human oral candidiasis in HIV-infected patients forthe first time. This also includes the expression of SAP2 duringhuman oral infections. Previously, this fact was questionable,because an acidic pH range was necessary for the induction andactivity of Sap2 in several in vitro studies (Hube, 1996); however,Sap2 may act in acidic microniches within the normally neutralmilieu of the oral cavity (Ruchel et al, 1991). Such a possiblemicroniche with an acidic pH range might be demonstrated inFig 3. The protein keratin is an important differentiation productof the human epidermis and represents the main component ofortho- or parakeratotic corneocytes. During this differentiationprocess, cells of stratified squamous epithelia become filled mainlywith fibrous and amorphous proteins and enveloped by a thickenedand resistant plasma membrane. Keratinolytic enzymes are necessaryfor the ability of C. albicans to invade corneocytes. In 1984,production of a keratinolytic proteinase was demonstrated whenC. albicans was cultivated in a medium containing human stratumcorneum as a nitrogen source (Negi et al, 1984). It was suggestedthat this proteinase may play an important role in superficialinfection by affecting the human corneocytes (Negi et al, 1984).The presence of Sap immunoreactivity directly at the site ofkeratinolytic degradation of the host during the infection process,suggests an important role of this virulence factor for the develop-ment of oral candidiasis in vivo. This provides a rationale for thedevelopment of Sap inhibitors as anti-mycotics for clinical use(Abad-Zapatero et al, 1996).

The authors thank E. Januschke (Ludwig-Maximilians-University, Munich,Germany) for excellent technical assistance and M. Roecken (University of Munich,Germany) for critical reading of the manuscript.

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