Candida of Oral EpitheliumThe colonization and invasion of various animal oral mucosae by Candida...

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INFECTION AND IMMUNITY, July 1980, p. 252-260 Vol. 29, No. 1 0019-9567/80/07-0252/09$02.00/0 Candida albicans Ultrastructure: Colonization and Invasion of Oral Epithelium JULIE A. HOWLETT AND CHRISTOPHER A. SQUIER* Department of Oral Pathology and Oral Diagnosis and The Dows.Institute for Dental Research, College of Dentistry, The University of Iowa, Iowa City, Iowa 52242 The colonization and invasion of various animal oral mucosae by Candida albicans were examined in an organ culture model. Scanning and transmission electron microscopy of the oral epithelium between 12 and 30 h after inoculation with the fungus revealed the morphological relationships between host and parasite. Examination of the fungi in thin sections showed five distinct layers in the cell wall of C. albicans within the epithelium, but changes were evident in the organization and definition of the outer cell wall layers in budding hyphae and in hyphae participating in colonization and invasion of the epithelial cells. Adherence of the fungus to the superficial cells of the oral mucosa appeared to involve intimate contact between the epithelial cell surface and the deeper layers of the fungal cell wall. During invasion a close seal was maintained between the invading hyphae and the surrounding epithelial cell envelope, there being no other evidence of damage to the host cell surface except at the site of entry. Within the epithelial cells there was only occasional loss of cytoplasmic compo- nents in the vicinity of the invading hyphae. These findings would suggest that enzymatic lysis associated with the invasive process is localized and that the mechanical support provided by surface adherence and the intimate association between the fungus and the epithelial cell envelope may permit growth of Candida on through the epithelium. Candida infection of skin and mucosa may be considered as occurring in three phases: first, adherence and colonization of the tissue surface (23); next, invasion of the superficial epithelial cells to establish an intracellular habitat (25); and finally, the provocation of an inflammatory response in the underlying tissues (28). The ad- herence of Candida albicans to epithelial cells has been demonstrated in vitro (22, 23) and, as has been shown for certain bacteria (14, 29), is probably mediated by the interaction of surface components of both the epithelial and microbial cells. However, the indigenous bacterial flora of the oral cavity may suppress colonization of Candida by competing successfully for receptor sites on the epithelial cells, and such selective adherence may play a significant role in protect- ing the mucosa from candidal infection (17). The differing affinity of various types of epithelial surfaces for C. albicans may also be responsible for the tissue and host tropism of the fungi (23). Although there has been progress in defining receptors involved in adherence of a variety of bacterial organisms to epithelium (3, 29), the identification of receptors involved in fungal ad- herence has only just begun (J. C. Lee and R. D. King, Abstr. Annu. Meet. Am. Soc. Microbiol. 1979, F50, p. 371). There are few reports on the mechanism of fungal invasion of animal cells, and the evidence supports the role of both hydrolytic enzyme activity (31, 32, 39) and mechanical force (10) in the invasion of host tissues by fungal pathogens. Pugh and Cawson (31), studying the invasion of chicken chorioallantoic membrane by C. albi- cans, have suggested that phospholipase A may participate in this process, but whether this or other hydrolytic enzymes play a part in invasion of the superficial cells of skin and oral mucosa has not been established. The experiments to be described here examine the ultrastructural relationship between C. al- bicans and the oral epithelium during the stages of fungal colonization and early invasion. These stages are not easy to investigate in humans, where clinically recognizable lesions usually rep- resent well-established infections, and in animal models in which clinical detection of the infected tissues is difficult (17). The host-parasite rela- tionship between C. albicans and epithelial tis- sues can be studied in explants of neonatal oral mucosa without the complicating effects of the normal microbiological flora and host defense mechanisms. Previous studies with both patho- genic and nonpathogenic species of Candida have validated this system as a reliable model 252 on May 5, 2021 by guest http://iai.asm.org/ Downloaded from

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Page 1: Candida of Oral EpitheliumThe colonization and invasion of various animal oral mucosae by Candida albicans were examined in an organ culture model. Scanning and transmission electronmicroscopyoftheoral

INFECTION AND IMMUNITY, July 1980, p. 252-260 Vol. 29, No. 10019-9567/80/07-0252/09$02.00/0

Candida albicans Ultrastructure: Colonization and Invasionof Oral Epithelium

JULIE A. HOWLETT AND CHRISTOPHER A. SQUIER*Department of Oral Pathology and Oral Diagnosis and The Dows.Institute for Dental Research, College of

Dentistry, The University of Iowa, Iowa City, Iowa 52242

The colonization and invasion of various animal oral mucosae by Candidaalbicans were examined in an organ culture model. Scanning and transmissionelectron microscopy of the oral epithelium between 12 and 30 h after inoculationwith the fungus revealed the morphological relationships between host andparasite. Examination of the fungi in thin sections showed five distinct layers inthe cell wall of C. albicans within the epithelium, but changes were evident inthe organization and definition of the outer cell wall layers in budding hyphaeand in hyphae participating in colonization and invasion of the epithelial cells.Adherence of the fungus to the superficial cells of the oral mucosa appeared toinvolve intimate contact between the epithelial cell surface and the deeper layersof the fungal cell wall. During invasion a close seal was maintained between theinvading hyphae and the surrounding epithelial cell envelope, there being noother evidence of damage to the host cell surface except at the site of entry.Within the epithelial cells there was only occasional loss of cytoplasmic compo-nents in the vicinity of the invading hyphae. These findings would suggest thatenzymatic lysis associated with the invasive process is localized and that themechanical support provided by surface adherence and the intimate associationbetween the fungus and the epithelial cell envelope may permit growth ofCandida on through the epithelium.

Candida infection of skin and mucosa may beconsidered as occurring in three phases: first,adherence and colonization of the tissue surface(23); next, invasion of the superficial epithelialcells to establish an intracellular habitat (25);and finally, the provocation of an inflammatoryresponse in the underlying tissues (28). The ad-herence of Candida albicans to epithelial cellshas been demonstrated in vitro (22, 23) and, ashas been shown for certain bacteria (14, 29), isprobably mediated by the interaction of surfacecomponents of both the epithelial and microbialcells. However, the indigenous bacterial flora ofthe oral cavity may suppress colonization ofCandida by competing successfully for receptorsites on the epithelial cells, and such selectiveadherence may play a significant role in protect-ing the mucosa from candidal infection (17). Thediffering affinity of various types of epithelialsurfaces for C. albicans may also be responsiblefor the tissue and host tropism of the fungi (23).Although there has been progress in definingreceptors involved in adherence of a variety ofbacterial organisms to epithelium (3, 29), theidentification of receptors involved in fungal ad-herence has only just begun (J. C. Lee and R. D.King, Abstr. Annu. Meet. Am. Soc. Microbiol.1979, F50, p. 371).

There are few reports on the mechanism offungal invasion of animal cells, and the evidencesupports the role of both hydrolytic enzymeactivity (31, 32, 39) and mechanical force (10) inthe invasion of host tissues by fungal pathogens.Pugh and Cawson (31), studying the invasion ofchicken chorioallantoic membrane by C. albi-cans, have suggested that phospholipase A mayparticipate in this process, but whether this orother hydrolytic enzymes play a part in invasionof the superficial cells of skin and oral mucosahas not been established.The experiments to be described here examine

the ultrastructural relationship between C. al-bicans and the oral epithelium during the stagesof fungal colonization and early invasion. Thesestages are not easy to investigate in humans,where clinically recognizable lesions usually rep-resent well-established infections, and in animalmodels in which clinical detection of the infectedtissues is difficult (17). The host-parasite rela-tionship between C. albicans and epithelial tis-sues can be studied in explants of neonatal oralmucosa without the complicating effects of thenormal microbiological flora and host defensemechanisms. Previous studies with both patho-genic and nonpathogenic species of Candidahave validated this system as a reliable model

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C. ALBICANS ORAL COLONIZATION AND INVASION 253

for investigating the colonization and invasion oforal mucosa by C. albicans (18, 19).

MATERIALS AND METHODSOrgan culture system. Tissues were obtained

from Sprague-Dawley rats and New Zealand whiterabbits aged between 0 and 4 days. Orthokeratinizedmucosa was taken from the dorsal surface of rattongue, parakeratinized tissue was from the dorsalsurface of rabbit tongue, and nonkeratinized tissue wasfrom the buccal mucosa of rabbit. Explants of oralmucosa about 2 mm square were maintained in vitroin a chemically defined nutrient medium containingpenicillin and streptomycin as described previously(18).Fungal inoculation. Strains of C. albicans 3091A,

3118C, and 3153 were obtained from the MycologicalReference Laboratory at the London School of Hy-giene and Tropical Medicine, London, England. Theseare all strains which have been isolated from cases ofhuman oral candidiasis. The strains were stored as

freeze-dried samples and before experiments were

maintained on Sabouraud dextrose agar at 37°C andsubcultured every 4 days. The identity of C. albicanswas confirmed by germ tube formation in serum, chla-mydospore production on cornmeal agar, and sugar

fermentations. Two methods of inoculation of theexplants were used; one involved application of a fluidmicroinoculum containing 25 to 50 viable yeast cellsin 1 of sterile saline (18), and the other used piecesof fine sterile chrome nickel wire to inoculate theexplants with a microcolony of organisms under a

dissecting microscope (19).Harvesting and fixation. Infected explants were

maintained in vitro for 12 to 30 h and harvested atregular intervals and fixed by the following methods.For scanning electron microscopy, specimens were

fixed in 1% glutaraldehyde in 0.2 M phosphate bufferfor 0.5 h and then in 3% glutaraldehyde in the same

buffer for 48 h at room temperature. The tissues were

subsequently washed in distilled water for 1 h anddehydrated in graded ethanols with amylacetate as

common solvent for transfer to liquid CO2. Criticalpoint drying was carried out in a Polaron E 3000apparatus (Polaron Equipment Ltd., Watford, Eng-land) by the method of Anderson (1). The specimenswere mounted on aluminum studs with silver dag andsputter coated with gold in a Polaron Sputter CoaterE5000 in a low-pressure argon atmosphere; coatingthickness was between 20 and 50 nm. The specimenswere viewed in a Cambridge Stereoscan 600 scanningelectron microscope at accelerating voltages of 15 or

25 kV.For transmission microscopy, specimens were either

fixed in 3% glutaraldehyde in 0.1 M s-collidine bufferat pH 6.8 for 18 to 20 h and postfixed in 1% osmiumtetroxide in the same buffer at pH 7.4 for 20 h (12) or

fixed in 2.5% glutaraldehyde in 0.05 M phosphatebuffer at pH 7.4 at 4°C for 2 h, buffer washed, andpostfixed in 2% osmium tetroxide in the same bufferfor 0.5 h at room temperature (35). All specimens were

rinsed in water, dehydrated in graded ethyl alcohols,and transferred to dry acetone and embedded in Epon812. Ultrathin sections were cut on an LKB-Huxley

Microtome and were stained with aqueous uranyl ace-tate and lead citrate by the method of Reynolds (34)and examined in either an AEI EM6B or a Siemens101 electron microscope at 60 kV.

RESULTS

The behavior of the three strains of Candidaused in all the investigations was similar, and sono distinction will be made between them in thedescription that follows. The illustrations allshow strain 3153.

In the scanning electron microscope, blasto-spores of C. albicans appeared randomly distrib-uted on the surface of the epithelium, and inmany cases hyphae could be seen extendingfrom the parent blastospore as germ tube for-mation proceeded. The surface of the blasto-spores was generally smooth, although occa-sional truncated bud scars could be seen andhyphal elements often had irregular massescoating their surface (Fig. 1). Colonization wasevident on both the superficial and deep surfacesof the epithelial squames. No alteration of theepithelial cell surface was visible in the vicinityof fungal colonies or in the immediate area offungal contact, and it was not possible to deter-mine how firmly the blastospores adhered to thecell membranes. There was no evidence to sug-gest that blastospores were able to penetrate theepithelial cell, and only the hyphal tips pene-trated the cell membrane; in some cases longhyphae appeared to colonize the surface of theepithelial cells without invading them. At sitesof hyphal penetration of the epithelial cell, therewas little disturbance of the epithelial cell mem-brane except for some loss of surface ridgingand, similarly, there were no changes in thestructure of the penetrating hyphae where theyentered the epithelial squames (Fig. 1). On oc-casion, hyphae passed entirely through the sur-face cells.Examination of the infected explants with the

transmission electron microscope showed fungito be present both intracellularly and extracel-lularly throughout the epithelium. No differencewas seen in the pattern of colonization or inva-sion in the various types of epithelia examined.A fairly uniform population of circular or ovoidtransactions of C. albicans was present in thesections, making it difficult to distinguish be-tween sections through spherical blastosporesand tubular hyphae (Fig. 2). However, becauseonly the hyphae could be seen penetrating theepithelium in the scanning electron microscopepreparations, the majority of intracellular fungalprofiles are likely to represent hyphae, althoughthis could be verified only when longitudinalsections were encountered; in such sections, hy-

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254 HOWLETT AND SQUIER

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FIG. 1. Scanning electron micrograph showing C. albicans colonizing and invading keratinized rat tonguemucosa. The blastospores (B) have developed germ tubes and the extending hyphae are invading thesuperficial squames (S. bud scar). Marker represents 1 um; magnification, x37,000. Inset: thejunction betweena hypha and the epithelial cell membrane. Irregular masses ofmaterial can be seen coating the surface of thefungus (D). Marker represents 0.1 tMm; magnification, x84,000.

phal cross walls were often seen (Fig. 2). Inheavily invaded tissues, intracellular hyphalforms appeared to be germinating, and buds andsporelike forms were seen adjoining the parentcells.Although some differences could be seen in

the definition of the fungal cell wall with the twofixation regimens used, the contrast of the inter-nal organelles and cytoplasmic membrane struc-tures was always low, fungal contents often hav-ing a uniformly dense appearance with organ-elles being apparent only as faint outlines. It wasimpossible to relate fungal cell wall thickness tofungal form or maturation of the hyphae, butfive distinct layers were usually evident in thefungal cell wall, regardless of whether Candidawas located intracellularly or extracellularly(Fig. 3). A fibrillar-floccular exterior layer wasparticularly pronounced in those explants whichhad undergone prolonged fixation, but theamount of floccular material varied considerablyand appeared to be related to the activity of thefungus. When Candida was located extracellu-

larly among the surface squames, the outermostfloccular layer was often very distinct. Beneaththe floccular layer was a layer of highly electron-dense material, whereas the third layer consistedof filament bundles orientated parallel to thesurface, the fourth contained dispersed materialof moderate electron density, and the fifth wasan electron-dense zone approximating theplasma membrane.

Structural changes in the cell walls were ob-served in hyphae which appeared to be undergo-ing lateral budding within the tissues. At theputative site of germination, the majority of thecell wall consisted of a layer of dispersed, mod-erately electron-dense material which seemed tooriginate in the fourth layer of the mature hy-phal wall (Fig. 4). The fibrous third layer wasinterrupted, whereas the outer layers merged toform a homogeneous electron-dense floccularlayer. In no case did these outer layers disappear,as has been described in budding yeast cells (6).The Candida often assumed a very close rela-tionship with the epithelial cells and showed a

INFECT. IMMUN.

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VOL. 29, 1980 C. ALBICANS ORAL COLONIZATION AND INVASION 255

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FIG. 2. Transmission electron micrograph showing C. albicans invading rabbit tongue mucosa. Littledisturbance of host cells by the fungus is seen, but there appears to be a marked variation in the definition ofthe outer fungal cell walls. Cross u'alls can be seen in some mycelial elements (C.), although most hypaarcut in cross section. Marker represents 5 ,tm; magnification, x 7,200.

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256 HOWLETT AND SQUIER

tight approximation of the fungal cell walls tothe undulations of the epithelial cell surfaces. Inthis situation, the floccular outer layer appearedto condense or disperse so that contact betweenthe epithelial cell membrane and the inner layersof the cell wall existed (Fig. 5). However, nobreakdown of the epithelial cell membrane wasevident unless it was in direct contact with thefungal cell wall, in which case there was onlyminimal disturbance of the epithelial cell mem-brane by the invading hypha and it remainedtightly opposed to the fungal cell wall where thehypha entered or left the epithelial cell (Fig. 6).The hyphae within the squames lay free in thecytoplasm, the tonofilaments frequently abut-ting directly against the fungal wall. The finelyfloccular outer layer could often be seen aroundintracellular fungal hyphae and appeared to bein intimate contact with the host cell cytoplasmand tonofilaments, sometimes extending into thesurrounding cell cytoplasm for a considerable

INFECT. IMMUN.

distance (Fig. 7). Occasionally, hyphal profileswere surrounded by an electron-lucent zone ofvariable size (Fig. 8), but this was not a consist-ent enough finding to be characteristic of theintracellular fungus.

DISCUSSIONThere have been several studies of the estab-

lished host-parasite relationship in human su-perficial candidiasis, and it is now well acceptedthat C. albicans is able to inhabit both the intra-and extracellular environment of the epithelialtissues (7, 25, 27, 38). Many of the observationsmade in the present study are consistent withthese existing reports, but the more interestingand novel findings concern features of the earli-est stages in the establishment of the host-par-asite relationship, involving both adherence andcell invasion.The adherence ofmicroorganisms to host cells

is probably the initial event occurring in the

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FIG. 3. Five distinct layers may be distinguished in the fungal cell wall seen in this section of a fungus inthe extracellular space of the epithelium. Marker represents 0.5 jm; magnification, x54,000.

FIG. 4. Where fungal elements within the epithelium appear to be budding, changes are evident in theorganization of the cell wall. This fungal profile shows an outgrowth in which cell wall development appearsto be proceeding (arrow) 1 ,um; magnification, x25,600.

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C. ALBICANS ORAL COLONIZATION AND INVASION 257

colonization process in many microbial infec-tions (13). C. albicans has been shown to adhereto the oral epithelial cells of rodent (23) and tohuman buccal (22) and vaginal cells (R. D. King,A. L. Morris, R. L. Taylor and E. E. Moody,Abstr. Annu. Meet. Am. Soc. Microbiol. 1977,F23, p. 122). It is likely that specific receptorsare present on the surface of both the epithelialcell and the fungal cell (14, 29). Lee and Kinghave attempted to characterize the adherence ofC. albicans to vaginal epithelial cells and claimthat the sites on the surface of the blastosporewhich mediate attachment differ markedly fromthose described for other microbial agents (J. C.Lee and R. D. King, Abstr. Annu. Meet. Am.Soc. Microbiol. 1978, F36, p. 319, and 1979, F50,p. 371).

Indigenous microbes attaching to the surface

of the gastrointestinal tract have been dividedinto those that induce no changes in the struc-ture of the epithelial cell membranes and thosethat bring about obvious alterations in the mem-branes of the cells to which they attach (36).The absence of any disturbance of the epithelialcell membranes in the proximity of the fungalcells described in the present study suggests thatC. albicans in the oral cavity belongs to theformer category. Although adherence of the fun-gus to the epithelial cell membrane may initiatechanges associated with invasion, this did notappear to be represented by actual membranebreakdown, and the scanning electron micro-scope evidence indicated that penetration of theepithelial cell by the hyphal tip occurs at somedistance from the site of attachment of the par-ent blastospore. Thus, the two events may be

-.4

.5FIG. 5. Outer layer of the fungal cell wall is often very distinct in fungi among the surface squames, except

where close contact exists between the fungal cell wall and surface of the epithelial squames. Here a junctionappears to form between the epithelial cell envelope and the second or third layers of the fungal cell wall(arrow). Such sites may be associated with the receptors mediating adherence of the fungus to the host cellsurface. Marker represents 1 tum; magnification, x20,000.

FIG. 6. Penetration of epithelial squames (S) by hyphae produces minimal disturbance of the host cellmembranes and cytoplasm and tonofilaments abut directly against the fungal cell wall. Marker represents 1,um; magnification, x20,000.

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258 HOWLETT AND SQUIER

independent of one another. Whether the pene-trating hyphal tip must adhere before invasionis not known.The importance of an understanding of the

cell wall structure of C. albicans would seem tolie in the significance of its antigenic componentsand its associated immunogenicity and enzymeactivity. Fixation is a key factor in the ultrastruc-tural demonstration of fungal cell wall detail; inthe present study primary fixation with glutar-aldehyde, especially when prolonged, gave gooddefinition of the cell wall. One of the problemsof investigating the structure of the wall in ul-trathin sections during colonization and invasionof the epithelial tissues is that of distinguishingbetween blastospores and hyphae on the basisof sectional profile. As mentioned in the results,there was no evidence to suggest that the blast-ospores could invade the epithelial cells directly,and previous histological studies of oral mucosalexplants infected with C. albicans have shownthe hyphal form to predominate within the epi-thelium in this model (18). However, the pres-

ence of intraepithelial blastospores cannot betotally discounted, for a lateral budding of themycelial forms was seen in both the previoushistological studies (18) and in the present ultra-structural investigations.

C. albicans growing within the epitheliumshowed a multilayered cell wall similar to thatdemonstrated by Djaczenko and Cassone forblastospores in culture (9). However, a markedvariation in appearance and width of the outercell wall layers was seen in the fungal profileswithin the tissues. Although there is some con-flict in the literature as to the delineation of thevarious layers in the cell wall (7, 9, 30), it isaccepted that some structural alterations occuras a result of growth (6, 8, 37) and as a conse-quence of the nutrient conditions available (5).Cassone et al. (6) reported the blastospore cellwall to be about twice the width of the mycelialwall in C. albicans growing in culture, and Chat-taway et al. (8) showed changes in the width ofthe wall of both forms during maturation. Suchchanges may well account for some of the vari-

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FIG. 7. Although the outer fungal cell wall becomes less conspicuous when Candida is in close associationwith the epithelial cell membrane, once entry into the cell has occurred the outer layer is again distinctlyvisible, and fibrillar material often extends some distance into the host cell cytoplasm (arrow). Markerrepresents 1 ,im; magnification, x15,000.

FIG. 8. Although the majority of sections of intracellular fungi show close contact between the epithelialcell contents and the fungal cell walls, a loss of material around individual fungal profiles is occasionallyseen (arrow). Marker represents 2 tum; magnification, x 10,000.

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C. ALBICANS ORAL COLONIZATION AND INVASION 259

ation seen in the width of the cell wall of thefungi within the oral epithelium, because thesections of oral mucosa obviously contain fungiat different stages of development. Variation inthe organization of the cell wall on germinationhas also been described by Cassone et al. (6) andScherwitz et al. (37) for Candida in culture, andsimilar structural changes in the third and fourthlayers of the cell wall were evident in some ofthe fungi in the present studies. However,whereas both Cassone et al. (6) and Scherwitzet al. (37) reported the disappearance of theouter floccular layer in budding cells in culture,this layer was never absent in our material andwas often more pronounced at the sites whereexpansion and bud formation appeared to beoccurring in the intracellular fungi. The moststriking changes in the outer layers of the wallof the invading fungi in the present study coin-cided with the establishment of an intimate re-

lationship between fungi and epithelial cells.Thus, changes occurring in the cell wall may notonly be associated with the development andgrowth of Candida but could also reflect eventsinvolved in the colonization and invasion of tis-sues.The mechanisms by which fungi gain entry

into the cell have still not been definitely estab-lished. Most intracellular parasites enter hostcells as a result of phagocytosis, which may bea host cell or parasite dependent process (26).Although phagocytosis of C. albicans by thecells of the reticuloendothelial system is wellestablished (2, 4), the absence of any membraneenclosing the intracellular fungi and the inabilityof terminally differentiated epithelial cells toparticipate in this activity (40) would suggestthat a different process is occurring during fun-gal invasion of the oral epithelium. A number ofworkers claim that fungal entry is dependentupon hydrolytic enzyme activity (31, 33). Elec-tron cytochemical studies by Pugh and Cawson(31) implicate phospholipase A in the invasionof the respiratory-type epithelium of the chickenchorioallantoic membrane by C. albicans. Theseworkers have suggested that enzyme activitymay be both widespread, facilitating fungal in-vasion by weakening the surface epithelial cells,as well as localized, thereby permitting mem-

brane penetration and intracellular invasion. Inthe present studies there is little evidence tosupport a role for widespread enzyme effects onthe epithelial cells of oral mucosa underlyingcolonies of C. albicans. Epithelial cell mem-

branes were only seen to be interrupted at theactual site of contact with the fungal cell wall,and no changes in the membranes were seen

which could be attributed to diffuse enzymeactivity from the surface fungi. However, the

phenomenon of generalized membrane damagewas proposed with a chorioallantoic epithelialmodel which has little resemblance to the differ-entiated epithelium of epidermis and oral epi-thelium. It is likely that the well-establishedphysical and chemical modifications of the cellmembranes of the superficial cell layers of bothkeratinized (21) and nonkeratinized (11) epithe-lium may protect them from the effects of gen-eralized enzyme activity, at least during the ini-tial stages of the colonization process. This re-sistance on the part of the epithelium may alsoaccount for the lack of penetration of the oralepithelium by blastospores.The extremely close relationship established

between plasma membrane and fungal cell wallevident in the earliest stages of hyphal invasionin the present study indicates that enzymaticbreakdown of the epithelial membrane, if occur-ring at all, must be closely related to the pointof contact between the membrane and the hy-phal tip and presumably associated with theouter portion of the cell wall. However, althoughhistochemical evidence supports the role ofphospholipase in the penetration of respiratorytype epithelium (31), the function of this enzymeis somewhat enigmatic in a keratinized epithe-lium, such as that of skin or regions of the oralmucosa, where biochemical evidence suggests anabsence of phospholipids in the superficial cellmembranes (15, 16). Presumably, other hydro-lytic enzymes which have been isolated in asso-ciation with C. albicans (20) may function in thepenetration of the thickened cell membranes ofthese cells. The only evidence for possible en-zymatic lysis of cytoplasmic components in thisstudy was the electron lucency around certainhyphae (Fig. 8) which has been reported in otherstudies (24, 25, 27). On the other hand, mechan-ical factors may play a role in the penetrationprocess. The binding of yeasts and epithelialcells during adherence and the close approxi-mation of the fungal cell wall and epithelial cellcomponents during the invasive process wouldundoubtedly provide anchorage for the hyphaeand allow for growth onward through the epi-thelial tissues. Thus, although the present studysupports the concept of a localized enzyme-me-diated penetration by the growing hyphal tip ofC. albicans, it also underscores the possible roleof the outer cell wall not only as a frameworkfor enzyme activity but also as a mechanicalsupport for the parasite entering the epithelialcells.

ACKNOWLEDGMENTS

This work was supported by the Dows Dental ResearchFund and by Public Health Service grant 5-501-445313-17from the National Institutes of Health.

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LITERATURE CITED

1. Anderson, T. F. 1951. Techniques for the preservation ofthree dimensional structure in preparing specimens forthe electron microscope. Trans. N.Y. Acad. Sci. 13:130-134.

2. Arai, T., Y. Mikami, and K. Yokoyama. 1977. Phago-cytosis of Candida albicans by rabbit alveolar macro-phages and guinea pig neutrophils. Sabouraudia 15:171-177.

3. Bartelt, M. A., and J. L. Duncan. 1978. Adherence ofgroup A streptococci to human epithelial cells. Infect.Immun. 20:200-208.

4. Belcher, R. W., J. F. Carney, and F. G. Monahan.1973. An electron microscopic study of phagocytosis ofC. albicans by polymorphonuclear leucocytes. Lab. In-vest. 29:620-627.

5. Cassone, A., D. Kerridge, and E. F. Gale. 1979. Ultra-structural changes in the cell wall of C. albicans follow-ing cessation of growth and their possible relationshipto the development of polyene resistance. J. Gen. Mi-crobiol. 110:339-349.

6. Cassone, A., N. Simonette, and V. Strippoli. 1973.Ultrastructural changes in the wall during germ-tubeformation from blastospores of C. albicans. J. Gen.Microbiol. 77:417-426.

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