Ultrastructural Imaging of Candida albicans Adhesionto Rat Genital Epithelium through Scanningand Transmission Electron Microscopy

Edilson Damke,1 Agenor Storti-Filho,1 Mary M.T. Irie,1 Márcia A. Carrara,1 Marcia R. Batista,1

Lucélia Donatti,2 Luciene S.A. Gunther,1 Eliana V. Patussi,1 Terezinha I.E. Svidzinski,1

and Márcia E.L. Consolaro1,*

1Department of Clinical Analysis, State University of Maringá, Av. Colombo, 5790, 87020-900, Maringá, Paraná, Brazil2Department of Cell Biology, Federal University of Paraná, Curitiba, 85131-990, Paraná, Brazil

Abstract: The adhesion of Candida albicans to the genital epithelium has not been fully investigated in vivo. Theobjective of this study was to evaluate the ultrastructural aspects of C. albicans adhesion in the lower genitalsystem of female Wistar rats through scanning and transmission electron microscopy. The genital infectionpersisted until the end of the experiment, and all rats showed the same adhesion aspects. Various associatedyeast/hyphae were observed in the lumen and adhered both at the vaginal and endocervical levels where thefungal filamentation process occurred. In the vaginal epithelium, closely adhered yeasts were observed asstretched strands bridging between yeasts and the epithelium surface. Different stages of the adhesion, whereyeasts internalized into the epithelial cell inside a cytoplasmic vacuole, resembling endocytosis, and a widefibrillar-floccular, glycocalyx-like layer on the yeasts were observed. On the endocervix, the adhesion occurredbetween the cilia. In the uterine body, only a yeast-like form was observed with superficial contact. This studyreached the initial goal of demonstrating an experimental model for in vivo studies. Continuation of this line ofresearch is important for studies of vulvovaginal candidiasis.

Key words: Candida albicans, adhesion in vivo, ultrastructure, genital epithelium

INTRODUCTION

Some aspects of the adhesion of Candida albicans to thegenital epithelium have not been fully investigated in vivo.Adherence to host tissues and production of extracellularenzymes are two of the most important attributes of thevirulence of C. albicans ~Calderone & Fonzi, 2001!. Adher-ence of yeast to the vaginal mucosa is an essential step inmicrobial colonization and a key event in the initiation ofvulvovaginal candidiasis ~VVC!. Prior colonization is a pre-disposing factor for the infectious process ~Kamai et al.,2002!. Because of the importance of adherence, some invivo and in vitro studies are being developed to characterizethe adherence of C. albicans to cell ~palatal and oral epithe-lia! and inanimate surfaces ~Jabra-Rizk et al., 2001; Shinet al., 2002; Irie et al., 2006!. However, with regard to thevaginal epithelium, there is still much to be understoodabout the in vivo adherence and penetration of Candida sp.and its influence on the pathogenesis of VVC.

VVC is an infectious process caused by the abnormalgrowth of yeasts in the mucosa of the female genital tract. It

is an infection of the vulva and vagina caused by yeasts thatnormally inhabit the vaginal mucosa, but which may be-come pathogenic in predisposing conditions ~Sobel, 2007!.C. albicans is the main agent responsible for 80 to 90% ofVVC ~Sobel, 1993; Pichová et al., 2001!. According to Mardhet al. ~2002!, approximately three-quarters of all women willexperience an episode of VVC at least once in their lives,and 40–50% of them will have more than one attack.Because it afflicts millions of women annually causing greatdiscomfort, interfering with sexual and affectional relations,and impairing work performance, VVC is considered animportant world health issue ~Consolaro et al., 2005!.

To aid in these aspects, we studied ultrastructural as-pects of C. albicans adhesion in the lower genital system offemale Wistar rats through scanning electron microscopy~SEM! and transmission electron microscopy ~TEM!.

MATERIALS AND METHODS

Animals

In this study, 20 adult female Wistar rats weighing 200–300 g, aged about three months, supplied by the CentralAnimal House of the State University of Maringá, Paraná,

Received July 23, 2009; accepted January 19, 2010*Corresponding author. E-mail: melconsolaro@yahoo.com.br

Microsc. Microanal. 16, 337–345, 2010doi:10.1017/S1431927610000164 Microscopy AND

Microanalysis© MICROSCOPY SOCIETY OF AMERICA 2010

Brazil ~UEM!, were employed. This research was approvedby the Committee on Ethical Conduct in the Use of Ani-mals of UEM ~Protocol no. 013/2006, statement no. 050/2006!. We used groups of five rats twice in two differentexperiments.

Experimental Vaginal Infection

The infection model was established as previously describedby De Bernardis et al. ~2006!, with some adaptations. Toinduce the infection, the state of prolonged pseudo-estrus isessential. Briefly, the rats were given subcutaneous injec-tions of estradiol valerate ~Sigma, Switzerland! dissolved insesame seed oil, 0.2 mg/week/rat, administered as fractionsthree times/week for four weeks. The hormonal activity waschecked by analysis of the cells from the vaginal fluid insmears stained by Papanicolaou ~Pap! ~Bibbo, 1997!. Pseudo-estrus was considered to be established when a predomi-nance of enucleated cornified cells was observed ~Mandl,1951!.

We used a strain of C. albicans isolated from a VVCpatient identified in the Laboratory of Medical Mycology ofUEM, where it has been stored, since its identification, in10% glycerinated water at �208C. The yeast isolate wasidentified through classical methods ~Kurtzmann & Fell,1998! and also by rDNA sequencing ~Sugita et al., 2002!. Asuspension of the yeast was prepared in a concentration of5 � 108 yeasts/mL counted in a Neubauer chamber.

To establish the infection, 80 mL of the yeast suspen-sion was introduced intravaginally one week after the begin-ning of the hormonal treatment. The development of theinfection was assessed after seven days with cultures ofvaginal secretions for yeasts in Sabouraud Dextrose Agar~SDA; Sigma Chemical, St. Louis, MO, USA! supplementedwith 50 mg/mL of chloramphenicol ~Sigma Chemical! for24–48 h at 258C. The maintenance of the infection wasassessed weekly for three weeks after induction. After thefungal growth in SDA, the number of yeasts was counted~colony-forming units, CFU/mL!, and the infection wasconsidered positive with CFU � 100 yeasts/mL.

Light Microscopy

Vaginal fluid was obtained with a plastic pipette after admin-istration of 20 mL sterile saline to the vagina of the rats. Adrop of this material was prepared as smears on glass slidesand stained for Papanicolaou ~Pap! ~Sigma Chemical!. Afterhormone injection, hormonal activity was checked weeklyby analysis of the vaginal-fluid cells in smears stained byPap ~Bibbo, 1997! and observed by optical microscopyusing a Nikon Eclipse E100 with 10� and 40� objectives.Pseudo-estrus was considered to be established when apredominance of enucleated cornified cells was observed~Mandl, 1951!.

In addition to the culture for assessing the developmentand maintenance of the infection, the yeasts/hyphae-likeform and vaginal cells were also observed in Pap smears by

light microscopy. Yeast-like forms of Candida sp. were ob-served in Pap smears as ovoid or round cells, 3–6 mm indiameter. They occurred isolated or in small groups at theperiphery of squamous cells, or in clusters close to leuko-cytes and cellular debris. Budding yeasts were also observed.The filamentous types, represented by hyphae-like forms,appeared as straight or curved filaments of variable lengthsand generally segmented. The appearance of mixed types~yeasts and filaments! was common ~Bibbo, 1997!.

For histopathology, tissue samples from the vagina anduterine cervix and body were prepared and sectioned in anultramicrotome using glass and diamond blades. This pro-cedure is described below in the Transmission ElectronMicroscopy section. The ultrathin sections were mountedon glass slides, stained by silver impregnation ~Gomori-Grocott! ~Sigma Chemical!, and examined under the lightmicroscope with 10� and 40� objectives.

Scanning Electron Microscopy

Three weeks postinfection all infected rats were killed withan overdose of anesthetics ~Ketamine and Xylazine, Parke-Davis, Morris Plains, NJ, USA!. The vagina and uterinecervix and body were removed for electron microscopy analy-sis. For SEM, tissue pieces were washed, fixed in a solutionof 2.5% glutaraldehyde in 0.1 M cacodylate buffer ~SigmaChemical!, and dehydrated in an ascending alcohol series.The critical point was obtained in a Balzers CPD-010 ~Bal-zers Instruments, Balzers, Liechtenstein! with carbonic gas.Metallization in gold was carried out in a Balzers SCD-030~Balzers Instruments!. The vagina and uterine cervix andbody pieces of all rats were observed and photographed witha JEOL-JSM 6360 LV scanning electron microscope ~JEOLLtd., Tokyo, Japan! at the Electron Microscopy Center, Fed-eral University of Paraná.

Transmission Electron Microscopy

For TEM, tissue pieces were removed and fixed in Kar-novsky’s fixative ~Karnovsky, 1965!. The vagina and uterinecervix and body pieces were washed three times for 10 minin 0.1 M cacodylate buffer, pH 7.2, at 48C. Next, the materialwas postfixed in 2.0% osmium tetroxide in 0.1 M cacodylatebuffer, pH 7.2, for 1 h. En bloc staining was done with 2.0%uranyl acetate for 2 h. The material was dehydrated in anascending alcohol series and then placed in acetone. Infiltra-tion and embedding was in Epon-812 resin @Electron Micros-copy Sciences ~EMS!, EMBed-812 Embedding Kit - Kit Cat.Þ14120# . Sections were cut with a Sorval Porter BlumMT-2 ultramicrotome using glass and diamond blades.Ultrathin sections were contrasted in an aqueous solutionof 2.0% uranyl acetate and lead nitrate/acetate. Materialfrom all the rats was observed under a JEOL 1200EX IItransmission electron microscope at the Electron Micros-copy Center, Federal University of Paraná.

338 Edilson Damke et al.

RESULTS

Confirmation of the Infection

Cultures of vaginal secretions were positive ~CFU � 100yeasts/mL! for all rats in the weeks of the experimentsdemonstrating the persistence of the infection. All ratsshowed the same adhesion aspects in electron microscopy,and therefore photographs from only one rat are presentedhere.

Pap staining of the rat vaginal smears showed pseudo-estrus ~an abundance of enucleated cornified cells! ~Fig. 1A,B!and the presence of yeasts and hyphae-like forms in vaginalsecretions from all rats ~Fig. 1C!. Pseudo-estrus was alsoobserved by SEM ~Fig. 2!, histopathology ~Fig. 3!, and TEM~Fig. 4! as enucleated cornified cells and thickened epithelium.

By histopathology it was possible to observe the layersand integrity of the vaginal epithelium ~Fig. 3A,B! and theinfection in the vaginal surface and secretions ~Fig. 3C,D!after three weeks of the experiment. Figure 3C shows vari-ous forms of yeasts, and Figure 3D shows yeasts and one

hyphae-like form. The infection was more difficult to ob-serve by histopathology than Pap staining.

General Adhesion to the Lower Genital System

Various associated yeast/hyphae-like forms were observedat both the vaginal and endocervical levels three weeksfollowing induction of the infection. The structures wereseen in the lumen of these two regions ~Figs. 3C,D, 4B,C, 8!and also adhered to the epithelia ~Figs. 4–9!. In the uterinebody we observed only yeast-like forms of the fungus~Figs. 10, 11!.

It was possible to demonstrate the process of in vivofilamentation of C. albicans by the hyphae-like form ~Figs. 3D,4A,B! that was observed on the surface and within the

Figure 1. Light microscopy of Papanicolaou staining in a vaginal smear from rats infected by C. albicans observedwith a light microscope. A, B, C: Pseudo-estrus—predominance of cornified cells ~irregular cells without a nucleus!.C: Positive infection by C. albicans ~yeast-like form* and hyphae-like form**!. Scale bars: ~A! 50 mm, ~B, C! 15 mm.

Figure 2. Scanning electron micrograph showing the vaginal epi-thelium of female Wistar rats after administration of 0.2 mg/rat/week of estradiol valerate. The epithelium is composed of enucleatedcornified epithelial cells in pseudo-estrus. Scale bar: 50 mm.

Figure 3. Light microscopy of histopathology smears for vaginalepithelium after experimental infection by C. albicans in Wistarrats. A,B: The vaginal epithelium in layers, suggesting integrityand a good state of preservation. C: Several yeast-like forms.D: One hyphae-like* form in addition to yeast forms. Scale bars:~A! 50 mm, ~B–D! 15 mm.

Imaging of Candida albicans Adhesion In Vivo 339

epithelia at the vaginal and endocervical levels. Budding ofthe yeasts was also observed ~Fig. 4B,D!. The fungus re-mained in the epithelium nearly up to the lamina propria~Figs. 4A, 7A!.

Vaginal Adhesion

Figure 5 shows yeasts closely adhered to the vaginal cells~Fig. 5A,B!; areas of contact appeared as regions of continu-ity resembling interdigitations and fibrils connecting C.albicans to the epithelium ~Fig. 5B!. In Figure 5C a fibrillar-floccular layer spreads from the cell wall of C. albicans,especially where it contacts the epithelium. Figure 6 alsoshows the intimate association among yeasts and the epithe-lial cells. In Figure 6A yeasts are in contact with the vaginalepithelium in three stages ~a, b, and c!, from the initialadhesion contact ~a! until the interiorization into the epithe-lium ~b, c!. The images shown in Figure 6B suggest that asthe adhesion process evolves, a growing accommodation onthe epithelial surface takes place, with the yeast first nestlingon it and then penetrating into it.

This interaction is best visualized through TEM ~Fig. 7!.In Figure 7A yeasts can be seen nestled in the vaginalepithelium reaching the basal epithelial layer of the tissue.In Figure 7B a yeast cell is shown internalized in a cytoplas-mic vacuole of an epithelial cell as a remnant of theendocytotic process. Both yeast/hyphae-like forms show a

glycocalyx with high electron density external to the cellwall during adherence to the epithelium ~Fig. 7D, in greaterdetail!. The beginning of the internalization of the filamen-tous type is shown in Figure 7C.

Figure 4. Transmission electron micrograph showing C. albicansin Wistar rat vagina. Many associated yeast*/hyphae-like** formsand structures of the yeasts were observed at the vaginal level threeweeks after the induction of the infection. The structures were seenwithin the epithelium ~A!, in the vaginal lumen ~B, C!, and adheredto the surface of the epithelium ~C, D!. The fungus remained in theepithelium nearly up to the lamina propria ~black arrows!. Scalebars: ~A, B! 1 mm, ~C, D! 0.5 mm.

Figure 5. Scanning electron micrographs showing the surface ofthe vaginal epithelium of Wistar rats in pseudo-estrus infected byC. albicans. Some yeast-like forms* can be observed adhered ~A! tothe epithelial cells and ~B! in intimate adherence, where areas ofcontact appeared as regions of continuity, with interdigitationsand fibrils connecting C. albicans to the epithelium. A possiblefibrillar-floccular layer spread over the cell wall ~white arrow! of C.albicans, especially at the site of contact with the epithelium ~C!.An apparent intimate association with the epithelial cell can beseen. Scale bars: ~A! 50 mm, ~B! 20 mm, ~C! 10 mm.

340 Edilson Damke et al.

Endocervical Adhesion

C. albicans, especially the hyphae-like forms, also adhered tothe epithelium of the endocervical canal ~Fig. 8A,B!. Theconnecting fibrils between the yeasts and the surface of theendocervical epithelium can be observed. However, thesestructures, in this epithelium, were not as long and finger-like as those occurring during the adhesion process in thevaginal epithelium, and their morphology also appearedmore irregular ~Fig. 6!.

In this epithelium, characteristically abundant, longcilia were observed by TEM ~Fig. 9A,B!. It was difficult toobserve the yeast-like forms because the external layer inthese structures ~glycocalyx! was poorly developed or moredifficult to visualize by TEM ~Fig. 9B,C!. It was possible to

observe the yeast-like forms adhered in a site of adhesion tothe epithelium ~Fig. 9A–C!.

Endometrial Adhesion

The process of C. albicans adhesion to the endometrialepithelium is more superficial and involves less intimatecontact. The yeast-like form showed its characteristic mor-phology, adhered in an arrangement similar to that ofcolonies ~Fig. 10A–C!. The yeasts adhered, but with fewinterdigitations and more external positioning relative tothe epithelial surface ~Fig. 11C! in comparison to the adhe-sion in the vaginal epithelium ~Figs. 5, 6!. The adhesionappears scanty and stretched, bridging between the yeastsand epithelial surfaces. In Figure 11, adhered yeasts are seenwith uniform and characteristic shapes, and the epithelialcells exhibit cilia. The external glycocalyx layer is almostimperceptible ~Fig. 11A–C!. Note in Figure 11D a yeast-likeform within the epithelium with sparse intracellular mate-rial and a less electron-dense aspect suggestive of cellularinviability. This suggests that the adhesion process in the

Figure 6. Scanning electron micrographs showing the surface ofthe vaginal epithelium of Wistar rats in pseudo-estrus infectedwith C. albicans in different stages of the process of adherence andpenetration. A: Yeasts in ~a! initial contact with the vaginal epithe-lium; ~b! more advanced stage of adhesion, and ~c! almost penetrat-ing the epithelium. B: Yeasts of ~a! and ~c! in detail. Scale bars: ~A,B! 10 mm.

Figure 7. Transmission electron micrographs showing the vaginalepithelium of female Wistar rats in pseudo-estrus and details of theinfection by C. albicans of this epithelium. The integrity of theepithelium can be seen, as well as the yeasts ~*! that penetrated andnestled near the basal layer. A: The fungus remained in the epithe-lium nearly up to the lamina propria ~white arrow!. B: Yeast-likeform completely internalized within an epithelial cell, with a sur-rounding structure resembling a cytoplasmic vacuole. C: A yeast-like form is beginning filamentation and epithelial penetration.D: Yeasts showing an external glycocalyx with high electron densityexternal to the cell wall adhering to the vaginal epithelium. Scalebars: ~A! 2 mm, ~B, D! 0.2 mm, ~C! 0.5 mm.

Imaging of Candida albicans Adhesion In Vivo 341

uterine body is superficial and that the penetration of thefungus, if it occurs, involves dead yeasts.

DISCUSSION

Although rats are a good experimental model for the studyof oral and vaginal candidiasis, they have not previouslybeen used to determine the adhesion characteristics of

Figure 8. Scanning electron micrograph showing the surface ofthe endocervical epithelium of female rats in pseudo-estrus in-fected with C. albicans. A, B: Extensive filamentation of the yeastscan be observed, with hyphae-like forms ~white arrows! in thelumen of the entire endocervical canal. C: The yeast-like ~*! formappears as shorter stretched strands ~red arrows! bridging betweenthe yeasts and the endocervical epithelium surface. Scale bars:~A, B! 50 mm, ~C! 20 mm.

Figure 9. Transmission electron micrographs showing the endocer-vical epithelium of female Wistar rats in pseudo-estrus, and detailsof C. albicans adhesion. A–C: The endocervical epithelium appearsundamaged and preserved, with characteristic abundant long cilia~black arrows!. The yeast-like forms ~*! adhered in a site of adhe-sion to the epithelium ~A–C!. Scale bars: ~A! 2 mm, ~B) 1 mm,~C! 0.2 mm.

342 Edilson Damke et al.

Candida spp. in the lower reproductive system. The presentstudy describes ultrastructural aspects of C. albicans adhe-sion to the vaginal and uterine endocervical and bodyepithelium in a Candida-induced disease in vivo.

The model of female rat infection used was satisfactory.Electron microscopy showed a thickened vaginal epitheliumresulting from the action of estrogen during pro-estrouswith abundant enucleated cells. Prolonged pseudo-estrus is

required for development of VVC in female rats ~Clemonset al., 2004!. The endocervical and endometrial epitheliumshowed characteristic cilia in a good state of preservation.Fungi were observed in the vaginal, endocervical, and endo-metrial lumens, as well as on the surface of and withinthe epithelia. This phenomenon was less pronounced in theendocervical and endometrial epithelia. Curiously, in theendocervix the yeast-like forms took on a quite variedmorphology, possibly to facilitate adhesion to the epithe-lium in the spaces between the cilia.

It was possible to observe the characteristics of C.albicans adhesion to the vaginal, endocervical, and endo-metrial epithelia. Surprisingly, fungi were detected in allthree compartments of the genital system including theuterine body. This pathology has previously been reportedas an infection only of the vulva and vagina ~Sobel, 2007!.Our studies also showed yeasts with filamentation ~hyphae-like forms! penetrating the vaginal and endocervical epithe-lia and/or totally nestled on them. Yeast-like forms were alsoobserved without filamentation or in structures completelyinserted in or integrated with the epithelium. This findingcontrasts with reports of other investigators ~Howlett &Squier, 1980; Jayatilake et al., 2005!, who believed thatepithelial cells cannot be pierced by yeasts, but only by theends of growing hyphae-like forms. Nevertheless, the depen-

Figure 10. Scanning electron micrographs showing the surface ofthe endometrial epithelium of female rats in pseudo-estrus in-fected with C. albicans. Yeast-like forms can be seen, devoid offilamentation, adhered like colonies* ~A–C!, and adhered to theepithelium with few interdigitations and a more external locationrelative to the epithelial surface ~C!. The adhesions appear scantyand stretched ~white arrows! bridging between yeasts and epithe-lium surfaces. Scale bars: ~A! 50 mm, ~B) 10 mm, ~C! 2 mm.

Figure 11. Transmission electron micrographs showing the epithe-lial surface covered with cilia ~black arrows!, characteristic of theendometrial epithelium of female Wistar rats. C. albicans can beobserved in detail adhered ~*! to the infected epithelium ~A–C!.Note in panel D the completely internalized yeast-like form with aless electron-dense aspect, suggesting scanty intracellular materialand therefore cellular inviability. Scale bars: ~A! 0.5 mm, ~B–D)0.2 mm.

Imaging of Candida albicans Adhesion In Vivo 343

dence of the invasion on the ability of the fungal isolate toconvert its growth to the hyphae shape seems to be true inesophageal tissue and in endothelial cells ~Phan et al., 2000;Bernhardt et al., 2001!. However, in the endometrial epithe-lium we observed only yeast-like forms of the fungus.

The adhesion to vaginal epithelium occurred as anintimate association among yeasts and the epithelial cells,and apparently the process is very strong. It was also possi-ble to observe different stages of adhesion at the vaginallevel, and the fibrillar-floccular layer ~glycocalyx! of thefungus seems to thicken as the adhesion evolves, in additionto the marked and progressive epithelial depression, resem-bling cavitation. Jayatilake et al. ~2005! also observed cavita-tion in the oral epithelium where hyphae penetrate, and Rayet al. ~1984! observed the same in corneocytes. These latterauthors suggested that cavitation is the result of the actionof acid proteases of Candida, which degrade the epithelialsurface. Therefore, it seems that this process is also inti-mately linked to vaginal infection.

The mechanisms by which C. albicans gains entry intodifferent host epithelia are unclear. Most intracellular patho-gens enter cells by endocytosis, and it is evident that phago-cytes such as reticuloendothelial cells engulf C. albicans~Marodi, 1997; Kaposzta et al., 1999; Jayatilake et al., 2005!.Although epithelial or endothelial cells are not phagocyticper se, they can engulf bacteria through a similar mecha-nism known as cellular internalization ~Dehio et al., 1997;Palmer et al., 1997!. Interestingly, we noted under TEM,some vaginal epithelial cells forming cytoplasmic processesencircling yeasts that resembles this endocytic process. Thepresent findings are consistent with previous work of Filleret al. ~1995! who demonstrated endocytosis of C. albicans incultured human vascular endothelial cells. Another ultra-structural study ~Drago et al., 2000!, where C. albicans wasincubated with human buccal and vaginal cells and culturedHeLa cells, showed that all three cell types can internalizeyeast in a manner similar to endocytosis. Recently, Jayatilakeet al. ~2005! demonstrated the internalization of the yeast inreconstituted human oral epithelium. Our results indicatethat the process of endocytosis by epithelial cells seems tooccur in vivo as well as in the experimental model. Dragoet al. ~2000! noted that the importance of this process isrelated to the possibility that epithelial cells represent a“reservoir” of C. albicans, which could explain recurrentyeast infections. The low therapeutic efficacy observed insome infections could be due to the difficulty of the anti-mycotic in penetrating into infected cells, which protectsthe yeast from drug action. In the endometrial epithelia,internalized yeasts were rarely observed. When they wereobserved, they showed sparse intracellular material and aless electron-dense aspect, suggesting that the adhesion pro-cess in the uterine body is superficial and that penetrationof the fungus, if it occurs, destroys the microorganism.

The present study is among the first to provide ultra-structural evidence of fimbriae-mediated Candida adhesionto vaginal epithelium in vivo in Candida-induced disease.

The adhesins recognize and bind to particular receptorsexpressed on the eukaryotic cell membrane. Morphologi-cally, Candida adhesins are integrated in the fimbriae. Themajor structural unit of C. albicans fimbriae consists of80–85% carbohydrate ~primarily D-mannose! and 10–15%protein, so that the fimbriae are biochemically analogous tobacterial fimbriae ~Yu et al., 1994!. Because carbohydratesare not stained by routine contrasting substances, the glyco-calyx cannot be easily imaged in TEM without the use ofspecial staining techniques. Vitkov et al. ~2002! reportedthat in some instances control stains, such as rutheniumred, that are used to color fimbriae can indicate a glycoca-lyx, similar to the fibrillar-floccular layer described by otherauthors ~Jayatilake et al., 2005!. However, our results dem-onstrated yeasts with a fibrillar-floccular layer, probablyrepresented by fimbriae, adherent to the vaginal epitheliumwithout specialized staining. Montes and Wilborn ~1968!suggested that the partial visualization of Candida glycoca-lyx in non-ruthenium-red-staining specimens could resultfrom staining of the proteinaceous part of the fimbriae orproteins adsorbed onto them. Even though we were able toimage the glycocalyx in these experiments, there is a needfor more in vivo studies with specialized glycocalyx stainingto study endocervical and endometrial adhesion of poorlydeveloped yeasts on these tissues.

CONCLUSIONS

This is one of the first studies to provide in vivo ultrastruc-tural evidence of the adhesion of C. albicans to the vaginal,endocervical, and endometrial epithelia. Important charac-teristics of the process of fungus-host interactions in thelower genital system were described. Additional in vivostudies, including descriptions of other species of C. albi-cans, are essential to substantiate our results and to clarifyadditional aspects of the pathogenesis of VVC. Finally,considering that few studies have investigated the in vivoadhesion aspects of the fungi, this work attained the initialgoal of demonstrating an experimental model for this re-search in female Wistar rats.

REFERENCES

Bernhardt, J., Herman, D., Sheridan, M. & Calderone, R.~2001!. Adherence and invasion studies of Candida albicansstrains, using in vitro models of esophageal candidiasis. J InfectDis 184, 1170–1175.

Bibbo, M. ~1997!. Comprehensive Cytopathology. Philadelphia, PA:W.B. Saunder Company.

Calderone, R.A. & Fonzi, W.A. ~2001!. Virulence factors ofCandida albicans. Trends Microbiol 9, 327–335.

Clemons, K.V., Spearow, J.L., Parmar, R., Espiritu, M. & Ste-vens, D.A. ~2004!. Genetic susceptibility of mice to Candida

344 Edilson Damke et al.

albicans vaginitis correlates with host estrogen sensitivity. InfectImmun 72~8!, 4878–4880.

Consolaro, M.E.L., Albertoni, T.A., Svidzinski, A.E., Peralta,R.M. & Svidzinski, T.I.E. ~2005!. Vulvovaginal candidiasis isassociated with the production of germ tubes by Candidaalbicans. Mycopathol 159, 501–507.

De Bernardis, F., Lucciarini, R., Boccanera, M., Amantini, C.,Arancia, S., Morrone, S., Mosca, M., Cassone, A. & San-toni, G. ~2006!. Phenotypic and fuctional characterization ofvaginal dendritic cells in a rats model of Candida albicansvaginitis. Infect Immun 74, 4282–4294.

Dehio, C., Meyer, M., Berger, J., Schwarz, H. & Lanz, C. ~1997!.Interaction of Bartonella henselae with endothelial cells resultsin bacterial aggregation on the cell surface and the subsequentengulfment and internalization of the bacterial aggregate by aunique structure, the invasome. J Cell Sci 110, 2141–2154.

Drago, L., Bárbara, M., De Vechi, E., Bonaccorso, C., Fassina,M.C. & Gismondo, M.R. ~2000!. Candida albicans cellularinternalization: A new pathogenic factor? Int J Antim Ag 16,545–547.

Filler, S.G., Swerdloff, J.N., Hobbs, C. & Luckett, P.M. ~1995!.Penetration and damage of endothelial cells by Candida albi-cans. Infect Immun 63, 976–983.

Howlett, J.A. & Squier, A. ~1980!. Candida albicans ultrastruc-ture: Colonization and invasion of oral ephitelium. Infect Im-mun 29, 252–260.

Irie, M.M.T., Consolaro, M.E.L., Guedes, T.A., Donatti, L.,Patussi, E.V. & Svidzinski, T.I.E. ~2006!. A simplified tech-nique for evaluating the adherence of yeasts to human vaginalepithelial cells. J Clin Lab Anal 20, 195–203.

Jabra-Rizk, M.A., Falkler, W.A., Jr., Merz, W.G., Baqui, A.A.M.A.,Kelley, J.I. & Meiller, T.F. ~2001!. Cell surface hydrofobicityassociated adherence of Candida dubliniensis to human bucalephitelial cells. Rev Iberoam Micol 18, 17–22.

Jayatilake, J.A., Saramanayake, Y.H. & Saramanayake, L.P.~2005!. An ultrastructural and a cytochemical study of candidalinvasion of reconstituted human oral epithelium. J Oral PatholMed 34, 240–246.

Kamai, Y., Kubota, M., Hosokawa, T., Fukuoka, T. & Filler,S.G. ~2002!. Contribution of Candida albicans ALS1 to thepathogenesis of experimental oropharyngeal candidiasis. InfectImmun 70, 5256–5258.

Kaposzta, R., Marodi, L., Hollinshead, M., Gordon, S. & DaSilva, R.P. ~1999!. Rapid recruitment of late endossomes andlysossomes in mouse macrophages ingesting Candida albicans.J Cell Sci 112, 3237–3248.

Karnovsky, M.J. ~1965!. A formaldehyde-glutaraldehyde fixativeof high osmolarity for use in electron microscopy. J Cell Biol27, 137–138.

Kurtzmann, C.P. & Fell, F.W. ~1998!. The Yeast. A TaxonomiaStudy. Amsterdam: Elsevier.

Mandl, A.M. ~1951!. The phases of the estrous cycle in the adultwhite rat. J Exp Biol 28, 576–584.

Mardh,P.A.,Rodrigues,A.G.,Genc,M.,Novikova,N.,Martinez-de-Oliveira, J. & Guaschino, S. ~2002!. Facts and myths onrecurrent vulvovaginal candidosis: A review on epidemiology,clinical manifestations, diagnosis, pathogenesis and therapy. IntJ STD AIDS 13, 522–539.

Marodi, L. ~1997!. Local and systemic host defense mechanismsagainst Candida: Immunophatology of candidal infections. Pae-diatr Infect Dis J 16, 795–801.

Montes, L.F. & Wilborn, W.H. ~1968!. Ultrastructural features ofhost-parasite relationship in oral candidiasis. J Bacteriol 96,1349–1356.

Palmer, L.M., Reilly, T., Utsalo, S.J. & Bonnenberg, M.S.~1997!. Internalization of Escherichia coli by human renal ephi-telial cells is associated with tyrosine phosphorylation of spe-cific host cell proteins. Infect Immun 65, 2570–2575.

Phan, Q.T., Belanger, P.H. & Filler, S.G. ~2000!. Role of hyphalformation in interactions of Candida albicans with endothelialcells. Infect Immun 68, 3485–3490.

Pichová, I., Pavlicková, J., Dostál, J., Dolejsi, E., Hrusková-Heindigsfeldová, O., Weber, J., Ruml, T. & Soucek, M.~2001!. Secreted aspartic protinases of Candida albicans, Can-dida tropicalis, Candida parapsilosis and Candida lusitaniae:Inhibition with peptidomimetic inhibitors. Eur J Biochem 268,2669–2677.

Ray, T.L., Digre, K.B. & Payne, C.D. ~1984!. Adherence of Can-dida species to human epidermal corneocytes and bucal muco-sal cells: Correlation with cutaneous pathogenicity. J InvestDermatol 83, 37–41.

Shin, J.H., Kee, S.J., Shin, M.G., Kim, S.H., Shin, D.H., Lee, S.K.,Suh, S.P. & Ryang, D.W. ~2002!. Biofilm production by isolatesof Candida species recovered from nonneutropenic patientscomparison of bloodstream isolates with isolates from otherssources. J Clin Microb 40, 1244–1256.

Sobel, J.D. ~1993!. Candidal vulvovaginitis. Clin Obstet Gynecol36, 153–212.

Sobel, J.D. ~2007!. Vulvovaginal candidosis. Lanc 369, 1961–1971.Sugita, T., Kurosaka, S., Yajitate, M., Sato, H. & Nishikawa,

A. ~2002!. Extracellular proteinase and phospholipase activityof three genotypic strains of a human pathogenic yeast, Can-dida albicans. Microbiol Immunol 46, 881–883.

Vitkov, L., Krautgartner, W.D., Hannig, M., Weitgasser, R. &Stoiber, W. ~2002!. Candida attachment to oral ephitelium.Oral Microbiol Immunol 17, 60–64.

Yu, L., Lee, K.K., Ens, K., Doig, P.C., Carpenter, M.R., Staddon,W. & Hodges, R.S. ~1994!. Partial characterization of a Can-dida albicans fimbrial adhesion. Infect Immun 62, 2834–2842.

Imaging of Candida albicans Adhesion In Vivo 345