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DOI 10.1007/s11046-010-9373-7
Received: 1 February 2010 / Accepted: 30 September 2010 / Published online: 22 October 2010
Springer Science+Business Media B.V. 2010
Abstract Commensal yeast Candida causes opportunistic infections ranging from superficial lesions to
disseminated mycoses in compromised patients.
Superficial candidiasis, the commonest form of
candidal infections, primarily affects the mucosa
and the skin where Candida lives as a commensal.
Conversion of candidal commensalism into opportunism at the fungalepithelial interface is still illdefined. Nevertheless, fungal virulence mechanisms
such as adhesion to epithelia, morphogenesis, production of secretory hydrolytic enzymes, and phenotypic switching are thought to contribute in the
process of pathogenesis. On the other hand, host
responses in terms of immunity and local epithelial
responses are actively involved in resisting the fungal
challenge at the advancing front of the infection.
Ultrastructural investigations using electron microscopy along with immunohistochemistry, cytochemistry, etc. have helped better viewing of Candidahost
interactions. Thus, studies on the ultrastructure of
superficial candidiasis have revealed a number of
fungal behaviors and associated host responses such
as adhesion, morphogenesis (hyphae and appresoria
formation),
thigmotropism,
production
and
Introduction
J. A. M. S. Jayatilake (&)
Division of Microbiology, Department of Oral Medicine
and Periodontology, Faculty of Dental Sciences,
University of Peradeniya, Peradeniya, Sri Lanka
e-mail: sumedhaj@pdn.ac.lk
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Methods
State of knowledge in the field of ultrastructural
pathology of superficial candidiasis on the basis of
selective review of English literature in various
sources was critically analyzed. These literature
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Laboratory Techniques
Conventional light microscopy shows that candidal
blastospores as well as hyphae penetrate into the
epithelium during superficial candidiasis. However,
fine features of these interactions are visualized using
transmission and scanning electron microscope.
Transmission (TEM) and scanning electron microscopy (SEM) provide bi- and tri-dimensional views of
tissue specimens affected by the fungi, respectively.
However, ultrastructural investigations of Candida
are hampered particularly by the technical difficulties
in processing yeasts for electron microscopy [12, 13].
Precise imaging of yeasts depends on the specific
fixatives, and staining chemicals used in the preparation of specimens for microscopy. Commonly used
fixatives for yeasts are potassium permanganate and
osmium tetroxide, and the latter is popular for
excellent results. In particular, osmium tetroxide
provides better contrast for intracellular organelles
of fungal elements interacting with the animal or
human tissues [14]. Intracellular organelles particularly nucleoli are not clearly visible when Candida is
fixed in potassium permanganate solution [15].
Tissues and fungi for SEM are generally fixed with
osmium tetroxide prior to gold coating for final
observations.
Various staining chemicals such as uranyl acetate,
lead citrate, and ruthenium red are useful in differentiation of fungal elements within the tissues by
TEM. Ruthenium red specifically stains yeast cell
wall polysaccharides, and it is useful in detailed
visualization of the outer fibrillar layer of candidal
cell wall [16]. Interestingly, this stain has produced
contrasting visuals of extracellular polysaccharides
associated with the yeast cell wall [17]. Additionally,
alkaline bismuth staining that can be used to stain
polysaccharides in electron microscopic studies is
another substitute for ruthenium red [18].
Furthermore, cytochemical and immunohistochemical methods have also been utilized in the
exploration of the invasive phase of Candida species
on tissue samples. In a pioneering study using
cytochemical methods, Pugh and Cawson [19] examined PL activity of C. albicans in vitro. In order to
visualize localized activity of candidal PL, these
investigators introduced lecithin and lead nitrate into
the reaction medium. Lead precipitates due to
interactions with the fatty acids derived by the action
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Host responses
Phagocytosis/cellular internalization
Cavitations/depressions
Persorption
Morphogenesis
Lysis of tonofilaments
Thigmotropism
Appressoria
Detachment of desmosomes
Irregular cells
Extracellular enzymes
SAPs
PL
Phynotypic switching
Rough and smooth colonies/cells
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study to examine candidal invasion of female genitourinary tract, Garcia-Tamayo et al. [49] examined
exudates collected from the vagina and the uterine
cervix of ten patients suffering from genital candidiasis using ruthenium red stain. Their observations
revealed that mucopolysaccharides were present in
the glycoprotein coat of fungi lying both within and
outside of the epithelial cells. Their SEM observations also demonstrated bud scars and glycoproteinaseous substances with a floccular appearance on the
surface of the invading fungi.
Korting et al. [50] who pioneered ultrastructural
studies of candidal invasion of reconstituted epithelia
found high affinity of Candida blastospores toward
the desmosomes of the reconstituted human epidermis. These investigators surmised that Candida cells
adhere to surface epithelial cells through the surface
fibrillar layer. Recently, using ruthenium red stain,
Vitkov et al. [51] demonstrated a fibrillar extracellular coat (glycocalyx) over the candidal cell wall
attached to the human oral epithelium affected by
denture stomatitis. It was also found that the invading
blastospores are internalized by the oral epithelial
cells, and remarkably, such internalized fungal elements were lacking a fibrillar coat comparable with
the previous observations of Rajasingham and Cawson [47]. However, Vitkov et al. [51] suggested that
the extracellular fibrillar coat acted as an adhesive
apparatus (fimbria-mediated epithelial attachment)
exhibited by C. albicans during pathogenesis. In
addition, post-mortem biopsies of various parenchymal organs, including kidney, liver, spleen, heart,
esophagus, and rectum of disseminated candidiasis
patients, have demonstrated a thick fibrillar outer coat
in blastospores multiplying within the tissues [52].
These investigators have argued that the fibrillar coat
is derived from the epithelial fibrin. As such, the
exact origin and the function of the candidal fibrillar
coat should be further investigated to describe its role
in the pathogenic process.
Numerous secretions present at the epithelial
surface affect the homeostasis of the normal microbial flora. In an early attempt to explore the behavior
of Candida with gastrointestinal epithelium, Pope
and Cole [53] observed differential candidal attachments with the mouse gut epithelium after intragastric inoculation of two C. albicans isolates to infant
mice. There were Candida blastospores attached to
the keratinized stomach epithelial surface mixed with
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Hyphal Invasion
Histopathology of mucosal candidiasis is characterized by the invasion of hyphae (true and pseudo) into
the superficial epithelium [60]. It has been demonstrated that hyphal mutants of C. albicans that do not
produce hyphae are unable to produce invasive
candidiasis in mice [61] as well as in tissue cultures
[62]. On the other hand, non-albicans Candida
species without hyphae were non-invasive in experimental human oral candidiasis produced on reconstituted human oral epithelium [63]. These
observations indicate that the conversion of blastospore into filamentous hyphae plays an important role
in candidal virulence. Ultrastructural investigations
of superficial candidiasis have pointed out that
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human oral epithelium [42] and rabbit tongue mucosal explants [43].
Appressoria
Appressoria are bulging structures that are seen at the
tips of the hyphae of some plant pathogenic fungi, and
they generate physical forces to facilitate tissue
invasion by the fungi into various substrates [72].
Ultrastructural investigations of superficial candidiasis
too have revealed bulging structures at the hyphal tips
resembling appressoria [47, 73]. These investigators
have suggested that bulging appressoria of Candida act
as pre-penetrative structures during pathogenesis. In a
comprehensive study of ultrastructural features of the
invasive phase of Candida in vaginal candidiasis,
Rajasingham et al. [73] examined sixty-three vaginal
scrapings of acute vaginal candidiasis patients and
noticed appressorial bulge at the tips of the invading
candidal hyphae. Furthermore, ultrastructure of those
appressoria indicated numerous cell organelles including mitochondria, endoplasmic reticulum, ribosomes,
and vesicles denoting an increased metabolic activity.
Hence, it was postulated that candidal appressoria
could generate increased turgor pressure at the hyphal
tips to facilitate penetration of the host epithelium
resembling some plant pathogenic fungi. Moreover,
Rajasingham et al. [73] surmised that physical forces of
the candidal appressoria act in tandem with the fungal
enzymes released at the tips of hyphae in invasive
pathogenesis. In a similar experiment, Rajasingham
[14] has demonstrated bulging appressoria at the
penetrating hyphae and associated extracellular material that superimposed the enzymatic activity in both
oral and vaginal candidiasis. In addition, some investigators have suggested that appressoria provide
anchorage effect besides acting as penetrative apparatus for invading fungi within the oral environment [64].
Extracellular Enzymes
Candida species produce several extracellular
enzymes, and the most important enzymes that are
responsible for virulence are secretory aspartyl proteinases (SAP), lipases, and phospholipases (PL).
Apart from facilitating the nutrient supply, these
hydrolases are involved in candidal: (1) invasion by
digesting host cell membranes, (2) adhesion by
degrading host cell surface molecules, and (3)
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Phenotypic Switching
Triggered by numerous environmental factors, Candida species undergo random alterations in colony
morphologies. This process includes an epigenetic
change of colony morphologies and precisely known
as phenotypic switching [83]. For the first time,
Slutsky et al. [33] observed spontaneous switching of
colony phenotypes (star, ring, irregular wrinkle, hat,
stipple, and fuzzy) of C. albicans grown on amino
acid-rich agar, and it was surmised that phenotypic
switching has a direct effect on candidal virulence.
Furthermore, ultrastructural observations of white
and opaque phenotypes of C. albicans have revealed
structural and morphological changes of the yeast cell
organelles including the nucleus suggesting that
phenotypic switching could affect the behavior of
the organism [84]. However, phenotypic switching
differs from morphogenesis (yeast-hyphal transition),
as it is assigned to describe the spontaneous morphological changes of the colonies of growing
Candida on solid media [85]. Finally, it has now
been proposed that switching as a mechanism of
accelerated microevolution allowing survival of the
fittest in new microniches of the growth environment
[11].
However, the relevance of phenotypic changes of
C. albicans with the Candidahost interactions has
not been well established. In an experiment to
determine the relationship between phenotypic
switching and the candidal adhesion to porcine
buccal epithelial cells using SEM, Vargas et al. [86]
demonstrated that smooth yeast colonies are more
adherent compared with irregular wrinkle, star, and
revertant smooth phenotypes. Their investigations
also revealed that differential fungalepithelial cell
interactions were based on the phenotypes. However,
further studies are necessary to ascertain the links
between phenotypic switching and the pathogenesis
of candidiasis.
Host Responses
Combinations of innate and adaptive immune mechanisms are known to act synergistically in candidiasis
[87]. Ultrastructural investigations using Candidaaffected tissue specimens have enormously helped
identify multiple host responses at the invasive
Phagocytosis
Histopathology of mucosal candidiasis is characterized by fungal invasion of the epithelium in both
blastospore and hyphal forms. On the other hand,
there are huge aggregations of polymorphonuclear
leukocytes (PMNL) within and underneath the epithelium leading to microabscess formation in mucosal candidiasis [88]. At present, it is well accepted
that PMNL and macrophages engulf invading fungal
elements as a part of normal protective mechanism.
In an early experiment to observe interaction between
C. albicans and human PMNL, Belcher et al. [89]
have demonstrated phagocytosis of Candida yeasts
by the PMNL cells. Besides, they noticed numerous
lysosomes of PMNL participate in candidal killing.
This study has also shown that single PMNL could
engulf multiple yeasts. Most interestingly, Belcher
et al. [89] pointed out that PMNL engulf yeast cells
and form phagolysosomes within 1560 min in vitro.
These observations were later reconfirmed by Marrie
and Costerton [17] who demonstrated phagocytosis
of yeasts by PMNL in pathological samples collected
from both oral and urinary candidiasis patients. In an
attempt to visualize the process of yeast phagocytosis
by macrophages, Kaposzta et al. [90] noticed pseudopodia formation by mouse macrophages to trap
Candida blastospores regardless of latters viability.
It was also confirmed that resulting phagosomes
rapidly attracted endosomes and lysosomes to form
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Conclusions
Extensive research over the past decades on the
ultrastructure of superficial candidiasis has elicited a
variety of fungalhost interactions at the invasive
interface. Putative virulence attributes of Candida are
broadly assigned to adhesion, morphogenesis, production of hydrolytic enzymes, and phenotypic
switching. Epithelial and immune cells of the host
respond differentially to candidal challenge in the
mucosal environment full of commensal organisms.
Ultrastructural investigations are tremendously useful
in gaining more insights into these pathological
mechanisms (Table 1).
Cell wall of Candida plays an important role in its
survival on various epithelia. Its unique structure with
polysaccharide fibrils and hydrolytic enzymes
enhances resistance against dislodging forces of
physical and chemical nature in the host niches. This
fibrillar coat arising from the cell wall is a significant
feature of yeasts in close proximity with the epithelial
cells. Epithelial cells show cavitations or depressions
in relation to yeasts contact. These phenomena are
attributed to the activity of lytic enzymes such as
SAP and PL of the invading fungi. Phagocytosis of
yeasts by epithelial as well as immune cells is
important in arresting the spread of infection. Yet,
unharmed fungal elements surviving within the
phagocytic cells may facilitate recurrence of infection, as the intracellular organisms can reactivate
when the suitable conditions supervene. Most of the
ultrastructural studies focused on fungalhost relationships in superficial candidiasis testify the importance of hyphal formation by Candida in invasive
pathogenesis. Hyphae act as invasive apparatus for
fungi by generating physical forces and lytic
enzymes. Moreover, they are capable of detecting
suitable sites for invasion such as breaches and
micropores in susceptible epithelia by a process
known as thigmotropism. Formation of appressoria at
the hyphal tips further provides penetrative appendages for the pathogen. Many ultrastructural studies
have shown epithelial tissues respond to these
pathogenic stimulants by cell rupture and alterations
of the tissue integrity.
One of the most significant findings of the
ultrastructural studies on superficial candidiasis is
the involvement of extracellular enzymes at the
Candida invasive front. In early studies using SEM
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