Advances in Microbiology, 2013, 3, 1-6
Published Online December 2013 (
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Ultrastructural Analysis of in Vitr o Adherence and
Production of Acid Proteases by Clinical Isolates of
Candida parapsilosis Sensu Stricto Following Growth in the
Presence of Keratinous Substrates from Human Source
Ana Flávia L. Specian1, Luciana Furlaneto-Maia2, Célia G. T. J. Andrade3,
Marcia Cristina Furlaneto1*
1Department of Microbiology, Paraná State University at Londrina, Londrina, Brazil
2Technological Federal University of Paraná, Londrina, Brazil
3Electronic Microscopy and Microanalysis Laboratory, Paraná State University at Londrina, Londrina, Brazil
Email: *
Received September 25, 2013; revised October 25, 2013; accepted November 2, 2013
Copyright © 2013 Ana Flávia L. Specian et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Candida par a p silosis is an increasingly important human pathogen. However, little is known about its potential to cause
disease. The aims of the present study were to analyse the production of acid proteinases by clinical isolates of C.
parapsilosis in the presence of different keratinous substrates from human sources (stratum corneum, nail and hair) and
to verify the capability of yeast cells to adhere and grow as biofilm on these substrates. By scanning electron micros-
copy, it was observed that all C. parapsilosis sensu stricto isolates adhered to the keratinous substrates. For the isolate
recovered from onychomycosis, the cell population attached to stratum corneum and hair keratin consisted mainly of
blastoconidia. Differently, on nail keratin, pseudohyphae production was observed. Overall, there was a loose associa-
tion between yeast cells and keratinous substrates. However, on stratum corneum, flocculent extracellular material was
seen evolving cells from the onychomycosis isolate by forming a biofilm-like structure. The isolates recovered from
onychomycosis and cutaneous lesion produced higher amount of acid proteinases in medium supplemented with nail
keratin and stratum corneum keratin, respectively, than that in salt medium (absence of keratin). Furthermore, no dif-
ferences were observed in the amount of acid proteinases produced by the isolate recovered from tracheal secretion in
the media tested (absence and presence of keratin substrates). The information derived from this study will further our
understanding of acid proteinase production by C. parapsilosis isolates an d provide an insight into pathogenic mecha-
nisms in C. parapsilosis particularly from isolates recovered from superficial mycoses.
Keywords: Adherence; Keratinic Substrates; SEM; Acid Proteinase
1. Introduction
Candida parapsilosis is an opportunistic yeast pathogen
that colonizes human skin and can spread nosocomially
through hand carriage [1,2]. Over the past decade, the
incidence of C. parapsilosis has dramatically increased.
The yeast can cause candidiasis that can vary from rela-
tively mild skin mycoses to life-threatening systemic or
disseminated disease (reviewed in van Asbeck et al. [3]).
Concerning superficial mycoses, C. parapsilosis has
gained increasing recognition worldwide as the most
common etiological agent causing Candida onychomy-
cosis (reviewed in Trofa et al. [4]). In Brazil, C. parapsi-
losis is the first or second most common cause of ony-
chomycosis lesions [5-8]. For C. parapsilosis the colo-
nized normal skin presumably serves as a reservoir of
infection for the nails. Recently, we showed the capabil-
ity of C. parapsilosis isolates exhibiting distinct pheno-
types to grow as biofilm on human nail surfaces [9].
Several virulence factors of C. parapsilosis have been
proposed, including adhesion, biofilm formation and
secretion of hydrolases such as secreted aspartic pro-
teinases (Saps) (reviewed in Trofa et al. [4]). We previ-
*Corresponding author.
ously reported the production of proteinases and haemo-
lytic factor by isolates of C. parapsilosis obtained from
distinct clinical sources [10]. More recently, we have
demonstrated that C. parapsilosis sensu stricto secretes a
hemolytic factor into culture medium [11].
However, in contrast to the species Candida albicans,
virulence traits of C. parapsilosis have not been exten-
sively studied. Few studies have been undertaken to
evaluate the adhesion ability of clinical strains of C.
parapsilosis [9,12-14]. Furthermore, the relationship be-
tween C. parapsilosis virulence and proteinase pheno-
type is still unclear.
In this study, we investigated for the first time the in
vitro adherence pattern and production of acid proteases
by clinical isolates of C. parapsilosis in the presence of
keratinous substrates from human source, e.g., stratum
corneum, nail and hair.
2. Materials and Methods
2.1. Candida Isolates and Identification
Isolates of C. parapsilosis sensu stricto included in this
study were recovered from fingernail onychomycosis
(isolate 150.06), cutaneous candidiasis (isolate 220.07)
and tracheal secretion (isolate 205.06) [15]. The identity
of isolates was determined by the PCR technique as de-
scribed by França et al. [10] using C. parapsilosis (for-
merly C. parapsilosis group I) specific primers for URA3
gene (orotidine-5’-phosphate decarboxylase) [16] that
allows to distinguish C. parapsilosis sensu stricto from
the cryptospecies belonging to the C. parapsilosis com-
2.2. Preparation of Keratin Substrates
For the substrate stratum corneum, fragments of human
sole from healthy volunteer were prepared as described
previously [17] with modifications. The fragments were
soaked in ethanol for 96 h following washes with steril-
ized distillated water until a clean solution was obtained.
The fragments were dried at 50˚C, grounded to a powder
in liquid nitrogen and dried again at 50˚C.
The substrates nail and hair were prepared as de-
scribed previously [18] with modifications. Human’s hair
and nail from healthy volunteer were cut to obtain small
fragments ranging from 0.5 to 1 cm of size. The frag-
ments were defatted by soaking for 4 d in chloroform-
methanol 1:1 (v/v). The solvent was changed once a day.
The fragments were then thoroughly washed with steril-
ized distillated water and dried for 3 d at 50˚C. The nails
fragments were grounded to a powder in presence of liq-
uid nitrogen and dried again at 50˚C. The prepared sub-
strates were autoclaved for 5 min at 115˚C and then
added to previously sterilized basal minimal medium.
2.3. Growth Profiles Determination and
Proteinase Production
The isolates were pre-cultured in Sabouraud dextrose (4%
dextrose, 1% peptone, 1% yeast extract) liquid medium
and grown in submerged culture (180 rpm) at 37˚C for 18
h. For enzyme production, cell suspensions (104 cells/ml)
were inoculated in basal minimal medium (MM) [19],
with modifications, containing (g/l): K2HPO4—1.5 g;
MgSO4·7H2O—0.025 g; CaCl2—0.025 g; FeSO4·7H2O—
0.015 g, ZnSO4·7H2O—0.005 g and 1% glucose, and
MM amended with either 0.5% (w/v) stratum corneum,
hair and nail (MM + SC, MM + H and MM + N, respec-
tively) and grown in submerged culture (180 rpm) at
37˚C for up to 10 days. Media were buffered using a cit-
rate buffer (0.1 M citric acid; 0.1 M sodium citrate), pH
5.0. Following growth, yeast cells were harvested by
centrifugation at 8000 g for 20 min at 4˚C. The super-
natants obtained were stored at 20 ˚C and assayed for
protease activity. For growth rate assessment samples
were removed at 12-h intervals. The growth rate was
determined by haemocytometric counts. All experiments
were repeated three times, and the results represent mean
values SD.
2.4. Proteinase Assay
Proteolytic activity was determined as previously de-
scribed [20] with modifications, using haemoglobin
(Sigma; St. Louis, MO) as substrate. Each assay included
50 l of culture supernatant and 0.2 ml of 20 mM citrate
buffer, pH 4.0, containing haemoglobin (0.5 mg/ml).
After incubation at 37˚C for 2 h, the reaction was stopped
with trichloroacetic acid (TCA) 5% on ice, following
incubation at 4˚C for 1 h. The mixture was centrifuged at
4000 g for 10 min at 4˚C. After this, three aliquots (150
μl each) of the reaction mixture were transferred to wells
on a microtiter plate containing 100 μl of a Coomassie
solution (0.025% Coomassie brilliant blue G-250,
11.75% ethanol, and 21.25% phosphoric acid). After 10
min to allow dye binding, the plate was read on an Asys
HiTech UVM 340 microplate reader at an absorbance of
595 nm. Protease activity was calculated based on the
absorbance difference between samples and controls. The
control samples were added supernatants that were im-
mediately treated with TCA. One unit (U) of proteolytic
activity was defined as the amount of enzyme that caused
an increase of 0.001 in absorbance unit, under standard
assay conditions. The proteolytic activity is expressed as
2.5. Scanning Electron Microscopy
To verify the adhesion pattern of yeast cells with human
keratin substrates, samples were fixed in 2.5% glutaral-
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dehyde (Electron Microscopy Sciences) in 0.1 M phos-
phate buffer, pH 7.2, followed by incubation at 4˚C for
18 h. Then, the samples were carefully washed with 0.1
M phosphate buffer, pH 7.2. Post-fixation was carried
out for 1 h at 25˚C with 1% osmium tetroxide in 0.1 M
phosphate buffer. Samples were gently dehydrated in
graded ethanol, critical point-dried in CO2 (BALTEC
DCP 030 Critical Point Dryer), coated with gold (BAL-
TEC SDC 050 Sputter Coater) and viewed in a FEI
Quanta 200 Scanning Electron Microscope.
between the organism’s virulence characteristics and the
host’s response. Compared with C. albicans, little is spe-
cifically known regarding virulence factors in C. parap-
silosis sensu stricto.
Adherence is essential for members of the genus Can-
dida to develop their pathogenic potential since it trig-
gers the process that leads to colonization and allows
their persistence in the host. For instance, more patho-
genic Candida isolates showed higher adherence capac-
ity on human oral and epithelial cells [21]. According to
these authors, epithelial cell variability played a critical
role in the adherence phenomenon [21].
2.6. Statistical Analysis
In our study, we analysed the in vitro pattern of ad-
herence of C. parapsilosis sensu stricto cells to human
keratinised substrates, i.e., on soft keratin (cutaneous
stratum corneum—the outermost layer of skin) and hard
keratin (nail and hair). By scanning electron microscopy
it was observed that all C. parapsilosis isolates adhered
to the keratinous substrates (Figure 1). On hair frag-
ments, the adherent cells are seen only within the follicle
cortex and the number of cells adhered to this substrate
varied among isolates (Figure 1(c)).
All the experiments were repeated three times and assay
was performed in duplicate. Tukey test was used to de-
termine statistical significance. P < 0.05 was considered
statistically significant.
3. Results and Discussion
3.1. In Vitro Adherence Pattern of C. parapsilsois
Cells to Human Keratinous Substrates
Yeast pathogenicity arises through complex interactions
Figure 1. Electron micrographs showing the in vitro adherence pattern of Candida parapsilsois cells to human keratinous sub-
strates. (a) Stratum corneum, (b) Nail and (c) Hair. (1) Isolate 150.06, (2) Isolate 220.07 and (3) Isolate 205.06. Magnification
of 3000×. Extracellular material (arrows) is seeing evolving cells from the isolate 150.06 (a1).
SEM analysis revealed that the onychomycoses isolate
presented different morphological pattern according to
the substrate that they were in contact. For instance, cells
adhered to stratum corneum and hair keratin consisted
mainly of cells in the budding-yeast phase of growth
(blastoconidia) (Figures 1(a1) and (c1)). Differently, on
nail keratin pseudohyphae production was observed (Fig-
ure 1(b1)), a pattern that could indicate that this situation
favours cellular morphologies with capacity for tissue
invasion. This data extend our previous observation that
different profiles of biofilm formation by C. parapsilosis
occurred as function of the keratinous substrate [9].
For the other isolates the cellular population consisted
of blastoconidia and pseudohyphae on all substrates ana-
lysed, with exception of the isolate 205.06 (tracheal se-
cretion) on hair keratin (Figure 1(c3)). Intraspecific dif-
ferences in adherence to polystyrene have been described
among clinical isolates of C. parapsilosis obtained from
distinct body sites [12]. More recently, reconstituted hu-
man epithelium (RHE) has been used to study in vitro
colonization by C. parapsilosis complex [13,14]. Ac-
cording to these authors, the extent of surface coloniza-
tion on RHE by C. parapsilosis was strain dependent.
Overall, there was a loose association between yeast
cells and keratinous substrates. However, on stratum cor-
neum flocculent extracellular material was seen evolving
cells from the onychomycoses isolate by forming a
biofilm-like structure (Figure 1(a1)). This feature was
not observed on the other two sources of human keratin
(nail and hair).
It has been showed that adhesion [22] and ability to
grow as biofilms [23] on abiotic surfaces are especially
important for outbreaks of C. parapsilosis infections (re-
viewed in Trofa et al. [4]). This is the first report of ul-
trastructural features related to adhesion of C. parapsilo-
sis isolates associated with skin and nail infections to
distinct keratinised substrates from human source.
3.2. Acid Proteinases Production
In this study, we evaluated for the first time the growth
profile and the production of acid proteinases by C.
parapsilosis using human keratin as sole source of nitro-
gen. The isolates tested presented similar trend of growth
rate in keratin-supplemented media. However, the fungal
growths were more profusely in stratum corneum-supple-
mented medium than in hair- and nail-supplemented me-
dium (cell density reached 108 cells/ml and 107 cells/ml,
respectively), probably due to differences in keratin struc-
ture and the degree of cross-linkages by disulfide and
hydrogen bonds.
The results obtained (Figure 2 ) showed that C. parap-
silosis sensu stricto isolates produced proteinases in all
tested media. The isolate recovered from onychomycosis
Figure 2. Measurement of secreted acid proteinase activity
on haemoglobin in clinical isolates of Candida parapsilosis
obtained from onychomycosis (150.06), cutaneous candidi-
asis (220.07) and tracheal secretion (205.06). After growth
in basal minimal medium (MM) and MM supplemented
with stratum corneum (MM + SC), MM supplemented with
hair (M + H) and MM supplemented with nail (MM + N)
for 10 d at 37˚C, the cultures were harvested and the spent
culture media were then tested to degrade soluble haemo-
globin. The proteolytic activity was determined as described
in Material and methods and is reported as arbitrary units
(U/ml). Standard errors of the means for three measure-
ments are presented as bars. *P < 0.05 for MM + N vs MM
(isolate 150.06) and MM + SC vs MM (isolate 220.07). P <
0.05 for isolate 150.06 vs 205.06 (MM + N medium).
produced higher amount of acid proteinases (P < 0.05) in
medium supplemented with nail keratin (440 U/ml) than
in salt medium (absence of keratin) (120 U/ml). No dif-
ferences (P > 0.05) were observed on proteinase produc-
tion in the presence of the other two sources of keratin
(stratum corneum and hair). For the isolate 220.07 (cuta-
neous lesion) the production of acid proteinases was
higher in medium supplemented with stratum corneum
keratin (440 U/ml) than in salt medium (120 U/ml) (Fig-
ure 2). These data suggest that the source of keratin
seems to be correlated to the induction of acid pro-
teinases in an isolate dependent manner.
Differently, no differences were observed in the
amount of acid proteinases produced by the isolate re-
covered from tracheal secretion in the media tested (ab-
sence and presence of keratin substrates) (Figure 2).
Furthermore, when compared proteinase production by
Candida isolates after growth in the same source of kera-
tin, e.g., in nail keratin medium, the isolate recovered
from onychomycosis exhibited higher proteinase activity
(P < 0.05) than isolate obtained from tracheal secretion,
suggesting that the potential of C. parapsilosis nail iso-
late to cause onychomycoses may be associated with acid
proteinase production.
It has been reported that the expression of genes en-
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coding aspartic proteinases (Saps) varied among different
clinical isolates of C. parapsilosis complex when grown
in contact with human oral epithelium [14]. According to
these authors there is a trend relating Sap production and
site of isolates recovering.
In C. albicans, the most discussed hydrolytic enzymes
are secreted aspartic proteinases (Saps), which are one of
the well-known virulence factors of this species (re-
viewed in Schaller et al. [24]). According to Monod and
Borg-von [25] Saps play a role in fungal adherence and
invasion of skin by C. albicans. The occurrence of Saps
has been previously demonstrated in C. parapsilosis iso-
lates obtained from distinct clinical samples [10,26-28]
and it has being suggested that Saps are associated in
superficial, but not with systemic invasion, caused by C.
parapsilosis [26]. They observed the induction of Saps
from C. parapsilosis cultivated in media containing bo-
vine serum albumin (BSA) as a nitrogen source. Consid-
ering the clinical point view, it is questionable whether
these enzymes could have the required function of di-
gesting keratinised tissue for parasitism. Recently we
showed that the production of Saps, in BSA inducing
medium, by C. parapsilosis isolates obtained from nail
and skin was less expressive compared to isolates ob-
tained from blood and tracheal secretion [10]. Although
it has been established that C. parapsilosis is an oppor-
tunistic pathogen related to the skin surface, and is also
emerging as an important cause of onychomycosis, as far
we know, in the present study we describe for the first
time the production of acid proteases by isolates of C.
parapsilosis recovered from superficial mycoses in the
presence of keratinous substrates obtained from human
sources. Nevertheless, the properties of the individual
proteins that presumably account for the virulence of C.
parapsilosis have not yet been elucidated to date.
4. Conclusion
In conclusion, ultrastructural investigations of the in-
terface of C. parapsilosis and the keratinised substrates
from human source reveal important features, which may
help to clarify the pathogenesis of superficial candidiasis.
Our findings indicate the need for investigation of a pos-
sible involvement of acid proteinase in the onychomyco-
sis and cutaneous lesion due to this species.
5. Acknowledgements
This work was supported by Conselho Nacional de De-
senvolvimento Científico e Tecnológico (CNPq)—Brazil,
Fundação Araucária—Paraná-Brazil and PROPPG/UEL-
Brazil. A.F.L.S. was fellowship-holder of Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior
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