Ultrastructural Analysis of in Vitro Adherence and Production of Acid Proteases by Clinical Isolates of <i>Candida parapsilosis</i> Sensu Stricto Following Growth in the Presence of Keratinous Substrates from Human Source

doi:10.4236/aim.2013.38A001

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Advances in Microbiology, 2013, 3, 1-6

Published Online December 2013 (http://www.scirp.org/journal/aim)

http://dx.doi.org/10.4236/aim.2013.38A001

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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: *[email protected]

Received September 25, 2013; revised October 25, 2013; accepted November 2, 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

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.

A. F. L. SPECIAN ET AL.

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-

plex.

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

U/ml.

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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A. F. L. SPECIAN ET AL.

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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

(a)

(b)

(c)

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).

A. F. L. SPECIAN ET AL.

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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A. F. L. SPECIAN ET AL. 5

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

(CAPES)—Brazil.

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