1
Full title: Comparative adherence of Candida albicans and Candida dubliniensis to
2
human buccal epithelial cells and extracellular matrix proteins
3
4
Short title: Adhesion of Candida albicans and Candida dubliniensis
5
6
Rachael P. C. Jordan1,2*, David W. Williams2, Gary P. Moran1, David C. Coleman1, Derek J.
7
Sullivan1
8
9
1
Microbiology Research Unit, Division of Oral Biosciences, Dublin Dental University
10
Hospital, University of Dublin, Trinity College, Dublin 2, Ireland.
11
2
12
and Life Sciences, Cardiff University, Heath Park, Cardiff, CF14 4XY, UK.
Tissue Engineering and Reparative Dentistry, School of Dentistry, College of Biomedical
13
14
*Correspondence: Dr. Rachael P. C. Jordan, Tissue Engineering and Reparative Dentistry,
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School of Dentistry, College of Biomedical and Life Sciences, Cardiff University, Heath
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Park, Cardiff, CF14 4XY, UK.
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Tel: +44 (0)29 2074 6464
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Fax: +44 (0)29 2074 8168
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E-mail: jordanrp@cardiff.ac.uk
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Keywords: Candida dubliniensis; Candida albicans; adhesion; buccal epithelial cells;
22
extracellular matrix proteins.
23
1
1
ABSTRACT
2
Candida albicans and Candida dubliniensis are very closely related pathogenic yeast
3
species. Despite their close relationship, the former is a far more successful coloniser and
4
pathogen of humans. The purpose of the current study was to investigate if the disparity in
5
the virulence of the two species could be attributed to differences in their ability to adhere to
6
human buccal epithelial cells (BECs) and/or extracellular matrix proteins. When grown
7
overnight at 30°C in Yeast Extract Peptone Dextrose (YEPD), genotype 1 C. dubliniensis
8
isolates were found to be significantly more adherent to human BECs than C. albicans or C.
9
dubliniensis genotypes 2-4 (P < 0.001). However, when the yeast cells were grown at 37°C,
10
no significant difference between the adhesion of C. dubliniensis genotype 1 and C. albicans
11
to human BECs was observed, and C. dubliniensis genotype 1 and C. albicans adhered to
12
BECs in significantly greater numbers than the other C. dubliniensis genotypes (P < 0.001).
13
Using surface plasmon resonance analysis, C. dubliniensis isolates were found to adhere in
14
significantly greater numbers than C. albicans to type I and IV collagen, fibronectin, laminin,
15
vitronectin and proline-rich peptides. These data suggest that C. albicans is not more
16
adherent to epithelial cells or matrix proteins than C. dubliniensis and therefore other factors
17
must contribute to the greater levels of virulence exhibited by C. albicans.
18
2
1
INTRODUCTION
2
Candida dubliniensis is a germ tube-positive, chlamydospore-producing yeast species
3
that was originally identified in oral samples from HIV-infected individuals [1]. Nucleotide
4
sequence analysis of the rRNA internally transcribed sequence has shown that C. dubliniensis
5
can be separated into four genotypes (1-4), with genotype 1 being the most prevalent [2].
6
Phylogenetic analysis reveals that C. dubliniensis is very closely related to Candida albicans
7
[1, 3], which is considered to be the most pathogenic Candida species, and given the close
8
relationship between the two species, it is not surprising that they share many phenotypic
9
properties [4]. However, despite their very close relatedness, epidemiological and infection
10
model data indicate that C. albicans is far more prevalent and significantly more pathogenic
11
than C. dubliniensis [5, 6, 7, 8, 9, 10, 11, 12].
12
Candida albicans is routinely found in over 50% of cases of systemic infection,
13
whereas C. dubliniensis has only been found in at most 2-3% of such cases [13, 14, 15].
14
Similarly, in a recent comprehensive study of the epidemiology of oropharyngeal candidiasis,
15
C. albicans was found in 62% of patients, whilst C. dubliniensis was found in only 12% [16].
16
This disparity in prevalence is not due to any differences in antifungal drug resistance
17
between the two species and instead is most likely due to differences in virulence [17]. The
18
reasons why C. dubliniensis is substantially less pathogenic than C. albicans are currently
19
unclear. However, although both species have the ability to produce hyphae it has been
20
proposed that the increased capacity of C. albicans to filament under a wider range of
21
environmental conditions is likely to contribute to its higher level of virulence [18, 10, 4].
22
One of the most fundamental microbial virulence factors is the ability of the
23
microorganism, including yeasts, to recognise and adhere to host cells and tissue [19, 20].
24
Generally, pathogenicity correlates positively with adherence to host cells, with C. albicans
25
being widely recognised as the most adherent and pathogenic yeast species [21, 22]. Candida
3
1
albicans shows a remarkable ability to adhere to cells, tissues, components of the
2
extracellular matrix (ECM) and abiotic surfaces [22, 23, 24, 20, 25], and uses a repertoire of
3
glycoprotein adhesins to interact with its human host. Filamentous forms of C. albicans are
4
considered more adherent than the yeast form and adhere to a greater variety of substrates
5
[21, 26].
6
Despite a high level of sequence homology and synteny in the C. albicans and C.
7
dubliniensis genomes, a key difference in gene content is the presence of a truncated HWP1
8
gene and the absence of ALS3 in C. dubliniensis [3, 27]. Both genes encode adhesive
9
glycoproteins and are thought to play an important role in C. albicans pathogenesis [28, 29].
10
Numerous studies have compared the adhesion of C. albicans and C. dubliniensis to
11
human cells, tissue and abiotic surfaces [30, 5, 31, 32, 24, 20]. The results of these studies are
12
generally conflicting, most likely due to differences in the methods and conditions used,
13
including the use of uncharacterised C. dubliniensis isolates. Growth temperature has been
14
previously suggested to affect the ability of C. albicans and C. dubliniensis to adhere to
15
human cells [33, 34, 35], however the results from individual studies are again conflicting.
16
The ECM is defined as the non-cellular component within tissues and organs that
17
provides essential physical scaffolding for the cellular constituents as well as initiating crucial
18
biochemical and biomechanical cues for tissue morphogenesis, differentiation and
19
homeostasis [36]. Most ECM proteins are large and complex, with multiple distinct domains,
20
and are highly conserved among different taxa [37]. Collagens provide the scaffold for the
21
attachment of other ECM components [38]. Collagen type I is the predominant collagen and
22
an organic component of dentine [39, 40]. Collagen may be denatured to gelatine by acids
23
and enzymes, particularly in caries and C. albicans has the ability to bind to the native and
24
denatured form of collagen type I [40]. Collagen type IV is the primary collagen found in
25
extracellular basement membranes and a major component of dermal–epidermal junctions
4
1
[41]. Gao et al. [42] found that type IV collagen expression changed from a brown linear
2
staining along the basement membrane to thin and discontinued in candidal leukoplakia. This
3
type of change may be related to basement membrane destruction by Candida indicating that
4
collagen type IV may be a target for candidal adhesion. Laminins are large ECM proteins
5
found in basement membrane and are involved in tissue morphogenesis, homeostasis and
6
structural integrity [43, 44]. Pärnänen et al. [45] demonstrated that C. albicans and C.
7
dubliniensis could degrade laminin causing functional disturbances in basement membrane
8
integrity, possibly allowing candidal invasion into tissues. Fibronectin is a large dimeric
9
protein component of the ECM of developing tissues and healing wounds. It is essential for
10
blood vessel morphogenesis [46, 47], and serves as a molecular bridge between the collagen
11
scaffold and other ECM components [37]. Using surface plasmon resonance (SPR), Donohue
12
et al. [48] demonstrated that the C. albicans Als1 protein bound to laminin and fibronectin
13
with micromolar affinity. Vitronectin is a glycoprotein found in serum and ECM, with
14
highest levels evident at sites of tissue damage. Vitronectin contributes to tissue remodeling
15
and healing by regulation of proteolysis, cell adhesion, migration, and survival in the injured
16
tissue [49, 50]. Santoni et al. [51] demonstrated that C. albicans adhered to vitronectin, and
17
vitronectin increased adherence of C. albicans to cultured macrophages [52]. Proline-rich
18
peptides (PRPs) are secreted from the parotid and submandibular/sublingual glands and
19
constitute 8.6% – 11% of whole saliva [53, 54]. PRPs are divided into acidic and basic
20
families; acidic PRPs adhere strongly to recently cleaned teeth providing a binding site for
21
bacteria and although basic PRPs do not adhere to teeth, they can bind to bacteria [54].
22
As adherence to host cells and tissue is an essential early step in the establishment of
23
infection, the present study comprehensively compares the ability of C. dubliniensis and C.
24
albicans to adhere to human buccal epithelial cells (BECs) using a collection of well-
25
characterised C. dubliniensis strains. In addition, the ability of these two species to adhere to
5
1
a range of ECM proteins using SPR was also assessed. It was anticipated that through such
2
analyses, insight into the reasons for the disparity in the pathogenicity of these two Candida
3
species would be obtained.
4
5
MATERIALS AND METHODS
6
Candida species and strains
7
The C. albicans and C. dubliniensis strains used in this study are described in Table 1.
8
The C. dubliniensis isolates used were selected to represent genotypes 1-4 (including the
9
genotype 4 variant, 4v [2, 64]).
10
Candida growth conditions
11
For the human buccal epithelial cell (BEC) adhesion assay, C. albicans (n=6) and
12
C. dubliniensis (n=21) strains (Table 1) were cultured overnight at either 30°C or 37°C in 50
13
ml of Yeast Extract Peptone Dextrose (YEPD) or Yeast Extract Peptone Galactose (YEPGal)
14
in an orbital incubator (Gallenkamp, Model G25) at 200 rpm.
15
For analysis of candidal interaction with ECM proteins, C. albicans (n=12) and
16
C. dubliniensis (n=9) strains (Table 1) were cultured without agitation in 10 ml YEPD
17
overnight at 30°C.
18
Human BEC adhesion assay
19
Adhesion of Candida blastospores to human BECs was measured using a method
20
adapted from a previously described protocol by Murphy and Kavanagh [65]. BECs were
21
collected from age- and gender-matched healthy adult human volunteers using sterile swabs
22
(Venturi Transystem, Brescia, Italy) by gently rubbing the inside of the buccal cavity with the
23
swab. Ethical permission was obtained from the Trinity College Dublin Faculty of Health
24
Sciences Research Ethics Committee. BECs from multiple volunteers were pooled in 10 ml
25
sterile Phosphate Buffered Saline (PBS; Oxoid), centrifuged at 760 × g for 5 min at room
6
1
temperature (Eppendorf 5804 centrifuge, Eppendorf, Hamburg, Germany) and then washed
2
twice with 10 ml sterile PBS. This cell suspension was adjusted to 2 × 105 cells/ml using a
3
Neubauer improved bright line haemocytometer (Hausser Scientific, Horsham, PA, USA)
4
and a Nikon Eclipse E600 microscope (Nikon Corp., Tokyo, Japan) which was fitted with a
5
super high pressure mercury lamp (Nikon). Candida cells were cultured (as described above
6
at either 30°C or 37°C) and harvested by centrifugation at 2,100 × g for 5 min at room
7
temperature. The cultured Candida were washed twice with 10 ml sterile PBS and re-
8
suspended in sterile PBS to provide a final yeast cell density of 1 × 107 cells/ml. Freshly
9
prepared Candida (1 ml) and BEC suspensions (1 ml) were then pooled, giving a ratio of
10
50:1 yeast cells: BECs, and incubated in an orbital incubator at 200 rpm for 2 h at the same
11
temperature as the yeast cells were previously cultured at (i.e. either 30°C or 37°C).
12
Following incubation, BECs with adherent yeast cells were collected by filtering the sample
13
through a hydrophilic, polycarbonate membrane (12 μm pores; Millipore, Cork, Ireland) and
14
washed gently, twice with 10 ml sterile PBS to remove non-adherent yeast cells. The
15
polycarbonate membrane was removed from the filter holder with receiver (Nalgene
16
Labware, part of Thermo Fisher Scientific Inc.) and BECs were transferred to a glass
17
microscope slide by placing the polycarbonate membrane face down on the glass microscope
18
slide. The membrane was then removed from the slide and discarded. Samples were air dried
19
before being stained for 30 s with crystal violet and rinsed with a decolouriser (Sigma-
20
Aldrich Ltd., Gillingham, Dorset, UK). The number of yeast cells adhering to each of 100
21
single human BECs was measured in triplicate using light microscopy.
22
Measurement of the interaction between Candida cells and ECM proteins by surface
23
plasmon resonance
24
The interaction between individual ECM proteins (human collagen type I [66], human
25
collagen type IV [66], bovine fibronectin (Sigma), human laminin (Sigma), human
7
1
vitronectin (Sigma) and PRPs [67]) and C. albicans and C. dubliniensis, was investigated by
2
real-time biomolecular interaction analysis (BIA) using a BIAcore 3000 system (BIAcore
3
AB, Uppsala, Sweden) and CM3 sensorchips (BIAcore). An overview of how BIAcore
4
methodology works is described by Jason-Moller et al. [68]. To obtain PRPs, stimulated
5
parotid saliva was obtained from 10 volunteers (5 male, 5 female) using modified Carlson-
6
Crittenden Cups. Saliva production was stimulated using 1% citric acid and approximately
7
2ml of saliva was collected per volunteer. Combined parotid saliva was clarified by
8
centrifugation, added to equal volumes of Tris/HCl buffer (20mM Tris/NaCl, pH 8.0; 0.5M
9
NaCl) and filtered through 0.22 µl filters. Protein fractions were separated using a sephacryl
10
S200 gel filtration column which was connected to a calibrated fast performance liquid
11
chromatography (FPLC) system and protein fractions were identified by absorbance peaks at
12
OD280. Protein fractions were dialysed for 5 days against distilled water containing protease
13
inhibitors before being lyophilized. Lyophilized samples were reconstituted in sample buffer
14
(0.062M Tris/HCl, pH 6.8; 10% glycerol, 2% sodium dodecyl sulphate, 5% 2-β-
15
mercaptoethanol, 0.002% bromophenol blue) and protein fractions were separated by
16
electrophoresis using 10-15% gradient polyacrylamide gels [67]. The CM3 sensorchip has a
17
short dextran matrix and is designed for working with large molecules and whole cells.
18
Individual ECM proteins (human collagen type I (pH 4, 100 μg/ml), human collagen type IV
19
(pH 5, 40 μg/ml), bovine fibronectin (pH 4, 40 μg/ml), human laminin (pH 4, 20 μg/ml),
20
human vitronectin (pH 4, 40 μg/ml) and PRPs isolated from human parotid saliva (100
21
μg/ml)) were prepared in 10 mM sodium acetate buffer (BIAcore). These ligands were
22
immobilised on the sensorchip surface of flow cell 2 and 4, with equivalent controls (flow
23
cell 1 and 3, respectively), allowing two separate ligands to be covalently coupled to each
24
sensorchip. Immobilisation was achieved via amino coupling by first injecting 35 μl of a N-
25
hydroxysuccinimide
(NHS;
BIAcore)/1-ethyl-3-(3-dimethylaminopropyl)
8
carbodiimide
1
hydrochloride (EDC; BIAcore) mixture over the sensorchip to generate active ester groups.
2
After activation, injection of the ECM protein ligand in appropriate pH buffer was applied to
3
the surface. A targeted immobilisation level of 2,500 resonance units (RU) was pre-selected
4
using the BIAcore Wizard software (BIAcore) for fibronectin, laminin and vitronectin. An
5
immobilisation level of 2,000 RU was selected for collagen type IV and PRPs. Collagen type
6
I was immobilised using a timed injection over 4 min with a flow rate of 10 μl/min. Finally,
7
deactivation of excess reactive groups on the sensorchip surface involved a 7 min pulse
8
injection of 1 M ethanolamine hydrochloride pH 8.5 (BIAcore). The high ionic strength of
9
this solution also removed non-covalently bound material from the surface. Flow cells 1 and
10
3 were used as in-line references for flow cells 2 and 4 respectively, where the blank surface
11
was exposed to amine coupling in the absence of ligand. Candida cells (cultured as described
12
above at 30°C) were diluted to a concentration of 1 × 108 cells/ml in HBS-EP buffer and were
13
injected over the various ligand-prepared surfaces at 5 μl/min for 4 min. The Candida/ligand
14
interaction resulted in an increase in the SPR signal, measured as RU. Residual bound
15
Candida cells were removed by injection (5 μl/min for 2 min) of 50 mM sodium hydroxide
16
(Fisher Scientific). Each isolate was analysed on two separate occasions.
17
18
Statistical analysis
19
Data were analysed and graphically depicted using GraphPad Prism® Software
20
Version 4.00 (GraphPad Software Inc., La Jolla, CA, USA) for Windows. Analysis was
21
conducted using the mean, standard error of the mean (SEM), one-way analysis of variance
22
(ANOVA) with Tukey’s multiple comparison post-test and unpaired 2-tailed t-test. Where
23
samples were found to have unequal variances, unpaired, 2-tailed, t-tests with Welch’s
24
correction were performed.
25
9
1
RESULTS
2
Adherence to human BECs
3
When Candida cells were cultured overnight at 30°C in YEPD, C. dubliniensis
4
genotype 1 isolates (n=5) adhered to human BECs in significantly higher numbers than C.
5
albicans (P < 0.001; n=6) and the other C. dubliniensis genotypes (P < 0.001; genotype 2
6
(n=5); genotype 3 (n=5), genotype 4 (n=4) and genotype 4v (n=2)). There was no significant
7
difference in adhesion to BECs between genotypes 2, 3, 4, 4v and C. albicans (P > 0.05; Fig.
8
1). When the Candida cells were cultured at 37°C overnight there was a general reduction in
9
adherence of all C. dubliniensis genotypes, and, in contrast to the experiment where the cells
10
were cultured at 30°C, there was no significant difference between the adhesion of C.
11
dubliniensis genotype 1 and C. albicans to BECs (P > 0.05; Fig. 1). It was also evident that
12
C. dubliniensis genotype 1 and C. albicans adhered in significantly higher numbers to BECs
13
than the other C. dubliniensis genotypes (P < 0.001; Fig. 1). Culturing Candida overnight at
14
37°C in YEPGal rather than YEPD also resulted in C. dubliniensis genotype 1 adhering to the
15
BECs at significantly higher numbers than the other genotypes of C. dubliniensis and C.
16
albicans (P < 0.001, except C. dubliniensis genotype 4v P < 0.01), although there was no
17
significant difference between the adhesion of C. dubliniensis genotypes 2, 3, 4, 4v and
18
C. albicans (P > 0.05) to BECs (Fig. 1). There was a general increase in the adhesion to
19
BECs of all C. dubliniensis genotypes tested when cultured in YEPGal at 37°C compared to
20
YEPD at the same temperature, however, the opposite was true for C. albicans.
21
Interaction of Candida with ECM proteins
22
The mean RU values ± SEM for adhesion of C. dubliniensis and C. albicans when
23
cultured at 30°C to the six ECM proteins indicated that the C. dubliniensis isolates tested
24
adhered to all six ECM proteins in significantly higher levels than the C. albicans isolates
25
(collagen type 1, P < 0.005, Fig. 2A; collagen type IV, P < 0.05, Fig. 2B; fibronectin, P <
10
1
0.0001, Fig. 2C; laminin, P < 0.001, Fig. 2D; PRPs, P < 0.0001, Fig. 2E; vitronectin, P <
2
0.05, Fig. 2F). These data also suggest that both species adhere most readily to laminin, with
3
lowest adherence evident with collagen type I and vitronectin.
4
5
DISCUSSION
6
Adherence of Candida to host tissue is seen as an essential early step in the
7
establishment of infection [69], and previous studies indicate that pathogenicity correlates
8
positively with adherence to host cells, with the most adherent Candida species, C. albicans
9
also considered the most pathogenic [21, 22]. Given the conflicting data obtained in previous
10
studies of comparative adherence of C. albicans and its closest relative C. dubliniensis to
11
human cells, the purpose of this current study was to definitively compare adhesion of these
12
two species using a comprehensive selection of well-characterised isolates representative of
13
all C. dubliniensis genotypes. Adhesion of C. albicans and C. dubliniensis to human BECs
14
was investigated under various growth conditions and the ability of these two species to
15
adhere to a range of ECM proteins was also assessed in real-time using SPR.
16
Candida dubliniensis genotype 1 and C. albicans showed a similar ability to adhere to
17
BECs when cultured at 37°C in glucose and adhered to BECs at significantly higher levels
18
than the other genotypes of C. dubliniensis. When cultured at 30°C however, a general
19
increase in adherence of the C. dubliniensis genotypes to BECs occurred, with the notable
20
difference that, at this temperature, C. dubliniensis genotype 1 cells adhered to the BECs at
21
significantly higher levels than C. albicans. Growth in galactose at 37°C also resulted in an
22
increase in adhesion by C. dubliniensis, with genotype 1 cells again adhering to the BECs in
23
significantly greater numbers than the other genotypes of C. dubliniensis and C. albicans.
24
Other groups have examined the comparative adhesion of C. albicans and C. dubliniensis to
25
BECs and while our data are in general agreement with those of McCullough et al. [30] and
11
1
Gilfillan et al. [5] they disagree with those of Vidotto et al. [31]. However, it should be noted
2
that none of these studies used strains representative of the four C. dubliniensis genotypes
3
which our data show have significantly different abilities to adhere to BECs. Growth
4
temperature is known to affect the cell surface hydrophobicity of dimorphic yeasts such as C.
5
dubliniensis and C. albicans and this may be a contributory factor in the differential
6
adherence ability of C. dubliniensis at various temperatures [33, 34, 35, 70]. The increased
7
capacity of C. dubliniensis to adhere to BECs when incubated at 30°C compared to 37°C may
8
suggest that C. dubliniensis could have an advantage in colonising the upper trachea which
9
has a temperature between 29°C and 32°C [71]. The fact that yeasts of C. albicans and C.
10
dubliniensis genotype 1 (the most prevalent genotype) show similar levels of adhesion to
11
BECs at 37°C suggests that other factors, such as the ability to produce hyphae, might be
12
responsible for the enhanced capacity of C. albicans to colonise and infect the oral cavity.
13
Although we attempted to investigate the comparative adhesion of C. albicans and C.
14
dubliniensis hyphae to BECs and other cell types this was not possible due to difficulty in
15
generating matching levels of hyphae in the two species and to the co-aggregation of germ
16
tubes and hyphae which prevented making accurate inocula.
17
The BIAcore system has previously been used to measure interactions between
18
proteins and other small molecules such as nucleic acids [72], lipids [73], and other proteins
19
[74]. It has also been used to measure interactions between bacteria and proteins [75, 76, 77],
20
however to date, only one other study using whole Candida cells as an analyte for the
21
BIAcore biosensor has been performed which characterised the interaction between
22
medically relevant Candida species and mannose-binding lectin [78]. In order to colonise and
23
infect human tissue, pathogenic Candida spp. have to be able to recognise and adhere to
24
ECM proteins [79]. The ability of Candida to recognise and adhere to a wide variety of
25
proteins of the ECM increases its potential to colonise and infect different anatomical niches.
12
1
In this study, we have shown that C. albicans and C. dubliniensis bind to the ECM proteins,
2
collagen type I, collagen type IV, laminin, fibronectin, vitronectin and PRPs. Comparing the
3
mean adherence of C. dubliniensis and C. albicans yeasts cultured at 30 °C to all six ECM
4
proteins we found that C. dubliniensis yeasts adhered to the proteins at a significantly higher
5
level than C. albicans yeasts, with laminin being the matrix protein to which both species
6
best adhered. In contrast, using proteins immobilised on plastic, Klotz [80] found that C.
7
albicans adhered most avidly to collagen type IV, followed by laminin and fibronectin.
8
The data presented show that C. dubliniensis yeast cells, in particular genotype 1,
9
were at least as adherent to human BECs, and that C. dubliniensis yeasts were even more
10
adherent to the ECM proteins tested, than C. albicans yeasts. Since it was not possible for us
11
to examine the comparative adherence of hyphae belonging to the two species, it is very
12
difficult to determine how important this might be in vivo. However, these findings may help
13
explain why genotype 1 is the most prevalent C. dubliniensis genotype in humans. The data
14
also suggest that a lower ability of yeast cells to adhere to host tissue is unlikely to be
15
responsible for the relatively poor capacity of C. dubliniensis to colonise and infect humans.
16
Instead, C. albicans has other attributes which very likely confer a competitive advantage,
17
including the ability to produce hyphae under a wider range of nutrient concentrations, and a
18
higher tolerance of environmental stresses such as temperature and oxidative stress. In
19
addition, comparative genomic analysis also suggests that C. albicans hyphae are equipped
20
with a superior arsenal of adhesins than C. dubliniensis hyphae [4, 25]. In particular, C.
21
dubliniensis has no ortholog for the hypha-specific genes ALS3 or HYR1 and encodes a
22
truncated version of HWP1. All three of these genes are believed to encode important
23
virulence factors and have been particularly associated with adherence and/or biofilm
24
formation. Indeed, it has already been shown that germinating C. albicans cells are more
25
adherent and invasive than germinating C. dubliniensis cells in an ex vivo oral infection
13
1
models [18]. Therefore, C. albicans may be a more successful commensal and pathogen of
2
humans than C. dubliniensis due to its enhanced capacity to produce hyphae and to the
3
presence of additional adhesins on these hyphae that enhance tissue recognition and adhesion.
4
Therefore, the disparity in virulence between the two species is likely due to the enhanced
5
capacity of C. albicans to adapt to the ever changing and challenging environmental
6
conditions encountered in various niches in the human body by rapidly changing morphology
7
between yeast and hyphal forms.
8
9
ACKNOWLEDGEMENTS
10
We would like to thank Professor Victor Duance of the Connective Tissue Biology
11
Laboratories, Cardiff School of Biosciences, The Sir Martin Evans Building, Museum
12
Avenue, Cardiff CF10 3AX, UK who provided the collagens used in the BIAcore
13
experiments, and Dr. Claire Price, formerly of The Department of Tissue Engineering and
14
Reparative Dentistry, School of Dentistry, College of Biomedical and Life Sciences, Cardiff
15
University, Heath Park, Cardiff CF14 4XY, UK who provided the sensor chip to which PRPs
16
had previously been coupled. This project was supported by the Microbiology Research Unit,
17
Division of Oral Biosciences, Dublin Dental University Hospital, University of Dublin,
18
Trinity College, Dublin 2, Ireland and the Health Research Board; grant number
19
RP/2004/226.
20
Conflict of Interest
21
None.
22
23
REFERENCES
24
1. Sullivan DJ, Westerneng TJ, Haynes KA, Bennett DE, Coleman DC. Candida
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dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species
14
1
associated with oral candidosis in HIV-infected individuals. Microbiol 1995 141:
2
1507–1521.
3
2. Gee SF, Joly S, Soll DR, et al. Identification of four distinct genotypes of Candida
4
dubliniensis and detection of microevolution in vitro and in vivo. J Clin
5
Microbiol 2002 40: 556–574.
6
3. Moran G, Stokes C, Thewes S, et al. Comparative genomics using Candida albicans
7
DNA microarrays reveals absence and divergence of virulence-associated genes in
8
Candida dubliniensis. Microbiol 2004 150: 3363–3382.
9
10
11
12
13
14
4. Moran GP, Coleman DC, Sullivan DJ. Candida albicans versus Candida dubliniensis:
Why is C. albicans more pathogenic? Int J Microbiol 2012: 205921
5. Gilfillan GD, Sullivan DJ, Haynes K, et al. Candida dubliniensis: phylogeny and
putative virulence factors. Microbiol 1998 144: 829–838.
6. Vilela MM, Kamei K, Sano A, et al. Pathogenicity and virulence of Candida
dubliniensis: comparison with C. albicans. Med Mycol 2002 40: 249–257.
15
7. Moran GP, MacCallum DM, Spiering MJ, Coleman DC, Sullivan DJ. Differential
16
regulation of the transcriptional repressor NRG1 accounts for altered host-cell
17
interactions in Candida albicans and Candida dubliniensis. Mol Microbiol 2007 66:
18
915–929.
19
8. Stokes C, Moran GP, Spiering MJ, et al. Lower filamentation rates of Candida
20
dubliniensis contribute to its lower virulence in comparison with Candida albicans.
21
Fungal Genet Biol 2007 44: 920–931.
22
9. Asmundsdóttir LR, Erlendsdóttir H, Agnarsson BA, Gottfredsson M. The importance
23
of strain variation in virulence of Candida dubliniensis and Candida albicans: results
24
of a blinded histopathological study of invasive candidiasis. Clin Microbiol Infect
25
2009 15: 576–585.
15
1
10. Spiering MJ, Moran GP, Chauvel M, et al. Comparative transcript profiling of
2
Candida albicans and Candida dubliniensis identifies SFL2, a C. albicans gene
3
required for virulence in a reconstituted epithelial infection model. Eukaryot Cell
4
2012 9: 251–265.
5
11. Koga-Ito CY, Komiyama EY, Martins CA, et al. Experimental systemic virulence of
6
oral Candida dubliniensis isolates in comparison with Candida albicans, Candida
7
tropicalis and Candida krusei. Mycoses 2011 54: e278–e285.
8
12. Owotade FJ, Patel M, Ralephenya TR, Vergotine G. Oral Candida colonization in
9
HIV-positive women: associated factors and changes following antiretroviral therapy.
10
J Med Microbiol 2013 62: 126–132.
11
13. Kibbler CC, Seaton S, Barnes RA, Gransden WR, et al. Management and outcome of
12
bloodstream infections due to Candida species in England and Wales. J Hosp Infect
13
2003 54: 18–24.
14
15
16
17
14. Odds FC, Hanson MF, Davidson AD, et al. One year prospective survey of Candida
bloodstream infections in Scotland. J Med Microbiol 2007 56: 1066–1075.
15. Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public
health problem Clin Microbiol Rev 2007 20: 133–163.
18
16. Patel PK, Erlandsen JE, Kirkpatrick WR, et al. The changing epidemiology of
19
oropharyngeal candidiasis in patients with HIV/AIDS in the era of antiretroviral
20
therapy. AIDS Res Treat 2012 2012: 262471
21
17. Moran GP, Sullivan DJ, Henman MC, et al. Antifungal drug susceptibilities of oral
22
Candida dubliniensis isolates from human immunodeficiency virus (HIV)-infected
23
and non-HIV-infected subjects and generation of stable fluconazole-resistant
24
derivatives in vitro. Antimicrob Agents Chemother 1997 41: 617–623.
16
1
18. O'Connor L, Caplice N, Coleman DC, Sullivan DJ, Moran GP. Differential
2
filamentation of Candida albicans and Candida dubliniensis is governed by nutrient
3
regulation of UME6 expression. Eukaryot Cell 2010 9: 1383–1397.
4
19. Sullivan DJ, Moran GP, Pinjon E, et al. Comparison of the epidemiology, drug
5
resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans.
6
FEMS Yeast Res 2004 4: 369–376.
7
20. Romeo O, De Leo F, Criseo G. Adherence ability of Candida africana: a comparative
8
study with Candida albicans and Candida dubliniensis. Mycoses 2010 54: e57–61.
9
21. Calderone RA, Braun PC. Adherence and receptor relationships of Candida albicans.
10
11
12
13
14
Microbiol Rev 1991 55: 1–20.
22. Cannon RD, Chaffin WL. Oral colonization by Candida albicans. Crit Rev Oral Biol
Med 1999 10: 359–383.
23. Li X, Yan Z, Xu J. Quantitative variation of biofilms among strains in natural
populations of Candida albicans. Microbiol 2003 149: 353–362.
15
24. Biasoli MS, Tosello ME, Luque AG, Magaró HM. Adherence, colonization and
16
dissemination of Candida dubliniensis and other Candida species. Med Mycol 2010
17
48: 291–297.
18
25. Bürgers R, Hahnel S, Reichert TE, et al. Adhesion of Candida albicans to various
19
dental implant surfaces and the influence of salivary pellicle proteins. Acta Biomater
20
2010 6: 2307–2313.
21
26. Umeyama T, Kaneko A, Watanabe H, et al. Deletion of the CaBIG1 gene reduces
22
beta-1,6-glucan synthesis, filamentation, adhesion, and virulence in Candida albicans.
23
Infect Immun 2006 74: 2373–2381.
17
1
27. Jackson AP, Gamble JA, Yeomans T, et al. Comparative genomics of the fungal
2
pathogens Candida dubliniensis and Candida albicans. Genome Res 2009 19: 2231–
3
2244.
4
28. Staab JF, Ferrer CA, Sundstrom P. Developmental expression of a tandemly repeated,
5
proline-and glutamine-rich amino acid motif on hyphal surfaces on Candida albicans.
6
J Biol Chem 1996 271: 6298–6305.
7
8
29. Hoyer LL. The ALS gene family of Candida albicans. Trends Microbiol 2001 9: 176–
180.
9
30. McCullough M, Ross B, Reade P. Characterization of genetically distinct subgroup of
10
Candida albicans strains isolated from oral cavities of patients infected with human
11
immunodeficiency virus. J Clin Microbiol 1995 33: 696–700.
12
31. Vidotto V, Mantoan B, Pugliese A, et al. Adherence of Candida albicans and
13
Candida dubliniensis to buccal and vaginal cells. Rev Iberoam Micol 2003 20: 52–54.
14
32. He XY, Meurman JH, Kari K, Rautemaa R, Samaranayake LP. In vitro adhesion of
15
16
17
Candida species to denture base materials. Mycoses 2006 49: 80–84.
33. Hazen KC, Wu JG, Masuoka J. Comparison of the hydrophobic properties of Candida
albicans and Candida dubliniensis. Infect Immun 2001 69: 779–786.
18
34. Jabra-Rizk MA, Falkler WA, Jr, Merz WG, et al. Cell surface hydrophobicity-
19
associated adherence of Candida dubliniensis to human buccal epithelial cells. Rev
20
Iberoam Micol 2001 18: 17–22.
21
35. Samaranayake YH, Samaranayake LP, Yau JY, et al. Adhesion and cell-surface-
22
hydrophobicity of sequentially isolated genetic isotypes of Candida albicans in an
23
HIV-infected Southern Chinese cohort. Mycoses 2003 46: 375–383.
24
25
36. Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. J Cell Sci
2010 123: 4195–4200.
18
1
37. Chagnot C, Listrat A, Astruc T, Desvaux M. |Bacterial adhesion to animal tissues:
2
protein determinants for recognition of extracellular matrix components. Cell
3
Microbiol 2012 14: 1687–1696.
4
38. Orgel JP, Antipova O, Sagi I, et al. Collagen fibril surface displays a constellation of
5
sites capable of promoting fibril assembly, stability, and hemostasis. Connect Tissue
6
Res 2011 52: 18–24.
7
8
39. Makihira S, Nikawa H, Tamagami M, et al. Bacterial and Candida adhesion to intact
and denatured collagen in vitro. Mycoses 2002 45: 389–392.
9
40. Makihira S, Nikawa H, Tamagami M, Hamada T, Samaranayake LP. Differences in
10
Candida albicans adhesion to intact and denatured type I collagen in vitro. Oral
11
Microbiol Immunol 2002 17: 129–131.
12
13
41. Abreu-Velez AM, Howard MS. Collagen IV in normal skin and in pathological
processes. N Am J Med Sci 2012 4: 1–8.
14
42. Gao Y, Liu X, Han Z. Type IV collagen and C-erbB-2 expression in oral candidal
15
leukoplakia. Zhonghua Kou Qiang Yi Xue Za Zhi 1999 34: 325–327. [in Chinese]
16
43. Pradhan S, Farach-Carson MC. Mining the extracellular matrix for tissue engineering
17
applications. Regen Med 2010 5: 961–970.
18
44. Hamill KJ, Kligys K, Hopkinson SB, Jones JC. Laminin deposition in the
19
extracellular matrix: a complex picture emerges. J Cell Sci 2009 122: 4409–4417.
20
45. Pärnänen P, Kari K, Virtanen I, Sorsa T, Meurman JH. Human laminin-332
21
22
23
24
25
degradation by Candida proteinases. J Oral Pathol Med 2008 37: 329–335.
46. Astrof S, Hynes RO. Fibronectins in vascular morphogenesis. Angiogenesis 2009 12:
165–175.
47. To WS, Midwood KS. Plasma and cellular fibronectin: distinct and independent
functions during tissue repair. Fibrogenesis Tissue Repair 2011 4: 21.
19
1
48. Donohue DS, Ielasi FS, Goossens KV, Willaert RG.The N-terminal part of Als1
2
protein from Candida albicans specifically binds fucose-containing glycans. Mol
3
Microbiol 2011 80: 1667–1679.
4
5
6
7
49. Tsuruta Y, Park YJ, Siegal GP, Liu G, Abraham E. Involvement of vitronectin in
lipopolysaccaride-induced acute lung injury. J Immunol 2007 179: 7079–7086.
50. Madsen CD, Sidenius N. The interaction between urokinase receptor and vitronectin
in cell adhesion and signalling. Eur J Cell Biol 2008 87: 617–629.
8
51. Santoni G, Spreghini E, Lucciarini R, Amantini C, Piccoli M. Involvement of αvβ3
9
integrin-like receptor and glycosaminoglycans in Candida albicans germ tube
10
adhesion to vitronectin and to a human endothelial cell line. Microb Pathog 2001 31:
11
159–172.
12
52. Limper AH, Standing JE. Vitronectin interacts with Candida albicans and augments
13
organism attachment to the NR8383 macrophage cell line. Immunol Lett 2004 42:
14
139–144.
15
16
17
18
53. Huq NL, Cross KJ, Ung M, et al. A review of the salivary proteome and peptidome
and saliva-derived peptide therapeutics. Int J Pept Res Ther 2007 13: 547–564.
54. Levine M. Susceptibility to dental caries and the salivary Proline-Rich Proteins Int J
Dent 2011, Article ID 953412.
19
55. Gillum AM, Tsay EY, Kirsch DR. Isolation of the Candida albicans gene for
20
orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and
21
E. coli pyrF mutations. Mol Gen Genet 1984 198: 179–182.
22
56. Gallagher PJ, Bennett DE, Henman MC, et al. Reduced azole susceptibility of oral
23
isolates of Candida albicans from HIV-positive patients and a derivative exhibiting
24
colony morphology variation. J Gen Microbiol 1992 138: 1901–1911.
20
1
2
3
4
57. Pinjon, E. Investigation into the molecular mechanisms of intraconazole resistance in
Candida dubliniensis. PhD thesis, University of Dublin, Trinity College, 2003.
58. McCourtie J. Douglas LJ. Relationship between cell surface composition, adherence,
and virulence of Candida albicans. Infect Immun 1984 45: 6–12.
5
59. Boerlin P, Boerlin-Petzold F, Durussel C, et al. Cluster of oral atypical Candida
6
albicans isolates in a group of human immunodeficiency virus-positive drug users. J
7
Clin Microbiol 1995 33: 1129–1135.
8
60. Al Mosaid A, Sullivan DJ, Polacheck I, et al. Novel 5-flucytosine-resistant clade of
9
Candida dubliniensis from Saudi Arabia and Egypt identified by Cd25 fingerprinting.
10
J Clin Microbiol 2005 43: 4026–4036.
11
61. Pinjon E, Sullivan D, Salkin I, Shanley D, Coleman D. Simple, inexpensive, reliable
12
method for differentiation of Candida dubliniensis from Candida albicans. J Clin
13
Microbiol 1998 36: 2093–2095.
14
62. Polacheck I, Strahilevitz J, Sullivan D, et al. Recovery of Candida dubliniensis from
15
non-human immunodeficiency virus-infected patients in Israel. J Clin Microbiol 2000
16
38: 170–174.
17
18
63. Al Mosaid A, Sullivan DJ, Coleman DC. Differentiation of Candida dubliniensis
from Candida albicans on Pal's agar. J Clin Microbiol 2003 41: 4787–4789.
19
64. McManus BA, Coleman DC, Moran G, et al. Multilocus sequence typing reveals that
20
the population structure of Candida dubliniensis is significantly less divergent than
21
that of Candida albicans. J Clin Microbiol 2008 46: 652–664.
22
23
65. Murphy AR, Kavanagh KA. Adherence of clinical isolates of Saccharomyces
cerevisiae to buccal epithelial cells. Med Mycol 2001 39: 123–127.
21
1
66. Duance VC. Purification of collagen types from various tissues. In: Harris ELV,
2
Angal S, eds. Protein Purification Applications: A Practical Approach, Oxford,
3
England: IRL Press, 1990: 137–142.
4
5
6
7
8
9
67. Price CL. The development of a silicone rubber surface inhibitory to the adherence of
micro-organisms. PhD Thesis, University of Wales, College of Medicine. 2004
68. Jason-Moller L, Murphy M, Bruno J. Overview of Biacore systems and their
applications. Curr Protoc Protein Sci 2006 Chapter 19:Unit 19.13.
69. Yang YL. Virulence factors of Candida species. J Microbiol Immunol Infect 2003 36:
223–228.
10
70. Hazen KC. Relationship between expression of cell surface hydrophobicity protein 1
11
(CSH1p) and surface hydrophobicity properties of Candida dubliniensis. Curr
12
Microbiol 2004 48: 447–451.
13
14
71. McFadden ER, Jr, Pichurko BM, Bowman HF, et al. Thermal mapping of the airways
in humans. J Appl Physiol 1985 58: 564–570.
15
72. Di Primo C, Lebars I. Determination of refractive index increment ratios for protein-
16
nucleic acid complexes by surface plasmon resonance. Anal Biochem 2007 368: 148–
17
155.
18
73. Sun YH, Shen L, Dahlbäck B. Gla domain-mutated human protein C exhibiting
19
enhanced anticoagulant activity and increased phospholipid binding. Blood 2003 101:
20
2277–2284.
21
74. Surinya KH, Forbes BE, Occhiodoro F, et al. An investigation of the ligand binding
22
properties and negative cooperativity of soluble insulin-like growth factor receptors. J
23
Biol Chem 2008 283: 5355–5363.
24
25
75. Clyne M, Dillon P, Daly S, et al. Helicobacter pylori interacts with the human singledomain trefoil protein TFF1. Proc Natl Acad Sci USA 2004 101: 7409–7414.
22
1
76. Kinoshita H, Uchida H, Kawai Y, et al. Quantitative evaluation of adhesion of
2
lactobacilli isolated from human intestinal tissues to human colonic mucin using
3
surface plasmon resonance (BIACORE assay). J Appl Microbiol 2007 102: 116–123.
4
5
77. Nobbs AH, Vajna RM, Johnson JR, et al. Consequences of a sortase A mutation in
Streptococcus gordonii. Microbiol 2007 153: 4088–4097.
6
78. Damiens S, Danze PM, Drucbert AS, et al. Characterization of the recognition of
7
Candida species by mannose-binding lectin using surface plasmon resonance. Analyst
8
2013 138: 2477–2482.
9
79. Singh B, Fleury C, Jalalvand F, Riesbeck K. Human pathogens utilize host
10
extracellular matrix proteins laminin and collagen for adhesion and invasion of the
11
host. FEMS Microbiol Rev 2012 36: 1122–1180.
12
13
80. Klotz SA. Adherence of Candida albicans to components of the subendothelial
extracellular matrix. FEMS Microbiol Lett 1990 56: 249–254.
14
23
1
FIGURE LEGENDS
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Figure 1. Adhesion of Candida dubliniensis and Candida albicans yeast cells to human
3
buccal epithelial cells (BECs). The results of the adhesion of each genotype of
4
C. dubliniensis (genotype 1 (n = 5), genotype 2 (n = 5), genotype 3 (n = 5), genotype 4 (n =
5
4), genotype 4v (n = 2)) and of C. albicans (n = 6) were averaged to give the mean number of
6
adherent Candida ± SEM per human BEC for each genotype of C. dubliniensis and of
7
C. albicans. Candida cells were cultured overnight at 30°C or 37°C in YEPD, and at 37°C in
8
YEPGal. Adhesion experiments were conducted using the same incubation temperature as the
9
overnight culture. Experiments were performed in triplicate.
10
11
Figure 2. Mean resonance unit value indicating adhesion of Candida dubliniensis and
12
Candida albicans to extracellular matrix (ECM) proteins. The results of the binding of
13
C. dubliniensis (n = 9) and of C. albicans (n = 12) to collagen type I (A), collagen type IV
14
(B), fibronectin (C), laminin (D), PRPs (E) and vitronectin (F) on at least two separate
15
occasions were averaged to give the mean number of resonance units indicative of adhesion ±
16
SEM for C. dubliniensis and C. albicans.
17
24