FEMS Yeast Research, 15, 2015, fov088
doi: 10.1093/femsyr/fov088
Advance Access Publication Date: 21 September 2015
Research Article
RESEARCH ARTICLE
Growth of Candida albicans in human saliva is
supported by low-molecular-mass compounds
Marianne Valentijn-Benz, Kamran Nazmi, Henk S. Brand, Wim van ’t Hof
and Enno C. I. Veerman∗
Section of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), Vrije Universiteit and
Universiteit van Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, the Netherlands
∗ Corresponding author: Academic Centre for Dentistry Amsterdam (ACTA), Department of Oral Biochemistry, Gustav Mahlerlaan 3004, 1081 LA
Amsterdam, the Netherlands. Tel: +31-20-5980883; E-mail: [email protected]
One sentence summary: The yeast Candida albicans can thrive on salivary constituents as nutrients, to maintain itself in the oral cavity.
Editor: Carol Munro
ABSTRACT
Saliva plays a key role in the maintenance of a stable oral microflora. It contains antimicrobial compounds but also
functions as a substrate for growth of bacteria under conditions of low external nutrient supply. Besides bacteria, yeasts, in
particular Candida albicans, commonly inhabit the oral cavity. Under immunocompromised conditions, instantaneous
outgrowth of this yeast occurs in oral carriers of C. albicans, suggesting that this yeast is able to survive in the oral cavity
with saliva as sole source of growth substrate. The aim of the present study was to identify the salivary constituents that
are used by C. albicans for growth and survival in saliva. In addition, we have explored the effect of growth in saliva on the
susceptibility of C. albicans to histatin 5, a salivary antifungal peptide. It was found that C. albicans was able to grow in
human saliva without addition of glucose, and in the stationary phase could survive for more than 400 h. Candida albicans
grown in saliva was more than 10 times less susceptible for salivary histatin 5 than C. albicans cultured in Sabouraud
medium.
Keywords: saliva; C albicans; glucose; culturing; histatin; peptides
INTRODUCTION
The numbers of microorganisms living within the body of the
average healthy adult human outnumber human cells 10 to 1.
One of the most heavily colonized parts of the human body is
the oral cavity, which harbors more than 108 microorganisms.
Although saliva contains several antimicrobial systems (Nieuw
Amerongen and Veerman 2004; Van ‘t Hof et al. 2014), it obviously
does not render the mouth aseptic, but rather seems to support
the maintenance of a multispecies benign oral microflora. In this
process saliva can play a key role, because on one hand it contains a wide variety of defensive systems that protect oral tissues against excessive microbial colonization, whereas on the
other hand it is a source of (glyco)proteins and peptides potentially serving as growth substrate for microorganisms under
conditions of low external nutrient supply. It is conceivable that
microorganisms with the ability to survive and proliferate on
these substrates under nutrient-poor conditions have an ecological advantage. Indeed, various oral bacteria are able to grow
in vitro to a certain extent in saliva or agar media prepared from
heated saliva (Van der Hoeven and Camps 1991; De Jong et al.
1997; Shelburne et al. 2005). A number of studies have demonstrated that oral bacteria are capable of utilizing the complex
carbohydrates of human salivary mucins as a nutrient source
(Wickstrom and Svensater 2008; Wickstrom, Hamilton and
Received: 13 April 2015; Accepted: 18 September 2015
C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Yeast Research, 2015, Vol. 15, No. 8
Svensater 2009; Wickstrom et al. 2009). This process requires a
wide variety of glycosidases.
Besides bacteria, yeasts, in particular Candida albicans, commonly inhabit the oral cavity. Candida albicans is present in the
oral cavity of 40–60% of the general population (Samaranayake,
Leung and Jin 2009), yet uncontrolled outgrowth of C. albicans occurs much less frequently and is related to several, often hostrelated, factors. For instance, suppression of the immune system, e.g. by inhalation of corticosteroids, frequently results in instantaneous outgrowth of the yeast in oral carriers of C. albicans.
This suggests that under healthy conditions C. albicans is able to
survive in a dormant state in the oral cavity. Previous studies
suggest that C. albicans is not able to thrive on human saliva in
the absence of added glucose, suggesting that, unlike bacteria,
the yeast cannot use salivary constituents as growth substrate.
Recently, however, it was described that C. albicans can survive
in tap water containing trace amounts of saliva (Gerhardts et al.
2012).
Because of the presumably suboptimal growth conditions in
the oral cavity, C. albicans present in this natural ecological habitat will likely differ from those cultured in vitro. This may have
profound effects on their virulence as well as on their resistance against innate immune proteins. For instance, it has been
demonstrated that, when the energy metabolism is suppressed,
the yeast’s resistance against antimicrobial peptides, including
histatins and defensins, is enhanced (Veerman et al. 2007). The
present study was undertaken to explore whether C. albicans is
able to remain viable in saliva as sole nutrient source, and to
which extent this affects its susceptibility to histatin 5, a salivary antifungal peptide. We found that C. albicans was able to
grow in human saliva without addition of glucose, and in the
stationary phase could survive for more than 400 h. In addition,
C. albicans cultured in saliva was more than 10 times less susceptible for salivary histatin 5 than those cultured in Sabouraud
medium.
MATERIALS AND METHODS
Collection of saliva from healthy human volunteers
Saliva was collected on ice, without conscious stimulation, from
healthy volunteers (3 males, 2 females, 23–61 y) according to the
method of Navazesh et al. (1992). In this method, saliva is allowed
to accumulate in the floor of the mouth and the subject spit out
in ice-chilled test tubes every 60 s. Collection of saliva was done
under a VUmc Ethical Committee approved protocol (OB 96 01).
After collection, the sample was homogenized on a Vortex mixer
for 1 min to decrease the viscosity, and subsequently clarified
by centrifugation at 10 000 g for 10 min to remove bacteria and
cellular debris. The supernatant (CHWS) was directly used or
stored at −20o C until use. In some experiments, pooled clarified human whole saliva (CHWS) was sterilized by incubation
in boiling water for 15 min. Sterilized pooled CHWS was frozen
in 1 ml aliquots at −20◦ C and thawed immediately before use.
Secretions from the parotid glands (PAR) were collected using
Lashley cups as described previously (Veerman et al. 1996). Submandibular/sublingual (SM/SL) and palatal (PAL) secretions were
collected using custom-made devices, as described previously
(Veerman et al. 1996).
Growth of C. albicans in human saliva
Candida albicans (ATTC 10231) was cultured aerobically at 30o C
on Sabouraud dextrose agar plates (SDA, Oxoid, Hampshire, UK).
Yeast was inoculated in 25 ml of Sabouraud dextrose broth (SDB,
Oxoid) in a 100 ml Erlenmeyer flask. After 20 h of growth at 30o C,
1 ml of this suspension was subcultured for 1–2 h in 20 ml of
SDB, to obtain a mid-log phase culture. After harvesting, cells
were washed three times in 1 mM PBS and resuspended in saliva
in Falcon culture tubes with caps loosely tightened, under gentle shaking (30o C). Aliquots were taken at indicated time points,
homogenized on a Vortex mixer to disrupt aggregates and OD600
was measured. For determination of CFU, samples were diluted
100-fold, homogenized on a Vortex mixer and plated on SDA.
The plates were incubated at 30◦ C and after 2 or 3 days colonies
were counted.
Biochemical analysis
Salivary glucose was determined with an enzymatic method
(Boehringer Mannheim, Mannheim, Germany) according to the
manufacturer’s instructions.
Sialyl-Lewisa and sulfo-Lewisa antigens on salivary mucins
were determined by ELISA as described previously (Veerman
et al. 1997). In short, saliva samples obtained before and after
growth were 2-fold serially diluted in 0.1 M NaHCO3 , (pH = 8.0),
starting from 1:200. After incubation overnight at 4o C, plates
were washed, and subsequently incubated with MAb E9 (antisialyl-Lewisa ) or MAb F2 (anti-sulfo-Lewisa ). Bound antibodies
were detected using anti-mouse immunoglobulin conjugated to
HRP in combination with OPD/H2 O2 .
SDS-PAGE analysis
Saliva
Saliva samples, before and after growth of C. albicans, were supplemented with four times concentrated sample-solubilizing
buffer (50 mM Tris-HCl, pH 6.8, containing 1% SDS and 10
mM DTT), boiled for 5 min and examined by SDS-PAGE on
precast 4–20% polyacrylamide gradient gels (Novex, Life Technologies, Bleiswijk, the Netherlands). After electrophoresis, gels
were protein-stained with Coomassie Brilliant Blue R250 in
10% (v/v) acetic acid and carbohydrate-stained using the Periodic Acid Staining (PAS) reagent. Gels were destained in
10% acetic acid.
Dialysis of saliva
Heat-treated CHWS was placed into dialysis tubings (Spectra/Por, Spectrum Europe, Breda, the Netherlands), with cut-off
values of 8000–10 000 Da, and dialyzed against a 10-fold excess
of saliva buffer (1 mM phosphate, 20 mM KCl, 1 mM CaCl2 , pH
7.0) with three buffer changes. The dialyzed saliva (‘retentate’)
was inoculated with C. albicans to a starting OD600 of ∼0.2. For 72
h, growth of the yeast was monitored by measuring the OD600 at
regular intervals.
In separate experiments, the dialyzable fraction of saliva
(‘dialysate’) was obtained by dialyzing 10 ml heat-treated CHWS
against 3 × 100 ml of a volatile dialysis buffer consisting of 0.1 M
(NH4 )2 CO3 (pH 7.2). After overnight dialysis, the three portions of
volatile buffer containing the dialyzed material were pooled and
lyophilized. For use in the experiments, this material was reconstituted into the original volume (10 ml) of either saliva buffer or
of the non-dialyzable fraction of saliva (‘retentate’). These solutions were also tested for their capability to support growth of C.
albicans, as described above.
Valentijn-Benz et al.
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Sensitivity of C. albicans to salivary histatin 5
Sensitivity of C. albicans to the membrane-disruptive activity
of histatin 5 was determined by monitoring the fluorescence
enhancement of propidium iodide (PI, Invitrogen, Breda, the
Netherlands) in peptide-treated cells, as described previously
(Veerman et al. 2007). The membrane impermeant dye PI only
enters membrane-compromized cells, after which the fluorescence of this probe is enhanced by 20- to 30-fold due to its binding to nucleic acids. Cells were grown for 24 h in saliva or SBD
medium, harvested by centrifugation and incubated with 5 mM
potassium phosphate, pH 7.0 (PPB), supplemented with 5 mM
NaCl or NaN3 at a density of 2 × 107 cells ml−1 . After 30 min,
PI was added to a final concentration of 10 μM and cells were
transferred to wells containing equal volumes of serially diluted
histatin 5 (0–100 μM) in the corresponding buffer. Development
of fluorescence was for each peptide concentration monitored
in a Fluostar Galaxy microplate fluorimeter (BMFG Labtechnologies, Offenburg, Germany) at 5 min intervals for 1 h, at excitation
and emission wavelengths of 544 and 620 nm, respectively.
Statistical analysis
Data are presented as means ± standard error, and were statistically analyzed with IBM SPSS Statistics version 21.0 (IBM
Corp., Armonk NY, USA; Chicago, USA), using one-way or twoway ANOVA. All levels of significance were set at P < 0.05.
RESULTS
To examine the ability of C. albicans to use human saliva as
growth substrate, CHWS from several donors was inoculated
with C. albicans cells, grown in a mid-log phase on SDA (Fig. 1). As
a control, cells were incubated in PBS and saliva buffer. A starting
cell density of approximately 2 × 106 cells ml−1 was used, corresponding to an OD600 of ∼0.08. At different time points, aliquots
were taken for OD600 measurement and determination of CFUs
by plating. Because C. albicans tended to clump in whole saliva,
it was difficult to correlate OD600 measurements with CFUs. Vigorous vortexing of the suspensions, before measurement and
culturing, improved the correlation and was therefore routinely
done. Four hours after inoculation with C. albicans OD600 and
CFUs started to increase (Fig. 1). This continued for approximately 20 h, after which the levels remained constant.
Although interindividual variations were present, CHWS of
all individuals tested supported growth of C. albicans (two-way
ANOVA, P < 0.0005). Growth of yeast was confirmed by microscopic inspection, showing that at the end of the incubation
only yeast cells, often in large aggregates, were present in saliva.
Three species of Candida (C. albicans, C. glabrata and C. tropicalis)
exhibited similar growth patterns on saliva (not shown). In the
controls (saliva buffer either with or without glucose), OD600 and
CFUs gradually decreased and after 70 h virtually no viable yeast
cells were present as determined by culturing.
Next we examined the effect of the inoculum density on the
growth of C. albicans (Fig. 2). CHWS of one donor was inoculated with different quantities of C. albicans to OD600 values of
0.027, 0.125 and 0.45, respectively. After different periods of time,
aliquots were taken for OD600 measurement and determination
of CFUs. Irrespective of the initial density, each of the suspensions had reached approximately the same OD600 after 40 h suggesting that growth was supported to a certain limit. To examine if C. albicans could persist in saliva for a prolonged period
of time, the growth of the yeast in saliva was monitored for up
Figure 1. Growth pattern of C. albicans in salivas from different donors. Candida albicans cells, grown in a mid-log phase on SDA, were inoculated in saliva collected
from different donors, in PBS and in saliva buffer (SB). Starting cell density was
2 × 106 cells ml−1 (OD600 of ∼0.08). A–E: saliva from five different donors. Saliva
buffer: 2 mM phosphate, 0.1 mM MgCl2 , 2 mM KSCN, 50 mM KCl (pH 6.8). At
the indicated time points, aliquots were taken. At different time points, aliquots
were taken for OD600 measurement (1A) and determination of CFUs by plating
(1B). Values are means ± standard error of three separate incubations. Growth in
saliva differed significantly from the growth in the control (saliva buffer) (twoway ANOVA, all P < 0.0005).
Figure 2. Effect of inoculum density on growth of C. albicans in saliva. Saliva of
one donor (final volume 2 ml) was inoculated with C. albicans to starting OD600
values of 0.027, 0.125 and 0.45. At the indicated time points, aliquots were taken
for determination of OD600 .
to 600 h (Fig. 3). Because of the large volume required, in this
experiment a pooled saliva batch was used, obtained from six
donors. To lower the risk of bacterial infection, it was sterilized
by incubation in boiling water for 15 min. Control experiments
revealed that sterilization of saliva had no effect on the growth
of C. albicans (not shown). After approximately 20 h, the culture
entered the stationary phase, which was maintained for at least
600 h. After 200 h, part of the saliva-grown cells was harvested by
centrifugation and resuspended in a batch of fresh saliva. This
restored growth to an approximately two times higher level, as
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FEMS Yeast Research, 2015, Vol. 15, No. 8
Figure 3. Prolonged growth of C. albicans in saliva. Candida albicans cells, grown in
a mid-log phase on SDA, were inoculated in 9 ml of pooled heat-treated CHWS,
obtained from six donors. Incubations were carried out in triplicate. Starting
density: approximately 1 × 106 cells ml−1 . At different time points, aliquots were
taken for OD600 measurement and CFU determination. After 7 days (arrows),
2 × 3 ml aliquots of the suspension were taken and centrifuged to separate
spent saliva and C. albicans. Upper curve: C. albicans in a new batch of heattreated CHWS. Middle curve: prolonged cultivation in original (spent) CHWS.
Lower curve: spent CHWS inoculated with fresh C. albicans. Values are mean ±
standard error. ∗ P < 0.0005 (vs t = 0 h); # P < 0.0005 (vs t = 168 h) (ANOVA).
evidenced by an increase in both OD600 and CFUs. Inoculation
of the resulting supernatant (spent CHWS) with a fresh batch
of C. albicans did not induce a further increase in C. albicans
(Fig. 3). Spent CHWS supplemented with 100 μM glucose and a
mixture of all amino acids at a physiological concentration of
20 μM (Brand et al. 1997) did not support growth of C. albicans
(not shown). The combined results of Figs 2 and 3 indicate that
CHWS is a source of growth substrates on which C. albicans can
thrive. Furthermore, C. albicans in the stationary growth phase
survived in CHWS for more than 600 h (Fig. 3). In contrast, in
mineral buffers (PBS or saliva buffer) CFU as well as OD600 decreased after 20 h.
Mucins do not support growth of C. albicans
Next we wanted to identify the salivary constituents responsible for the saliva-supported growth of C. albicans. Whole saliva
originates from various glandular sources, each of which contributes a characteristic set of (glyco)proteins and peptides
(Veerman et al. 1996). We examined if secretions directly collected from the parotid (PAR), palatal (PAL) and submandibular (SM) glands supported growth of C. albicans (Fig. 4). Of the
glandular secretions, SM supported growth to the largest extent
Figure 4. Growth pattern of C. albicans on secretions from individual salivary
glands. Candida albicans cells, grown in a mid-log phase on SDA, were inoculated
in different glandular secretions collected from one donor. Starting cell density
was approximately 1 × 106 cells ml−1 (OD600 of approximately 0.08). At different
points, time aliquots were taken for determination of CFUs. Values are mean of
triplicates ± standard error. CHWS: cleared whole saliva; PAL: Palatal saliva; PAR:
parotid saliva; SM: submandibular saliva. # P = 0.45; ∗∗ P = 0.01; ∗∗∗ P = 0.0005.
(P = 0.0005), followed by PAL (P = 0.001). In PAR, no significant
growth occurred (P = 0.45). Chemical analysis of the glucose content in CHWS after growth revealed that after culturing of C. albicans, the glucose concentration had dropped to 2 μM, indicating
that salivary glucose had been used as growth substrate. Still,
there was no clear correlation between the growth supporting
properties of the different salivas and their glucose concentration. For instance, the glucose concentrations in CHWS and PAR
were comparable, still, hardly if any growth occurs on PAR. On
the other hand, the growth on CHWS (10 μM glucose) was clearly
higher than on PAL (110 μM glucose). Furthermore, after supplementing spent CHWS or PAR with 100 μM glucose, still no
growth of C. albicans was observed (not shown). Because CHWS,
PAL and SM, but not PAR, contain MUC5B, we hypothesized that
C. albicans might utilize these heavily (>90%) glycosylated glycoproteins as carbohydrate source, similar to bacteria (Van der
Hoeven and Camps 1991; Wickstrom and Svensater 2008). We
thus examined if mucin carbohydrates were degraded during
growth of C. albicans (Fig. 5), by monitoring the sialic acid content of mucins. Hydrolysis of sialic acid, the terminal residue of
the carbohydrate side chains, is the first step in the degradation
of complex carbohydrates, making the remainder of the chain
accessible for enzymes. To examine if breakdown of the carbohydrate moiety of MUC5B occurred during growth of C. albicans
on (heat-sterilized) CHWS, aliquots were taken before and after 7
days of growth and tested in ELISA with mAb E9, directed against
sialic acid containing epitopes (Veerman et al. 1997). Previously, it
has been found that the E9 epitope is a sensitive marker for bacterial degradation of carbohydrates in mucins (Nieuw Amerongen et al. 1993). As a control mAb F2 was used. This antibody
recognizes the sulfo-Lewisa epitope, which is more robust than
Valentijn-Benz et al.
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Figure 6. SDS PAGE analysis of CHWS before and after growth of C. albicans. CHWS
was incubated with or without C. albicans for 7 days. Left panel: PAS staining; –:
no pretreatment of CHWS; 100◦ C, heat-sterilization; C. albicans: inoculated with
C. albicans; day 0, day 7: before, respectively, after 7 days incubation at 30◦ C.
∗
; untreated, infected saliva.
compounds in the mass range of 1000–2000 Da had disappeared
after growth of Candida (Fig. 7).
Dialysis of CHWS removes the growth-supporting
compounds
Figure 5. Effect of C. albicans on the concentration of MUC5B-associated carbohydrate epitopes. Heat-treated CHWS was incubated with or without C. albicans
for 7 days. Before and after culturing the concentration of sulfated and sialylated
Lewisa was determined by ELISA. (A) Sulfo-Lewisa ; diamonds: before culturing;
filled circles: heat-treated CHWS after 7 days incubation with C. albicans; open
circles: untreated (infected) CHWS incubated in parallel without C. albicans. (B)
Sialo-Lewisa ; diamonds: before culturing; filled circles: after 7 days incubation
with C. albicans; open circles: untreated (infected) CHWS after 7 days without
C. albicans.
sialyl Lewisa (Veerman et al. 1991, 1997). After 7 days of growth of
C. albicans, levels of both E9- and F2-epitopes were not changed
(Fig. 5). In a batch of non-sterilized saliva incubated in parallel, a
bacterial infection developed over this period, as was confirmed
by visual and microscopic inspection. In this sample, the sialylLewisa epitope was barely detectable after 7 days, whereas the
level of sulfo-Lewisa was virtually unchanged (Fig. 5). This illustrates the sensitivity of the E9 epitope for breakdown by bacterial enzymes. Analysis of the saliva protein composition before and after growth of C. albicans using SDS PAGE confirmed
that little if any degradation of mucins or of other salivary proteins occurred during growth of Candida in heat-treated CHWS
(Fig. 6). On the other hand, SDS PAGE analysis of the infected
saliva batch revealed a decrease in the intensity of bands corresponding to mucin and other specific proteins. To explore if
changes occurred in low-molecular-mass salivary constituents,
which are not detected by SDS PAGE, in another experiment
CHWS was analyzed by MALDI-MS. This revealed that a series of
To further characterize the putative growth substrates in saliva,
we fractionated saliva by dialysis using a membrane with a
cut-off of approximately 8000 Da and subsequently tested the
high- and low-molecular-mass fractions individually. After reconstitution to the original volume of either water or retentate,
the dialyzed fraction supported growth of C. albicans (Fig. 8). The
non-dialyzable fraction (retentate) no longer supported growth.
This again underlines that the growth of C. albicans on saliva
depends on low-molecular-mass constituents. Saliva contains
a variety of low-molecular-mass compounds including glucose,
amino acids and ammonia, which are potential growth substrates for C. albicans. Together these results suggest that besides glucose, other low-molecular-mass compounds, presumably peptides, are required for growth of C. albicans in saliva.
Effect of saliva culturing on susceptibility to salivary
histatins
In a first attempt to examine phenotypic consequences of
growing on a low-energy substrate such as saliva, we tested the
sensitivity of C. albicans towards histatin 5, an innate immunity
peptide of saliva, with potent in vitro membrane-disruptive properties against C. albicans. The sensitivity of C. albicans to histatin
5 was determined using the viability probe PI (Fig. 9). When the
membrane is compromised, PI can enter the cell, after which its
fluorescence is enhanced 20- to 30-fold due to its binding to nucleic acids. These experiments revealed that saliva-grown C. albicans was more than 10 times less susceptible to salivary histatin
5 than C. albicans cultured in Sabouraud medium (Fig. 8). The
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FEMS Yeast Research, 2015, Vol. 15, No. 8
Figure 9. Candidacidal effects of histatin 5 towards C. albicans cultured in CHWS.
Candida albicans cells were grown in CHWS or SDB for 24 h. Cells (2 × 107 ml–1 )
were incubated with PPB supplemented with 5 mM NaCl or NaN3 . After 30 min,
PI was added and cells were transferred to wells containing equal volumes of
serially diluted histatin 5 in the corresponding buffer. Development of fluorescence was monitored for each concentration of the peptides at 5 min intervals
at λexc 485 nm and λem 620 nm. The figure shows the fluorescence, expressed in
arbitrary units (AU), after 1 h of incubation. Values are mean of duplicate measurements ± standard error.
DISCUSSION
Figure 7. MALDI-MS analysis of the low-molecular-weight fraction of saliva before (upper panel) and after growth (lower panel) of C. albicans for 2 days.
Figure 8. Effect of dialysis of CHWS on growth of C albicans. Candida albicans cells
were inoculated in CHWS fractionated by dialysis. CHWS: starting material (control); R: dialyzed CHWS (retentate); D: dialyzed material (dialysate), reconstituted
into the original volume of water; R+D: dialysate reconstituted into the original
volume of retentate.
decreased sensitivity to histatin 5 may be due to a diminished
energy content of yeast cells grown on saliva as sole source of
nutrients. Previous studies have revealed that under low-energy
conditions, C. albicans is virtually completely insensitive to histatin 5 (Veerman et al. 2007).
In previous studies, it was found that supplementation with
relatively high amounts of glucose (20 mM) turns saliva into
a suitable growth medium for C. albicans (Samaranayake et al.
1994; Lenanderlumikari and Johansson 1995). The present study,
however, demonstrates that saliva by itself provides sufficient
nutrients for the long-term survival of C. albicans in the oral
cavity. The availability of nutrients in saliva seems to be a
growth-limiting factor, but even when saliva was depleted of nutrients, C. albicans survived for more than 400 h. In vivo saliva
is continuously replenished, ensuring a constant availability of
nutrients for growth and survival of C. albicans.
Our data suggest that C. albicans, in contrast to oral bacteria (Van der Hoeven and Camps 1991; Wickstrom and Svensater
2008; Wickstrom et al. 2009), is by itself not able to utilize the
carbohydrate moiety of salivary mucins as carbon source. (Figs 5
and 6). It can be envisaged, however, that in vivo, where C. albicans is often found in polymicrobial biofilms with bacteria (Rafay,
Homer and Beighton 1996; Harriott and Noverr 2011), it can take
advantage of glycosidases released by bacteria.
It has been shown that C. albicans can use host proteins
as source of nitrogen by secreting aspartic proteinases (Zaugg
et al. 2001), which degrade proteins in peptides that are taken
up into the cell by specific transporters (Morschhaeuser 2011;
Dunkel et al. 2013, 2014). In the present study, however, we
did not find evidence for substantial degradation of salivary
proteins by C. albicans. Rather it seems that growth substrates
for C. albicans reside in the low-molecular-mass, dialyzable
fraction of saliva (Fig. 7). This comprises a number of potential low-molecular carbon and nitrogen sources for C. albicans,
including glucose (Gough et al. 1996; Andersson et al. 1998;
Luke et al. 1999), lactate (Ene et al. 2013), amino acids Brand
et al. (1997), oligopeptides (Payne, Barrettbee and Shallow 1991;
Dunkel et al. 2013) and ammonium ions (Morschhaeuser 2011;
Navarathna et al. 2012; Dunkel et al. 2014). Although salivary
Valentijn-Benz et al.
glucose was decreased after growth of C. albicans, several observations suggest that it is not the only growth limiting factor in saliva: (i) supplementation of spent saliva with physiological amounts of glucose did not restore growth; and (ii) there
was not a clear correlation between the glucose level in different glandular secretions and their growth supporting abilities.
This indicates that besides glucose other presumably nitrogencontaining nutrients determine the growth and survival of C.
albicans in CHWS. A preferred nitrogen source of C. albicans
that is present in sufficient amounts in saliva is ammonium
ions (Oliver, Silver and White 2008). The ammonium concentration of saliva varies between 2 and 5 mM (Huizenga and Gips
1982), which is well in the range required for growth of C. albicans (Vidotto et al. 1991). However, heat treatment and freezedrying, treatments by which volatile compounds such as ammonia are removed from saliva, had no discernible effect on
the growth of C. albicans in either saliva or the reconstituted
dialysate. The MALDI-MS analysis suggests as possible nutrients oligopeptides, which are either secreted directly in saliva
or originate from breakdown of proteins by bacterial and host
proteases.
The current finding that C. albicans could survive in saliva
for more than 400 h is in line with previous observations that
saliva promotes survival and proliferation of Candida species in
tap water (Barbot et al. 2011; Gerhardts et al. 2012). This is remarkable as saliva is generally considered a hostile environment for C. albicans because of the presence of antimicrobial
proteins, including histatin 5. In the present study, we found,
however, that growth in saliva reduced the susceptibility to histatin 5 more than 10-fold. This is in line with a previous study
reporting that C. albicans grown under nitrogen-limiting conditions (with BSA as sole nitrogen source) was 4- to 16-fold less
susceptible to amphotericin B and nystatin than when grown in
a preferred nitrogen source (Oliver, Silver and White 2008). The
underlying mechanism for the increased resistance of Candida
that has been cultured in saliva is not known, but we envisage
that the energy charge of Candida is diminished under these conditions. It has been shown that under low energetic conditions
Candida becomes resistant against a variety of membrane-active
antimicrobial peptides, including histatin 5 (see e.g. Koshlukova
et al. 1999; Veerman et al. 2007). Together with the fact that
the candidacidal activity of whole saliva is partially masked
by calcium ions, this may provide an explanation for the ability of C. albicans to colonize the oral tissues, which may even
be enhanced under conditions when the oral immunity is
compromised.
Conflict of interest. None declared.
REFERENCES
Andersson AB, Birkhed D, Berntorp K, et al. Glucose concentration in parotid saliva after glucose/food intake in individuals
with glucose intolerance and diabetes mellitus. Eur J Oral Sci
1998;106:931–7.
Barbot V, Migeot V, Rodier MH, et al. Saliva promotes survival
and even proliferation of Candida species in tap water. FEMS
Microbiol Lett 2011;324:17–20.
Brand HS, Jörning GG, Chamuleau RA, et al. Effect of a proteinrich meal on urinary and salivary free amino acid concentrations in human subjects. Clin Chim Acta 1997;264:37–47.
De Jong MH, Van der Hoeven JS. The growth of oral bacteria on
saliva. J Dent Res 1987;66:498–505.
7
Dunkel N, Biswas K, Hiller E, et al. Control of morphogenesis,
protease secretion and gene expression in Candida albicans
by the preferred nitrogen source ammonium. Microbiology
2014;160:1599–608.
Dunkel N, Hertlein T, Franz R, et al. Roles of different peptide
transporters in nutrient acquisition in Candida albicans. Eukaryot Cell 2013;12:520–8.
Ene IV, Cheng SC, Netea MG, et al. Growth of Candida albicans cells
on the physiologically relevant carbon source lactate affects
their recognition and phagocytosis by immune cells. Infect
Immun 2013;81:238–48.
Gerhardts A, Hammer TR, Balluff C, et al. A model of the
transmission of micro-organisms in a public setting and
its correlation to pathogen infection risks. J Appl Microbiol
2012;112:614–21.
Gough H, Luke GA, Beeley JA, et al. Human salivary glucose analysis by high-performance ion-exchange chromatography and
pulsed amperometric detection. Arch Oral Biol 1996;41:141–5.
Harriott MH, Noverr MC. Importance of Candida-bacterial
polymicrobial biofilms in disease. Trends Microbiol
2011;19:557–63.
Huizenga JR, Gips CH. Determination of ammonia in saliva using indophenol, an ammonium electrode and an enzymatic
method – a comparative investigation. J Clin Chem Clin Bio
1982;20:571–4.
Koshlukova SE, Lloyd TL, Araujo MWB, et al. Salivary histatin 5
induces non-lytic release of ATP from Candida albicans leading to cell death. J Biol Chem 1999;274:18872–9.
Lenanderlumikari M, Johansson I. Effect of saliva composition
on growth of Candida-albicans and Torulopsis glabrata. Oral Microbiol Immun 1995;10:233–40.
Luke GA, Gough H, Beeley JA, et al. Human salivary sugar clearance after sugar rinses and intake of foodstuffs. Caries Res
1999;33:123–9.
Morschhaeuser J. Nitrogen regulation of morphogenesis and
protease secretion in Candida albicans. Int J Med Microbiol
2011;301:390–4.
Navarathna DHMLP, Lionakis MS, Lizak MJ, et al. Urea amidolyase (DUR1,2) contributes to virulence and kidney pathogenesis of Candida albicans. PLos One 2012;7:e48475.
Navazesh M, Mulligan RA, Kipnis V, et al. Comparison of whole
saliva flow rates and mucin concentration in healthy Caucasian young and aged adults. J Dent Res 1992;71:1275–8.
Nieuw Amerongen AV, Strooker H, Henskens YM, et al. Biochemical changes in (glyco)proteins of saliva of epileptic patients.
Ann NY Acad Sci 1993;694:283–5.
Nieuw Amerongen AV, Veerman ECI. Saliva–the defender of the
oral cavity. Oral Dis 2002;8:12–22.
Oliver BG, Silver PM, White TC. Polyene susceptibility is dependent on nitrogen source in the opportunistic pathogen Candida albicans. J Antimicrob Chemoth 2008;61:1302–8.
Payne JW, Barrettbee KJ, Shallow DA. Peptide-substrates rapidly
modulate expression of dipeptide and oligopeptide permeases in Candida albicans. FEMS Microbiol Lett 1991;79:
15–20.
Rafay AM, Homer KA, Beighton D. Effect of mucin and glucose
on proteolytic and glycosidic activities of Streptococcus oralis.
J Med Microbiol 1996;44:409–17.
Samaranayake LP, Leung WK, Jin LJ. Oral mucosal fungal infections. Periodontol 2000 2009;49:39–59.
Samaranayake YH, Macfarlane TW, Samaranayake LP, et al.
The in-vitro proteolytic and saccharolytic activity of Candida species cultured in human saliva. Oral Microbiol Immun
1994;9:229–35.
8
FEMS Yeast Research, 2015, Vol. 15, No. 8
Shelburne SA, Granville C, Tokuyama M, et al. Growth characteristics of and virulence factor production by group A
Streptococcus during cultivation in human saliva. Infect Immun 2005;73:4723–31.
Van ‘t Hof W, Veerman EC, Nieuw Amerongen AV, et al. Antimicrobial defense systems in saliva. Monogr Oral Sci 2014;24:
40–51.
Van der Hoeven JS, Camp PJM. Synergistic degradation of mucin
by Streptococcus-oralis and Streptococcus sanguis in mixed
chemostat cultures. J Dent Res 1991;70:1041–4.
Veerman ECI, Bolscher JGM, Appelmelk BJ, et al. A monoclonal
antibody directed against high M(r) salivary mucins recognizes the SO3–3Gal beta 1–3GlcNAc moiety of sulfo-Lewis(a):
a histochemical survey of human and rat tissue. Glycobiology
1997;7:37–43.
Veerman ECI, Valentijn-Benz M, Nazmi K, et al. Energy depletion protects candida albicans against antimicrobial peptides by rigidifying its cell membrane. J Biol Chem 2007;282:
18831–41.
Veerman ECI, Valentijn-Benz M, Van den Keijbus PAM, et al.
Immunochemical analysis of high-molecular-weight human
salivary mucins (MG1) using monoclonal antibodies. Arch
Oral Biol 1991;36:923–32.
Veerman ECI, van den Keijbus PAM, Vissink A, et al. Human glandular salivas: Their separate collection and analysis. Eur J
Oral Sci 1996;104:346–52.
Vidotto V, Ochoa LG, Cortes JM, et al. Optimal concentration
of ammonium ion in a minimal synthetic medium for the
growth of Candida albicans. Mycopathologia 1991;113:139–42.
Wickstrom C, Hamilton IR, Svensater G. Differential metabolic
activity by dental plaque bacteria in association with two
preparations of MUC5B mucins in solution and in biofilms.
Microbiol-SGM 2009;155:53–60.
Wickstrom C, Herzberg MC, Beighton D, et al. Proteolytic
degradation of human salivary MUC5B by dental biofilms.
Microbiol-SGM 2009;155:2866–72.
Wickstrom C, Svensater G. Salivary gel-forming mucin MUC5B –
a nutrient for dental plaque bacteria. Oral Microbiol Immun
2008;23:177–82.
Zaugg C, Borg-von Zepelin M, Reichard U, et al. Secreted aspartic proteinase family of Candida tropicalis. Infect Immun
2001;69:405–12.