Basic Research—Biology
Differential Proteinase Patterns among Candida albicans
Strains Isolated from Root Canal and Lingual Dorsum:
Possible Roles in Periapical Disease
Tatiana Teixeira Miranda, MSc, PhD,* Cristina R. Vianna, MSc, PhD,†
Leonardo Rodrigues, MSc, PhD,* Carlos Augusto Rosa, MSc, PhD,* and Ary Corr^ea, Jr, MSc, PhD*
Abstract
Introduction: Proteinases play pivotal roles in Candida
albicans infections. Although the yeast can colonize the
pulpal environment, there is no information about the
enzymatic profile of this organism. This in vitro study
aimed to determine the proteolysis levels and to investigate differences in the expression of aspartyl proteinase
genes (Sap 1, Sap 2, and Sap 4) among various root canal
strains and clinical isolates from the lingual dorsum.
Methods: The extracellular proteinase activity of
104 C. albicans samples isolated from the lingual
dorsum and from necrotic root canals was measured
with respect to bovine serum albumin degradation after
5 days of incubation at 37 C. We used reversetranscription polymerase chain reaction, a highly sensitive
method, to detect messenger RNA transcripts of aspartyl
proteinase genes (Sap 1, Sap 2, and Sap 4). The C. albicans strain SC 5314 was used as a positive control for
both experiments because it is recognized as being highly
proteolytic. All tests were performed in triplicate.
Results: Regardless of the isolation site, all C. albicans
strains produced an opaque precipitation halo around the
colonies, indicating some proteinase activity. However,
the production of proteinase on the plates was significantly greater (P < .05) by the endodontic samples. Sap
2 was the most commonly expressed gene in all samples.
Among the root canal samples, the detection of Sap 1
transcripts was always associated with the expression
of Sap 2 and Sap 4. Sap 4 gene expression was detected
in all root canal samples. The simultaneous expression of
the 3 investigated Sap genes (Sap 1, Sap 2, and Sap 4)
was more common in strains isolated from the lingual
dorsum (50%) than in those isolated from root canals
(29.4%). Conclusions: The increased proteolytic activity
as well as the distinct pattern of Sap expression observed
among the root canal samples may suggest a pathogenic
role for C. albicans in endodontic infections. (J Endod
2015;41:841–845)
Key Words
Aspartyl proteinases, Candida albicans, root canal, virulence factors
C
andida albicans is a commensal yeast that colonizes human mucosal surfaces
such as the vaginal and gastrointestinal tracts. However, this yeast can cause
both superficial and life-threatening systemic infections in a compromised host. This
duality of C. albicans is directly related to the expression of several virulence factors,
among which secretory aspartyl proteinases (Saps), encoded by a family of 10 genes,
have long been recognized as a major cause of yeast virulence (1, 2).
Saps 1 to 10 range between 35 and 50 kd in size and account for all the extracellular proteolytic activity of C. albicans. Sap genes are differentially regulated depending
on the surrounding environment. The expression of Saps 1–3 has been mainly observed
in the yeast phase, whereas Saps 4–6 are expressed in the hyphal phase. Sap 7 expression has been detected in mouse models but not under any in vitro conditions. Sap 8 is
transiently expressed in yeast and epithelial models. Sap 9 and Sap 10, which encode
glycosylphosphatidylinositol-anchoring domains, are expressed under many conditions and are believed to maintain cell wall integrity through the post-translational
processing of cell wall proteins. All types of Sap genes are suggested to contribute to
various virulence processes, such as adhesion, invasion, degradation of host proteins,
and macrophage escape. Therefore, it has been suggested that the virulence of C. albicans strains correlates with the level of proteolysis activity as well as with the number of
Sap genes (3–6).
Previous studies have reported the presence of C. albicans in 7%–26% of infected
root canals (7–11). Its presence has been commonly associated with persistent cases of
apical periodontitis (12, 13). Although many studies have sought to investigate the
frequency of yeasts in root canal infections, the role of Candida species in the
endodontic environment at the onset of periapical disease remains unclear.
The regulation of Sap secretion and Sap gene expression has been extensively evaluated in different C. albicans strains (14–16). Nevertheless, there is no information
about whether C. albicans isolated from endodontic infections expresses distinct
patterns of Sap genes and whether it secretes different amounts of Saps. Thus, we
hypothesized root canal strains would also have the ability to secrete proteolytic
enzymes as reported for isolates from other yeast infections, which would contribute
to colonization and survival in endodontic harsh ecologic conditions. The
production of aspartyl proteases would increase their pathogenic potential.
This in vitro study aimed to determine the proteolysis levels and to investigate differences in the expression of aspartyl proteinase genes among various root canal strains
From the *Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte; and †Department of General Education,
Federal Center of Technological Education, Varginha, Minas Gerais, Brazil.
Address requests for reprints to Dr Tatiana Teixeira Miranda, Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Antonio Carlos 6627, Pampulha, CEP31270-901, Belo Horizonte, MG, Brazil. E-mail address: [email protected]
0099-2399/$ - see front matter
Copyright ª 2015 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2015.01.012
JOE — Volume 41, Number 6, June 2015
Candida albicans Strains
841
Basic Research—Biology
and clinical isolates from the lingual dorsum. Sap 1, Sap 2, and Sap 4
genes were selected as a members of 2 Sap subfamilies, Sap 1–3 and
Sap 4–6, respectively, which differ in their biological properties and potential roles in different types of C. albicans infections.
Materials and Methods
Clinical Material
Patients seeking treatment at the Endodontic Department of the
Federal University of Minas Gerais, Belo Horizonte, Brazil, were
selected. A detailed medical and dental history was obtained from
each patient. Patients who had received antibiotic and antifungal treatment during the previous 6 months or who had a systemic disease were
excluded. The Ethical Committee in Research of the Federal University of
Minas Gerais approved the protocol describing the specimen collection
for this investigation, and all patients were informed of the method, objectives, and risks of the procedures. Patients who met the inclusion
criteria and agreed to take part in the experiment were asked to sign
the written informed consent form.
Sampling Procedure and Yeast Isolation
A total of 168 patients without pain (94 women and 74 men)
from 20–65 years of age were selected. One hundred eighty-four teeth
with intact pulp chambers and no cracks and therefore with no direct
oral pulpal communication but presenting with caries, pulpal necrosis, and radiographic evidence of apical periodontitis were selected
from the group and microbiological samples collected. Pulpal necrosis was confirmed through heat, cold, and electric pulp sensibility
tests. Based on the periodontal evaluation, the study population was
considered healthy. Normally, a single tooth was sampled from
each patient; however, in 16 patients, 2 teeth were sampled. For
the statistic analysis of yeast frequency, 16 teeth were randomly
excluded; therefore, each patient contributed only 1 tooth. All the
teeth were subject to the initial standard endodontic procedure in
which a rubber dam was used for the complete isolation of the target
tooth from the oral cavity and the root canal was accessed using a
sterilized dental drill without irrigation. Decontamination of the crown
and the surgical field was accomplished by the sequential application
of 30% hydrogen peroxide, 5% iodine dye, and 5% sodium thiosulfate
solutions; all solutions were left in place for 1 minute. The aseptic status of the crown was confirmed by rubbing sterilized swabs along the
crown and then using the swabs to streak modified Sabouraud agar
(2% glucose, 1% peptone, 0.5% yeast extract, 2% agar, and
100 mg/L chloramphenicol). After intracoronal access, the canals
were irrigated with sterile saline to remove debris. Samples were
collected from root canals flooded with sterile absorbent paper cones
that remained inside the root canal for 1 minute (8). The samples
were collected aseptically in triplicate. In each case, a single root canal was sampled in order to restrict the microbial evaluation to a single ecologic environment. In multirooted teeth with periapical lesions,
only the widest root canal was selected. The paper points were transferred immediately aseptically to tubes containing 4 mL modified Sabouraud broth with 100 mg/L chloramphenicol. After sampling, the
teeth were restored temporarily using a standard cement and a
base of zinc oxide. The tubes were immediately transported to the laboratory and left at room temperature for 48 hours. After incubation,
aliquots of 0.1 mL were transferred to modified Sabouraud agar with
100 mg/L chloramphenicol, and the plates stood for 5 days at room
temperature.
Sterile swabs were used to collect samples from the dorsum of the
tongue from each patient during the treatment. The swab was inserted
inside the mouth, and the collection tip was rubbed along the tongue.
842
Miranda et al.
The material was streaked directly on modified Sabouraud agar with
100 mg/L chloramphenicol and incubated for 5 days at room temperature (9). Each patient contributed only 1 tooth and 1 tongue sample.
Yeast Strains
Yeasts were characterized by the standard methods of Yarrow. The
taxonomic keys of Kurtzmann and Fell (17) were used for species identification. The polymerase chain reaction (PCR) fingerprint technique
was used to confirm identification of all the yeasts.
In total, 115 yeast colonies were obtained. Those yeasts identified
as C. albicans isolated from the lingual dorsum (n = 70) and necrotic
root canals (n = 34) were selected for this study. The C. albicans strain
SC 5314, previously recognized as a virulent strain (18, 19), was used as
a positive control.
Assessment of C. albicans Extracellular Proteolytic
Activity on Albumin Agar Plates
The extracellular proteinase activity of C. albicans isolates was
analyzed in terms of bovine serum albumin (BSA) degradation according to a previously described protocol (20). Briefly, 5 mL of
each C. albicans strain at 107 colony formed unit (CFU)/mL was
spot inoculated on plates containing 0.15% yeast nitrogen base
(Difco, Detroit, MI), 0.2% BSA (Sigma-Aldrich), 2% glucose
(Sigma-Aldrich), and 2% agar (Sigma-Aldrich) at a pH of 5.0. Plates
were incubated at 37 C for 5 days. Each isolate was tested in triplicate. After incubation, the plates were fixed with 20% trichloroacetic acid (Sigma-Aldrich) and stained with 0.5% amido black
(Sigma-Aldrich). Decolorization was performed with acetic acid.
The zone of clearance around the colony indicated enzyme production and was recorded as the proteinase zone (PZ). Using a caliper
ruler, the proteinase activity was calculated as the ratio of the diameter of the colony to the diameter of the proteolytic unstained zone
and was scored in 3 categories:
Index zero: PZ = 1 mm (strain without enzymatic activity)
Index 1: 1 mm > Pz > 0.64 mm (strain with some enzymatic activity)
Index 2: Pz # 0.64 mm (strain with high enzymatic activity)
Culture Growth Conditions to Identify Aspartyl
Proteinase RNA Transcripts of C. albicans Strains
C. albicans strains were initially grown in YPD broth containing 1% yeast extract (Sigma-Aldrich), 1% peptone (Difco), and 2%
glucose (Sigma-Aldrich) at 37 C overnight. After this incubation
period, 50 mL YPD-grown culture was centrifuged (2000g for
10 minutes). The yeast cells were washed twice in phosphate buffered saline (pH = 7.5) and used to inoculate, at a final concentration of 105 cells/mL, yeast nitrogen base-BSA medium, which
contained 0.17% yeast nitrogen base without amino acids or ammonium sulfate (Difco), 0.2% glucose (Sigma-Aldrich), and 0.1% BSA
(Sigma-Aldrich) at a pH of 5.5. Cultures (200 mL) were incubated
at 37 C in a rotary shaking incubator (200 rpm). Sap 1, Sap 2, and
Sap 4 RNA transcripts were investigated after 48 hours of C. albicans growth because a good expression profile of Sap 1, Sap 2,
and Sap 4 was detected in C. albicans SC5314. Culture aliquots
were taken, and the optical density values at 540 nm (Shimadzu
Corporation, Kyoto, Japan) did not differ among strains at 48 hours
after inoculation. Culture aliquots (35 mL) were transferred to RNAfree centrifuge tubes containing RNAse-free ice-cold water and
centrifuged (2000g for 5 minutes at 4 C). The supernatant was decanted, and the cells were resuspended in 1 mL RNAse-free ice-cold
JOE — Volume 41, Number 6, June 2015
Basic Research—Biology
TABLE 1. Primer Sequences of Used Genes
Gene
Sense and antisense
Base pairs of amplicon size
Sap-1
50 TCA ATC AAT TTA CTC TTC CAT TTC TAA CA30
50 CCA GTA GCA TTA ACA GGA GTT TTA ATG A30
50 AAC AAC AAC CCA CTA GAC ATC ACC30
50 TGA CCA TTA GTA ACT GGG AAT GCT TTA GGA30
50 CAT TCA TTC CTT TAA TAC CGA CTA TC30
50 GGT AAC AAA CCC TGT AGA TCT TTT AAC30
50 GAT TTT GTC TGA ACG TGG TAA CAG30
50 GGA GTT GAA AGT GGTT TGG TCA ATA C30
161
Sap-2
Sap-4
Act-1
178
156
304
bp, base pairs of amplicon size.
water and transferred to microcentrifuge tubes where RNA extraction was conducted.
RNA Extraction and Complementary DNA Synthesis
for Reverse-transcriptase PCR
RNA was extracted using Trizol (Life Technologies, Grand Island,
NY), and reverse transcription was performed to obtain complementary
DNA (cDNA). Cells were resuspended in 1 mL Trizol containing 0.3 g
glass beads (250 mm) and vortexed for 10 minutes. After being spun
for 3 minutes at 3000g, the aqueous phase was removed and extracted
twice in phenol/chloroform. The RNA was precipitated with 20 mL
96% ethanol and stored at 20 C. Precipitated RNA was pelleted
and resuspended in 20 mL RNAse-free water. Before performing reverse
transcription (RT), the RNA was purified twice using DNAse RQ1 RNasefree (Promega, Fitchburg, WI) to remove contaminating DNA. For the
cDNA synthesis, 3 mg purified RNA was added to 10 pmol antisense
primer for a specific Sap gene (Table 1), 1 mL deoxyribonucleotide
triphosphate (DNTP) (10 mmol/L), and 6 mL RNAse-free water and
was heated to 74 C in a thermocycler (Applied Biosystems 2720 Thermal Cycler; Life Technologies, Grand Island, NY) for 5 minutes. After
heating, 4 mL Moloney murine leukemia virus buffer (Promega),
4 mL Dithiothreitol (DTT) (0.1 mol/L), and 1 mL RNase out (Invitrogen,
Carlsbad, CA) were added to the reaction mixture. The mixture was
incubated at 37 C for 2 minutes. Then, 1 mL Moloney murine leukemia
virus reverse-transcriptase enzyme (Promega) was added to bring the
reaction mixture to a total volume of 20 mL. Once the enzyme was
added, the mixture was incubated at 42 C for 60 minutes. The enzyme
was inactivated at 70 C for 20 minutes.
Qualitative RT-PCR
The RT-PCR reaction was performed by adding the cDNA template
to the PCR mix, which contained a final concentration of: 1.5 mmol/L
MgCl2, 1 reaction buffer (200 mmol/L Tris-HCl [pH = 8.4],
500 mmol/L KCl [Taq Polymerase, Invitrogen]), 0.2 mmol/L each
DNTP, 10 pmol sense and antisense primers for a specific Sap gene,
and 1 U Taq DNA polymerase (Invitrogen). Taq DNA polymerase was
added to the reaction mixture after preincubating the mixture at 94 C
for 4 minutes. The primers and the annealing temperatures used for
touchdown cycling were previously described (21). The purified RNA
from each sample was confirmed to be DNA free by the absence of amplified product after performing PCR with complementary primers specific
to the C. albicans actin gene (Table 1). The amplified DNA products were
separated by electrophoresis on 1.8% agarose gels; Tris-borate-EDTA
was used as the running buffer, and a 1 Kb plus DNA ladder (Invitrogen)
was used as a molecular weight marker. After separation of the DNA, the
agarose gel was stained with ethidium bromide and visualized under an
ultraviolet transilluminator (ImageMaster VDS Apparatus; Pharmacia
Biotech, Uppsala, Sweden) using an image capture program.
Statistical Analysis
Quantitative data were analyzed using the statistical features of
GraphPad Prism Version 5.0 (GraphPad Inc, San Diego, CA).
One-way analysis of variance and the Tukey test were used to determine
significance, and P < .05 was considered to be statistically significant.
Results
Proteolytic Activity on the Plates
Regardless of the isolation site, all C. albicans strains produced an
opaque precipitation halo around the colony, indicating some proteinase
activity. Among the yeasts isolated from the lingual dorsum, the Pz values
ranged from 0.33–0.74. Thirty-nine of these samples (55.7%) showed Pz
values lower than or equal to 0.64, which corresponds to an enzymatic
activity index of 2, whereas for 31 samples (44.3%), the average Pz value
was 0.74, corresponding to an enzymatic activity index of 1 (Table 2). Pz
values below 0.64, indicating strong enzymatic activity (index 2), were
obtained for all root canal samples. The proteinase production was significantly greater (P < .05) in endodontic samples scored as index 2 than in
tongue strains with the same score (Fig. 1).
Expression of Sap 1, Sap 2, and Sap 4
by C. albicans Strains
All the studied Sap genes were expressed by the lingual and root
canal strains. The results revealed that Sap 1 was expressed more
p < 0.05
0.8
Lingual dorsum
Root canal
0.6
Pz
TABLE 2. Extracellular Proteinase Activity of C. albicans Strains
No. of isolates (%)
Score of proteinase activity
0.2
Isolation site
Negative
1+
2+
Lingual dorsum
Root canal
Strain SC5314
0 (0)
0 (0)
0 (0)
31 (44.3)
0 (0)
0 (0)
39 (55.7)
34 (100)
1 (100)
JOE — Volume 41, Number 6, June 2015
0.4
0.0
Index 2
Index 1
Figure 1. Pz averages produced by C. albicans strains in albumin agar plates.
Candida albicans Strains
843
Basic Research—Biology
TABLE 3. Sap Genes Expression in Candida albicans Strains
Gene expression
Isolation site
Sap 1 (%) Sap 2 (%) Sap 4 (%)
Lingual dorsum
Root canal
52 (74.3)*
10 (29.4)*
63 (90)
29 (85.3)
†
40 (57.1)
34 (100)†
Act 1
70 (100)
34 (100)
*Significantly different expression of Sap 1 gene (P < .05).
†
Significantly different expression of Sap 4 gene (P < .05).
frequently by commensal strains (74.3%) (P < .05). Sap 2 was expressed by a larger number of strains that were isolated either from
the lingual dorsum (90%) or the root canal (85.3%). A higher frequency of Sap 4 was observed in root canal isolates (100%) than in
tongue samples (57.1%) (P < .05) (Table 3).
Three different association patterns of Sap genes were found
among the strains. Ten isolates (29.4%) from an endodontic environment carried all the studied genes, whereas 40 lingual samples (57.1%)
showed the same association (P < .05). Among the root canal isolates,
Sap 1 expression was always related to the other studied genes. A Sap 1/
Sap 4 association was observed only in commensal strains (15.7%). A
Sap 2/Sap 4 association was observed in 19 root canal samples (55.9%)
and 15 lingual samples (21.4%) (P < .05) (Fig. 2).
Sap 4 was the only gene expressed by 5 root canal isolates
(14.7%). The exclusive expression of Sap 1 was verified in 1 lingual
sample (1.4%). Eight lingual strains (11.4%) expressed only Sap 2
(Fig. 2).
Discussion
The production of aspartyl proteinases is considered to be 1 of the
most important virulence factors in yeasts of the genus Candida. The
present study is the first to show that C. albicans strains isolated
from endodontic infections present different expression patterns for
the genes encoding Sap 1, Sap 2, and Sap 4 and different levels of enzymatic secretion in vitro compared with commensal isolates.
Because only teeth with intact pulp chambers were sampled, the
isolation of yeasts in root canals is intriguing and may be suggestive
of the capability of such microorganisms to invade the pulpal environment actively through dentinal tubules as other studies have already
indicated (12, 13) or be carried to the region passively via lateral
root canals, resorption lacunae, or periodontal sulci.
Strains isolated from the lingual dorsum and from the root canal
exhibited proteolytic activity on plates. In principle, this finding indicates that the ability to secrete aspartyl proteinases is an intrinsic char80
Lingual dorsum
Root canal
p<0.05
Isolates (N)
60
40
20
1
2
4
Sa
p-
Sa
p-
-4
Sa
p-
Sa
p
-4
1/
Sa
p
Sa
p-
2/
-2
Sa
p
1/
Sa
p-
Sa
p-
/S
a
p4
0
Figure 2. The number of C. albicans isolates in which Sap genes were detected individually or in combination.
844
Miranda et al.
acteristic of C. albicans yeast. However, when considering the primary
site of isolation of the yeast strains, the plate testing revealed significant
differences between the lingual and root samples in their ability to
degrade a protein substrate. In particular, samples isolated from root
canals generated larger albumin degradation halos. Our results are
in accord with those of Mardegan et al (22), who emphasized that
different ecosystems have specific characteristics that limit or induce
the expression of enzymes and generate different levels of secretion.
Similarly, Schaller et al (23) detected reduced levels of enzyme secretion among commensal isolates of C. albicans.
Kumamoto (24) suggested that the differential secretion of
enzymes by yeast of the genus Candida is directly related to the
need to overcome conditions imposed by the peculiarities of
each ecologic niche. In this respect, the presence of collagen as
the only available nutrient supply in root canals may favor
increased secretion of aspartyl proteinases by strains isolated
from endodontic environments.
Various studies (2, 6, 14, 16) have emphasized that the ability to
degrade protein substrates is an important contribution to the
establishment of C. albicans infections in various organic
environments. Specifically, the increased proteolytic activity among
strains isolated from necrotic root canals suggests that these microorganisms can participate in establishing and maintaining endodontic
infections although this remains to be shown experimentally.
Regarding the expression of the genes encoding Sap 1, Sap 2, and
Sap 4, the expression of Sap 1 was detected by RT-PCR in 74.3% of
commensal strains and in 29.4% of strains isolated from teeth with primary apical periodontitis. A study conducted by Kvaal et al (25) indicated
that Sap 1 plays an important role in the adherence of C. albicans to
different substrates. Although the mechanisms by which these proteins
contribute to the adhesive ability of yeast are not yet fully understood,
it is assumed that they act as ligands for cell surface molecules or modify
the structural conformation of proteins or surface ligands of the fungus
and of the host, resulting in changes in hydrophobicity (3).
The virulence potential attributed to aspartyl proteinase enzymes
would be markedly expanded if Sap 1 acted as an adhesin itself. In
our study, 57.1% of yeast samples isolated from the lingual dorsum
that expressed Sap 1 showed concomitant expression of the genes encoding Sap 2 and Sap 4. However, all the root canal isolates that expressed Sap 1 also expressed the other studied genes. Based on this
finding, we conjecture that Sap 1 has some function in the initial stages
of dentin colonization, possibly mediating the adhesion of C. albicans
yeast to teeth. The concomitant expression of Sap 1, Sap 2, and Sap 4 in
most, but not all, strains isolated from the lingual dorsum suggests that
only a group of microorganisms possessing these characteristics may be
able to penetrate the endodontic environment. In contrast to the results
of this study, the expression of the Sap 1 gene has often been correlated
with symptomatic infections (21, 26–29) and has not been detected
among commensal isolates of C. albicans.
The expression of Sap 2 was observed in 90% of the yeasts isolated
from the lingual dorsum and 85.3% of the strains isolated from root canals. Regardless of the site of isolation, Sap 2 has been shown to be preferentially expressed by yeast-forming fungi in media containing bovine
serum albumin as the protein substrate (14). Among the enzymatic
properties of Sap 2 described in the literature, the proteolysis of
collagen (30), the decreased efficiency of phagocytosis by polymorphonuclear neutrophils (3), and the activation of the proinflammatory cytokine interleukin-1 beta (31, 32) may contribute to bone resorption in
the periapex, thus playing an important role in the onset and
progression of apical periodontitis. The unique expression of Sap 2
in 11.4% of the commensal isolates indicates the existence of a
microbial group that has potential proteolytic capabilities but lacks
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Basic Research—Biology
the ability to penetrate the dentinal environment and participate in the
infectious process.
In our study, all the tested strains that were isolated from the root
canal expressed the gene that encodes Sap 4, which was the only gene
expressed by 14.7% of these microorganisms. According to Naglik et al
(3), the secretion of Sap 4 is concomitant with the formation of hyphae
and germ tubes by yeast of the species C. albicans. Moreover, the
expression of Sap 4 by all the strains isolated from teeth with primary
apical periodontitis suggests that this gene is important in colonization
of the root canal after exposure of the dentinal tubules and in the perpetuation of endodontic infections. This undifferentiated genetic pattern indicates that pleomorphic growth patterns are an important mechanism
in the colonization of the pulp environment via the dentinal tubules. The
concomitant expression of Sap 2 and Sap 4 in 55.9% of the strains isolated from root canals suggests a coordinated regulation of genes in
endodontic niches. In addition, Brown et al (33) argued that some
Sap genes may be indirectly regulated by the proteolytic activity of other
members of the same family of enzymes. Among the isolates obtained
from the lingual dorsum, 57.3% were found to express Sap 4, indicating
that commensal isolates also have the potential to invade the dentin.
Furthermore, the combined expression of the Sap 1 and Sap 4 genes
was only evident in yeasts isolated from the tongue (15.7%), suggesting
that they play a role in adhering to the oral epithelium at the expense of
colonizing tooth surfaces.
With regard to the production of extracellular proteinases by oral
isolates of C. albicans, the results of this study do not necessarily indicate that these enzymes are acting in vivo; rather, the results suggest only
that the potential exists for them to act. The proteolytic ability of yeasts of
the genus Candida in endodontic environment should be examined
further, particularly with in vivo experiments. In this context, the selective pressures exerted by the peculiarities of the endodontic environment
and the presence of other microbial types can modulate the secretion of
these enzymes. The detection of C. albicans in cases of endodontic failure might be partially explained by its proteolytic ability.
In conclusion, this in vitro study shows unequivocally that C. albicans strains isolated from the root canal exhibit high proteolytic activity and a distinct pattern of Sap expression. With these findings in
mind, it is reasonable to assume that C. albicans has adapted to this
niche by expressing a different set of SAP genes. Also, the ability to
degrade a protein substrate may suggest a pathogenic role for C. albicans in endodontic infections.
Acknowledgments
The authors thank Dr Patrick VanDijck, Catholic University of
Leuven, Leuven, Belgium, for providing Candida albicans strain
SC5314.
Supported by CNPq and FAPEMIG.
The authors deny any conflicts of interest related to this study.
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