Lasers Med Sci DOI 10.1007/s10103-015-1747-0
ORIGINAL ARTICLE
Evaluation of gene expression SAP5, LIP9, and PLB2 of Candida albicans biofilms after photodynamic inactivation Fernanda Freire 1 & Patrícia Pimentel de Barros 1 & Damara da Silva Ávila 1 & Graziella Nuernberg Back Brito 1 & Juliana Campos Junqueira 1 & Antonio Olavo Cardoso Jorge 1
Received: 2 July 2014 / Accepted: 19 March 2015 # Springer-Verlag London 2015
Abstract With the increasing number of strains of Candida ssp. resistant to antifungal agents, the accomplishment of researches that evaluate the effects of new therapeutic methods, like photodynamic inactivation (PDI), becomes important and necessary. Thus, the objective of this study was to verify the effects of the PDI on Candida albicans biofilms, evaluating their effects on the expression of the gene hydrolytic enzymes aspartyl proteinase (SAP5), lipase (LIP9), and phospholipase (PLB2). Clinical strains of C. albicans (n=9) isolated from patient bearers of the virus HIV and a pattern strain ATCC 18804 were used. The quantification of gene expression was related to the production of hydrolytic enzymes using the quantitative polymerase chain reaction (qPCR) assay. For PDI, we used laser-aluminum-gallium arsenide low power (red visible, 660 nm) as a light source and the methylene blue at 300 μM as a photosensitizer. We assessed two experimental groups for each strain: (a) PDI: sensitization with methylene blue and laser irradiation and (b) control: without sensitization with methylene blue and light absence. The PDI decreased gene expression in 60 % of samples for gene SAP5 and 50 % of the samples decreased expression of LIP9 and PLB2. When we compared the expression profile for of each gene between the treated and control group, a decrease in all gene expression was observed, however no statistically significant difference (Tukey’s test/p=0.12). It could be concluded that PDI (photosensitization with methylene blue followed by
* Fernanda Freire [email protected] 1
Department of Biosciences and Oral Diagnosis, Institute of Science and Technology, Universidade Estadual Paulista (UNESP), Francisco José Longo 777, São Dimas, São José dos Campos, São Paulo 12245-000, Brazil
low-level laser irradiation) showed a slight reduction on the expression of hydrolytic enzymes of C. albicans, without statistical significance.
Keywords Biofilm . Candida albicans . Genes of hydrolytic enzymes . Photodynamic inactivation . qPCR
Introduction In view of the increase in opportunistic infections [1–5] caused by species of the genus Candida in immunodepressed patients and the consequent emergence of strains that are resistant to conventional antifungal agents [6], new treatment options for oral candidosis are required to improve the therapeutic arsenal. In this respect, photodynamic inactivation (PDI) has been proposed as a new option to reduce yeasts of the genus Candida in the oral cavity [7, 8]. PDI is based on the concept that a nontoxic dye, known as a photosensitizer, may be located preferentially in certain tissues or cells and subsequently activated by the harmless visible light to produce reactive oxygen species that can kill cells that bind to the photosensitizer. Numerous published studies have demonstrated that PDI is highly effective in the inactivation of fungi in vitro [9–12]. Additionally, it is believed that the development of resistance by microorganisms PDI is an unlikely event since this is considered as a typical multitarget process, which is a difference between PDI and most antifungal agents [13]. This is the first study to report the action of PDI in the expression of three genes in Candida albicans biofilm related to virulence [14–16]. The goal of the study was to investigate whether PDI using methylene blue and low-power laser acts on the expression of SAP5, LIP9, and PLB2 genes in C. albicans biofilms.
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Materials and methods
Table 1
Code of the isolates used in the study
Origin
Code
ATCC 18804 Patient 1—oral candidiasis lesion Patient 2—oral candidiasis lesion Patient 3—oral candidiasis lesion Patient 4—oral candidiasis lesion Patient 5—oral candidiasis lesion Patient 6—oral candidiasis lesion Patient 7—oral candidiasis lesion Patient 8—saliva Patient 9—saliva
ATCC 1 14 21 31 40 60 70 76S 228S
Ethics committee The project for the collection of clinical isolates was approved by the Ethics Committees of Emílio Ribas Institute of Infectious Diseases (São Paulo, Brazil) and Institute of Science and Technology (UNESP/São José dos Campos Dental School). Isolates The isolates are stored in the Laboratory of Microbiology and Immunology, Institute of Science and Technology (UNESP/ São José dos Campos Dental School) (04569812.5.0000.0077). Nine clinical isolates of C. albicans and one C. albicans reference strain (ATCC 18804) were used. Table 1 shows the origin of the isolates and their codes. Biofilm formation For the biofilm formation, the methods described by Seneviratne et al. [17] and Costa et al. [18] were used with some changes and using 24-well flat-bottom plates (Costar Corning). The strains frozen at −75 °C were activated on Sabouraud dextrose agar (Himedia Laboratories Pvt. Ltd., India) containing chloramphenicol (Alamar Tecno Científica Ltda.) and maintained in an oven for 24 h at 37 °C. Next, a C. albicans inoculum was prepared in yeast nitrogen base (YNB; Himedia) broth supplemented with 100 mM glucose and diluted ten times in sterile distilled water and incubated in an oven for 16 h at 37 °C. The inoculum was then washed twice with sterile 0.9 % NaCl, and standardized suspensions containing 107 cells/mL YNB broth (10×) were prepared in a spectrophotometer (B582, Micronal, São Paulo, Brazil). One milliliter of the yeast suspension was added to the wells, and the plates were incubated in an oven for 1.5 h at 37 °C under shaking at 75 rpm (Quimis, Diadema, Brazil) for initial adherence. After this period, the microorganism supernatant was gently aspirated to remove nonadherent cells (planktonic forms), and each well was rinsed with 1 mL sterile 0.9 % NaCl saline. These steps were repeated twice for removal of nonadherent cells. Then, 1 mL yeast nitrogen base (YNB; Himedia) broth supplemented with 100 mM glucose was pipetted, and the plates were incubated at 37 °C under shaking at 75 rpm (Quimis, Diadema, Brazil) for 48 h. The broth was changed after 24 h. Two groups per strain were evaluated, and one plate was prepared for each group: (1) PDI: sensitization with methylene blue and laser irradiation and (2) control: no sensitization with methylene blue and no laser irradiation. The PDI group received 1-mL methylene blue solution, and the control group received 1 mL sterile 0.9 % NaCl. The plates were shaken for 5 min (pre-irradiation time) in an orbital shaker (Solab,
ATCC American Type Culture Collection
Piracicaba, Brazil). We performed two assays (one for the untreated group and the group treated) with ten isolated under the same conditions of biofilm formation. After the procedures, the samples were plated on Sabouraud agar for counting of colony-forming units (CFU/mL). These values were subjected to t test for statistical analysis. Photosensitizer and laser Methylene blue at a concentration of 300 μM was used for yeast sensitization. The dye solution was prepared freshly each time before use by dissolving the powder (SigmaAldrich, Steinheim, Germany) in sterile distilled water and filtering the solution through a 0.22-μm membrane filter (Millipore, São Paulo, Brazil). After filtration, the dye solution was stored in the dark [19]. An aluminum-gallium arsenide laser (AlGaAs; Laser Fácil, Clean Line, Taubaté, Brazil) was used as the light source. This laser emits continuous light at 660 nm (visible red), the wavelength corresponding to the maximum absorption of methylene blue. The following parameters were used: power of 35 mW, energy of 10 J, time of 285 s, irradiance of 13.78 mW/cm2, and light dose of 3.93 J/cm2. The biofilms were irradiated under aseptic conditions in a laminar flow hood protected from light. The tip was supported on the lid of a 24-well plate toward the bottom of the well where the biofilm was made (approximately 1 cm of distance). A matte black screen with an orifice whose diameter corresponded to the entry of the well was used to avoid the scattering of light. After irradiation of the biofilms, the sterile 0.9 % NaCl or dye solution was discarded and 1 mL TRIzol was added to the first well and then transferred to the following wells until complete removal of the biofilm. The biofilm solution with TRIzol was transferred to 2-mL microtubes, and the tubes were stored in a freezer at −75 °C for subsequent RNA extraction.
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Extraction and measurement of RNA RNA was extracted with the TRIzol kit (Ambion, Inc., AM1926) according to the manufacturer’s recommendations. The extracted RNA was quantified (ng/μL) in a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Absorbance ratios (260/280) of ~2.0 indicate the degree of purity necessary for good performance of the subsequent reactions. DNase treatment of RNA and reverse transcription polymerase chain reaction For removal of contaminating DNA, the extracted total RNA was treated with DNase I (Turbo DNase Treatment and Removal Reagents, AM 1907, Invitrogen) according to the manufacturer’s instructions. After DNase treatment, the RNA was transcribed to complementary DNA (cDNA) using the SuperScript® III FirstStrand Synthesis SuperMix Kit for reverse transcription PCR (Invitrogen) according to the manufacturer ’s instructions.
Table 2
Primers used for real-time PCR
Gene
Position
Reference
ACT1
FW-TTTCATCTTCTGTATCAGA GGAACTTATTT RV-ATGGGATGAATCATCAAAC CAGAG FW-TGCTTACTTATTGTTAGTTC AAGGTGGTA RV-CAACACCAACGGATTCCAA TAAA FW-CATCACAACCAGGTTCTAC AACCAAT RV-CTACTATTAGCAGCACCACCC FW-CCAGCATCTTCCCGCACTT RV-GCGTAAGAACCGTCACCATA TTTAA FW-TGAACCTTTGGGCGACAACT RV-GCCGCGCTCGTTGTTAA FW-CGCAAGTTTGAAGTCAGGAAAA RV-CCCACATTACAACTTTGGCATCT
Nailis et al. [21]
RPP2B
EFG1
SAP5
PLB2 LIP9
Nailis et al. [21]
Hnisz et al. [22]
Nailis et al. [21]
Nailis et al. [21] Nailis et al. [21]
PCR polymerase chain reaction, ACT β-actin, RPP2B cytosolic ribosomal acidic protein P2B, EFG1 transcription factor, SAP5 secreted aspartyl protease, PLB2 phospholipase B, LIP9 lipase, FW forward, RV reverse
Evaluation of the expression of the SAP5, LIP9, and PLB2 genes First, the efficiency of amplification of the ATCC strain and of isolates 1 and 14 (randomly selected) was tested for analysis of the following data: to evaluate the performance of the qPCR assay, to evaluate the best cDNA concentration to be used for qPCR, and to determine whether the target genes were expressed by the strains tested. Three serial base ten dilutions of the samples (1:1, 1:10, and 1:100) were tested in triplicate, and the three dilutions were used for construction of the efficiency curve. The expression of the SAP5, LIP9, and PLB2 genes in relation to a housekeeping gene of choice (ACT1, EFG1, or RPP2B) was evaluated in the clinical isolates and strain ATCC 18804. This evaluation was performed for strains submitted or not to PDI. The primers used for the SAP5, LIP9, PLB2, ACT1, and RPP2B genes have been described by Nailis et al. [18] and the primers for EFG1 by Hnisz et al. [20] and are listed in Table 2. Quantitative polymerase chain reaction The qPCR products were amplified for relative quantification of the expression of the SAP5, LIP9, and PLB2 genes. Gene expression is reported as the ratio of the concentration of the target genes, described above, in relation to the concentration of the housekeeping gene (ACT1, EFG1, or RPP2B) and represents the mean expression of different qPCR assays. In this study, to evaluate the efficiency of qPCR amplification and to select the best housekeeping gene, three housekeeping genes
were tested. This analysis was performed by the website http:// www.leonxie.com, which is evaluated by four different methods: Delta CT [21], BestKeeper [22], NormFinder [23], and Genorm [24]. An absolute standard curve was constructed for each primer to calculate its amplification efficiency. Ideally, the efficiency (E) of amplification should range from 90 to 100 % (−3.6> slope>−3.1) and is determined by the following equation: E ¼ 10−1=slope −1 100; Quantitative PCR was used to evaluate the quantity of the cDNA product during the exponential phase of the amplification reaction. The SYBR® Green fluorophore (Platinum® SYBR® Green qPCR SuperMix-UDG Kit, Invitrogen) was used as the detection system. The detection of fluorescence at the end of each PCR cycle permitted to monitor the increase in the amount of amplified cDNA. The data obtained were analyzed with the software of the real-time equipment based on the formula 2−ΔΔCT in comparison to a previously selected internal reference target, which results in a value of 1. Relative quantification is expressed as the variation in the expression level of the genes of interest (SAP5, LIP9, and PLB2) in the strains sensitized by PDI and in the untreated control (calibrator, normalized to 1). In each reaction, the qPCR block was filled, in triplicate, with the sensitized strains and contained specific primers for each target gene and primers for the endogenous control. An untreated control was included which served as calibrator.
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Relative quantification
Fig. 1 Reduction expressed as mean values CFU/mL (log10) and standard deviation in the viability of biofilm cells exposed to methylene blue photosensitizer (P+L+) relative to the control group (P−L−). The P value was 0.01
The results were obtained by comparison of the variation in Ct for the genes of interest in the sensitized strains and in the control strain and are shown in Figs. 3 and 4. The strains tested exhibited different expression profiles after PDI, with a reduction in the expression of the SAP5 gene in 60 % of the strains and in the expression of LIP9 and PLB2 in 50 % of the strains. Comparing the gene expression between the control group (P − L−) and the treated (P + L+) by statistical analysis (Tukey’s test), it was observed that the PDI reduced the gene expression; however, this reduction was not statistically significant (p=0.12) (Fig. 5).
Results Biofilm formation
Discussion
Comparing the CFU/mL between the control group (P−L−) and the treated (P+L+) by statistical analysis (t test), a statistically significant reduction after PDI procedures (p=0.01) was observed (Fig. 1). Efficiency of qPCR amplification The following parameters were observed in all efficiency curves: a slope~−3.32, correlation coefficient ~1.000, efficiency ~100 %, and Ct ≤32. These data are shown in Tables 3, 4, and 5. The best concentration to be used was 1:1 since the Ct value was within its acceptance threshold at this dilution. The PLB2 gene was not expressed in strain ATCC 18804 since its efficiency was beyond the acceptable (116.55), as shown in Table 3. This gene was not included in the relative comparison with strain ATCC 18804 in order to avoid an analysis with inconsistent values. Normalization After evaluation by four methods, Delta CT [21], BestKeeper [22], NormFinder [23], and Genorm [24], the website (http:// www.leonxie.com) recommends the ACT1 gene to perform the relative quantification (Fig. 2). Table 3 Slope, correlation, efficiency, and mean Ct at a 1:1 concentration of the ATCC strain Slope Correlation Efficiency Mean Ct at 1:1 concentration
PDI can be used as a coadjuvant for the treatment of candidiasis since studies have shown that this therapy can reduce the in vitro growth of these microorganisms [12, 17, 25, 26]. Pereira et al. [17] evaluated the in vitro effect of PDI using methylene blue (0.1 mg/mL, 300 μM) as a photosensitizer and low-level laser light (InGaAlP, 660 nm, 98 s) on individual and mixed biofilms of C. albicans, Staphylococcus aureus, and Streptococcus mutans. The number of CFU was counted after PDI and detachment of the biofilm. Greater reductions (log10) were observed for the biofilms of individual species (2.32–3.29) compared to mixed biofilms (1.00–2.44). These results indicate PDI mediated by methylene blue as a new resource for the control of oral biofilms. A statistically significant reduction between the untreated group and the treated group (t test/ p=0.01) was also observed in our study. We therefore chose to apply PDI to C. albicans biofilms using methylene blue and low-level laser irradiation in order to determine whether this treatment, in addition to reducing CFU, also acts on the expression of hydrolytic enzymes. The hydrolytic enzymes SAP5, LIP9, and PLB2 are important virulence factors of C. albicans. The enzyme SAP5 is able to degrade human proteins such as salivary antibodies, mucin, and collagen and to activate the production of other virulence factors by Candida [14], in addition to acting directly on the innate immune system, degrading components of the ACT1
RPP2B
EFG1
SAP5
LIP9
PLB2
−3.31 −0.998 100.5 27.01
−3.47 −0.997 94.17 20.58
−3.52 −1.000 92.34 24.46
−3.39 −1.000 97.23 27.14
−3.32 −0.999 100.08 27.65
−2.98 −0.967 116.55 28.73
Ct cycle threshold, ATCC American Type Culture Collection, ACT β-actin, RPP2B cytosolic ribosomal acidic protein P2B, EFG1 transcription factor, SAP5 secreted aspartyl protease, LIP9 lipase, PLB2 phospholipase B
Lasers Med Sci Table 4 Slope, correlation, efficiency, and mean Ct at a 1:1 concentration of isolate 1 ACT1 Slope Correlation Efficiency Mean Ct at 1:1 concentration
RPP2B
−3.47 −3.24 −0.995 −0.994 94.17 103.53 21.43 16.31
EFG1
SAP5
LIP9
PLB2
−3.55 −0.999 91.28 18.01
−3.47 −0.999 94.17 24.16
−3.47 −0.996 94.17 26.84
−3.47 −0.997 94.17 24.21
Ct cycle threshold, ACT β-actin, RPP2B cytosolic ribosomal acidic protein P2B, EFG1 transcription factor, SAP5 secreted aspartyl protease, LIP9 lipase, PLB2 phospholipase B
Fig. 2 Selection of the best housekeeping gene (ACT1, RPP2B, and EFG1) by the website (http://www.leonxie.com)
complement system such as C3b, C4b, and C5 [15]. Lipase (LIP) and phospholipase (PL) contribute to colonization and infection by degrading components of the host cell membrane [16]. The goal of the study is evaluate the expression of the SAP5, LIP9, and PLB2 genes in C. albicans biofilms after PDI, enzymes that play an important role in the virulence of this yeast. The expression of these enzymes in biofilms has been the subject of many studies. Nailis et al. [18] evaluated the expression of these enzymes during C. albicans biofilm formation on silicone in microtiter plates, in CDC reactors, on polyurethane in an in vivo subcutaneous catheter rat model and on mucosal surfaces in the reconstituted human epithelium (RHE) model. The authors observed more pronounced expression levels of SAP1 in the in vitro models, while the expression levels of SAP2, SAP4, and SAP6 were higher in the in vivo model. Furthermore, SAP5 was positively expressed in the in vivo and RHE models. For SAP9 and SAP10, the expression levels were similar in all models. PLBs were not significantly expressed in the biofilms, while LIP1–3, LIP5– 7, and LIP9–10 were highly expressed in the in vitro models. We did not use an in vivo model but studied clinical isolates from patients with HIV. Naglik et al. [14] used qPCR to identify which SAP genes are highly expressed and potentially contribute to infection with C. albicans in RHE in vitro and in vivo. SAP5 was the only gene that showed significant positive expression in the RHE model and was also highly expressed in isolates of
patients. These results encouraged the use of the enzyme SAP5 in the present study. Samaranayake et al. [27] cultured two C. albicans strains, SC5314 (wild type) and its mutant (HLC54), for 48 h under different conditions, including the presence or absence of silicone disks and human serum. The growth of planktonic and biofilm cells of both strains was monitored at three time points (90 min, 24 h, and 48 h) by a tetrazolium salt reduction assay and by scanning electron microscopy. Additionally, the expression of the virulence genes ALS3, HWP1, EAP1, ECE1, SAP1– SAP10, PLB1, PLB2, PLC, and PLD was analyzed by qPCR. In planktonic cells, expression of the ten SAP genes was initially higher in the wild-type strain, but these genes were highly expressed in the HLC54 strain by 48 h. Overexpression of the ALS3, HWP1, EAP1, ECE1, SAP1, SAP4, SAP6–SAP10, PLB1, PLB2 and PLC genes was observed for wild-type biofilms cultured on serum-treated silicon disks for at least one time point compared to biofilms grown on serum-free disks. The authors concluded that human serum stimulates the growth of C. albicans biofilms on silicone disks and upregulates the expression of virulence genes, particularly the adhesion genes ALS3 and HWP1 and the hydrolase-encoding genes SAPs, PLB1, and PLB2. These data indicate the susceptibility of the human host to colonization with C. albicans and the overexpression of virulence genes in C. albicans biofilms under the conditions studied. These observations support the importance of the present study which evaluated the response of the SAP5, LIP9, and PLB2 genes to PDI.
Table 5
Slope, correlation, efficiency, and mean Ct at a 1:1 concentration of isolate 14
Slope Correlation Efficiency Mean Ct at 1:1 concentration
ACT1
RPP2B
EFG1
SAP5
LIP9
PLB2
−3.39 −0.990 97.23 22.24
−3.34 −0.995 99.25 14.39
−3.60 −0.997 90.00 17.73
−3.29 −0.997 101.34 22.55
−3.34 −0.996 99.25 26.09
−3.28 −0.996 101.78 23.13
Ct cycle threshold, ACT β-actin, RPP2B cytosolic ribosomal acidic protein P2B, EFG1 transcription factor, SAP5 secreted aspartyl protease, LIP9 lipase, PLB2 phospholipase B
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Fig. 5 Statistical analysis of gene expression SAP5, LIP9, and PLB2. The P value was 0.12
Fig. 3 Value of gene expression in the PDI group compared to the control group, normalized to 1, of the Candida albicans lesion strains studied
The precise estimation of the efficiency of qPCR depends on a series of factors such as reagents, experimental design, quality of the sample, and analysis method. Low PCR efficiencies can reduce the accuracy of the replica of the sample and consequently result in poor quantification [28]. Nailis et al. [29] investigated the expression stability of eight potential housekeeping genes (ACT1, PMA1, RIP, RPP2B, LSC2, IMH3, CPA1, and GAPDH) used as reference genes for the study of gene expression in C. albicans biofilms and found the ACT1 gene to be the most stable, in agreement with the present results. In the present study, a difference in behavior was observed between the clinical C. albicans isolates and the reference strain tested. This behavioral difference has also been reported by Rahman et al. [30]. In that study, oral inoculation of the C. albicans reference strain SC5314 into immunocompromised rats induced, in most cases, transient colonization of the oropharynx without development of the disease. However, a C. albicans strain isolated from a patient with oropharyngeal candidiasis colonized the oropharynx of immunocompromised
Fig. 4 Value of gene expression in the PDI group compared to the control group, normalized to 1, of the Candida albicans saliva strains studied
rats, indicating that a clinical isolate can be more virulent than a reference strain. As can be seen in Figs. 3 and 4, the strains tested exhibited different expression profiles after PDI, with a reduction in the expression of the SAP5 gene in 60 % of the strains and in the expression of LIP9 and PLB2 in 50 % of the strains. Since the PLB2 gene did not show good efficiency, it was not included in the relative comparison with strain ATCC 18804, as explained above. The PDI application is the combination of laser and dye and so was the focus of this study. When we compared the expression profile for of each gene between the treated (P+L+ ) and control group (P−L−), a decrease in all gene expression was observed, however no statistically significant difference (Tukey’s test/p=0.12) (Fig. 5). Since other studies in our group [8, 31] showed no change in C. albicans colonyforming units (CFU/mL) in the groups only with laser (light and no methylene) or dye (methylene and no light) and due to the costs of the qPCR technique, we chose not to include these groups in the analyses. The clinical isolates selected for this study were obtained from different patients, and the overexpression of the genes in some isolates and their suppression in others may be a result of the immune status of each individual. This hypothesis is supported by the observation of Pierce and Kumamoto [32] who found that the expression of the EFG1 gene in mice infected with C. albicans differed depending on the immune status of the host. The demonstration that the expression and activity of EFG1 in C. albicans differ during colonization of healthy or immunocompromised mice shows that the microorganism adjusts its physiology when colonizing different hosts. Furthermore, the effects of a healthy host on a heterogenous population of C. albicans containing cells with different levels of EFG1 activity indicate that selective pressure in the host can alter the composition of the population, permitting the population to respond to the immune status of the host. In this study of nine clinical isolates, seven were collected from candidosis lesions of HIV-positive patients with CD4 cell count below 200 cells/mm3 and two were collected from the saliva of patients without Candida lesions and with CD4 cell count
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greater than 200 cells/mm3; however, no correlation between the sample expression profiles in relation to CD4 cell count for any of the genes evaluated was observed. The association between the clinical variables of HIV-positive patients and the oral level of Candida spp. has been frequently discussed; however, the results are still controversial. Some studies reported no association between Candida counts and CD4 lymphocytes [6, 33–36]; on the other hand, other research reported higher frequency of yeast isolation associated with reduced CD4 cell counts [37, 38]. This study was the first to evaluate the effect of PDI on these gene expressions; however, further studies should be conducted to elucidate what the role of PDI treatment and previous isolate condition in the modulation of gene expression in C. albicans is. Within the limits of this study, it could be concluded that PDI (photosensitization with methylene blue followed by low-level laser irradiation) showed a slight reduction on the expression of hydrolytic enzymes of C. albicans, without statistical significance.
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Acknowledgments This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil (Grant: 2012/09188-0).
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ORIGINAL ARTICLE
Evaluation of gene expression SAP5, LIP9, and PLB2 of Candida albicans biofilms after photodynamic inactivation Fernanda Freire 1 & Patrícia Pimentel de Barros 1 & Damara da Silva Ávila 1 & Graziella Nuernberg Back Brito 1 & Juliana Campos Junqueira 1 & Antonio Olavo Cardoso Jorge 1
Received: 2 July 2014 / Accepted: 19 March 2015 # Springer-Verlag London 2015
Abstract With the increasing number of strains of Candida ssp. resistant to antifungal agents, the accomplishment of researches that evaluate the effects of new therapeutic methods, like photodynamic inactivation (PDI), becomes important and necessary. Thus, the objective of this study was to verify the effects of the PDI on Candida albicans biofilms, evaluating their effects on the expression of the gene hydrolytic enzymes aspartyl proteinase (SAP5), lipase (LIP9), and phospholipase (PLB2). Clinical strains of C. albicans (n=9) isolated from patient bearers of the virus HIV and a pattern strain ATCC 18804 were used. The quantification of gene expression was related to the production of hydrolytic enzymes using the quantitative polymerase chain reaction (qPCR) assay. For PDI, we used laser-aluminum-gallium arsenide low power (red visible, 660 nm) as a light source and the methylene blue at 300 μM as a photosensitizer. We assessed two experimental groups for each strain: (a) PDI: sensitization with methylene blue and laser irradiation and (b) control: without sensitization with methylene blue and light absence. The PDI decreased gene expression in 60 % of samples for gene SAP5 and 50 % of the samples decreased expression of LIP9 and PLB2. When we compared the expression profile for of each gene between the treated and control group, a decrease in all gene expression was observed, however no statistically significant difference (Tukey’s test/p=0.12). It could be concluded that PDI (photosensitization with methylene blue followed by
* Fernanda Freire [email protected] 1
Department of Biosciences and Oral Diagnosis, Institute of Science and Technology, Universidade Estadual Paulista (UNESP), Francisco José Longo 777, São Dimas, São José dos Campos, São Paulo 12245-000, Brazil
low-level laser irradiation) showed a slight reduction on the expression of hydrolytic enzymes of C. albicans, without statistical significance.
Keywords Biofilm . Candida albicans . Genes of hydrolytic enzymes . Photodynamic inactivation . qPCR
Introduction In view of the increase in opportunistic infections [1–5] caused by species of the genus Candida in immunodepressed patients and the consequent emergence of strains that are resistant to conventional antifungal agents [6], new treatment options for oral candidosis are required to improve the therapeutic arsenal. In this respect, photodynamic inactivation (PDI) has been proposed as a new option to reduce yeasts of the genus Candida in the oral cavity [7, 8]. PDI is based on the concept that a nontoxic dye, known as a photosensitizer, may be located preferentially in certain tissues or cells and subsequently activated by the harmless visible light to produce reactive oxygen species that can kill cells that bind to the photosensitizer. Numerous published studies have demonstrated that PDI is highly effective in the inactivation of fungi in vitro [9–12]. Additionally, it is believed that the development of resistance by microorganisms PDI is an unlikely event since this is considered as a typical multitarget process, which is a difference between PDI and most antifungal agents [13]. This is the first study to report the action of PDI in the expression of three genes in Candida albicans biofilm related to virulence [14–16]. The goal of the study was to investigate whether PDI using methylene blue and low-power laser acts on the expression of SAP5, LIP9, and PLB2 genes in C. albicans biofilms.
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Materials and methods
Table 1
Code of the isolates used in the study
Origin
Code
ATCC 18804 Patient 1—oral candidiasis lesion Patient 2—oral candidiasis lesion Patient 3—oral candidiasis lesion Patient 4—oral candidiasis lesion Patient 5—oral candidiasis lesion Patient 6—oral candidiasis lesion Patient 7—oral candidiasis lesion Patient 8—saliva Patient 9—saliva
ATCC 1 14 21 31 40 60 70 76S 228S
Ethics committee The project for the collection of clinical isolates was approved by the Ethics Committees of Emílio Ribas Institute of Infectious Diseases (São Paulo, Brazil) and Institute of Science and Technology (UNESP/São José dos Campos Dental School). Isolates The isolates are stored in the Laboratory of Microbiology and Immunology, Institute of Science and Technology (UNESP/ São José dos Campos Dental School) (04569812.5.0000.0077). Nine clinical isolates of C. albicans and one C. albicans reference strain (ATCC 18804) were used. Table 1 shows the origin of the isolates and their codes. Biofilm formation For the biofilm formation, the methods described by Seneviratne et al. [17] and Costa et al. [18] were used with some changes and using 24-well flat-bottom plates (Costar Corning). The strains frozen at −75 °C were activated on Sabouraud dextrose agar (Himedia Laboratories Pvt. Ltd., India) containing chloramphenicol (Alamar Tecno Científica Ltda.) and maintained in an oven for 24 h at 37 °C. Next, a C. albicans inoculum was prepared in yeast nitrogen base (YNB; Himedia) broth supplemented with 100 mM glucose and diluted ten times in sterile distilled water and incubated in an oven for 16 h at 37 °C. The inoculum was then washed twice with sterile 0.9 % NaCl, and standardized suspensions containing 107 cells/mL YNB broth (10×) were prepared in a spectrophotometer (B582, Micronal, São Paulo, Brazil). One milliliter of the yeast suspension was added to the wells, and the plates were incubated in an oven for 1.5 h at 37 °C under shaking at 75 rpm (Quimis, Diadema, Brazil) for initial adherence. After this period, the microorganism supernatant was gently aspirated to remove nonadherent cells (planktonic forms), and each well was rinsed with 1 mL sterile 0.9 % NaCl saline. These steps were repeated twice for removal of nonadherent cells. Then, 1 mL yeast nitrogen base (YNB; Himedia) broth supplemented with 100 mM glucose was pipetted, and the plates were incubated at 37 °C under shaking at 75 rpm (Quimis, Diadema, Brazil) for 48 h. The broth was changed after 24 h. Two groups per strain were evaluated, and one plate was prepared for each group: (1) PDI: sensitization with methylene blue and laser irradiation and (2) control: no sensitization with methylene blue and no laser irradiation. The PDI group received 1-mL methylene blue solution, and the control group received 1 mL sterile 0.9 % NaCl. The plates were shaken for 5 min (pre-irradiation time) in an orbital shaker (Solab,
ATCC American Type Culture Collection
Piracicaba, Brazil). We performed two assays (one for the untreated group and the group treated) with ten isolated under the same conditions of biofilm formation. After the procedures, the samples were plated on Sabouraud agar for counting of colony-forming units (CFU/mL). These values were subjected to t test for statistical analysis. Photosensitizer and laser Methylene blue at a concentration of 300 μM was used for yeast sensitization. The dye solution was prepared freshly each time before use by dissolving the powder (SigmaAldrich, Steinheim, Germany) in sterile distilled water and filtering the solution through a 0.22-μm membrane filter (Millipore, São Paulo, Brazil). After filtration, the dye solution was stored in the dark [19]. An aluminum-gallium arsenide laser (AlGaAs; Laser Fácil, Clean Line, Taubaté, Brazil) was used as the light source. This laser emits continuous light at 660 nm (visible red), the wavelength corresponding to the maximum absorption of methylene blue. The following parameters were used: power of 35 mW, energy of 10 J, time of 285 s, irradiance of 13.78 mW/cm2, and light dose of 3.93 J/cm2. The biofilms were irradiated under aseptic conditions in a laminar flow hood protected from light. The tip was supported on the lid of a 24-well plate toward the bottom of the well where the biofilm was made (approximately 1 cm of distance). A matte black screen with an orifice whose diameter corresponded to the entry of the well was used to avoid the scattering of light. After irradiation of the biofilms, the sterile 0.9 % NaCl or dye solution was discarded and 1 mL TRIzol was added to the first well and then transferred to the following wells until complete removal of the biofilm. The biofilm solution with TRIzol was transferred to 2-mL microtubes, and the tubes were stored in a freezer at −75 °C for subsequent RNA extraction.
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Extraction and measurement of RNA RNA was extracted with the TRIzol kit (Ambion, Inc., AM1926) according to the manufacturer’s recommendations. The extracted RNA was quantified (ng/μL) in a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Absorbance ratios (260/280) of ~2.0 indicate the degree of purity necessary for good performance of the subsequent reactions. DNase treatment of RNA and reverse transcription polymerase chain reaction For removal of contaminating DNA, the extracted total RNA was treated with DNase I (Turbo DNase Treatment and Removal Reagents, AM 1907, Invitrogen) according to the manufacturer’s instructions. After DNase treatment, the RNA was transcribed to complementary DNA (cDNA) using the SuperScript® III FirstStrand Synthesis SuperMix Kit for reverse transcription PCR (Invitrogen) according to the manufacturer ’s instructions.
Table 2
Primers used for real-time PCR
Gene
Position
Reference
ACT1
FW-TTTCATCTTCTGTATCAGA GGAACTTATTT RV-ATGGGATGAATCATCAAAC CAGAG FW-TGCTTACTTATTGTTAGTTC AAGGTGGTA RV-CAACACCAACGGATTCCAA TAAA FW-CATCACAACCAGGTTCTAC AACCAAT RV-CTACTATTAGCAGCACCACCC FW-CCAGCATCTTCCCGCACTT RV-GCGTAAGAACCGTCACCATA TTTAA FW-TGAACCTTTGGGCGACAACT RV-GCCGCGCTCGTTGTTAA FW-CGCAAGTTTGAAGTCAGGAAAA RV-CCCACATTACAACTTTGGCATCT
Nailis et al. [21]
RPP2B
EFG1
SAP5
PLB2 LIP9
Nailis et al. [21]
Hnisz et al. [22]
Nailis et al. [21]
Nailis et al. [21] Nailis et al. [21]
PCR polymerase chain reaction, ACT β-actin, RPP2B cytosolic ribosomal acidic protein P2B, EFG1 transcription factor, SAP5 secreted aspartyl protease, PLB2 phospholipase B, LIP9 lipase, FW forward, RV reverse
Evaluation of the expression of the SAP5, LIP9, and PLB2 genes First, the efficiency of amplification of the ATCC strain and of isolates 1 and 14 (randomly selected) was tested for analysis of the following data: to evaluate the performance of the qPCR assay, to evaluate the best cDNA concentration to be used for qPCR, and to determine whether the target genes were expressed by the strains tested. Three serial base ten dilutions of the samples (1:1, 1:10, and 1:100) were tested in triplicate, and the three dilutions were used for construction of the efficiency curve. The expression of the SAP5, LIP9, and PLB2 genes in relation to a housekeeping gene of choice (ACT1, EFG1, or RPP2B) was evaluated in the clinical isolates and strain ATCC 18804. This evaluation was performed for strains submitted or not to PDI. The primers used for the SAP5, LIP9, PLB2, ACT1, and RPP2B genes have been described by Nailis et al. [18] and the primers for EFG1 by Hnisz et al. [20] and are listed in Table 2. Quantitative polymerase chain reaction The qPCR products were amplified for relative quantification of the expression of the SAP5, LIP9, and PLB2 genes. Gene expression is reported as the ratio of the concentration of the target genes, described above, in relation to the concentration of the housekeeping gene (ACT1, EFG1, or RPP2B) and represents the mean expression of different qPCR assays. In this study, to evaluate the efficiency of qPCR amplification and to select the best housekeeping gene, three housekeeping genes
were tested. This analysis was performed by the website http:// www.leonxie.com, which is evaluated by four different methods: Delta CT [21], BestKeeper [22], NormFinder [23], and Genorm [24]. An absolute standard curve was constructed for each primer to calculate its amplification efficiency. Ideally, the efficiency (E) of amplification should range from 90 to 100 % (−3.6> slope>−3.1) and is determined by the following equation: E ¼ 10−1=slope −1 100; Quantitative PCR was used to evaluate the quantity of the cDNA product during the exponential phase of the amplification reaction. The SYBR® Green fluorophore (Platinum® SYBR® Green qPCR SuperMix-UDG Kit, Invitrogen) was used as the detection system. The detection of fluorescence at the end of each PCR cycle permitted to monitor the increase in the amount of amplified cDNA. The data obtained were analyzed with the software of the real-time equipment based on the formula 2−ΔΔCT in comparison to a previously selected internal reference target, which results in a value of 1. Relative quantification is expressed as the variation in the expression level of the genes of interest (SAP5, LIP9, and PLB2) in the strains sensitized by PDI and in the untreated control (calibrator, normalized to 1). In each reaction, the qPCR block was filled, in triplicate, with the sensitized strains and contained specific primers for each target gene and primers for the endogenous control. An untreated control was included which served as calibrator.
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Relative quantification
Fig. 1 Reduction expressed as mean values CFU/mL (log10) and standard deviation in the viability of biofilm cells exposed to methylene blue photosensitizer (P+L+) relative to the control group (P−L−). The P value was 0.01
The results were obtained by comparison of the variation in Ct for the genes of interest in the sensitized strains and in the control strain and are shown in Figs. 3 and 4. The strains tested exhibited different expression profiles after PDI, with a reduction in the expression of the SAP5 gene in 60 % of the strains and in the expression of LIP9 and PLB2 in 50 % of the strains. Comparing the gene expression between the control group (P − L−) and the treated (P + L+) by statistical analysis (Tukey’s test), it was observed that the PDI reduced the gene expression; however, this reduction was not statistically significant (p=0.12) (Fig. 5).
Results Biofilm formation
Discussion
Comparing the CFU/mL between the control group (P−L−) and the treated (P+L+) by statistical analysis (t test), a statistically significant reduction after PDI procedures (p=0.01) was observed (Fig. 1). Efficiency of qPCR amplification The following parameters were observed in all efficiency curves: a slope~−3.32, correlation coefficient ~1.000, efficiency ~100 %, and Ct ≤32. These data are shown in Tables 3, 4, and 5. The best concentration to be used was 1:1 since the Ct value was within its acceptance threshold at this dilution. The PLB2 gene was not expressed in strain ATCC 18804 since its efficiency was beyond the acceptable (116.55), as shown in Table 3. This gene was not included in the relative comparison with strain ATCC 18804 in order to avoid an analysis with inconsistent values. Normalization After evaluation by four methods, Delta CT [21], BestKeeper [22], NormFinder [23], and Genorm [24], the website (http:// www.leonxie.com) recommends the ACT1 gene to perform the relative quantification (Fig. 2). Table 3 Slope, correlation, efficiency, and mean Ct at a 1:1 concentration of the ATCC strain Slope Correlation Efficiency Mean Ct at 1:1 concentration
PDI can be used as a coadjuvant for the treatment of candidiasis since studies have shown that this therapy can reduce the in vitro growth of these microorganisms [12, 17, 25, 26]. Pereira et al. [17] evaluated the in vitro effect of PDI using methylene blue (0.1 mg/mL, 300 μM) as a photosensitizer and low-level laser light (InGaAlP, 660 nm, 98 s) on individual and mixed biofilms of C. albicans, Staphylococcus aureus, and Streptococcus mutans. The number of CFU was counted after PDI and detachment of the biofilm. Greater reductions (log10) were observed for the biofilms of individual species (2.32–3.29) compared to mixed biofilms (1.00–2.44). These results indicate PDI mediated by methylene blue as a new resource for the control of oral biofilms. A statistically significant reduction between the untreated group and the treated group (t test/ p=0.01) was also observed in our study. We therefore chose to apply PDI to C. albicans biofilms using methylene blue and low-level laser irradiation in order to determine whether this treatment, in addition to reducing CFU, also acts on the expression of hydrolytic enzymes. The hydrolytic enzymes SAP5, LIP9, and PLB2 are important virulence factors of C. albicans. The enzyme SAP5 is able to degrade human proteins such as salivary antibodies, mucin, and collagen and to activate the production of other virulence factors by Candida [14], in addition to acting directly on the innate immune system, degrading components of the ACT1
RPP2B
EFG1
SAP5
LIP9
PLB2
−3.31 −0.998 100.5 27.01
−3.47 −0.997 94.17 20.58
−3.52 −1.000 92.34 24.46
−3.39 −1.000 97.23 27.14
−3.32 −0.999 100.08 27.65
−2.98 −0.967 116.55 28.73
Ct cycle threshold, ATCC American Type Culture Collection, ACT β-actin, RPP2B cytosolic ribosomal acidic protein P2B, EFG1 transcription factor, SAP5 secreted aspartyl protease, LIP9 lipase, PLB2 phospholipase B
Lasers Med Sci Table 4 Slope, correlation, efficiency, and mean Ct at a 1:1 concentration of isolate 1 ACT1 Slope Correlation Efficiency Mean Ct at 1:1 concentration
RPP2B
−3.47 −3.24 −0.995 −0.994 94.17 103.53 21.43 16.31
EFG1
SAP5
LIP9
PLB2
−3.55 −0.999 91.28 18.01
−3.47 −0.999 94.17 24.16
−3.47 −0.996 94.17 26.84
−3.47 −0.997 94.17 24.21
Ct cycle threshold, ACT β-actin, RPP2B cytosolic ribosomal acidic protein P2B, EFG1 transcription factor, SAP5 secreted aspartyl protease, LIP9 lipase, PLB2 phospholipase B
Fig. 2 Selection of the best housekeeping gene (ACT1, RPP2B, and EFG1) by the website (http://www.leonxie.com)
complement system such as C3b, C4b, and C5 [15]. Lipase (LIP) and phospholipase (PL) contribute to colonization and infection by degrading components of the host cell membrane [16]. The goal of the study is evaluate the expression of the SAP5, LIP9, and PLB2 genes in C. albicans biofilms after PDI, enzymes that play an important role in the virulence of this yeast. The expression of these enzymes in biofilms has been the subject of many studies. Nailis et al. [18] evaluated the expression of these enzymes during C. albicans biofilm formation on silicone in microtiter plates, in CDC reactors, on polyurethane in an in vivo subcutaneous catheter rat model and on mucosal surfaces in the reconstituted human epithelium (RHE) model. The authors observed more pronounced expression levels of SAP1 in the in vitro models, while the expression levels of SAP2, SAP4, and SAP6 were higher in the in vivo model. Furthermore, SAP5 was positively expressed in the in vivo and RHE models. For SAP9 and SAP10, the expression levels were similar in all models. PLBs were not significantly expressed in the biofilms, while LIP1–3, LIP5– 7, and LIP9–10 were highly expressed in the in vitro models. We did not use an in vivo model but studied clinical isolates from patients with HIV. Naglik et al. [14] used qPCR to identify which SAP genes are highly expressed and potentially contribute to infection with C. albicans in RHE in vitro and in vivo. SAP5 was the only gene that showed significant positive expression in the RHE model and was also highly expressed in isolates of
patients. These results encouraged the use of the enzyme SAP5 in the present study. Samaranayake et al. [27] cultured two C. albicans strains, SC5314 (wild type) and its mutant (HLC54), for 48 h under different conditions, including the presence or absence of silicone disks and human serum. The growth of planktonic and biofilm cells of both strains was monitored at three time points (90 min, 24 h, and 48 h) by a tetrazolium salt reduction assay and by scanning electron microscopy. Additionally, the expression of the virulence genes ALS3, HWP1, EAP1, ECE1, SAP1– SAP10, PLB1, PLB2, PLC, and PLD was analyzed by qPCR. In planktonic cells, expression of the ten SAP genes was initially higher in the wild-type strain, but these genes were highly expressed in the HLC54 strain by 48 h. Overexpression of the ALS3, HWP1, EAP1, ECE1, SAP1, SAP4, SAP6–SAP10, PLB1, PLB2 and PLC genes was observed for wild-type biofilms cultured on serum-treated silicon disks for at least one time point compared to biofilms grown on serum-free disks. The authors concluded that human serum stimulates the growth of C. albicans biofilms on silicone disks and upregulates the expression of virulence genes, particularly the adhesion genes ALS3 and HWP1 and the hydrolase-encoding genes SAPs, PLB1, and PLB2. These data indicate the susceptibility of the human host to colonization with C. albicans and the overexpression of virulence genes in C. albicans biofilms under the conditions studied. These observations support the importance of the present study which evaluated the response of the SAP5, LIP9, and PLB2 genes to PDI.
Table 5
Slope, correlation, efficiency, and mean Ct at a 1:1 concentration of isolate 14
Slope Correlation Efficiency Mean Ct at 1:1 concentration
ACT1
RPP2B
EFG1
SAP5
LIP9
PLB2
−3.39 −0.990 97.23 22.24
−3.34 −0.995 99.25 14.39
−3.60 −0.997 90.00 17.73
−3.29 −0.997 101.34 22.55
−3.34 −0.996 99.25 26.09
−3.28 −0.996 101.78 23.13
Ct cycle threshold, ACT β-actin, RPP2B cytosolic ribosomal acidic protein P2B, EFG1 transcription factor, SAP5 secreted aspartyl protease, LIP9 lipase, PLB2 phospholipase B
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Fig. 5 Statistical analysis of gene expression SAP5, LIP9, and PLB2. The P value was 0.12
Fig. 3 Value of gene expression in the PDI group compared to the control group, normalized to 1, of the Candida albicans lesion strains studied
The precise estimation of the efficiency of qPCR depends on a series of factors such as reagents, experimental design, quality of the sample, and analysis method. Low PCR efficiencies can reduce the accuracy of the replica of the sample and consequently result in poor quantification [28]. Nailis et al. [29] investigated the expression stability of eight potential housekeeping genes (ACT1, PMA1, RIP, RPP2B, LSC2, IMH3, CPA1, and GAPDH) used as reference genes for the study of gene expression in C. albicans biofilms and found the ACT1 gene to be the most stable, in agreement with the present results. In the present study, a difference in behavior was observed between the clinical C. albicans isolates and the reference strain tested. This behavioral difference has also been reported by Rahman et al. [30]. In that study, oral inoculation of the C. albicans reference strain SC5314 into immunocompromised rats induced, in most cases, transient colonization of the oropharynx without development of the disease. However, a C. albicans strain isolated from a patient with oropharyngeal candidiasis colonized the oropharynx of immunocompromised
Fig. 4 Value of gene expression in the PDI group compared to the control group, normalized to 1, of the Candida albicans saliva strains studied
rats, indicating that a clinical isolate can be more virulent than a reference strain. As can be seen in Figs. 3 and 4, the strains tested exhibited different expression profiles after PDI, with a reduction in the expression of the SAP5 gene in 60 % of the strains and in the expression of LIP9 and PLB2 in 50 % of the strains. Since the PLB2 gene did not show good efficiency, it was not included in the relative comparison with strain ATCC 18804, as explained above. The PDI application is the combination of laser and dye and so was the focus of this study. When we compared the expression profile for of each gene between the treated (P+L+ ) and control group (P−L−), a decrease in all gene expression was observed, however no statistically significant difference (Tukey’s test/p=0.12) (Fig. 5). Since other studies in our group [8, 31] showed no change in C. albicans colonyforming units (CFU/mL) in the groups only with laser (light and no methylene) or dye (methylene and no light) and due to the costs of the qPCR technique, we chose not to include these groups in the analyses. The clinical isolates selected for this study were obtained from different patients, and the overexpression of the genes in some isolates and their suppression in others may be a result of the immune status of each individual. This hypothesis is supported by the observation of Pierce and Kumamoto [32] who found that the expression of the EFG1 gene in mice infected with C. albicans differed depending on the immune status of the host. The demonstration that the expression and activity of EFG1 in C. albicans differ during colonization of healthy or immunocompromised mice shows that the microorganism adjusts its physiology when colonizing different hosts. Furthermore, the effects of a healthy host on a heterogenous population of C. albicans containing cells with different levels of EFG1 activity indicate that selective pressure in the host can alter the composition of the population, permitting the population to respond to the immune status of the host. In this study of nine clinical isolates, seven were collected from candidosis lesions of HIV-positive patients with CD4 cell count below 200 cells/mm3 and two were collected from the saliva of patients without Candida lesions and with CD4 cell count
Lasers Med Sci
greater than 200 cells/mm3; however, no correlation between the sample expression profiles in relation to CD4 cell count for any of the genes evaluated was observed. The association between the clinical variables of HIV-positive patients and the oral level of Candida spp. has been frequently discussed; however, the results are still controversial. Some studies reported no association between Candida counts and CD4 lymphocytes [6, 33–36]; on the other hand, other research reported higher frequency of yeast isolation associated with reduced CD4 cell counts [37, 38]. This study was the first to evaluate the effect of PDI on these gene expressions; however, further studies should be conducted to elucidate what the role of PDI treatment and previous isolate condition in the modulation of gene expression in C. albicans is. Within the limits of this study, it could be concluded that PDI (photosensitization with methylene blue followed by low-level laser irradiation) showed a slight reduction on the expression of hydrolytic enzymes of C. albicans, without statistical significance.
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Acknowledgments This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil (Grant: 2012/09188-0).
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