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Estimating the expression levels of genes controlling biofilm formation and evaluating the effects of different conditions on biofilm formation and secreted aspartic proteinase activity in Candida albicans and Saccharomyces cerevisiae: a comparative study
Beni-Suef University Journal of Basic and Applied Sciences volume 13, Article number: 49 (2024)
Abstract
Background
Characterization of yeast virulence genes is an important tool for identifying the molecular pathways involved in switching yeast virulence. Biofilm formation (BF) and secreted aspartic proteinase (SAP) activity are essential virulence factors that contribute to yeast pathogenicity.
Results
Four Candida albicans and two Saccharomyces cerevisiae strains were tested for BF and SAP activity under optimum conditions, and the expression levels of several genes controlling BF were quantified under the optimal conditions. Biofilm formation was assessed by the microplate method at different pH values, incubation times and culture media. Similarly, SAP activity was assessed at different pH values and incubation periods. The expression levels of nine genes were determined via qRT-PCR technique. All tests were carried out in triplicate, and the values presented as the means ± standard deviations and were analysed with the SPSS programme. Only C. albicans (1), C. albicans (2) and S. cerevisiae 43 formed biofilms. The optimal BF was obtained after culture in sabouraud dextrose broth with 8% glucose at pH 7.5, 4 and 6, respectively, for 48h. Candida albicans biofilm production was more significant than that of S. cerevisiae 43. Moreover, the SAP activity was estimated under the optimum conditions. All yeasts showed optimal SAP activity at pH 4, but astonishingly the SAP activity of S. cerevisiae 44 was higher than that of C. albicans. The expression levels of EFG1 and ZAP1 (transcription factors); ALS3, HWP1and YWP1 (adhesion genes); SAP1 and SAP4 (aspartic proteinase) in C. albicans (1); and FLO11 (adhesion gene) and YPS3 (aspartic proteinase) in S. cerevisiae 43 were quantified during biofilm development at different time intervals. The expression levels of EFG1, ALS3, YWP1, SAP1, SAP4, FLO11 and YPS3 were upregulated at 8 h, while that of ZAP1 was upregulated at 48 h. Only HWP1 was downregulated.
Conclusions
The findings of the present study may provide information for overcoming yeast BF and pathogenicity by regulating specific genes at specific times. Additionally, this study revealed the virulence of the commensal S. cerevisiae, which may take the pathogenicity direction as C. albicans.
1 Background
Fungal infections have frequently increased in recent years [1]. Fungal infections have emerged as a major important public health challenge [2]. The most opportunistic fungal pathogen that causes mucosal to systemic infections is C. albicans. It is responsible for about 70% of fungal infections worldwide. In recent decades, C. albicans has become linked to fatal invasive infections [3, 4].
The yeast pathogenicity mechanism is associated with many virulence factors. Candida albicans undergoes cellular differentiation and transcriptional reprogramming inside the host under the control of different genes to adapt to new habitats with various nutritional elements, pH values, and CO2 and O2 levels [4, 5]. Additionally, Candida albicans pathogenicity is attributed to adhesions to the cell surface, the production of hydrolytic enzymes and biofilm formation, which increase the opportunity of C. albicans to cause infection in various host habitats [6, 7].
On the other hand, Saccharomyces cerevisiae is a safe (nonpathogenic) fungus. It is the most explored microorganism in industrial aids and genetic research [8, 9]. Saccharomyces cerevisiae has traditionally been generally regarded as a safe (GRAS) microbe for nutritional purposes without considering its unfavourable attributes. However, this concept has changed because of the increasing number of infections associated with S. cerevisiae [10]. There is no doubt that these infections are opportunistic. However, studies have revealed that not all strains can cause infection even in the presence of favourable conditions [11]. The morphological change of S. cerevisiae from the yeast form to multicellular pseudohyphae promotes invasive growth [12, 13]. The virulence of S. cerevisiae is related to its ability to grow at high temperatures and to form pseudohyphae [14].
Many microbes, including yeasts, form biofilms as one of the major virulence factors. A biofilm is a community of microbes attached to biotic or abiotic surfaces embedded within an extracellular matrix (ECM) [15]. Candida albicans is one of the most common biofilm-forming yeasts [16, 17]. Candida albicans biofilm development includes four phases: adhesion, proliferation, maturation, and dispersion [18]. Likewise, S. cerevisiae has also been found to form biofilms but in only two stages (adhesion and maturation) [12, 19]. Additionally, S. cerevisiae has been explored as an attractive model for biofilm formation due to its short cell growth cycle, genetic tractability, and easy culture [12, 19].
The morphological transformation between yeast, pseudohyphae and hyphae during biofilm development is associated with pathogenicity and is controlled by many regulatory genes [20, 21]. In C. albicans, several genes are involved in biofilm formation; HWP1 (hyphal wall protein) and ALS3 (agglutinin-like sequence) are associated with biofilm formation by acting as complementary adhesions to biotic and abiotic surfaces [6, 22] and play roles in cell morphology [18, 23]. Additionally, SAP1 and SAP4 (secreted aspartic proteinase) are associated with the adherence process [24]. Moreover, secreted aspartic proteinase promotes the invasion of host tissues through the breakdown of cell surface proteins [25]. EFG1 (enhanced filamentous growth protein) is the central transcription regulator of biofilm formation in C. albicans [26]. Additionally, it is involved in the colonization process. Its expression is related to the host’s immune system state and confers to Candida albicans ability to switch from a commensal status to an opportunistic pathogen status [27, 28]. ZAP1 (zinc-responsive transcription factor) is involved in biofilm maturation by regulating the production of ECM [17, 29]. Additionally, YWP1 (yeast wall protein) plays a vital role in the dispersion phase [30]. The Saccharomyces cerevisiae gene FLO11 (flocculins) is associated with adhesion and biofilm formation, enabling yeast cells to adhere to surfaces and to each other [31, 32]. Moreover, the FLO11 protein forms a part of the ECM of S. cerevisiae mature biofilms [33].
Secreted aspartic proteinase activity is another major virulence factor due to its role in host defense protein hydrolysis and yeast adhesion during biofilm development [34, 35]. The expression of SAP genes leads to biofilm development and raises C. albicans pathogenicity. The variety of SAP genes in host tissues enables the utilization of various kinds of nitrogenous compounds in the host. Hence, the presence and expression of SAP genes provide certain adaptive advantages for Candida spp., especially in response to the selective pressure of antifungal agents [36]. In C. albicans, secreted aspartic proteinase is encoded by ten genes (SAP1 to SAP10) [24, 37]. Saccharomyces cerevisiae has yapsin gene homologues to SAP genes in C. albicans [38, 39]. Yapsin protein 3 (Yps3) shares the most peptide sequence like SAPs in C. albicans [39, 40].
Studying the molecular mechanism of yeast pathogenicity during virulence stages is very important for accurate diagnosis and improvement of therapeutic strategies. Therefore, the objective of this study was to quantify biofilm formation and secreted aspartic proteinase activity under optimum conditions for both C. albicans and S. cerevisiae and following up the temporal profiles of different gene expression levels during biofilm development: EFG1, ZAP1, ALS3, HWP1, YWP1, SAP1 and SAP4 in C. albicans and FLO11 and YPS3 in S. cerevisiae at different time intervals to determine the specific time required for gene activation and to investigate the relationship between biofilm formation stages and switching time of their virulence genes.
2 Methods
2.1 Yeast cultures
2.1.1 Candida albicans
Four identified Candida albicans strains, C. albicans (1), C. albicans (2), C. albicans (3) and C. albicans (4) were used in this study. They were obtained from the Nephrology Department at Theodor Bilharz Research Institute Hospital, Giza, Egypt [41]. They were identified using CHROM agar Candida, Cornmeal-Tween 80 agar and germ tube formation tests as well as biochemical characterization according to Kurtzman and Fell [42].
2.1.2 Saccharomyces cerevisiae
Two strains, S. cerevisiae 43 and S. cerevisiae 44, were obtained from the Mersin Culture Collection. The working cultures were kept at 4 °C on universal agar slants [43].
2.2 Estimation of biofilm formation
Biofilm formation was quantified by using a microplate method as described by Mohammed et al. [22] with some modifications. Yeasts grown overnight on sabouraud dextrose agar (SDA) (Oxoid) were suspended in sabouraud dextrose broth (SDB) with 8% glucose. Suspensions equivalent to 0.5 McFarland were prepared and diluted to 1:20 by SDB with 8% glucose after which 200 μl of each yeast suspension was pipetted into three wells. After 24 h of incubation at 37 °C, the broth medium was removed, and the wells were rinsed with 200 μl of phosphate-buffered saline (PBS). Adhered biofilms were stained with 200 μl of 0.1% crystal violet for 20 min followed by washing with PBS. Crystal violet was solubilized by the addition of 200 μl of acetone: ethanol mixture (20:80 v/v) for 10 min and the absorbance was determined using an ELISA reader (Bio Tek ELx808, USA) at a wavelength of 450 nm. Biofilm formation was detected as reported by Rodrigues et al. [44]. Uninoculated SDB was used as a negative control.
2.3 Optimization of some environmental conditions affecting biofilm formation
The optimization of pH, incubation time and culture media for biofilm formation was determined for the biofilm-forming yeast strains by utilizing SDB with 8% glucose over a wide pH range (4–8 with 0. 5 intervals) and different incubation periods (90 min and 8, 24 and 48 h) and two culture media, SDB with 8% glucose and Roswell Park Memorial Institute 1640 (RPMI 1640) (Caisson laboratories Inc., USA) as mentioned before.
2.4 Detection of secreted aspartic proteinase activity
The secreted aspartic proteinase activity of the tested yeast strains was estimated according to AKçağlar et al. [45] with slight modifications. Overnight-grown yeast on yeast extract peptone dextrose (YEPD) agar [46] at 37 °C was inoculated into a flask containing YEPD broth, and a suspension equivalent to 0.5 McFarland was prepared. Then sterile filter paper disks were dipped into this suspension and applied to bovine serum albumin agar (BSAA) plates. Dipped filter paper disks into uninoculated YEPD broth were used as negative controls. After incubation for 7 days at 28 °C, enzyme activity was recorded according to AKçağlar et al. [45].
2.5 Optimization of some environmental factors affecting secreted aspartic proteinase activity
The optimal environmental conditions, such as pH and incubation time, for SAP production were determined by utilizing BSAA over a wide pH range (4–8 with 0. 5 intervals) and different incubation periods (2, 3, 4, and 5 days) as mentioned before.
2.6 Gene expression analysis
From the previous procedures, C. albicans (1) and S. cerevisiae 43 strains were chosen to evaluate the temporal profile of biofilm-forming genes, as C. albicans (1) formed the strongest biofilm and S. cerevisiae 43 was the only S. cerevisiae strain that formed a biofilm. Gene expression analysis was carried out for the biofilm-forming cells under optimum conditions. Yeast suspensions equivalent to 0.5 McFarland were prepared and diluted to 1:20 using SDB with 8% glucose. A volume of 0.5 ml of yeast suspension was added to polystyrene tubes containing 4.5 ml of SDB with 8% glucose according to Shin et al. [47] and incubated for 90 min and 8, 24 and 48 h at 37 °C. Following incubation, the broth was aspirated, and the adhering biofilms were rinsed with PBS. Biofilm cells were recovered from the tubes according to the methods of Samaranayake et al. [48]. Ribonucleic acid was extracted using a TRIzol kit (Invitrogen Life Technologies, Catalogue # 15596-026) according to the manufacturer’s protocol [49]. Ribonucleic acid quantity and quality were assessed with a NanoDrop 2000c Spectrophotometer (Thermo Scientific, Q992, USA).
The extracted RNA (1µg) was treated with DNase I and 10X reaction buffer with MgCl2 (Thermo Fisher Scientific Inc. kit) followed by complementary DNA (cDNA) synthesis using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions [50] and utilized directly in the qRT-PCR assay. qRT-PCR was done utilizing Maxima SYBR Green/ROX qPCR Master Mix (2X) (Thermo Fisher Scientific Inc.) as described by Longo et al. [51]. Briefly,10 µl of Maxima SYBR Green/ROX qPCR Master Mix (2X), 0.25 μl for each primer, 2 μl of cDNA solution and 7.5 μl of nuclease-free water were added to each gene. Then, the microtubes were subjected to qRT-PCR (Bio-Rad, CFX Connect, Singapore). The amplification programme was run on a two-step cycling protocol: 1 cycle of initial denaturation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 60 s. The primers used were obtained from Macrogen, Korea (Table 1). The qRT-PCR products were analysed by amplification and melting curve analysis. The expression levels of genes were analysed for C. albicans and S. cerevisiae using CEF3 and ACT1 (actin) as housekeeping genes for C. albicans and S. cerevisiae, respectively. Gene expression levels were calculated in relation to 2− ΔΔ ct according to Livak and Schmittgen [52].
2.7 Statistical analysis
Each test was carried out in triplicate, and the acquired values are presented as the mean ± standard deviation (SD) according to SPSS version 16.0 [59]. A t test was used to compare biofilm formation between the positive yeast strain and negative controls and the expression levels of virulence-related genes. A p value < 0.05 indicated a statistically significant difference.
3 Results
3.1 Biofilm formation and optimization of factors affecting biofilm formation
Three out of six yeast strains, C. albicans (1), C. albicans (2) and S. cerevisiae 43, were able to form biofilms while C. albicans (3), C. albicans (4) and S. cerevisiae 44 were unable to form biofilms. Candida albicans (1) showed strong biofilm formation (OD 0.987 ± 0.02) and C. albicans (2) formed moderate biofilm (OD 0.257 ± 0.01), while S. cerevisiae 43 formed weak biofilm (OD 0.091 ± 0.01) (Fig. 1).
The optimization results of the three positive strains revealed that Candida albicans (1) and C. albicans (2) showed optimal and marked significant biofilm formation (ODs of 1.416 ± 0.07 and 0.298 ± 0.01, respectively) at pH 7.5 and pH 4, while S. cerevisiae 43 showed optimal biofilm formation (OD of 0.123 ± 0.02) at pH 6 (Fig. 2). The optimum incubation time for biofilm formation by the three strains was 48 h (Fig. 3). Additionally, SDB with 8% glucose was the optimum medium for biofilm formation by C. albicans (1) and C. albicans (2) strains (ODs of 1.416 ± 0.07 & 0.445 ± 0.03 and 0.298 ± 0.01 & 0.183 ± 0.03 in SDB with 8% glucose and RPMI 1640, respectively), while for S. cerevisiae 43, there was no significant difference in biofilm formation between the two media (ODs of 0.123 ± 0.03 and 0.129 ± 0.03 in SDB with 8% glucose and RPMI 1640, respectively).
3.2 Secreted aspartic proteinase activity and optimization of some factors affecting secreted aspartic proteinase activity
All the tested yeast strains were positive for SAP production. The diameter of the clear zones reflecting SAP activity among C. albicans strains ranged from 0.12 ± 0.03 to 0.18 ± 0.06 cm, while in S. cerevisiae, it ranged from 0.15 ± 0.05 to 0.48 ± 0.03 cm (Fig. 4). Secreted aspartic proteinase activity was detected at pH values ranging from 4 to 6 with 0.5 intervals. All yeast strains showed optimal SAP activity at pH 4. Notably, the highest secreted aspartic proteinase activity (1.02 ± 0.08 cm) was detected with S. cerevisiae 44 in comparison with C. albicans (3) (0.38 ± 0.08 cm) which showed the highest SAP activity among C. albicans strains (Fig. 5). Moreover, there was no clear measurement of SAP activity after 2, 3, 4 or 5 days of incubation.
3.3 Gene expression during different phases of biofilm formation in Candida albicans (1) and Saccharomyces cerevisiae 43
Since C. albicans (1) formed the strongest biofilm and S. cerevisiae 43 was the only S. cerevisiae strain that formed a biofilm, these strains were selected to estimate the temporal profile of biofilm-forming genes. The expression levels of EFG1 and ZAP1 which are transcriptional regulatory genes; ALS3, YWP1 and HWP1, which are adhesion genes; and SAP1 and SAP4, which are aspartic proteinase genes, were quantified in C. albicans (1), while the expression levels of the adhesion gene FLO11 and the aspartic proteinase gene YPS3 were quantified in S. cerevisiae 43. All these genes were quantified at 90 min, 8, 24 and 48 h of biofilm development in relation to the expression levels of the housekeeping genes CEF3 for C. albicans (1) and ACT1 for S. cerevisiae 43 using qRT-PCR. Cells grown for 90 min were used as a control since no biofilm had formed.
In C. albicans (1), the expression level of the EFG1gene showed a fluctuating pattern in which it was highly significantly upregulated by sevenfold at 8 h (7.53 ± 1.006) (p = 0.003) compared with the control, and a highly significant increase at 48 h was observed. However, after 24 h, there was no significant difference in gene expression compared with that at 8 h. For the ZAP1 gene, the expression level was directly proportional to the incubation time. The highest expression level was achieved at 48 h (1.73 ± 0.248) but this upregulation was not significant.
For the adhesion genes ALS3 and YWP1, 8 h had the highest expression level. The ALS3 gene expression level was positively regulated at 8 h (2.51 ± 0.247), but the difference was not significant. The YWP1 gene expression level was significantly upregulated at 8 h (4.74 ± 0.041) (p = 0.004) compared with that in the control group and gradually decreased with increasing time but was still greater than that in the control group. However, the HWP1 gene expression level gradually downregulated, and its expression level was less than that of the control, with no significant difference (Fig. 6).
In addition, the expression level of the SAP genes (SAP1 and SAP4) showed the highest expression level at 8 h. The SAP1 gene expression level was significantly upregulated at 8 h (5. 30 ± 0.394) (p = 0.003) compared with those in the control group and these values gradually downregulated with increasing time. However, the expression level of SAP4 fluctuated. It exhibited the highest level at 8 h (2.22 ± 2.273), but this difference was not significant (Fig. 6).
On the other hand, in S. cerevisiae (43), the expression level of the adhesion gene FLO11 was upregulated twofold, reaching the highest level at 8 h (2.67 ± 2.645), but it was not significant, and there was significant downregulation after 24 and 48 h (Fig. 7). However, the expression level of YPS3 fluctuated, as it was upregulated at 8 h and increased at 48 h to1.34 ± 2.336, with no significant difference, whereas after 24 h, there was a decrease in the gene expression level, which was less than the expression level of the control (Fig. 7).
4 Discussion
Biofilm formation is a main virulence trait for numerous microorganisms [15]. The National Institute of Health demonstrated that biofilms are the cause of 80% of all infections [18]. The formation of biofilms by yeast has gained attention, and many species can grow into communities [60, 61]. The development of biofilms is under the control of different genes [62].
The current study revealed that out of the six yeast strains, two C. albicans strains and one S. cerevisiae strain were able to form biofilms, while the remaining strains were negative for biofilm formation. Candida albicans (1) showed strong biofilm formation and C. albicans (2) formed moderate biofilm while S. cerevisiae 43 formed weak biofilm. These findings are similar to the findings of Ranjith et al. [53], who stated that 4 out of 7 C. albicans isolates were positive for biofilm formation. In addition, Speranza et al. [61] noted that S. cerevisiae strains had the ability to form biofilm. These findings are also supported by the study of Tefiani et al. [1], who found that 53.33% of C. albicans isolates displayed high biofilm formation, while 46.66% exhibited moderate biofilm formation. Like the present results, Chandra et al. [63] revealed that S. cerevisiae is a poorer biofilm producer than C. albicans.
Biofilm formation is affected by nutrient availability, pH, species, and other factors [64]. The influence of these parameters on adhesion could vary from one species to another [61]. C. albicans can adapt to a broad range of pH values and this characteristic is essential for modifying gene expression and morphological changes [65]. Interactions as hydrophobic and hydrogen bonding, covalent bonding, and ionic bonding are involved in adhesion and biofilm development. Thus, it is reasonable to assume that the pH of the medium influences the cell surface properties involved in adhesion and biofilm development [66].
The present study demonstrated that the three biofilm-forming yeast strains had the ability to form biofilms at a wide range of pH values (from 4 to 8). These findings are in agreement with the findings of Mba and Nweze [62], who found that changes in pH promote signalling pathways that enable the microbes to adapt to various pH values. Furthermore, the optimization results revealed that C. albicans (1), C. albicans (2) and S. cerevisiae 43 had optimal pH values of 7.5, 4 and 6, respectively, which indicates that each strain had its own optimal pH for biofilm formation. These findings agree with the findings of Speranza et al. [61], who found that the effect of pH depends on the strain, as they reported that S. cerevisiae strain 4 formed the highest level of biofilm at pH 4–5, while S. boulardii (subtype of S. cerevisiae) formed biofilm at pH 6. The effect of pH on biofilm development is strain dependent, which may be explained by the ability of various strains to utilize various pathways for morphogenesis transition under nutritional and environmental conditions [67]. Previously, Shih et al. [68] stated that nutrient consumption may result in the production of organic acids or other metabolic products and subsequently change the pH of the medium. Candida albicans (1) showed optimal biofilm formation at pH 7.5. Alshanta et al. [65] and Davis et al. [69] stated that C. albicans grows as a yeast at acidic pH and in a filamentous form at alkaline pH. The result for C. albicans (2) at pH 4 is in accordance with those of de Vasconcellos et al. [70] and Ferreira et al. [71], who observed that an acidic pH had a significant positive effect on biofilm development by Candida species.
The present results showed a fluctuating pattern of biofilm formation by C. albicans (1) at different pH values, as the OD representing biofilm formation of C. albicans (1) was high at pH 5–5.5, decreased at pH 6–6.5 and increased again at pH 7 to reach the highest OD at pH 7.5 (Fig. 2), which may be explained as suggested by Verstrepen et al. [72],who stated that surface proteins (involved in adhesion) may be inactive at certain pH values as a result of conformational changes that takes place when the electrostatic charge of surface proteins changes.
The incubation period is very important for the formation of mature biofilms. Adhesion is the first stage of biofilm formation; it takes approximately11h, and it is characterized by adherence and development into distinct microcolonies. The second stage (proliferation) takes 12-30h and is characterized by the formation of a bilayer of yeast, germ tubes and hyphae in addition to ECM production. In the maturation stage, the accumulation of a thick layer of ECM in which yeasts and hyphae are embedded is observed. Dispersion is the last stage in biofilm formation in which nonadherent cells are released to develop other biofilms [63, 73]. The present results showed that the incubation period had a significant effect on biofilm formation and that 48 h was the optimum time for biofilm formation by the three yeast strains compared to shorter incubation periods, revealing that biofilm production by C. albicans and S. cerevisiae strains increased with increasing incubation time. These results are supported by the study of de Barros et al. [28], who concluded that the biofilms produced by C. albicans at different incubation periods, 12, 24 and 48 h, increased with increasing time. Moreover, Serrano-Fujarte et al. [74] noted that biofilm formation significantly increased after a longer incubation time (48 h). Chandra et al. [63] found that in comparison with C. albicans biofilms, S. cerevisiae biofilms exhibited much less growth at 0, 12, 36, 48, 60 and 72 h.
Culture media are vital for the growth and development of microorganisms [75]. The availability of nutrients in growth media has been found to affect microbial adhesion to surfaces; thus, an increase in nutrient levels increases the microbial adhesion rate [76]. This study proved that SDB with 80 g/l glucose was the most suitable medium for C. albicans biofilm development. SDB with 8% glucose contains a high amount of glucose that provides energy for microorganism growth and contains peptone, which supplies vitamins, nitrogen, amino acids, minerals, and growth factors, as stated by PorfÃrio et al. [77]. Moreover, JastrzÄ™bska et al. [78] observed that biofilm formation increased with increasing sugar content in culture media. Furthermore, Santana et al. [79] stated that the ability of C. albicans to produce polysaccharides increases in response to high glucose levels, which modulates biofilm formation and matrix composition. In a related study, Van Nguyen et al. [80] stated that glucose regulates biofilm formation by modulating cell adhesion and allowing planktonic cells to be released from biofilm cells.
This study also revealed that there was no significant difference in S. cerevisiae 43 biofilm formation between SDB with 80 g/l glucose and RPMI 1640 media. RPMI 1640 medium contains 2 g/l glucose and high concentration of Arginine, Asparagine, Glutamine, vitamins, and inorganic salts, as described by Weerasekera et al. [81]. These findings are in agreement with Speranza et al. [61], who reported that S. cerevisiae formed the greatest amount of biofilm when it was grown in rich media containing yeast extract, glucose and peptone and formed the lowest amount of biofilm when it was grown in poor media containing only carbohydrate or nitrogen sources.
All yeast strains were positive for SAP production with optimal activity at pH 4, but their proteolytic activity differed from one strain to another, and surprisingly, S. cerevisiae 44 showed greater proteolytic activity than C. albicans strains. According to several studies, low pH is the optimum pH for the activity of Sap1-Sap3, and high pH is the optimum pH for the activity of Sap4–Sap6 in C. albicans [1, 82]. The Yps1 and Yps3 yapsins family of aspartic proteinases in S. cerevisiae are functional at pH 5–6 [39, 40, 83]. These results are in accordance with those of Germaine and Tellefson [84], who reported that pH 3.8 to 4.0 was the optimum pH for proteinase activity. The results of Younes et al. [85] support the present results, as S. cerevisiae has the ability to secrete proteinase at low pH. The present results also indicate that after 2, 3, 4 and 5 days of incubation, no detectable SAP activity was detected. This result confirmed that seven days is the optimal incubation period for SAP production, as stated by AKçağlar et al. [45].
The development of biofilms is controlled by the expression of different genes and takes place in a sequential process over a period of 24–48 h. Although the expression of genes linked to virulence in biofilms has been extensively examined in bacterial diseases, less is known about the expression of these genes in fungal biofilms [48].
The current results revealed that different genes are responsible for initial adherence and adhesion maintenance. Furthermore, the expression levels of genes vary depending on the gene and the time of biofilm development. This work studied and quantified the temporal expression level of the genes; EFG1, ZAP1, ALS3, YWP1, HWP1, SAP1, SAP4, FLO11 and YPS3, which are involved in the development of biofilm under optimum conditions. To form mature biofilms, cells must remain adhered to surfaces for a long time. The ability of C. albicans to form biofilms has been associated with the presence of transcriptional regulatory genes [28, 81], including EFG1 and ZAP1 [53]. The upregulation of these two genes is related to the increase in the incubation time which in turn caused an increase in biofilm formation by C. albicans, as reported above. Previously, de Barros et al. [28] studied the morphological progression of biofilm formation by C. albicans. A gradual increase in the biomass of the biofilm was detected by increasing the time from 0 to 48 h. Therefore, incubation time is a very important factor for the formation of mature biofilms. The early phase of biofilm formation (adhesion phase) takes approximately 11 h and is characterized by the adherence and development of distinct microcolonies [73]. In this respect, the current results showed that the EFG1, ALS3 and YWP1genes exhibited the greatest upregulation after 8 h. Additionally, biofilm formation by C. albicans is associated with the presence of the adhesion genes ALS3, YWP1 and HWP1 [30, 86]. These genes are also associated with the morphological transition from yeast to the hyphal form [30, 87]. The adherence gene EFG1 regulates the expression of HWP1 and ALS3, which function in cell invasion and biofilm formation [88]. Additionally, EFG1 regulates the expression of YWP1, which is an adhesion maintenance protein [89]. ALS3 is the most important member of the agglutinin-like sequence family involved in biofilm formation because it promotes cell adhesion [62]. The YWP1 gene is responsible for adherence to surfaces during the growth stage [89], and HWP1, which encodes the cell wall’s mannoprotein, also plays a critical role in the development of biofilms [86]. The present findings revealed that 8 h showed the highest expression level for ALS3. However, downregulation of HWP1 expression was observed throughout all time periods. These findings agree with the findings of de Barros et al. [28], who reported that ALS3 gene expression in C. albicans was upregulated at 12 h, and a significant gradual decrease was found at other time intervals (24 and 48 h).
Moreover, Bonfim-Mendonça et al. [24] found that ALS3 expression level in C. albicans isolated from a patient with recurrent vulvovaginal candidiasis increased from 2 to 6 h but decreased in C. albicans isolated from a patient with asymptomatic vaginal candidiasis at 6 h. Additionally, YWP1 gene expression was upregulated at 8 h, which agrees with the findings of McCall et al. [89], who noted that C. albicans strains with YWP1 exhibited a greater initial attachment rate than the wild-type strain and that YWP1had dual functions, as initial attachment, and adhesion maintenance. This result proved the importance of the YWP1 gene in the adhesion process. Moreover, Samaranayake et al. [48] observed that HWP1 expression was upregulated at 90 min and downregulated at 24 and 48 h on silicon biomaterial discs with or without serum. In contrast, Bonfim-Mendonça et al. [24] found that HWP1 expression in recurrent vulvovaginal candidiasis isolate increased from 2 to 6 h. ALS3 and HWP1 expression levels varied according to the isolate’s characteristics [24]. Furthermore, Pokhrel et al. [90] found that ALS3 and HWP1 were upregulated at 8 h. The ability of S. cerevisiae to form pseudofilaments, adhesion and biofilms is not related to the expression of these genes but is associated with the presence of FLO genes [13]. The FLO11 gene is among the FLO gene family in S. cerevisiae, and it is important for cell–cell and cell–surface adhesion by pathogenic yeast to adhere to abiotic surfaces [13]. The present findings revealed that the FLO11 gene was expressed at the highest level in S. cerevisiae after 8 h, which proves the significance of the FLO11 gene in the adhesion phase of biofilm formation. This result was previously reported by Yang et al. [91], who found that the FLO11 gene was upregulated throughout biofilm formation. Moreover, the absence of this gene prevents biofilm formation, as reported by McCall et al. [89], who found that S. cerevisiae with a deleted FLO11 gene fails to make initial attachments.
The maturation stage of biofilm formation is characterized by the accumulation of a thick layer of ECM in which yeasts and hyphae are embedded, which takes 38-72h (Cavalheiro and Teixeira, [73]. In this respect, the current findings revealed that the expression level of ZAP1 gene increased with increasing time and was positively regulated after 48 h. These findings are in accordance with the findings of Jastrzębska et al. [78] and Weerasekera et al. [81] who stated that ZAP1 regulates biofilm maturation and ECM accumulation. These findings are also in accordance with Ranjith et al. [53] who found that EFG1 is required for the maturation phase and that the ZAP1 gene expression level increases throughout the biofilm formation stages, with greater increases occurring at 48 and 72 h of biofilm development, and that ZAP1 gene may be needed for maturation.
Following biofilm maturation, many hydrolases are produced to facilitate host cell penetration and efficient nutrient acquisition. Secreted aspartic proteinase genes (SAP1 and SAP4) are among the SAPs family in C. albicans and have several functions during infection, such as hydrolysis of host tissue barriers, proteins during invasion, distortion of host defense molecules or nutrient acquisition [24, 92]. The present results showed that 8 h of treatment upregulated the expression of SAP1, SAP4 and YPS3. These findings confirm a previous report, showing that the upregulation of SAP1 expression in asymptomatic vaginal candidiasis isolate and of SAP4 in recurrent vulvovaginal candidiasis isolate was observed at 6 h and was associated with the adhesion step [24]. Therefore, SAP genes are associated with virulence processes, such as adhesion, invasion, and immune evasion.
The YPS3 gene is a member of the yapsin family in S. cerevisiae and contributes to proteolytic activity at the cell surface [39, 93]. Additionally, Mercurio et al. [39] stated that S. cerevisiae Yps3 protein has the highest peptide sequence like that of C. albicans SAPs. These results indicate that YPS3 might play a vital role in biofilm formation in S. cerevisiae as SAPs genes function in C. albicans. These findings revealed that the temporal changes in gene expression in response to the commensal S. cerevisiae occurred in the same way as those in response to the pathogenic C. albicans and may be involved in the pathogenesis.
5 Conclusions
This work may serve as a prospective objective for inhibiting the virulence process and yeast pathogenicity by downregulating the studied genes at specific times. Further studies will be crucial to determine whether S. cerevisiae uses biofilms and aspartic proteinase to initiate pathogenicity or accomplish other biochemical processes.
Availability of data and materials
All data generated or analysed in this study are involved in this manuscript.
Abbreviations
- ACT1:
-
Actin 1
- ALS3:
-
Agglutinin-like sequence 3
- BF:
-
Biofilm formation
- BSAA:
-
Bovine serum albumin agar
- CEF3:
-
Cassette protein translation elongation factor
- DNA:
-
Deoxyribonucleic acid
- ECM:
-
Extracellular matrix
- EFG1:
-
Enhanced filamentous growth protein 1
- ELISA:
-
Enzyme-linked immunosorbent assay
- FLO11:
-
Floculins 11
- HWP1:
-
Hyphal wall protein 1
- OD:
-
Optical density
- PBS:
-
Phosphate-buffered saline
- qRT-PCR:
-
Quantitative real-time polymerase chain reaction
- RPMI 1640:
-
Roswell park memorial institute 1640
- RNA:
-
Ribonucleic aid
- SAP:
-
Secreted aspartic proteinase
- SDA:
-
Sabouraud dextrose agar
- SDB:
-
Sabouraud dextrose broth
- SPSS:
-
Statistical package for the social science
- YEPD:
-
Yeast extract peptone dextrose
- YPS3:
-
Yapsins
- YWP1:
-
Yeast wall protein 1
- ZAP1:
-
Zinc-responsive transcription factor
References
Tefiani I, Seddiki SML, Mahdad YM, Boucherit-Otmani Z, Touil HFZ, Bessnouci C (2020) Hydrolytic activity and biofilm formation in clinical isolates of Candida albicans: the effect of changing pH and temperature. Acta Microbiol Hell 65(3):161–170
Guimarães CRE, de Freitas HF, Barros TF (2019) Upregulation of secreted aspartyl proteinase genes of fluconazole-sensitive Candida albicans isolates. Mol Biol Rep 46(6):6147–6154
Talapko J, Juzbašić M, Matijević T, Pustijanac E, Bekić S, Kotris I, Škrlec I (2021) Candida albicans—the virulence factors and clinical manifestations of infection. J Fungi 7(2):1–19
Rai LS, Wijlick LV, Bougnoux ME, Bachellier-Bassi S, d’Enfert C (2021) Regulators of commensal and pathogenic lifestyles of an opportunistic fungus Candida albicans. Yeast 138(4):243–250
Noble SM, Gianetti BA, Witchley JN (2017) Candida albicans cell-type switching and functional plasticity in the mammalian host. Nat Rev Microbiol 15(2):96–108
Mayer FL, Wilson D, Hube B (2013) Candida albicans pathogenicity mechanisms. Virulence 42:119–128
Zhao T, Zhang K, Shi G, Ma K, Wang B, Shao J, Wang T, Wang C (2022) Berberine inhibits the adhesion of Candida albicans to vaginal epithelial cells. Front Pharmacol 13:1–12
Branduardi P, Smeraldi C, Porro D (2008) Metabolically engineered yeasts: potential industrial applications. J Mol Microbiol Biotechnol 15(1):31–40
Fakruddin MD, Hossain M, Ahmed MM (2017) Antimicrobial and antioxidant activities of Saccharomyces cerevisiae IFST062013, a potential probiotic. BMC Complement Altern Med 17(1):1–11
Pérez-Torrado R, Querol A (2013) Opportunistic strains of Saccharomyces cerevisiae: a potential risk sold in food products. Front Microbiol 6(1522):1–5
Lopis S, Hernandez-Haro C, Monteoliva L, Querol A, Molina M, Fernandez-Espinar MT (2014) Pathogenic potential of Saccharomyces strains isolated from dietary supplements. PLoS One 9(5):1–21
Reynolds TB, Fink GR (2001) Bakers’ yeast, a model for fungal biofilm formation. Science 291:878–881
Bouyx C, Schiavone M, François JM (2021) FLO11, a developmental gene conferring impressive adaptive plasticity to the yeast Saccharomyces cerevisiae. Pathogens 10(11):1509
McCusker JH, Clemons KV, Stevens DA, Davis RW (1994) Saccharomyces cerevisiae virulence phenotype as determined with CD-1 mice is associated with the ability to grow at 42°C and form pseudohyphae. Infect Immun 62(12):5447–5455
Ponde NO, Lortal L, Ramage G, Naglik JR, Richardso JP (2021) Candida albicans biofilms and polymicrobial interactions. Crit Rev Microbiol 47(1):91–111
Nett J, Andes D (2006) Candida albicans biofilm development, modelling a host–pathogen interaction. Curr Opin Microbiol 9(4):340–345
Pereira R, Fontenelle RO, de Brito EHS, de Morais SM (2021) Biofilm of Candida albicans: formation, regulation and resistance. J Appl Microbiol 131(1):11–22
Xu Z, Huang T, Du M, Soteyome T, Lan H, Hong W, Peng F, Fu X, Peng G, Liu J, Kjellerup BV (2022) Regulatory network controls microbial biofilm development, with Candida albicans as a representative: from adhesion to dispersal. Bioengineered 13(1):253–267
Jiang Y, Liang C, Zhao W, Chen T, Yu B, Hou A, Zhu J, Zhang T, Liu Q, Ying H, Lui D, Sun W, Chen Y (2022) Cell cycle progression influences biofilm formation in Saccharomyces cerevisiae 1308. Microbiol Spect 10(3):1–11
Jacobsen ID, Hube B (2017) Candida albicans morphology: still in focus. Expert Rev Anti-Infect Therap 15(4):327–330
Felemban SM, Al-Zahrani HA, Fatimah A, Alshehrei F, Elsharawy NT (2019) Candida albicans characters, pathogenesis and effect of ZnO NPs in treatment of candidiasis. Biosci Res Art 16(4):3817–3833
Mohammed NA, Ajah HA, Abdulbaqi NJ (2021) Determination the gene expression levels of adhesins and extracellular enzymes genes in candida albicans biofilm producer by quantitative real time PCR technique (qRT-PCR). Indian J Forens Med Toxicol 15(2):1517–1527
Nobile CJ, Schneider HA, Nett JE, Sheppard DC, Filler SG, Andes DR, Mitchell AP (2008) Complementary adhesin function in C. albicans biofilm formation. Curr Biol 18(14):1017–1024
Bonfim-Mendonça PDS, Tobaldini-Valério FK, Capoci IR, Faria DR, Sakita KM, Arita GS, Svidzinski TI (2021) Different expression levels of ALS and SAP genes contribute to recurrent vulvovaginal candidiasis by Candida albicans. Future Microbiol 16(4):211–219
Silva NC, Nery JM, Dias AL (2014) Aspartic proteinases of Candida spp.: role in pathogenicity and antifungal resistance. Mycoses 57(1):1–11
Theberge S, Semlali A, Alamri A, Leung KP, Rouabhia M (2013) C. albicans growth, transition, biofilm formation, and gene expression modulation by antimicrobial decapeptide KSL-W. BMC Microbiol 13(1):1–14
Pierce JV, Kumamoto CA (2012) Variation in Candida albicans EFG1 expression enables host-dependent changes in colonizing fungal populations. mBio 3(4):1–8
de Barros PP, Rossoni RD, Ribeiro FDC, Junqueira JC, Jorge AOC (2017) Temporal profile of biofilm formation, gene expression and virulence analysis in Candida albicans strains. Mycopathologia 182(3–4):285–295
Gulati M, Nobile CJ (2016) Candida albicans biofilms: development, regulation, and molecular mechanisms. Microb Infect 18(5):310–321
Bachtiar BM, Fath T, Widowati R, Bachtiar EW (2020) Quantification and pathogenicity of Candida albicans in denture-wearing and nondenture-wearing elderly. Euro J Dentist 14(3):423–428
Brückner S, Schubert R, Kraushaar T, Hartmann R, Hoffmann D, Jelli E, Drescher K, Müller DJ, Essen LO, Mösch HU (2020) Kin discrimination in social yeast is mediated by cell surface receptors of the Flo11 adhesin family. Elife 9:1–26
Forehand AL, Myagmarsuren D, Chen Z, Murphy HA (2022) Variation in pH gradients and FLO11 expression in mat biofilms from environmental isolates of the yeast Saccharomyces cerevisiae. Microbiol Open 11(2):1–16
Zara G, Budroni M, Mannazzu I, Fancello F, Zara S (2020) Yeast biofilm in food realms: occurrence and control. World J Microbiol Biotechnol 36(9):1–10
Winter MB, Salcedo EC, Lohse MB, Hartooni N, Gulati M, Sanchez H, Takagi J, Hube B, Andes DR, Johnson AD, Craik CS, Nobile CJ (2016) Global identification of biofilm-specific proteolysis in Candida albicans. mBio 7(5):1–13
Dostál J, Brynda J, Vaňková L, Zia SR, Pichová I, Heidingsfeld O, LepÅ¡Ãk M (2021) Structural determinants for subnanomolar inhibition of the secreted aspartic protease Sapp1p from Candida parapsilosis. J Enzy Inhibit Med Chem 36(1):914–921
Bu QR, Bao MY, Yang Y, Wangm TM, Wang CZ (2022) Targeting virulence factors of Candida albicans with natural products. Foods 11(19):1–22
Naglik JR, Challacombe SJ, Hube B (2003) Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiol Mol Biol Rev 67(3):400–428
Krysan DJ, Ting EL, Abeijon C, Kroos L, Fuller RS (2005) Yapsins are a family of aspartyl proteases required for cell wall integrity in Saccharomyces cerevisiae. Eukaryot Cell 4(8):1364–1374
Mercurio K, Singh D, Walden E, Baetz K (2021) Global analysis of Saccharomyces cerevisiae growth in mucin. G3 (Bethesda) 11(11):1–15
Olsen V, Cawley NX, Brandt J, Egel-Mitani M, Loh YP (1999) Identification and characterization of Saccharomyces cerevisiae yapsin 3, a new member of the yapsin family of aspartic proteases encoded by the YPS3 gene. Biochem J 339(2):407–411
Ashour SM, Kheiralla ZM, Maklad SS, Ameen MR, Zaki SS (2015) Relationship between virulence factors of Candida species with candiduria and myeloperoxidase concentrations. Inter J Curr Microbiol Aappl Sci 4(1):108–123
Kurtzman CP, Fell JW (1998) The yeasts: a taxonomic study, 4th edn. Elsevier, Amsterdam, pp 891–947
Suzuki C, Yamada K, Okada N, Nikkuni S (1989) Isolation and characterization of hallo tolerant killer yeasts from fermented foods. Agric Biol Chem 53(10):2593–2597
Rodrigues LB, Dos Santos LR, Tagliari VZ, Rizzo NN, Trenhago G, de Oliveira AP, Goetz F, do Nascimento VP (2001) Quantification of biofilm production on polystyrene by Listeria, Escherichia coli and Staphylococcus aureus isolated from a poultry slaughterhouse. Brazil J Microbiol 41:1082–1085
AKçağlar S, Ener B, Töre O (2011) Acid proteinase enzyme activity in Candida albicans strains: a comparison of spectrophotometry and plate methods. Turk J Biol 35:559–567
Cassone A, De Bernardis F, Mondello F, Ceddia T, Agatensi L (1987) Evidence for a correlation between proteinase secretion and vulvovaginal candidiasis. J Infect Dis 156(5):777–783
Shin JH, Kee SJ, Shin MG, Kim SH, Shin DH, Lee SK, Suh SP, Ryang DW (2002) Biofilm production by isolates of Candida species recovered from non-neutropenic patients: comparison of bloodstream isolates with isolates from other sources. J Clin Microbiol 40(4):1244–1248
Samaranayake YH, Cheung BP, Yau JY, Yeung SK, Samaranayake LP (2013) Human serum promotes Candida albicans biofilm growth and virulence gene expression on silicone biomaterial. PLoS One 8(5):1–12
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162(1):156–159
Wiame I, Remy S, Swennen R, Sági L (2000) Irreversible heat inactivation of DNase I without RNA degradation. BioTechniques 29(2):252–256
Longo MC, Berninger MS, Hartley JL (1990) Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93(1):125–128
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25(4):402–408
Ranjith K, Chakravarthy SK, Adicherla H, Sharma S, Shivaji S (2018) Temporal expression of genes in biofilm-forming ocular Candida albicans isolated from patients with keratitis and orbital cellulitis. Invest Ophthalmol Vis Sci 59(1):528–538
Finkel JS, Mitchell AP (2011) Genetic control of Candida albicans biofilm development. Nat Rev Microbiol 9(2):109–118
Nobile CJ, Nett JE, Hernday AD, Homann OR, Deneault JS, Nantel A, Andes DR, Johnson AD, Mitchell AP (2009) Biofilm matrix regulation by Candida albicans. PLoS Biol 7(6):1–15
Nailis H, Coenye T, Van Nieuwerburgh F, Deforce D, Nelis HJ (2006) Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR. BMC Mol Biol 7(1):1–9
Sohn K, Urban C, Brunner H, Rupp S (2003) EFG1 is a major regulator of cell wall dynamics in Candida albicans as revealed by DNA microarrays. Mol Microbiol 47(1):89–102
Zhang D, Wang F, Yu Y, Ding S, Chen T, Sun W, Liang C, Yu B, Ying H, Liu D, Chen Y (2021) Effect of quorum-sensing molecule 2-phenylethanol and ARO genes on Saccharomyces cerevisiae biofilm. Appl Microbiol Biotechnol 105(9):3635–3648
SPSS version 16.0 (2007) Users guide, SPSS INC., Chicago
Sardi JDCO, Pitangui NDS, RodrÃguez-Arellanes G, Taylor ML, Fusco-Almeida AM, Mendes-Giannini MJS (2014) Highlights in pathogenic fungal biofilms. Revista Iberoamericana De Micolog. Highlights in pathogenic fungal biofilms. Rev Iberoam Micol 31(1):22–29
Speranza B, Corbo MR, Campaniello D, Altieri C, Sinigaglia M, Bevilacqua A (2020) Biofilm formation by potentially probiotic Saccharomyces cerevisiae strains. Food Microbiol 87(103393):1–10
Mba IE, Nwezem EI (2020) Mechanism of Candida pathogenesis: revisiting the vital drivers. Eur J Clin Microbiol Infect Dis 39(10):1797–1819
Chandra J, Kuhn DM, Mukherjee PK, Hoyer LL, McCormick T, Ghannoum MA (2001) Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol 183(18):5385–5394
Speranza B, Corbo MR (2017) The impact of biofilms on food spoilage. In: Bevilacqua A, Corbo MR, Sinigaglia M (eds) The microbiological quality of food. Woodhead Publishing, Lecce, pp 259–282
Alshanta OA, Albashaireh K, McKloud E, Delaney C, Kean R, McLean W, Ramage G (2022) Candida albicans and Enterococcus faecalis biofilm frenemies: when the relationship sours. Biofilm 4:1–13
Joseph CML, Kumar GR, Su E, Bisson LF (2007) Adhesion and biofilm production by wine isolates of Brettanomyces bruxellensis. Am J Enol Vitic 58:3
Zhu LB, Wang Y, Zhang ZB, Yang HL, Yan RM, Zhu D (2017) Influence of environmental and nutritional conditions on yeast–mycelial dimorphic transition in Trichosporon cutaneum. Biotechnol Biotechnol Equip 31(3):516–526
Shih IL, Tsai KL, Hsieh C (2007) Effects of culture conditions on the mycelial growth and bioactive metabolite production in submerged culture of Cordyceps militaris. Biochem Eng J 33:193–201
Davis D, Wilson RB, Mitchell AP (2000) RIM101-dependent and-independent pathways govern pH responses in Candida albicans. Mol Cell Biol 20(3):971–978
de Vasconcellos AA, Gonçalves LM, Cury AA, da Silva WJ (2014) Environmental pH influences Candida albicans biofilms regarding its structure, virulence and susceptibility to fluconazole. Microb Pathog 69:39–44
Ferreira C, Gonçalves B, Vilas Boas D, Oliveira H, Henriques M, Azeredo J, Silva S (2016) Candida tropicalis biofilm and human epithelium invasion is highly influenced by environmental pH. Pathog Dis 74:1–8
Verstrepen KJ, Derdelinckx G, Verachtert H, Delvaux FR (2003) Yeast flocculation: what brewers should know. Appl Microbiol Biotechnol 61:197–205
Cavalheiro M, Teixeira MC (2018) Candida biofilms: threats, challenges, and promising strategies. Front Med 5(28):1–15
Serrano-Fujarte I, López-Romero E, Reyna-López GE, MartÃnez-Gámez MA, Vega-González A, Cuéllar-Cruz M (2015) Influence of culture media on biofilm formation by Candida species and response of sessile cells to antifungals and oxidative stress. BioMed Res Inter. https://doi.org/10.1155/2015/783639
Gonçalves LNC, Costa-Orlandi CB, Bila NM, Vaso CO, Da Silva RAM, Mendes-Giannini MJS, Taylor ML, Fusco-Almeida AM (2020) Biofilm formation by Histoplasma capsulatum in different culture media and oxygen atmospheres. Front Microbiol 11:1455
Kumar CG, Anand SK (1998) Significance of microbial biofilms in food industry: a review. Int J Food Microbiol 42(1–2):9–27
PorfÃrio BEC, Ribeiro CL, Oliveira MDS, Ribeiro EL, Silva MDRR, Naves PLF (2017) Cell surface hydrophobicity and biofilm formation of Candida yeast species in different culture media. Biosci J 33(3):739–746
Jastrzębska A, Jakubowski WIT, Jastrzębski K, Walkowiak BO (2017) Evaluation of Candida albicans adhesion on AlSl-316 L and Ti6Al4V in changing environmental conditions. Eng Biomater 20(141):2–7
Santana IL, Goncalves LM, de Vasconcellos AA, da Silva WJ, Cury JA, Cury AA (2013) Dietary carbohydrates modulate Candida albicans biofilm development on the denture surface. PLoS One 8(5):e64645
Van Nguyen P, Plocek V, Váchová L, Palková Z (2020) Glucose, Cyc8p and Tup1p regulate biofilm formation and dispersal in wild Saccharomyces cerevisiae. NPJ Biofil Microbio 6(1):1–10
Weerasekera MM, Wijesinghe GK, Jayarathna TA, Gunasekara CP, Fernando N, Kottegoda N, Samaranayake LP (2016) Culture media profoundly affect Candida albicans and Candida tropicalis growth, adhesion and biofilm development. Mem Inst Oswaldo Cruz 111(11):697–702
Dabiri S, Shams-Ghahfarokhi M, Razzaghi-Abyaneh M (2016) SAP (1–3) gene expression in high proteinase producer Candida species strains isolated from Iranian patients with different candidosis. J Pure Appl Microbiol 10(3):1891–1896
Gagnon-Arsenault I, Parisé L, Tremblay J, Bourbonnais Y (2008) Activation mechanism, functional role and shedding of glycosylphosphatidylinositol-anchored Yps1p at the Saccharomyces cerevisiae cell surface. Mol Microbiol 69(4):982–993
Germaine GR, Tellefson LM (1981) Effect of pH and human saliva on protease production by Candida albicans. Infect Immun 31(1):323–326
Younes B, Cilindre C, Villaume S, Parmentier M, Jeandet P, Vasserot Y (2011) Evidence for an extracellular acid proteolytic activity secreted by living cells of Saccharomyces cerevisiae PlR1: impact on grape proteins. J Agric Food Chem 59(11):6239–6246
Nejatbakhsh S, Ilkhanizadeh-Qomi M, Razzaghi-Abyaneh M, Jahanshiri Z (2020) The effects of ellagic acid on growth and biofilm formation of Candida albicans. J Med Microbiol Infect Dis 8(1):14–18
Hoyer LL, Green CB, Oh SH, Zhao X (2008) Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene family a sticky pursuit. Med Mycol 46(1):1–15
Prawiro SR, Dewi A, Diniayuningrum A, Maghfiroh DO, Nurdiana N, Ndharti AT, Rahardjo B (2021) Propolis ethanolic extract influenced biofilm formation and gene expression level of EFG1 in Candida albicans. Am Inst Phys Conf Proceed 2353(1):0301191–0301196
McCall AD, Pathirana RU, Prabhakar A, Cullen PJ, Edgerton M (2019) Candida albicans biofilm development is governed by cooperative attachment and adhesion maintenance proteins. Nat Part J Biofil Microbiol 5(1):1–12
Pokhrel S, Boonmee N, Tulyaprawat O, Pharkjaksu S, Thaipisutikul I, Chairatana P, Ngamskulrungroj P, Mitrpant C (2022) Assessment of biofilm formation by Candida albicans strains isolated from hemocultures and their role in pathogenesis in the zebrafish model. J Fungi 8(10):1–11
Yang L, Zheng C, Chen Y, Ying H (2018) FLO genes family and transcription factor MIG1 regulate Saccharomyces cerevisiae biofilm formation during immobilized fermentation. Front Microbiol 9(1860):1–11
Odds FC (2008) Secreted proteinases and Candida albicans virulence. Microbiology 154(11):3245–3246
Egel-Mitani M, Flygenring HP, Hansen MT (1990) A novel aspartyl protease allowing KEX2-independent MFα propheromone processing in yeast. Yeast 6(2):127–137
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Conceptualization, supervision, data collection and resources were produced by SAT. Experimental design, data collection, methodology, interpretation of the results and writing original draft of the genetic work were achieved by ShSS. Experimental design, data collection, providing the tested yeast strains, methodology, interpretation of the results and writing original draft of the microbiology work were done by SSZ. Data collection, methodology and analysis were done by RGE. All authors read and approved the final manuscript.
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Sobieh, S.S., Elshazly, R.G., Tawab, S.A. et al. Estimating the expression levels of genes controlling biofilm formation and evaluating the effects of different conditions on biofilm formation and secreted aspartic proteinase activity in Candida albicans and Saccharomyces cerevisiae: a comparative study. Beni-Suef Univ J Basic Appl Sci 13, 49 (2024). https://doi.org/10.1186/s43088-024-00504-x
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DOI: https://doi.org/10.1186/s43088-024-00504-x